HORMONAL AND PHYSICAL INTERACTIONS WITH ACETYLCHOLINESTERASE AND CATIONS IN THE PORCINE BRAIN AND HYPOPHYSES IN A HOT HUMID CLIMATE BY DAVID OLUSOJI ADEJUMO B.SC. ANIMAL SCIENCE (IBADAN) M.Sc. ANIMAL SCIENCE (IBADAN) A THESIS IN THE DEPARTMENT OF ANIMAL SCIENCE SUBMITTED TO THE FACULTY OF AGRICULTURE IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHYLOSOPHY UNIVERSITY OF IBADAN AUGUST 1983 UNIVERSI Y OF IBADA LIBRARY i ABSTRACT Prenatal and Postnatal changes in the physiological development of the porcine brain were determined. The results indicated a decline in amniotic fluid volume with increased gestation length. Marked fetal brain and body development was observed between four and six weeks of gestation where mean embryo weight increased by 48,873% from four weeks to six weeks with a concomitant increase of 1,300% in mean fetal brain weight. Acetylcholinesterase (AChE) activity in the amniotic fluid declined progressively with age (r=-0.‘56) while AChE activity in the fetal brain increased with gestation length (r=0.88). Total protein in both the amniotic fluid and in fetal brain did not correlate significantly with gestation length (P>0.05). Specific acetylcholinesterase (SAChE) activities in the amniotic fluid declined significantly between four and six weeks of gestation while activity in the fetal brain increased significantly from 5.12 + 0.39 at 6 weeks to 21.54 + 2.6 at 12 weeks. Postnatally, AChE activity declined significantly with age in the pons, hypothalamus, midbrain, medulla and hypophyses while no significant cnanges were observed in the cerebellum, cerebral cortex and hippocampus. A significant rise was observed in the amygdala. Total protein increased significantly with age in all brain regions and hypophyses while SAChE ssrivities declined steadily with age. Significant and Positive correla­ tions were observed in the calcium and sodium content of the embryonic UNIVERSITY OF IBADAN LIBRARY ii brain while negative correlations were observed in the copper and zinc content. Postnatally, Positive correlations were observed in the calcium, magnesium, potassium, sodium, copper and zinc contents of the pons, cerebellum, medulla and midbrain. The effects of castration at different ages and hormonal therapy on brain and hypophyseal physiology of pigs were also evaluated. Castration significantly depressed AChE activity in the cerebellum, amygdala, hippocampus, hypothalamus, midbrain and medulla in all age groups except at 7-8 months of age while testosterone maintained AChE activity at levels similar to controls. The cortex was not significantly affected except in the Pre-weaners where a depression was recorded. AChE activity in the adenohypophyses of testosterone-treated castrates and controls were similar and inferior to the untreated castrates. Protein levels of all the brain regions and hypophyses of boars were depressed by castration. In addition treated castrates were still inferior to the controls. A decline was also observed in the concentrations of calcium, sodium, potassium, copper and zinc in castrated boars. Exogenous Progesterone or estradiol administration also significantly depressed AChE activity in the cerebellum, amygdala, hippocampus, midbrain and the medulla with the Progesterone - induced depression being significanlty lower than that caused by estradiol. Total protein in the brain regions of ovariectomized gilts was depressed by estrogen and progesterone but to varying extents. Progesterone tended to elevate magnesium and zinc in the amygdala UNIVERSITY OF IBADAN LIBRARY iii hippocampus and cerebellum while estradiol facilitated retention of copper and potassium in several brain regions and the neurohypophyses. Testosterone injection also significantly depressed AChE activity in all brain regions. However no significant differences were observed in the hypophyses. Testosterone further depressed total protein levels in the cerebellum, hypothalamus, cortex, medulla and elevated it in the pons. Testosterone injection in gilts also depressed calcium levels in all brain regions and hypophyses while causing a rise in the magnesium, zinc, and potassium levels in several regions. Lastly, heat stress caused significant increases in respiratory rates and rectal temperatures of heat-stressed boars. Heat stress also elevated AChE and SAChE activities in the Pons, cerebellum, amygdala, hippocampus and medulla. No significant effect was found in the cortex. Total protein levels in the heat-stressed pigs were generally inferior to the controls but the amygdala and cortex were unaffected. Heat stress significantly increased calcium, potassium levels and depressed magnesium, zinc and copper levels in several regions. Water deprivation also depressed AChE activity in the amygdala, medulla and hippocampus but no effect was observed in the cortex. Total protein levels were also depressed by water deprivation in several regions UNIVERSITY OF IBADAN LIBRARY iv whereas SAChE activities were elevated in water deprived animals above the controls. Water deprivation resulted in a decline in Calcium and Sodium levels of several regions while increases were recorded in magnesium, potassium and zinc concentrations in some brain regions and the hypophyses. UNIVERSITY OF IBADAN LIBRARY V ACKNOWLEDGEMENTS My eternal gratitude goes to Professor G.N. Egbunike, ray supervisor for the guidance, encouragement, invaluable help and thorough supervision he rendered to me during the course of this study. In the course of the project, I many times over, shamelessly exploited his infinite sense of tolerance and natural qualities of mind and character. I acknowledge with thanks, the interest and encouragement of Professor A.U. Mba, my former Head of Department, Professor J.A. Oluyemi, my present Head of the Department, Professor B.L. Fetuga of the postgraduate school and Dr. T.I. Dede of the reproductive physiology Unit. I am particularly indebted to Mr. Moshudi Oladeji for his excellent technical assistance and Messrs Taiye Omotosho, John Nwabueze, Sule Ayinla, Sola Adeoye and Theophilus "Baba" Otegbayo for their vital assistance in the management of the experimental animals. To Messrs 0. Amao, Wahab Busari and D. Laditi all of the slaughter house, I must express my profound gratitude for their untiring spirit during their overwhelming and physically exhausting task of slaughtering the staggering number of pigs used for this study. All my research colleagues particularly Mr. E.A. Agiang deserve a big "thank you" for the reciprocal camaraderie existing among us. I also deeply appreciate the vital support of my cousin Mr. Deji Adejumo of Thomas Wyatt, Lagos and the interest of Messrs J. Adewale Adejumo, Dan E. Adejumo and their families. UNIVERSITY OF IBADAN LIBRARY vi To Ifo Amata and Tina Fahm, so far away yet seemingly so near, I appreciate your sincere encouragement all along. My thanks are also due to Folakemi Fatona and Yetunde Ojo for being so nice and for the interest shown in my work. To my Uncle Dr. R.B. Alade and his wife, your invaluable contribution to my work is deeply appreciated. Due thanks also go to Chris Umebese and Carol Umebese for their understanding and friendship and to all others whose contribution I cannot mention for lack of space. I say 'thank you very much'. Upon all, I wish to thank God Almighty for giving me the good health and courage to carry the work through. - ADEJUMO D.O. AUGUST 1983 UNIVERSITY OF IBADAN LIBRARY vii CERTIFICATION I certify that this work was carried out by Mr. David Olusoji Adejumo in the Department of Animal Science, University of Ibadan. I B•Sc•, Ph.D. (Ibadan) Professor in the Department of Animal Science, University of Ibadan. September 1983. UNIVERSITY OF IBADAN LIBRARY viii D E D I C A T I O N This piece of work is dedicated to My mother, Mrs. C. Wuraola Adejumo , my Father, Rev. Cannon M. Oluyemi Adejumo and my brothers Babatunde (Sir T.) and Gboyega (Gbogus) Adejumo. UNIVERSITY OF IBADAN LIBRARY I X TABLE OF CONTENTS Page ABSTRACTS..................................................... i AKNOWLEDGEMENTS............................................... iv CERTIFICATION BY SUPERVISOR................................... vi DEDICATION.................................................... vii CHAPTER ONE: INTRODUCTION.......................................... 1 CHAPTER TWO: INTRODUCTORY LITERATURE REVIEW........................ 8 2.1 The Brain: Anatomical Differentiation and - functions.............................. 8 2.2 The Hypophyses................................ 14 2.3 The Transmitter Substance..................... 15 2.3.1 Biosynthesis of Acetylcholine................. 16 2.3.2 Gross function of Acetylcholine............... 18 2.3.3 Removal of Acetylcholine...................... 18 2.3.4 Structure of Acetylcholine.................... 19 2.3.5 Major factors affecting AChE Activity......... 20 2.3.6 Gross effects of AChE inhibition.............. 21 CHAPTER THREE: PRENATAL AND POSTINATAL CHANGES IN THE DEVELOPMENT OF - THE PORCINE BRAIN..................................... 22 3.1 Development of Fetal Brain DuringG estation.... 22 3.2 Neonatal Development and Sexual Differentiation - of the Brain.................................. 22 3.3 Mineral Composition of the Fetus.............. 27 3.4 Postnatal Development of the Pig Brain........ 28 3.5 Body Mineral Composition of the Pig Fetus...... 30 MATERIALS AND METHODS: 3.1.1 Animals and Management........................ 31 3.1.2 Tissue Processing............................. 32 3.3.1 Acetylcholinesterase Assay.................... 32 3.3.2 Procedure..................................... 33 3.4.1 Determination of Total Protein................ 34 3.5.1 Calculation for Specific AChE Activity........ 34 3.6 Mineral Analyses.............................. 35 3.7 Statistical Analyses.......................... 35 3.8 RESULTS....................................... 35 3.8.1 Intra-uterine Development..................... 36 3.8.2 AChE Activity, Protein Concentration and SAChE Activity in the Conceptus from Four Weeks to Twelve Weeks Gestational Age.................. 38 UNIVERSITY OF IBADAN LIBRARY X Page 3.8.3 Mineral Profile in the Fetal Brain............. 42 3.9 Changes in Acetylcholinesterase Activity and Cations in the Porcine Brain and Hypophyses from Day Old to Four Months of Age.................... 42 1 Postnatal Development.............. ............. 47 '.'.2 Age and Sex Differences on the Regional Distribu­ tion of AChE Activity and Cations of Porcine Brains at Day Old to Four Months of Age................. 51 1.2.0 DISCUSSION....................................... 93 C m R K R FOUR: TnE EFFECT OF AGE AT ORCHIDECTOMY AND TESTOSTERONE ON PORCINE BEHAVIOUR AND BRAIN AND HYPOPHYSEAL PHYSIOLOGY.... 106 4.1.1 INTRODUCTION..................................... 107 4.1.2 LITERATURE REVIEW................................ 108 4.1.3 Porcine Plasma Testosterone...................... 115 4.1.4 Possible Mechanisms of Hormonal Action........... 116 4.1.5 The Recepter Mechanisms.................. 117 4.1.6 The Role of Minerals on Testosterone Metabolism... 118 4.1.7 The Role of the Hypophyseal-Adrenal-Thyroid-Gona- dal Axis in Androgen metabolism.................. 120 4.1.8 MATERIALS AND METHODS............................ 121 4.1.9 Behavioural Tests................................ 122 RESULTS: 4.2.0 Effect of Pre-weaning Castration with or without Testosterone on Porcine Brain and Hypophyseal AChE Activity, Total Protein and SAChE Activity....... 123 4.2.1 Mineral Profile in the Brain and Hypophyses...... 125 4.2.2 Effect of Pre-versus Post-pubertal Castration with or without Testosterone Therapy on Aggressive Sex­ ual Behaviour in Male Pigs.......... ............. 130 4.2.3 Effect of orchidectumy at 3-4 months of Age and Testosterone on the Pig Brain and Hypophyseal Physiology....................................... 133 4.2.4 Mineral profile in the Brain and Hypophyses...... 135 4.3.0 Effect of Orchidectomy at 5-6 Months of Age with or without Testosterone on the Porcine Brain and Hypophyseal Acetycholinesterase level............. 139 4.3.1 Mineral Profile in the Brain and Hypophyses...... 141 4.4.0 Effect of Orchidectomy at 7-8 Months of Age and Testosterone on Porcine Brain and Hypophyses...... 146 4.4.1 Mineral Profile in the Brain and Hypophyses....... 149 DISCUSSION....................................... 153 4.5.0 Orchidectomy and Testosterone Therapy on Sexual Behaviour in Boars.................. ............. 153 4.5.1 AChE Activity, Total Protein and SAChE Activity in the Brain Regions............ 154 4.5.2 Mineral Profile in the Brain Regions............. 157 4.5.3 AChE Activity, Total Protein and SAChE Activity in Hypophyses....................... ............... 161 4.5.4 Mineral Profile in the Hypophyses.......... 162 UNIVERSI Y OF IBADAN LIBRARY xi .gaae, CHAPTER FIVE: EFFECT OF OVARIECTOMY WITH ESTRADIOL OR PROGRESTERONE ON PORCINE BRAIN AND HYPOPHYSEAL PHYSIOLOGY.................... 165 INTRODUCTION................................................ 166 5.1.0 Secretions of the Ovary........... 166 5.1.1 LITERATURE REVIEW .................................. 170 5.1.2 Effect of Ovarian Steroids on Ovariectomized Gilts... 175 5.1.3 Some Metabolic Effects of Ovarian Steroids on Mineral Metabolism.......................................... 178 5.1.4 Materials and Methods............................... 178 5.1.5 Statistical Analyses....................... 178 RESULTS............................................. 179 5.2.0 Effect of Ovariectomy with estradiol or Progesterone on Brain and Hypophyseal AChE Activity, Total Protein and SAChE Activity....... 179 5.2.1 Effect of Ovariectomy with Estradiol or Progesterone on the Mineral Profile of the Porcine Brain and Hypo­ physes.......... 182 5.3.0 DISCUSSION.......................................... 186 5.3.1 Mineral Profile in the Brain........................ 191 5.3.2 AChE Activity, Total Protein and SAChE Activity in the Hypophyses.......................................... 193 5.3.3 Mineral Profile in the Hypophyses................... 194 CHAPTER SIX: EFFECT OF TESTOSTERONE INJECTION OF THE AChE ACTIVITY AND CATIONS IN THE BRAIN AND HYPOPHYSES OF GILTS................. 196 6.1.1 INTRODUCTION........................................ 196 6.1.2 LITERATURE REVIEW................................... 196 6.2.1 MATERIALS AND METHODS............................... 200 6.2.2 STATISTICAL ANALYSIS................................ 200 RESULTS: 6.3.1 Effect of Testosterone Injection on the AChE Activity, Total Protein and SAChE Activity of the Brain and Hypophyses of Gilts................................. 200 6.3.2 Effect of Testosterone Injection on the Mineral Profile in the Brain and Hypophyses of Gilts.............. 202 6.4.1 DISCUSSION.......................................... 205 6.4.2 Mineral Profile in the Brain Regions................ 207 6.4.3 AChE Activity, Total Protein and SAChE Activity in the Hypophses....................................... 209 6.4.4 Mineral Profile in the Hypophyses.......... 210 CHAPTER SEVEN: INFLUENCE OF HEAT STRESS AND WATER DEPRIVATION ON PORCINE BRAIN AND HYPOPHYSEAL ACETYLCHOLINESTERASE AND CATIONS..... 212 INTRODUCTION................................................ 213 7.1.1 Stress............................................. 213 7.1.2 Heat Stress: Its Effect on Body Metabolism......... 213 7.1.3 Water Deprivation: Effects on Body Metabolism....... 214 UNIVERSITY OF IBADAN LIBRARY Xii .?.age LITERATURE REVIEW..................... 214 7.1.4 Heat Stress........................................ 213 7.1.5 Water Deprivation.................................. 219 7.".c Hormones and Electrolytes in water Metabolism...... 220 ~.;.1 MATERIALS AND METHODS.............................. 221 7._.2 Physiological and Climatic measurements............. 222 7.2.3 Statistical Analyses................................ 222 RESULTS........................................................ 222 -.3.1 Influence of Short-term Exposure to tropical sunlight on AChE Activity, Total Protein and SAChE Activity in the Porcine Brain and Hypophyses.......... 223 7.3.2 Influence of Short-term Exposure to tropical Sunlight on the Cations Concentration in the Porcine brain and Hypophyses..................... ..................... 223 7.4.1 Effect of Acute and Prolonged Heat Stress on the AChE Activity, Total Protein and SAChE Activity of the Porcine Brain and Hypophyses................. 228 7.4.2 Effect of Acute and Prolonged Head Stress on the MineralP rofile in the Brain and Hypophyses......... 230 7.5.1 Effect of Acute Water Deprivation on the Activity, Total Protein and SAChE activity in the Porcine - Brain and Hypophyses. ................................ 234 7.5.2 Effect of Acute Water Deprivation on the Mineral Profile in theP orcine Brain and Hypophyses......... 237 DISCUSSION: ................................................ 242 7.6.1 Heat Stress......................................... 242 7.6.2 Mineral Profile in the Brain and Hypophyses.......... 245 7.6.3 Water Deprivation................................... 246 7.6.4 Mineral Profile in the Brain andH ypophyses.......... 250 7.6.5 Summary............................................. 252 CHAPTER EIGHT: 8.1.0 CONCLUSIONS.......................................... 254 REFERENCES.......................................................... 259 APPENDICES.......................................................... 295 UNIVERSITY OF IBADAN LIBRARY x i i i LISTS OF TABLES TABLE TITLE PAGE 3.1 Changes in the Weights of the Reproductive Tract, Embryo and the Embryonic Brain During Gestation............... . 37 3.2 AChE Activities, Protein Concerntration and SAChE activities in the conceptus During Gestation......................... . 37 3.3 Regression Table of AChE Activity, Protein Concentration and SAChE Activity in the Amniotic fluid and Embryonic Brain on Age.......................... ............................... 39 3.4 Changes in the Mineral Profile of the Embryonic Brain during gestation..... .............................................. 39 3.5 Regression table of the Mineral content of the embryonic - Brain on Age................................................ 41 3.6 Regional distribution of AChE Activity, Protein content and SAChE activity in the Brain and Hypophyses.................. 48 3.7 Regional Distribution of Minerals in the Porcine Brain and Hypophyses........ 49 3.8 Post-natal Development in the Pig........................... 50 3.9 Effect of Sex on the Regional Distribution of AChE Activity, Protein content and SAChE activity of the Porcine Brain Regions at Different ages................................... 53 3.1.0 AChE Activity, Protein content and SAChE activity in the Porcine Brain at Different Ages............................. 54 3.1.1 Regression Table of AChE, TOTAL PROTEIN AND SAchE IN THE VARIOUS BRAIN REGIONS ON AGE................................ 55 3.1.2 Effect of Sex on the Regional Distribution of Cations in the Porcine Brain Regions at Different ages................. 65 3.1.3 Regional distribution of the Mineral Profile of Porcine Brain Regions at Different ages............................. 66 3.1.4 Regression Table of Mineral Concentration in the Brain Regions of Pigs on Age................. 67 3.1.5 Effect of Sex on the Development of AChE, total Protein and SAChE Activities in The Hypophyses with increasing Age......................................................... 72 3.1.7 Changes in the Cations content of porcine Hypophyses with Increasing Age 73 UNIVERSITY OF IBADAN LIBRARY x i v Effect of Sex on the Mineral Profile of Porcine Hypophyses at different Ages.......................................... . 74 Regression Table of Hypophyseal Cations, AChE, Total Protein, and SAChE content on Age........................... 92 The Effect of Pre-weaning Castration with or without Test- tosterone on acetylcholinesterase activity in the Pig........ 124 Effect of Pre-weaning Castration and Testosterone on the mineral profile in the Brain and Hypophyses of Pigs....... 127 Parameters of Aggression and Sexual behaviour displayed by pre-versus post-pubertally castrated male pigs compared to gonadally intact mates...................................... 128 The effect of orchidectomy at 3-4 months of Age with or - without Testosterone on Porcine Brain and Hypophyseal Physiology................................................... 134 Effect of orchidectomy at 3-4 Months of Age and Testosterone on the Mineral Profile of the porcine Brain and Hypophyses.... 137 The Effect of orchidectomy at 5-6 Months of Age and Testos­ terone on Porcine Brain and Hypophyseal physiology........... 140 Effect of orchidectomy at 5-6 months of Age and Testosterone on the Mineral Profile of the Porcine Brain and Hypophyses.... 144 The effect of orchidectomy at 7-8 months of Age and Testos­ terone on Porcine Brain and Hypophyseal physiology........... 147 Effect of orchidectomy at 7-8 months of Age and Testosterone on the mineral Profile of the Porcine Brain and Hypophyses.... 150 Effect of ovariectomy with Estradrol or Progesterone Therapy on Porcine Brain and Hypophyseal Physiology............... ... 130 Effect of ovariectomy with estradrol or Progesterone on the Calcium magnesium and zinc levels in the porcine brain and hypophyses.............. .......... .......................... 183 Effect of ovariectomy with estradrol or Progesterone Therapy on the Potassium, sodium and copper levels in the procione brain and Hypophyses.......... ............................... 184 Effect of Testosterone Injection the AChE activity, Total protein and SAChE activity in the Brain and Hypophyses of gilts........................................................ 201 Effect of Testosterone Injection on the Calcium, magnesium and Hypophyses of Gilts........... ....................... UNIVERSITY OF IBADAN LIBRARY XV Page 6.3 Effect of Testosterone injection on the Potassium, Sodium and copper levels in the Brain and hypophyses of Gilts........... 204 7.1.1 Changes in mean Temperature, Relative Humidity, Respiratory rate, rectal Temperature During the Exposure of Boars to direct sunshine....................... ...................... 222 7.1.2 Influence of Short-Term Exposure to Tropical sunlight on AChE activity Total Protein and SAChE activity in the Porcine Brain and Hypophyses.................. 224 7.1.3 Influence of Short-Term Exposure to Tropical Sunlight on the calcium, magnesium and zinc levels in the porcine Brain and Hypophyses.......... 225 7.1.4 Influence of Short-Term Exposure to Tropical sunlight on the potassium, sodium and copper levels in the porcine brain and Hypophyses................................................... 226 7.1.5 Effect of Acute and prolonged heat stress on the AChE activity, total protein and SAChE activity of the porcine Brain and hypo­ physes....................................................... 229 7.1.6 Effect of Acute and prolonged heat stress on the calcium, magnesium and zinc levels in the porcine brain and hypophses.. 231 7.1.7 Effect of Acute and Prolonged heat stress on the Potassium, Sodium and Copper levels in the procine brain and hypophyses.. 232 7.1.8 Effect of Acute water deprevation on the AChE Activity, Total protein and SAChE activity in the porcine brain and hypo­ physes....................................................... 236 7.1.9 Effect of Acute water deprivation on the Calcium, Magnesium and zinc levels in the porcine brain and hypophyses..... . 238 7.2.0 Effect of Acute water deprivation on the potassium, sodium and copper levels in the porcine brain and hypophyses............ 239 UNIVERSITY OF IBADAN LIBRARY x v i LIST OF FIGURES FIGURE TITLE Page 1. Diagramatic Representation of the porcine Brain............ 9 3.1 Relationship Between Gestation length and AChE Activities and Total Protein in the Embryonic Brain and Amniotic fluid...................................................... ZfO 3.2 Relationship Between Gestation length and SAChE Activities in the Embryonic Brain and Amniotic fluid..... . ............4% 3.3 Relationship Between Gestation length and Mineral concentra­ tions in the Embryonic Brain.................... 46 3.4 Relationship between Age and AChE Activities in Different Brain Regions................................................ 60 3.5 Relationship between Age and Total Protein in Different Brain Regions............................... 62 3.6 Relationship between Age and SAChE Activities in Different Brain Regions............................... 64 3.7 Relationship between Age and AChE Activities and Total Pro­ tein in the Hypophyses....................................... 7 ^ 3.8 Relationship) between Age and SAChE Activities in the Hypo­ physes .................................................. 76 3.9 Relationship between Age and Calcium concentrations in the Different Brain Regions...................................... 77 3.1.1 Relationship between Age and Magnesium concentrations in the Different Brain Regions..................................... 80 3.1.2 Relationship between Age and Potassium concentrations in the Different Brain Regions...................................... 82 3.1.3 Relationship between Age and Sodium concentrations in the Different Brain Regions..................................... 84 3.1.4 Relationship between Age and Copper concentrations in the Different Brain Regions..................................... 86 3.1.5 Relationship between Age and Zinc concentrations in the Different Brain Regions..... ................................ 89 3.1.6 Relationship between Age and Minerals concentrations in the Hypophyses.................................................. 9^ UNIVERSITY OF IBADAN LIBRARY 1 CHAPTER ONE INTRODUCTION The demand for pork as a source of protein and energy for the growing world population requires that production be increased in regions which at first glance do not seem suitable for pig keeping and economic pork production. However, the limited ability of the pig to withstand stress presents an obstacle to the attainment of these goals. About one third of the world's pig population is kept in the tropics at temperatures well above the comfort zone for sexually mature pigs. i.e. around 15®C (Steinbach, 1971). Also pigs have a higher body temperature and are less efficient than most other domestic animals in their rate of body heat loss. They are thus particularly susceptible to heat (Ingram, 1965). Ibadan, located in the rain forest zone has a climate characterized by annual temperature of 26.6°C and Relative humidity of 71 percent. Therefore, in order to make positive, practical suggestions to improve pig rearing in the tropics, a knowledge of the physiological reaction of pigs to changed environmental conditions is essential. To survive, an animal must positively react to changes in its environment. This necessitates mechanisms for detecting such changes and putting the appropriate responses into operation. UNIVERSITY OF IBADAN LIBRARY 2 The environment of an animal may be the chemical constituents or the external environment characterized by factors such as climate, disease, feed and management system. While it is well established that the normal growth of an animal hinges on provision of well prepared feeds that are balanced in terms of energy, protein, vitamins, minerals and other additives, it is also well known that the proper functioning of an animal absolutely depends on a sometimes precarious balance between the circulating hormones inside it and the external environmental factors. Thus it is not surprising that an animal that suffers from hormonal dysfunction may consume a lot of balanced feed and still be in a state of negative nitrogen balance. It is also clear that both the animal's internal environment as well as the external environment exert profound effects on the productivity of the animal. Physiological studies have revealed that cells function very well within narrow limits. Quite small fluctuations in osmotic pressure, temperature or the amounts of chemical substances can disrupt biochemical activities and in extreme cases may kill the cells altogether. It is now known that hormones and enzymes are of vital importance to the physiological integrity of the animal and do contribute to its productivity. It is also becoming increasingly apparent that nervous and endocrine systems both function to integrate the organism and are not so divergent and sharply delimited as was formerly assumed. UNIVERSITY OF IBADAN LIBRARY 3 In addition, the products of the coordinatory systems take part in every bodily function and exert gross effects on the mental states and behavioural patterns of the individual, and that important actions may be exerted during developmental stages as well as in mature organisms. Many endocrine glands, through their secretions affect the nervous system, on the other hand endocrine organs are frequently stimulated or inhibited by products of the nervous system. A common physiologic attribute of these two systems is their ability to synthesize and release chemical agents or neurotransmitter that are capable of taking part in the chemical integration of the animals, thus specialized nerve cells produce transmitters that act as chemical messengers either locally e.g. Acetylcholine (ACh) at synaptic junctions or at a distance e.g. oxytocin from the neurohypophysis. The brain, by virture of its position as being the most complex organ in the body enjoys considerable protection from abuse through the highly developed blood-cerebrospinal fluid barrier and the blood- brain barrier. However it is also known that the brain is one of the least resistant organs to stress and the final collapse of the brain function due to stress also marks the death of the animal. It is also established that the brain plays a modulating role on all body functions and through its control on the hypophyseal hormonal pathways keeps the level of hormonal production and metabolism of the other glands in check. The hypophyses, apart from being partly evolved from the UNIVERSITY OF IBADAN LIBRARY 4 hypothalamus is wholy subservient to the brain. It secretes at least nine hormones, six of which directly monitor the release of other endocrine glands concerned with the production of sex-hormones, growth and body metabolism. It is able to perform such a delicate and complex duty by means of its classic short-loop and long- loop feed back systems. There can no longer be any doubt that the relationship between the nervous and endocrine systems is one of reciprocity. The modern view is that the brain is not only an endocrine organ, but also a hormone target. These studies were therefore aimed at (1) Investigating the development of the pig fetal brain during gestation and after birth to four months of age. \ (2) Evaluating the effects of castration and ovariectomy on certain brain and pituitary gland functions. The study was further extended to evaluate the effect of hormonal replacement therapy on such functions. (3) Evaluating the effects of testosterone on the brain and pituitary gland function in intact gilts. (4) Investigating the effects of environmental stress such as heat exposure or water deprivation on the brain and pituitary gland functions. The parameters studied were: (i) Acetylcholinesterase (AChE) activity UNIVERSITY OF IBADAN LIBRARY 5 (ii) Protein concentration (iii) Cations concentration (Calcium, Magnesium, potassium, Sodium, Copper and Zinc). The tropical climate is essentially stressful and has direct effect on the productivity of the animal. In addition, the levels of circulating hormones, particularly the gonadal steroids have been known to adjust in response to stimuli arising from the exterior as well as from within the organism. It is thus essential that effects of these hormones and of their absence on AChE activities of the brain regions and hypophyses be monitored. The brain has been divided into certain regions because the organ itself is anatomically and functionally divided into many parts and although the functions of some brain regions overlap each other there is considerable evidence in support of the fact that there is marked differential distribution of enzymes, proteins, minerals and other chemical constituents in these regions. The protein and mineral(cations) concentrations of these brain regions and hypophyses were also monitored because environmental stimuli and hormones have been known to exert some effects on protein and mineral metabolism in the rat brain. Furthermore, the brain contains complex enzyme systems and these metals are either contained in the enzyme or are activated by them and in some cases the requirement is specific for a particular metal such that the removal of the metal completely inactivates the enzyme. The metals are also essential in the transmission of nervous impulse and their absence abolishes nervous transmission. UNIVERSITY OF IBADAN LIBRARY 6 Where possible, the study has been sex-specific because studies on animal behaviour have indicated sex differences within animals. Arnold, (1980) defined this "organizational hypothesis" as a quantitative or qualitative difference in the synaptic contacts made among neuronal populations involved in a behaviour. For purposes of clarity the studies have been reported in five chapters viz: Chapter three in the first part deals with the Ontogenic changes in AChE activity and mineral concentrations of the embryonic brain from four to twelve weeks of gestation. The second part deals with the development of the same parameters in the pig brain from birth to four months of age. Chapter four investigates the effects of prepubertal and post pubertal castration with or without testosterone therapy on the brain and hypophyseal AChE activity and cations. Chapter five considers the effects of ovariectomy with or without estradiol or progesterone therapy or the brain and hypophyseal AChE activity and cations in the gilt. Chapter six reports the effect of testosterone on the brain and hypophyseal AChE activity and cations in the gilt. Chapter seven deals with the effects of environmental stress on brain and hypophyseal AChE activity and cations. This is reported in three sections. UNIVERSITY OF IBADAN LIBRARY 7 The first two sections deal with short-term heat exposure of boars to sunlight while the third investigates the effects of acute water deprivation on the brain and hypophyseal parameters. UNIVERSITY OF IBADAN LIBRARY -8- CHAPTER TWO INTRODUCTORY LITERATURE REVIEW 2.1.1 THE BRAIN: ANATOMICAL DIFFERENTIATION AND FUNCTIONS For the purpose of this study, certain brain regions would be considered. The brain is an enlongate, oval body, widest in the caudal third (Fig. 2.1) It is anatomically and functionally divided into many parts but for the purpose of this study the regions investigated were: 1. Pons 2. Cerebellum 3. Amygdala 4. Hippocampus 5. Hypothalamus 6. Cerebral cortex 7. Mid Brain (mesencephalon) 8. Medulla oblongata. The heterogeneity of cell types and regional differences in the brain have a major influence on the movement of materials in and out of brain tissues and the functions of the various regions overlap each other. THE DIENCEPHALON: It includes the epithalamus, subthalamus and hypothalamus. The hypothalamus lies below or ventral to the thalamus and forms the floor and part of the inferior lateral walls of the third ventricle. UNIVERSITY OF IBADAN LIBRARY Fig. 2*1 • Diagramatic representation of the porcine brain. A: Dorsal view. B: Ventral view. C: Midsagittal view. 1: Cerebral cortex; 2: Cerebellum ; 3: Medulla oblongata; 4: Hypothalamus; 5: Amygdala; 6: Pons Varoli; 7: Mesencephalon (Midbrain); 8: Hippocampus with the arrow showing the point and direction of tracing from the septum pel lucidum From Egbunike 1981) ' UNIVERSITY OF IBADAN LIBRARY -10- The hypothalamus is one of the most important areas of the brain and regulates sexual behaviour. It is also believed that the behavioural components that accompany mating are regulated by the limbic system and the hypothalamus. It is now known that neurosecretory neurons in the hypothalamus synthetize at least nine peptide hypophysiotrophic hormones (releasing and inhibiting factors) and discharge them into the medial eminence (Schally et al. , 1977). Therefore it is the hypothalamus which provides a link between the brain and the hypophyses. Both the ventromedial hypothalamus and the lateral hypothalamus also help in regulating body weight (Xeesy and Powley, 1975). In addition, cholinergic compounds that influence the release of gonadotropins and prolactin are thought to exert at least a portion of their action indirectly through pathways which terminate in hypothalamus (Hahlweg and Jonkmann, 1932, Liberton and McCann, 1974). Also ACh* sensitive neurons in the lateral hypothalamus are specifically concerned with the regulation of glucagon synthesis (Matsushita et al., 1979). It is also considered that the hypothalamus, the anterior thalamic nuclei, the cingulate gyrus, the hippocamus and their interconnections may serve as a structural and functional unit for emotion. Neurons within the hypothalamus regulate the basal level of pituitary activity responsible for folllicular growth and estrogen secretion (Elerko, 1966; Everetts, 1969; Gorski, 1971a). The hypothalamus is also involved in the control of pain. UNIVERSITY OF IBADAN LIBRARY 2. 1.2 THE BRAIN STEM The brain stem is defined anatomically to include the medulla oblongata, pons and midbrain (Mesencephalon) 2.1.2.1 THE PONS The pons lies ventral to the cerebellum and anterior to the medulla from which it is seperated by a groove through which the abducen, facial and acoustic nerves emerge. The AChE-containing fibres of the fore brain are assumed to be derived in large part from the brain stem. These form the ascending cholinergic reticular system. Lewis and Shutte (1967) demonstrated the presence of considerable AChE-containing fibres in the rat hippocampus, brain stem, medial septa, mid brain to the thalamus, hind brain (cerebellum) cortex and amygdaloid bodies through various pathways (Gerebtzoff, 1959, Koelle, 1950). The pons integrates basic reflex behaviour and cruder intellectual functions. Hence damage to the pons usually results in facial paralysis. 2.1.2.2 MEDULLA OBLONGATA It is the pyramid-shaped portion of the brain stem between the spinal cord and the pons. The lower half contains a central canal. The medulla oblongata together with the pons are divided into the motor and sensory pathways. The medulla oblongata maintains nervous co­ ordination and muscle tone. It also co-ordinates reflexes concerned with respiration, swallowing, vomitting and cardiovascular controls. Other functions include regulation of blood pressure, salivation and response due to stimuli. UNIVERSITY OF IBADAN LIBRARY -12- 2.1.3 THE LIMBIC SYSTEM The limbic system is also referred to as rhinencephalon and it includes the amygdala, hippocampus, cingulate gyrus and septum. The amygdala is a small spherical gray mass located in the roof of the terminal part of the inferior horn of the lateral ventricle. The hippocampus extends along the interiomedian aspect of the temporal lobe from the area of the splenium of the corpus callosum to the Uncus (see Fig. 2.1). The limbic system has been demonstrated to influence gonadotropin secretion, the stimuli arriving from the amygdala being facilitatory and those of the hippocampus inhibitory (Koikegami et al., 1954, Bunn and Everett, 1957; Velasko and Taleisnik, 1969a, b, Kawakami, et al., 1971). Injection of cholinergic agents into either the hippocampus or the amygdala can induce seizures not produced by injections of control drugs (Grossman, 1963; McLean, 1955). Hippocampus pyramidal cells receive a cholinergic input from the septum. They also project back to the septum where they contribute to the feed back regulation of the septal cholinergic neurons (Lewis et al.,1967). Click et. al̂ (1973) also postulated that newly synthetized ACh released at the hippocampal synapse is essential for mice to learn a passive avoidance response. The hippocampus forms one of the richest cholinergic structures in the brain (Lewis and Shutte, 1967). The amygdala exerts a modulating-influence on the hypothalamic-hypophyseal system for the secretion of certain trophic hormones (Koikegami et al., 1954). UNIVERSITY OF IBADAN LIBRARY -13- 2.1.4 THE BRAIN It is also known as mesencephalon. It is a short portion of the brain between the pons and cerebral hemispheres. The mid brain controls the eye muscles, involuntary movements and posture. 2.1.5 CEREBRAL CORTEX The cerebral cortex controls all sensory, (olfactory, sight, smell etc.) and memory functions. It also controls all co-ordinated movements which include control of voluntary movements of the skeletal muscle. AChE is particularly active in the cingulate cortex and part of paleocortex - which are regions which regulate behaviour (Gerebtzoff, 1959). It is of interest to note that depriving the eyes of light reduces the amount of AChE in the retina (Glow and Rose, 1964) while a visually enriched environment combined with training in visual problems results in an increase in the cortical content of AChE (Holoway, 1966). Behavioural stimulation also depletes AChE activity in the visual cortex (Krech et at., 1966). 2.1.6 CEREBELLUM The cerebellum is a great oval folded dorsal expansion of the hind brain. It is located in the posterior fossa of the skull and seperated from the overlying cerebrum by an extension of dura matter, the tentorium cerebili. The cerebellum keeps the individual oriented in space by controlling balance and maintenance of posture, equilibirum and fine adjustments of movements. It also controls the anti-gravity muscles of the body (Chusid, 1970). UNIVERSITY OF IBADAN LIBRARY -14- Lewis and Shutte, (1967) demonstrated presence of considerable AChE activity in the rat hind brain and cerebellum among other brain parts. 2.2 THE HYPOPHYSES The pituitary glands or hypophyses are joined anatomically to the hypothalamus by a slender stalk and are believed to be subservient to and to have partly evolved from the hypothalamus. The hypophyses consists of an adenohypophysis and a neurohypo­ physis and these two subdivisions are distinctly different in embryonic origin and in histological composition. The hypophyses secrete at least nine hormones; six of these hormones are from the adenohypophysis. They all exert their effects indirectly by stimulating the functional activities of other endocrine glands. The hormones of the neurohypophysis (Vasopressin and oxytocin) are the products of hypothalamic secretory cells and are stored and released from the pars nervosa. All neurons within the hypothalamus regulate the basal level of pituitary activity responsible for gonadotropin secretion (Elerko, 1966; Everett, 1969). Apart from the gonadotropins, the adenohypophysis secretes two hormones; Adrenocorticotrophin (ACTH) and Thyrotrophin (TSH) which are useful parameters of emotional disturbances such as fear, anxiety (Dupont et al., 1973), or response to cold stress or non-specific stress (Fortier, 1973). For instance, Dupont et al (1971) a, b) found a positive correlation between passive avoidance learning and the plasma corticosterone concentration, used as an index of ACTH release in rats. UNIVERSITY OF IBADAN LIBRARY -15- Contrariwise, active avoidance learning proved inversely related to plasma corticosterone level and positively related to the plasma TSH concentra­ tion. It is also well known that the maintenance of a basic level of gonadotropin secretion depends on the adenohypophysis which in turn depends on its connection with the hypothalamus. The developmental changes of several enzyme and hormone systems in the rat brain from fetal life to birth and onwards with particular emphasis on their contributions to sexual differentiation have been investigated by a number of workers. Before the roles of these enzymes and hormones are reviewed it is necessary to review the role of the enzyme investigated in this study. 2.3 THE TRANSMITTER SUBSTANCE The transmitter substance at the majority of synapses is acetylcholine (ACh) which is the acetyl derivative of chlorine which may be considered to be a substituted ammonium hydroxide compound thus: UNIVERSITY OF IBADAN LIBRARY -16- H-N-H. OH Ammonium hydroxide CH. CHn Crt,-Nt-CH„2. C' -H^.CH^.OH OH'* ‘*2 2 ’ C H 3“ N+- C H 2 .C H 2 .0 .C 0 .C H 30H" c h 3 CH-, choline Acetyl choline Acetycholine has been found to be present in three distinct subcellular compartments namely in nerve endings, axons and bodies of nerve cells (de Robertis, 1964). A further candidate for consideration as a transmitter in the central nervous system on the grounds of its presence in brain extracts is nora drenaline. Nerve fibres which form and release acetylcholine as transmitters are called cholinergic while those which form and release an adrenaline­ like substance (now known to be non-adrenaline) are known as adrenergic. 2.3.1 BIOSYNTHESIS OF ACETYLCHOLINE Acetylcholine is synthesized by a specific enzyme choline acetylase or choline acetyltransferase which occurs in all cholinergic neurons. UNIVERSITY OF IBADAN LIBRARY -17- In addition to choline acetylase, free choline, acetylco-enzyme A, Adenosine triphosphate (ATP) and glucose are required. The synthesis takes place in the mitochondria (Ganong, 1979). Acetylcholine is therefore the mediator at all synapses between preganglionic and postganglionic fibres of the autonomic nervous system, at the myoneural junction and all post ganglionic para-sympathetic and sympathetic endings. On the other hand, noradrenaline is the mediator at most post ganaglionic sympathetic endings in the autonomous nervous system (Ganong, 1979). The mode of action of ACh can either be muscarinic or nicotinic depending on the ganglionic location of the cholinergic neurons. The biosynthesis and catabolism of ACh is as shown below: CHOLINE Acetyl - COA choline acetyl transferase CH-* C - 0 - CH? C H , -> N + - CH-j / \ CH3 Cfi3 Acetylcholine Acetylcholinesterase Cho'vline Acetate UNIVERSITY OF IBADAN LIBRARY -18- 2.3.2 GROSS FUNCTION OF ACETYLCHOLINE Acetylcholine has been known to be responsible for aggressive behaviour in cats and local infusion of ACh produces renal vasolidation in rat kidney (Daugherty et a_l., 1968). ACh also causes excitation of the heat production pathway with inhibition of the heat loss pathway (Findlay and Thompson, 1968). This action is also considered to be temperature regulatory and is discussed in more detail in subsequent chapters. Newly synthesized ACh appears to be more readily released on nerve stimulation than depot or stored ACh. About half of the choline produced by AChE activity is re-utilized to synthesize new ACh (Collier and Ilson, 1977). Other extra-neurotransmitter activity of ACh are: 1) Stimulation of inorganic phosphate into phospholipids. 2) Release of thiamine (Itokama and Cooper 1970). 3) Release of amines (Catecholamines) from chromaffin cells. 4) Ciliary motility of respiratory oesophageal tracts. 2.3.3 REMOVAL OF ACETYLCHOLINE As a result of its role in stimulating nervous impulses in organisms, acetylcholine must be rapidly removed to prevent the body from being in a state of constant excitation. The removal of ACh is brought about by two major pathways. 1) DIFFUSION: In a few sites, diffusion is almost rapid enough to account for the rate of decay in action of ACh. However, in most sites, there is a barrier to free diffusion of ACh. UNIVERSITY OF IBADAN LIBRARY -19- 2) ACETYLCHOLINESTERASE (AChE) HYDROLYSIS OF ACh: AChE is a glycoprotein enzyme (Leuzinger and Baker, 1967, Ciliv and Ozano, 1972) and is partly associated with the smooth endoplasmic reticulum of cholinergic axons (Kasa, 1970; Tennyson et al., 1968, Somogyi et al., 1975). The mode of action of AChE involves the hydrolysis of ACh into its physiologically inactive products: acetate and choline. 2.3.4 STRUCTURE OF ACETYLCHOLINE Two separate areas can be distinguished on the active surface of AChE. These consist of an anionc (N+ ATTRACTIVE) site and an esteratic (ester-binding) site. (Augustinsson, 1963). The structure is given below: The anionic site is capable of coulombic interaction with the trimethyl ammonium group of acetylcholine and this interaction facilitates a favourable orientation on the enzyme surface so that the hydrolysis of ACh is carried out by the esteratic site. The hydroxyl of serine is the main functional group of the esteratic site (Kabachuik et al. 1970). UNIVERSITY OF IBADAN LIBRARY -20 Since it is also known that the functional groups of the esteratic and anionic sites that react directly with the substrate are probably very similar, if not identical in the two main types of cholinesterases: Specific ChE (E.C. 3.1.1.7) and Pseudo ChE (E.C. 3.1.1.8), it is therefore logical to suppose that the type of cholinsterases is determined not by differences in the structure of the active centres but by some differences in the structure of various areas around the active centres. 2.3.5 MAJOR FACTORS AFFECTING AChE ACTIVITY The following factors have been known to influence AChE activity: INHIBITORS One of the most fruitful studies of AChE has centered on its inhibition by a variety of chemical agents and drugs called "anticholinesterases". The administration of such highly specific AChE inhibitors results in the accumulation of AChE and arrest of synaptic transmission. The death of an animal treated in vivo with such agents is associated with a complete inhibition of its brain AChE. In vitro, the addition of such an inhibitor to the medium bathing the nerves results in an alteration of the action potential (Mazur and Harrow, 1966). Such inhibitors include physostigmine (eserine) and neostigmine which are reversible inhibitors. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor and is the most powerful inhibitor even at concentrations as low as 1 x 10”^M. It is also a powerful nerve poison but the kidney possesses a mechanism for detoxifying it (Mazur and Harrow, 1966). UNIVERSITY OF IBADAN LIBRARY -21- 2.3.6 GROSS EFFECTS OF AChE INHIBITION Since the destruction of AChE prolongs the effects of ACh, persons suffering from AChE inhibition may exhibit convulsive contractions of all muscles upon the slightest stimulation. AChE inhibition in rats reduced food intake, and increased the level of water intake (Adams, 1983). It also reduced the level of spontaneous activity. Cholinesterase inhibition also produced increased sexual behaviour with induced aggressive behaviour in conscious rats (Hoyland et al., 1970; Ferguson et al., 1970) and also increased locomotor activity (Fibiger and Campbell, 1971). Inhibition by eserine has been known to cause gross emotional, autonomic and motor phenomena such as agitation, fear-like responses, anger, rage, itching, respiratory embarrasment, salivation, ataxia, tremor, circling and clonic-tonic convulsions in rabbits (Beleslin et al., 1973). Studies by Kozar et al (1976) indicate that recovery of AChE activity in some brain areas after chemical depression preceeds the return of the relevant behaviour to base-line level. UNIVERSITY OF IBADAN LIBRARY 22 CHAPTER THREE PRENATAL' AND POSTNATAL' CHANGES IN THE DEVELOPMENT OF THE PORCINE BRAIN LITERATURE REVIEW 3.1. DEVELOPMENT OF FETAL BRAIN DURING GESTATION The somatic stability or vigour of newborn animals is associated with their physiological development during pregnancy. Several workers have emphasized the need for a study of the changes taking place in the body composition of pig fetuses. Such knowledge enhances correct feeding of the pregnant sow as well as that of the piglets after birth. Warwick (1928), Mitchell et al (1931), Urbany (1952) and Pomery (1960) have made detailed studies of fetal weight gains during pregnancy. Their results indicate that very little fetal weight gains occur up to the 50th day of pregnancy. More recent result from Padalikova etal (1972) and Newland and Davis (1974) indicate that by the 80th day of pregnancy, the fetus weighs up to 30 - 4035 of its final weight. They further linked this with a very rapid development during the last 30 days of intra uterine life. On the other hand, Day (1972) reported that nearly complete morphologic development takes place around mid-gestation. 3.2. NEONATAL DEVELOPMENT AND SEXUAL DIFFERENTIATION OF THE BRAIN Information is still very scanty on the metabolic activities occuring in the brain of the fetal pig. However, some considerable UNIVERSITY OF IBADAN LIBRARY 23 amount of work has been done on other species particularly the rat. This may not however be consolatory because the work of Reis et al (1977) indicates that some of these events during fetal growth are species-specific and are not paralleled by observations made in other species. For instance, McEwen (1978,a,b) observed that during the late fetal life in the rat, the brain undergoes a series of changes which appear to be related to its susceptibility to hormones in undergoing sexual differentiation. These include: (1) Final cell divisions of neurons of hypothalamus and the preoptic area. (2) Onset of testosterone secretion (3) Appearance of the enzymes involved in testosterone metabolism and (4) Perinatal appearance and increase in the concentration of steroid receptors. The research also revealed that although testosterone is the major secretory product of the testes involved in brain sexual differentation, It does not seem to be testosterone which actually brings about sexual differentation but rather a metabolite produced in some cases within the brain itself. The brain also contains enzymes for producing the two major metabolites of testosterone which are 5 dihydrotestosterone (DHT) and estradiol. In the guinea pig, in the early fetal life, the cerebral cortex contains low but constant concentrations of respiratory enzymes e.g. Cytochrome C. UNIVERSITY OF IBADAN LIBRARY 24 The concentration of these enzymes increase sharply at the time of morphologic differentiation and the onset of electrical activity in the nerve cells. The adult level is reached or approximated at birth. The same pattern between functional development and brain enzyme concentrations was observed with cholinesterase and carbonic anhydrase. Colenbrander et al(l978) during their study with the pig observed that fetal serum testosterone concentrations are elevated between 40 to 60 days post coitum and decreased between 60 to 100 days. Moon and Hardy (1973) however reported that in the fetal pig, the leydig cells are highly differentiated between 33 to 40 days post coitus. However, it has not actually been established that the same hormone is responsible for sex differentiation of the brain in all animals, e.g in song birds, androgens(and presumably androgen receptors) are involved in the differentiation of the brain's capacity to produce song (Nottebohm, 1980). Yet in the quail, estrogens may be involved in the differentiation of reproductive behaviours (Adkins, 1973, Hutchson, 1978). In certain mammals, (the guinea pig and rhesus monkey) the androgen pathway appears to be essential for sexual differentiation of masculine sexual behaviour (Gold foot etal 1975) where as the estrogen pathways appears to be involved in masculinization of hamsters (Paup etal 1974) and in rats(Booth, 1978). Thus with respect to the suppression of feminine characteristics UNIVERSITY OF IBADAN LIBRARY 25 (defeminization), the estrogen pathway appears to be of primary importance in a number of mammalian species (guinea pig and rat), (Gold foot _etal 1975, McEwen etal 1977). The involvement of one of these two pathways is based upon a variety of evidence. First the efficacy of DHT in producing brain sexual differentiation is an index of androgen pathway involvement. Since DHT is not aromatizable, the efficacy of estrogens in producing sexual differentiation in brain is an indication of estrogen pathway involvement. Second, the action of various inhibitors provides complementary data to the actions of the various antagonists although there are complications in the use of inhibitory drugs. For the androgen pathway, the anti-androgen flutamide (Neri, 1977) and Cyprotene acetate (CA)(Neumann and Steinbeck, 1974) are effective antagonists. Progesterone, a preferential substrate for 5- reductase (Massa and Martini, 1971) may also have anti-androgenic activity. The responsiveness of developing neural tissue to androgens and estrogens is determined by the appearance of receptor systems during the perinatal period. The estrogen receptor system of the rat brain becomes detectable about fetal day 17(Vito etal, 1979) and increases rapidly during 1 or 2 days before birth and in the 5 or 6 days right after birth (Maclusky etal 1979 a,b). This increase coincides with the onset of the critical period for the defeminizing aspect of rat brain sexual differentation and it may represent a critical and even rate-limiting step in this process (McEwen, 1980 b). The progestin receptor system is below the limits of detection in the rat brain at birth but increases rapidly during the first 10 days of life in parallel with the estrogen receptor system (Maclusky and UNIVERSITY OF IBADAN LIBRARY 26 McEwen, 1980). Thus these receptors are present during the time that progestins have their blocking action over Exogenous estrogen(E2) and Testosterone(T)-induced sexual differentiation (Kind and Maqueo, 1965, McEwen etal, 1979). By the end of the first 10 days of life, estrogen inducibility of the progestin receptor system is begining to emerge (Maclosky and McEwen, 1980). Evidence from research on mammlian brains show that the peak of cell divisions of hypothalamic and preoptic area neurons occur prior to fetal day 17, which is also before the critical period (Ifft, 1972). This implies that changes involved in sexual differentiation are located in these two brain areas. However, it is inadisable to rule out changes in other brain regions in which some cell division may be occuring during the critical period. The pioneering work of Dorner and Stavdt (1968, 1969) and more recent work (Staudt and Dorner, 1976) have indicated the presence of anatomical sex differences in rats with reports that there is a sex difference in the size of nuclei in neurons in the preoptic area, the anterior and ventromedial hypothalamus and amygdala. Dyer and his co-workers (1976) using electrophysiological techniques demonstrated that in intact male rats, neurons of the preoptic area which project to the medial basal hypothalamus receive more synaptic connections from the amygdala than do similar neurons in intact females. Hence neonatally- castrated males are similar to intact females in this regard and neonatally androgenized females are intermediate between males and females. Raisman and Field (1971, 1973) provided the first siginificant anatomical evidence for an organizational sex-difference in the brain. They found a sexual dimorphism in the number of synapses on dendritic spines stemming from non-amygdaloid afferents in the preoptic area of UNIVERSITY OF IBADAN LIBRARY 27 the rat. This difference was reversed by neonatal androgenization of females or neonatal castration of males. A second dimorphism in the dorso medial preoptic area has also been reported by Greenough etal (1977) who detected a sex difference in the topographical distribution of golgi-stained dendrites in the hamster which can also be manipulated appropriately by neonatal steroid treatments. Ryan and Arnold (1979) found that there are sexual differences in topographical distributions of AChE and catecholamines in the brain. Such findings were the first step toward using histochemical and biochemical measures of cholinergic and catecholaminergic function to study the normal development of this area and how to explore how certain experimental interventions disturb this normal development. It is however the view of Gorski (1973) that the production by the neonatal testes of a substance, presumably androgen is the factor which determines the course of development of the brain. 3.3. MINERAL! COMPOSITION OF THE FETUS. Few studies have been made of the mineral content of pig fetuses. Mitchell etal (1931), Urbany (1932), Salmon-legagneur (1968) all found an increase in the crude ash content of the pig fetus from the early fetal life to 112th day of pregnancy. Mitchell etal (1931) also reported an increase in calcium content with intrauterine age. During the period, calcium content increased steadily from 0.26g/kg wet matter at 31 days to 2.5-2.7g on 56th day reaching 3.6g on 70th day and suddenly doubling to 8.2g on the 98th dday. Shortly before parturition, the level rose to 10.9g/kg wet matter.(wm) The same trend was recorded for phosphorus. Sodium concentration UNIVERSITY OF IBADAN LIBRARY 28 was found to decrease with gestation length while potassium content rose (Pomeroy, 1960). Magnesium in the 43-day old fetus was O.llg/kg wm (Urbany 1952) and tripled by 113th day to 0.31g/kg wm (Pomeroy 1960). Nitrogen (as on index of protein content) increased rapidly between 30 days to 45 days of pregnancy stabilizing thereafter till the 90th day after which it increased till parturition time. Hurley (1981) found that fetuses with zinc deficiency during prenatal development results in fetal abnormalities. 3.4. POSTNATAU DEVELOPMENT OF THE PIG BRAIN AND HYPOPHYSES In the adult guinea pig brain, metabolic activity is highest in the cerebral cortex and cerebellum. The high energy requirement of most pontions of the brain is related to the transport of ions, the synthesis of ACh and the metabolism of glutamic acid. Report by Reis etal(1977) indicate that age-dependent changes occur in the concentrations of neurotransmitters in the brain and the adrenals of the rat. Further study also indicated that some of these changes during aging may be a species-specific event. Cotman etal(1978), found that AChE activity and AChE levels in the hippocampus of both young and adult rats are similar. A contradictory report by Schreff etal(1980) states that a progessive decline of AChE activity occurs in the rat brain regions with age. According to Curtis etal (1967), developmental age may not necessarily coincide with chronological age. UNIVERSITY OF IBADAN LIBRARY 29 There is also a conflict as to the relationship between birth weight and chemical maturity. Curtis etal (1967) found negative correlation between the two while Widdowson (1950) found a positive correlation and Pomeroy (1960) could not detect any significant correlation. Halasz etal (1968), 1971, a,b) showed that the development of the rat brain takes place to a great extent after birth and also that a large number of cells in the cerebellum and cerebrum are formed after birth. Age-related studies on the endocrine system suggest that the endocrine system is involved in the ageing process(Ascheim, 1976, Oilman, 1970, Mills and Mahesh, 1978, Finkelstein etal 1972). \ However, Morrison etal(1981) disagreed with these views when he reported an increase in growth hormone release in rams with increasing age. An interesting comparison between chronological development in humans with that of sheep was carried out by Broody (1945) who calculated that 28 months of age in sheep is chronologically equivalent to 24 years of age in humans. The pineal gland and the amygdala have been implicated in playing some role in puberty (Relkin, 1971). Colenbrander etal (1978) observed a rise in serum testosterone level in the pig from birth till the 3rd week of age declining thereafter until the 18th week when it started rising again. This rise continued until the second month followed by another decline probably accdmpanied by other biochemical and morphologic changes. The dependence of AChE system on age has been discovered for some UNIVERSITY OF IBADAN LIBRARY 30 time (Moudgil and kanungo, 1973) and Davies(1979) observed a decline in AChE activity in human brain with age. AChE activity of cerebral hemisphere of normal rat is highest at 9 weeks and decreases thereafter. No change was reported in the cerebellum (Moudgil and kanungo, 1973). They further postulated that since the 9-week old rat is still in the learning phase, a higher activity of AChE at this stage may facilitate the learning process. The decrease in the enzyme activity in the adult and old rats may also be due to a loss of nerve cells. Also, the rate of synthesis of the enzyme may decrease after the growth period. The constant different activity level recorded in the cerebellum suggests a differential metabolic activity of different parts of the brain as a function of age. In pigs, the establishment of synaptic junctions and glial cell multiplication followed by myelination takes place during the period of "brain growth spurt" which in pigs occur during the first five post-natal weeks (Davison and Dobbing, 1968). Kova'cs(1971) also noted that the rate of protein synthesis in rat brain varies with age. In the cerebrum and cerebellum, the highest rate was followed by a decrease. Moudgil etal(1973) observed peak levels of AChE in the cerebral cortex of rats at 9 weeks of age followed by a 50% decrease at 29 weeks and 65% decrease thereafter. 3.3. BODY MINERAL! COMPOSITION OF THE PIG FETUS Brooks etal (1962) found positive correlations between potassium concentration, ether extract, ash and protein content with developmental maturity, potassium content (Curtis etal 1967) has been suggested as an indication of the fat-lean composition of the whole body because they observed a positive correlation between potassium and moisture, lean content and protein concentration and a negative correlation with fat content. UNIVERSITY OF IBADAN LIBRARY 31 Calcium fluctuates although it tends to decrease with increasing body weight (Freese 1958). Other minerals were found to be fluctuating (Manners and McCrea, 1963, Kirchgessner and Kellner, 1972 and Spray and Widdowson, 1950). MATERIAUS AND METHODS. Two experiments were conducted. The first was concerned with the development of the brain and hypophyses functions during gestation while the second looked into the postnatal changes in the same parameters. 3.1.1. ANIMAUS AND MANAGEMENT The Uarge White pigs used in these experiments were already adapted to the climatic conditions of Tbadan, housed in dwarf-walled, concrete- floored, corrugated iron-roofed pens and generally managed as already described (Egbunike and Steinbach, 1972). In the first experiment, 15 sows bred and ascertained to be pregnant were slaughtered 4, 6, 8, 10, and 12 weeks post-coitum. Each slaughter group comprised three sows. After slaughter, the reproductive tracts were removed in toto and taken to the laboratory for processing. In the second experiment, 40 Uarge White piglets were selected at birth with both sexes equally represented. They were allowed to remain with their dams with no creep feeding until they were weaned at five weeks (except those that were sacrificed at day old). UNIVERSITY OF IBADAN LIBRARY 32 Thereafter, they were fed with grower's ration ad libitum (Egbunike, 1973). Four piglets per sex were slaughtered at day-old, one month, two months, three months and four months of age. 3.1.2. TISSUE PROCESSING. After the animals in the first experiment had been slaughtered, the whole reproductive tract was weighed, opened longitudinally and amniotic fluid collected into sample tubes. Thereafter, the embryos were removed and separately homogenized whole (4-week-old embryos). With older embryos, the brain was carefully dissected from the Skull, freed from adhering meninges and blood clot and homogenized whole (15o w/v) in 0.1m ice-cold phosphate buffer (PH 7.4) using a potter-Elvehjem glass-glass homogenizer. With the animals slaughtered postnatally, the heads were quickly saWn open and samples were taken from the following regions of the brain as described by Egbunike(1981). Hypothalamus, cerebellum, Cerebral cortex, amygdala, midbrain (Mesencephalon), pons, hippocampus and medulla oblongata. The adenohypophyses and neupohypophyses were also removed for processing. All brain and hypophyseal samples were then homogenized (13S w/v) as stated earlier. To ensure minimal decline of enzyme activity, all samples were analysed within 5 hours after homogenization, for the determination of the minerals, aliquots of samples were stored frozen at -20°C for analyses. 3.3.1. ACETYL! CHOU INESTERASE ASSAY The method used was a modification of the colorimetric method of Ellman etal (1961) which measured the rate of hydrolysis of acetylthiocholine iodide substrate to thiocholine and acetate using 5:3-dithiobis-2-nitrobenzoate (DTN8: Aldrich chemical company) as the colour reagent. UNIVERSITY OF IBADAN LIBRARY 33 TEST PRINCIPLE AChE (1) H20 + (CH3)3NCH2CH2SC0CH3 > (CH3)NH2CH2SH CH3C00- Acetylthiocholine Thiocholine acetate (11) (CH3)3 NCH2 CH2S- + DTNB 2-nitro-5-mercapbenzoate thiocholine. 3.3.2. PROCEDURE The reaction mixture in a glass cuvette contained 2.6 ml of 0.1m phosphate buffer, 0.4ml of the tissue homogenate and 0.1ml of DTNB. The cuvette was then inverted with the cap on to mix the contents and inserted in an Eppendorf Photometer (1101M) as a blank. The cuvette was then taken out and the reaction started by adding 0.02ml of 0.075M substrate(sigma) to the mixture. The contents were then quickly mixed and the increase in absorbance over 4 minutes was measured at 405nm. The rate of hydrolysis was calculated as follows: R A 1 -4 A / x = 5.74(10 ) /CO -4 1.36(10 ) (400/3120) cO Where R = Rate of hydrolysis per minute. A = change in absorbance per minute. CO = Original concentration of tissue (lOmg in 1ml) UNIVERSITY OF IBADAN LIBRARY 34 With the dilution rate used, the change in optical density was multiplied by a constant 14.35 to give the AChE activity as Umole/g wet weight/min. 3.4.1 DETERMINATION OF TOTAU PROTEIN The biuret method of Weichselbauin (1946) was used. Test Principle: Protein forms a coloured complex with cupric ions in an alkaline medium. Procedure: 5.0 ml of the biuret solution was added to a clean class test-tube followed by 0.1ml of the tissue homogenate. The contents were then mixed and incubated for 30 minutes with occasional shaking at a room temperature of about 25 A glass cuvette containing about 3ml of the biuret solution with 0.1ml of distilled water was used as a blank. About 3ml of the test sample was poured into a clean glass cuvette and the absorbance measured against the reagent blank at Hg 546hm. The protein concentration (C) of the sample was determined by multiplying the absorbance by a constant 19 obtained from a regression curve and was expressed in g/100 ml. 3.5.1 CAUCUUATION FOR SPECIFIC AChE ACTIVITY The AChE activity of the sample was divided by its UNIVERSITY OF IBADAN LIBRARY 35 total protein concentration to give the specific AChE activity in Umole/g protein/min. 3.6. MINERAL! ANAUYSES The mineral contents of the samples were determined by the standard procedures of Willis (1961, 1962), David(1958,1960) and Gatehouse and Willis (1961). Calcium, Magnessium, Copper, Iron and Zinc were determined by flaming in a Perkin-Elmer atomic absorption spectrophotometer 703 using different lamps. Sodium and Potassium were determined by a Corning 400 flame photometer. All assays were expressed in parts per million (ppm or mg/litre). ' 3.7. STATISTICAL! ANAUYSIS All results were subjected to a multi-factor analysis of variance by a digital stored computer. Treatment means + standard error of means (S.E.M) were compared using the studentized least significant difference method (Steel and Torrie, 1960). Where necessary, correlation and regression analyses were also carried out relating AChE activities, Protein concentrations and cation levels with gestational age. 3.8. RESUUTS A decline in amniotic fluid volume with gestation was observed from about ten weeks onwards. At four weeks of gestation, the brains of the fetuses could not be dissected out, hence, the whole fetus was UNIVERSITY OF IBADAN LIBRARY 36 used for the biochemical assay. It was also observed that fetuses at the upper end of the uterine horns (near the uterotubal junction) were slightly heavier than those at the cervical end but these differences were not significant (P>0.05) By the sixth week of gestation, the fetuses had developed enough for the brains to be taken out and by 8 weeks, they could be clearly differentiated into females and males. 3.8.1. INTRA UTERINE DEVELOPMENT The reproductive tract weights, embryo weights and brain weights from four to 12 weeks of gestation are summarized in table 3.1. The reproductive tract maintained a steady increase in weight with advancing gestation. Thus by six weeks, the reproductive tract had increased by 7.80S while mean embryo weight increased by 48,878S from a mere 0.06 + 0.03g at four weeks to 27.43 + 1.47g at 6 weeks and mean brain weight increased by 300.OS. At eight weeks of gestation mean reproductive tract weight increased by 114,7S while mean embryo weight increased by 132.00S and mean brain weight by 1,082.00S. By ten weeks, of mean reproductive tract weight was 8.28 + 0.18g showing an increase of 343S while mean embryo weight increased by 326.40S and mean brain weight by 139.IS. At twelve weeks, mean reproductive tract weight showed only a slight increase of 4.4S but mean embryo weight still recorded a substantial 102S increase with brain weight declining by 2.2S. From the foregoing, it is evident that rapid development of the embryo occuring between the 4th and 6th weeks of gestation. It also marks a period of spectacular brain spurt which continues progressively until 10 weeks UNIVERSITY OF IBADAN LIBRARY 37 TABLE 3.1 CHANGES IN THE WEIGHTS OF THE REPRODUCTIVE TRACT, EMBRYO AND THE EMBRYONIC BRAIN DURING GESTATION, (means + S.E.M). LENGHT OF |REPRODUCTIVE TRCT EMBRYO WEIGHT EMBRYONIC BRAIN GESTATION WEIGHT (kg) (g) WEIGHT (g) 4 Weeks ! 0.06 0.06 (0.03) (0.03) 6 Weeks 27.43 0.78 (0 .002) 8 Weeks 9.27 tl'M) 10 Weeks 271.94 8 : $ (11.48) 12 Weeks m v (0.89) TABLE 3.2 CHANGES IN AChE ACTIVITY, PROTEIN CONCENTRATION AND SAChE ACTIVITY IN THE CONCEPTUS DURING GESTATION AChE ACTIVITY PROTEIN CONCENTRATNj SAChE ACTIVITY LENGHT OF Amniot-jEmbryo- Amniot-j Embryo-!Amniot-j Embryo­ GESTATION 1C nic ic nic ic nic Fluid iBrain Fluid Brain 'Fluid 'Brain 4 Weeks 0.492 0.11 10.23 4769 77735 (0.03) (0?09) [0.04) (0 .02) (1.68) (0.34) 6 Weeks 0.19 1.16 0 0.23 1.53 5.13 (0.01) 0.07) ('a!3i) (0.21) (0.39) 8 Weeks 0.21 .34 0.12 1.86 11.42 (0.004) 0.09) (0 .01) „ ,(0.19) (1.50) 10 Weeks 0.22 1.71. 0. .,14" (0.007) (0.IT) 12 Weeks 0.26 3.26 S ? i S 0 2 ) j » i l i 4> 1 4 (0.06)' 1(O0..c96) i(0^02) i(0.04) Uo.73) ! (2.50) * Values are means and the standard errors are in parentheses. UNIVERSITY OF IBADAN LIBRARY 38 of age. It is also worth mentioning that while mean embryo weight and mean reproductive tract weights increase very linearly up to 12 weeks of gestation, brain growth rate had reduced between the tenth and twelfth week of gestation. 3.8.2 AChE ACTIVITIES, PROTEIN CONCENTRATION AND SAChE ACTIVITIES IN THE CONCEPTUS FROM FOUR WEEKS TO TWEUVE WEEKS OF GESTATION Table 3.2 Shows mean AChE activities, protein concentrations and SAChE activity in the conceptus. AChE activity in the amniotic fluid decreased very sharply from 0.49 + 0.03 at four weeks to 0.19 + 0.01 at six weeks and thereafter maintained a fairly steady profile to twelve weeks of age. This is further shown by the highly significant but negative correlation coefficient of -0.56 (P<0.05) between amniotic AChE and gestation length (Table 3.3) and the regression curve in fig. 3.1. AChE activity in the embryo brain increased linearly from 1.16 + 0.06 at 6 weeks to 3.26 + 0.96 at 12 weeks with a highly significant and positive correlation coefficient of 0.88 (P<0.001) (table 3.3). regression between embryobrain AChE and gestation (fig. 3.1). UNIVERSITY OF IBADAN LIBRARY 39 TABLE 3-3 REGRESSION TABLE OF AChE ACTIVITY, PROTEIN CONCENTRATION AND SAChE ACTIVITY IN THE AMNIOTIC FLUID AND EMBRYONIC BRAIN ON AGE Y I x !PREDICTION EQUATION r ! SIG. AChE ACTIVITY j AGE ! y=a+bx 1 Amniotic fluid y=-0.453-0.022x ± f ! # Embryonic Brain 11 J y=-0.38+0.64x 0.88 ##*1 1 1 PROTEIN CONCENTRATION | ! ! Amniotic fluid 11 | y=0.11=0.004x 0.31 ] n.s Embryonic Brain 1 j y=0.21-0.004x -0.11 j n.s SAChE ACTIVITY 11 I1 11 Amniotic fluid 11 ! y=-3.90-0.52x -0.541 ! Embryonic Brain 1 y=-0.88 + 3-03x 0.86 [ **#* * = PC0.05, ** = P<0.01, *** = PC0.001, n.s=P>0.05. TABLE 3.4 CHANGES IN THE MINERAL PROFILE OF THE EMBRYONIC BRAIN DURING GESTATION * LENGHT OF CALCIUM j MAGNESIUM j POTASSIUM j SODIUM COPPER j ZINC GESTATION ! I I 4 Weeks 1.61 1 . 3 4 15.61 0.51 10.72 (0 .01) (0.50) (0.004) (0.04) 6 Weeks ' .502 ».493t3) 26.09 0 . 0 0 0.46 0.46 0 . 02) ;o . 0 2 ) (0.58) .08) (0.01) (0.02) 8 Weeks i?02 16.64 26.7 0.23 0.44 0% (0.08) (0.54) 1.11, (0.005) (0.01) 10 Weeks 18.19 45.5C 0.11 (6.5)3) 1.55 0.35(0.05) (0.25) ,.2.10) (0.003) (0.04) 12 Weeks 1.41 2 0 . 9 0 0.12 0.40 (0 .01) (0.58) (0.002) 1(0.01) * Values are means and the standard errors are in parentheses. UNIVERSITY F IBADAN LIBRARY 4-0 1.00 , o .io . _ IHBPYOinC BRAIN |o .* o . . . a * | 0.40 0 .2 0 T ■ 0.2I-O.OO<«* -J----- 1----- 1----- 1----- (- GRSTATION LENGTH (WEEKS) i.o K.cr 6.o 8.o io.o i?..o GESTATION LENGTH (WEEKS) GESTATION LENGTH (WEEKS) 1.0 %iO 6.0 *.0 10.0 12.0 GESTATION LENGTH (WEEKS ) F i g . ' 3«li Relationship Between Gestation length a n d AChfi Activities and Total Protein in the Embryonic Brain and Amniotic Fluid. L C hl uaol/g/ain AChT u*el/c/«l» UNIVERSITY OF IBADAN LIBRARY TABLE 3.5 REGRESSION TABLE OF THE MINERAL CONTENT OF THE EMBRYONIC BRAIN ON GESTATION MINERAL (y)!AGE(x)i Y = a+bx !PROBABILITY Calcium Mafgnesium (P>0.005)Po»t;assium (P>0.005) Sodium 101 Copper Zinc Protein concentration in the amniotic fluid tended to increase with increasing intra uterine age but the correlation coefficient is weak and non significant (table 3*3 and fig. 3*1). Protein concentration in the embryonic brain tended to decline in a non consistent trend evidenced by a very weak and negative correlation coefficient of —0.114(P>0.05; table 3*3) and a non significant regression curve in fig 3.1. Specific acetylcholinesterase activity in the amniotic fluid declined significantly (P<0.05) from 4.69 + 1.68 at four weeks to 1.53 + 0.21 at six weeks and this was somewhat maintained till twelve weeks of gestation. Hence, the correlation coefficient of -0.54 is negative and significant. Specific acetylcholinesterase activity in the embryonic brain increases very significantly from 5,13 + 0.39 at 6 weeks to 21.54 + 2.60 at 12 weeks, with a highly significant and positive correlation coeficient of 0.86 (P<0.001) and a positive regression curve (Table 3.3 and fig 3.2 respectively) No significant differences were observed in all the above parameters with regards to position of embryos in the uterine horn (P>0.05) UNIVERSITY OF IBADAN LIBRARY 3.8.3. MINERAL! PROFIUE IN THE FETAL! BRAIN DURING GESTATION Tables 3.4 and 3.5 summarize the mineral profiles of the embryonic brain of the pig with intra uterine age. There is an increase in calcium content in the embryo brain with gestational age (r=0.761, P<0.001). With magnesium there was a slight increase in the magnesium content of the embryonic brain from 4 weeks of gestation till about 10 weeks after which it declined. The correlation coefficient is therefore negative but not significant (r=0.25, P>0.05). Potassium did not exhibit a particularly consistent trend and the correlation coefficient was therefore not significant (r=0.099, P>0.05). Sodium on the other hand maintained slight increases in concentration with intra-uterine age and was borne out by a positive and significant correlation coefficient (r=0.793, PC0.001). Copper and Zinc showed steady decline in concentration with intra-uterine age and the correlation coefficients were negative and significant (r=-0.948 and -0.772, P<0.001, respectively). Fig. 3.3 illustrates there relationships. 3.9 CHANGES IN ACETVUCHOUINESTERASE ACTIVITY AND CATIONS IN THE PORCINE BRAIN AND HYPOPHYSES FROM DAY OUD TO FOUR MONTHS OF AGE 3.9.1 REGIONAL! DISTRIBUTION OF AChE IN THE BRAIN Table 3.6 Shows the general profile of AChE activity, protein concentration and SAChE activities of the brain and hypophyses. AChE activity was highest in the amygdala, mid-brain and medulla oblongata, lowest in the cerebral cortex and cerebellum but intermediate in the hippocampus, hypothalamus and pons. On the other UNIVERSITY OF IBADAN LIBRARY 43 hand protein concentration was highest in the cerebellum, pons, medulla oblongata and hypothalamus, lowest in the cerebral cortex and generally stable and medium in the midbrain, hippocampus and amygdala. Consequently SAChE activity was highest in the midbrain, pons and hypothalamus, lowest in the cerebral cortex and medium in the other brain regions. 3.9.2 HYPOPHYSEAL! AChE AChE activity in the adenohypophysis was higher than in the neurohypophysis but the difference was not significant (P>0.05). Protein concentration was however significantly higher in the adenohypophysis than in the neurohypophysis. (P<0.05). SAChE activity was also higher (P<0.05) in the adenohypophysis than in the neurohypophysis (table 3.6). 3.9.3 MINERAL' PROFILES IN THE BRAIN Table 3.7 Shows the concentration of minerals in the various brain regions. Calcium, Magnesium, Potassium, Copper and Zinc were more concentrated in the pons, cerebellum, cerebral cortex and medulla oblongata than in the other brain regions. Sodium did not display any particularly consistent trend. 3.9.4 HYPOPHYSEAL MINERAL PROFILES Calcium, Potassium and Zinc were higher in the adenohypophysis than in the neurohypophysis (P<0.05) while Sodium, Magnesium and Copper were similar (table 3.7). UNIVERSITY OF IBADAN LIBRARY - kk - 19.00 — i I ;i ■ j i pf [ i i> I i i I F i g . SAChE Activities in the Embryonic Brain UNIVERSITY OF IBADAN LIBRARY - 45 - Fig.3*3. Relationship Between Gestation Length and Mineral Concentrations in the Embryonic Brain. U C a le lu n (p p » ) » .g n .« iu » (PP»)NIVERSITY OF IBADAN LIBRARY 1.00.__ EMBRYONIC BRAIN 0. 80. 0. G O . *•9 5.6 7*2 8.8 10.4 12 .0 GESTATION LENGTH « 3.3( Continued) UN ZINCI (Vppm) COPPER (p p n )ERSITY OF IBADAN LIBRARY 47 3.1.0.1. POST NATAU DEVELOPMENT Live weight changes from birth to four months of age are displayed in table 3.8. Also displayed are absolute and relative brain weights, of the brain paired adrenal glands and thyroid glands. Mean birth weight was 1.08 + 0.104 kg which increased very rapidly to 6.07+0.47 kg by one month of age followed by a more gradual increase to 33.36 kg at four months of age. Brain weight similarly increased in a more linear fashion from 31.48 + 2.06 g at birth to 30.19 + 1.76g at one month of age to 78.71 + 3.82g at 4 months. A consistent decrease in relative brain weight with increasing age was also observed. Paired adrenal weight showed a very rapid increase from 0.13 + 0.003g at birth to 0.49 + 0.063g at one month thereafter followed by a more gradual increase to 1.07 + 0.03 at four months of age. A consistent decline was also observed in relative adrenal weight. The thyroid gland showed a sharp growth-spurt from 0.18 + 0.002 at birth to 0.67 + 0.19 at one month and 4.37 + 0.52g at four months. Relative thyroid gland weights were steady and not affected by increase in age. UNIVERSITY OF IBADAN LIBRARY TABLE 3.6 REGIONAL DISTRIBUTION OF AChE ACTIVITY, PROTEIN CONTENT AND SAChE ACTIVITY IN THE BRAIN AND HYPOPHYSES (means ± S.E.M). BRAIN REGION AChE ACTIVITY PROTEIN CONC. [SAChE ACTIVITY b a [ ac Pons 4.69 + 0.75 0.30 + 0.11 [28.36 + 12.40 d a ! d Cerebellum 2.83 + 0.39 0.38 + 0.19 [22.03 + 5.09 a ab ! " b Amygdala 6.05 + 1.19 0.21 + 0.06 !33.93 + 7.29 c ab i d Hippocampus 4.06 + 0.28 0.23 ± 0.07 [23-92 + 5.80 b a ! c Hypothalamus 4.85 + 0.43 0.32 ± 0.08 [26.23 + 11.82 e b Q Cerebral cortex 1.56 + 0.10 0.16 + 0.04 [11.57 + 1.95 a ab | a Mid Brain 6.34 + 0.32 0.23 + 0.03 [36.98 + 10.36 Medulla a a l d< - oblongata 6.43 + 0.84 0.38 + 0.09 [25.76 + 13.28 11 HYPOPHYSES 11 a a Adenohypophysis 1.43 + 0.36 0.34 + 0.13 [10.11 ± 4.96 a b [ b Neurohypophysi s 1.09 + 0.19 0.12 + 0.02 ! 8.05 + 3.12 *Values in the same vertical column differently superscripted differ significantly. (PC0.05). UNIVERSITY OF IBADAN LIBRARY •i9 TABLE 3.7 REGIONAL DISTRIBUTION OF MINERALS IN THE PORCINE BRAIN AND HYPOPHYSES, (means + S.E.M) BRAIN REGIONS 1 CALCIUM I MAGNESIUM ! POTASSIUM ! b 1I ac 1 a Pons 2.05 + 0.15 11.54 + 0.09 j 28.17 + 3-29a 1 a ab Cerebellum 2.42 + 0.12 ', 1.57 + 0.12 j 27.16 + 2.34 c c b Amygdala 1.85 + 0.10 J 1.23 + 0.14 J24.55 + 2.66 d be I ab Hippocampus 1.67 + 0.12 j 1.46 + 0.09 25.00 + 3.07 c I * ac ab Hypothalamus |1.88 + 0.09 j 1.52 + 0.03 J 26.00 + 2.61 bd ab Cerebral Cortex !1.66 + 0.19 j 1-36 + 0.15 J 26.15 + 2.89 c d — a Mid Brain 1.84 + 0.24 J 1.29 + 0.05 J 28.18 + 3.37 Medulla b b ab - Oblongata j 2.08 + 0.26 j 1.45 + 0.03 J25.96 + 3.56 HYPOPHYSES I 1I 1 Adenohypophysis 12.66 + 0 .263 ! 2-10 + 0. 043 J 21.80 + 2. 59a b a I b Neurohypophysis l2.07 + 0-23 i 1-98 + 0.25 ! 15.95 + 1.43 BRAIN REGIONS i SODIUM ! COPPER ! ZINC ab 11 b 1I b Pons 538.09+3.70 0.14 + 0.01 0.39 + 0.04 ac a 1 a Cerebellum 545.07+6.62 Jlo . 1 6 + 0.02 | 0.49 + 0.08be I b b Amygdala 530.90+3.13 0.13 + 0.01 0.39 + 0.02 b I b l c Hippocampus 529-32+22.87 J o . 13 + 0.02K J 0.29 + 0.02be K Hypothalamus 537.46+4.42 J o . 13 + 0.02 J 0.43 + 0.05 be b b Cerebral cortex 534.25+2.81 J o . 14 + 0.01 I 0.45 + 0.06ac I b 1 b Mid Brain 548. 45+6.79 |0 .13 + 0.01 f 0.42 + 0.07b a a Medulla j 534. 55+3.11 0.16 + 0.03 | 0.49 + 0.08 - Oblongata 11 1 HYPOPHYSES I 1 1 a 1| a 1I a Adenohypophysis 546.00+3.98 0.21 + 0.022 2.20 + 0.42 a I a b Neurohypophysis ! 535. 12t 4 88 J O . 22 + 0.01 ! 0.48 + 0.04 Values in the same vertical column differently superscripted difer significantly (PC0.05). UNIVERSITY OF IBADAN LIBRARY 50 TABLE 3.8 POST-NATAL DEVELOPMENT IN THE PIG. ’’Age j s i x ” LIVE - j Relative WEIGHT(KG) ! Male ! Female mean [weights % e! Day Old |1.12+0.14 1.05+0.72 I . 081+0.45 One Month j6.02+1.10 6.11+0.44 6.07 -t1.02C Two Months|11.75+2.63 10.75+1.71 I I . 25+2.12° t 3 Months [22.47+0.81 21.05+1.65 21.76+1.63^ 4 Months |34.60*1.34 32.10+1.73 33-36t1.14c BRAIN Day Old j31.65+1.87 31. 18+2. 18131. 48+2.07 3 -65+0.06 WEIGHT(g) One Month |51.72+1.03 48. 6 6 +2 . 49!50. 1 9±1 -76 [ o . 83+0.05 Two Monthsj69.05+3.65 67. 07+4. 93[ 6 8 . 0 6 +3 . 8 0 ! o . 6 0 +0 . 0 2 3 Months |71.89+1.77 71. 97+2. 04[7 1 . 93+1.45 [ o . 33+0 . 0 7 4 Months [81.97+3.82 75. 45+3. 81[78. 71+4.00 [ o . 23+0 . 08' PAIRED I |1 I e ADRENAL , a[Day Old [0.13+0.05 [0.15+0.04 0.13+0.05 0.01+0.004 WEIGHT j 1 d ' b One Month [0.58+0.15 [0.40+0.06 0.49+0.06 0.008+- 0.0007C [Two Months J 0.58+0.06 [0.61+0.015 [0.59+0.04 [0.005+0.0004 1 b dc [3 Months [0.81+0.05 [0.80+0.06 0.81+0.07 0.004+0.0006A [4 Months jl.05+0.07 [1.08+0.06 [l.07±0.03 [0.003+0.0005 THYROID |I e a GLAND WT. |Day Old [6.175+0.004 [0.18+0.003 0.02+0.003 [one Month [0.73+0.31 [0.61+0.13 [0.67+0.19 ]o.01+0.006 [Two Months [1.40+0.36 [1.29+0.26 [1.44+0.28 [0.01+0.005 b [3 Months [2.38+0.22 [2 .61+0 .3 0 [2.50+0.27 [0 .01+0 .006 ~ a 1 [4 Months [4.26±0.52 [4.48+0.52 !4.37+0 52a !0 .01+0 .00) No Sex differences were observed. Values in the same vertical column bearing different superscripts are significantly different IP<0.05). UNIVERSITY OF IBADAN LIBRARY OOJTO• OO O• 51 3.1.0.2 AGE AND SEX DIFFERENCES IN THE REGIONAL! DISTRIBUTION OF AChE ACTIVITY AND CATIONS OF PORCINE BRAINS FROM DAY OUD TO FOUR MONTHS OF AGE Tables 3.9 and 3.10 show the effect of increasing age and sex on the regional distribution of AChE protein and SAChE in the porcine brain. Table 3.11 shows the various correlation coefficients corresponding to the different brain regions. AChE activity declined significantly (P<0.05) with age from day old to four months of age in the pons, hypothalamus, mid brain and medulla oblongata. The correlation coefficients were -0.89(P<0.001), -Q.69(P<0.001), -0.49(P<0.Q5) and 0.81(P<0.001) respectively and the corresponding regression curves are displayed in figures 3.4 and 3.5. It is worth mentioning that in the brain regions mentioned above, the AChE activities were a bit similar at two and three months of age. The amygdala on the other hand displayed a significant rise in AChE activity with age (r=0.906, P<0.001), while the Cerebellum and Cerebral cortex displayed non-significant increase in AChE activity with age. Sex influences were only observed in the Pons, hypothalamus and medulla oblongata (table 3.9) where the males were superior to the females at certain ages (P<0.05). No significant differences were observed in the cerebellum, amygdala, hippocampus and midbrain while the cerebral cortex did not exhibit a consistent trend. Age/Sex interaction was evident in the medulla oblongata and pons with the males having significantly higher AChE activities than the females at day old (P<0.05). UNIVERSITY OF IBADAN LIBRARY 52 3.1.0.3 PROTEIN CONCENTRATION A significant rise in protein content with age from day old to four months of age was observed in all the brain regions. Generally protein content at four months was higher than other age groups while values recorded at two and three months were similar and superior to protein concentrations at day old and one month of age. The various correlation coefficients and regression curves are displayed in table 3.10 and figure 3.5. Sex differences were not very evident but some age/sex interactions were observed in the amygdala where males had higher protein levels at one to four months of age and in the hippocampus where the four-month males had higher protein levels than the females (P<0.05). 3.1.0.4 SAChE ACTIVITY Significant age differences were observed in the various brain regions. Table 3.9 shows that SAChE activity was highest at day old followed by a sharp drop by one month of age thereafter declining steadily. The only exceptions were the cerebellum and the amygdala. The cerebellum increased from 16.90 at Day old to 32.83 at 2 months of age declining to 7.53 at four months of age. The amygdala also showed a steady rise from 22.91 at day old to 58.11 at two months followed by a steady decrease to 20.13 at four months. UNIVERSITY OF IBADAN LIBRARY 53 TABLE 3.9 EFFECT OF SEX ON THE REGIONAL DISTRIBUTION OF ACHE ACTIVITY, PROTEIN CONTENTS AND SAChE ACTIVITY OF THE PORCINE BRAIN REGIONS AT DIFFERENT AGES A G E B R A I N R E G I 0 N S HYPOTHALAMUS CEREBRAL l MID BRAIN lMEDULLA AChE ACTIVITY PONS lCEREBELLUM AMYGDALA HIPPOCAMPUS -CORTEX l l -OBLONGATA Male lFemalelMale 1 female 1 Male 1 Female 1 Male 1 Female Male [Female Male lFemale[Male lFemalelMale [Female Day Old 7-93 l6.84 11.61 11.80 1.99 12.41 13-78 13-85 ; 6.39 1 5.45* 1.22 [ 1.51*1 6.53 1 6.22 l10.38 1 7.50* One month 4.55 14.39 13-44 13 -95 4.93 l4.65 14.05 13-75 6.08 1 5.17* 1.85 l 1.96 l 7.55 1 6.98 1 7.45 1 7-90 Two months 4.85 13-73* 13-15 13-23 8.13 l7-27 14.18 14.87 1 3-61 l 4.05 1.20 [ 1.55*1 6.19 l 6.03 1 4.54 1 5.02 Three months 4.05 14.44 12.87 13-41 7.11 17.11 [4.63 14.73 1 4.08 [ 4.22 1.87 i 1.26*1 6.03 l 6.02 1 5.14 l 5.45 Four months 3-09 13-05 12.43 12.42 8.66 [8.35 13-28 13.93 j 4.74 l 4.47 1.58 l 1.58 I 6.50 l 5.33 l 5.12 1 5.84 S.E.M 0.20 11 0.19 0.23 11 0.25 -19 0.06 li 0.33 1 0.23 0 PROTEIN Day Old 0.09 10.10 10.11 10.09 0.10 10.09 10.09 10.10 ! 0.09 l 0.09 0.09 l 0.08 l 0.09 l 0.09 [ 0.17 1 0.09 One month 0.19 10.19 10.14 10.20 0.13*10.12* 10.12 10.20 1 0.12 l 0.28 0.12 [ 0.35 l 0.11 ! 0.30 1 0.37 1 0.46 Two months 0.20 10.23 10.10 10.10 0.19 10.10* 10.23 10.16 0.36 ! 0.37 0.13 i 0.10 l 0.33 l 0.21 [ 0.45 1 0.22 Three months 0.24 10.25 [0.11 10.13 0.30 10.24* 10.23 [0.29 0.46 i 0.44 0.13 l 0.12 l 0.23 l 0.25 1 0.51 l 0.58 Four months 0.58 [0.51 0.45 l0.40* l0.47* [ 0.69 i 0.60 S.E.M. 0.01 1 10.33 10.31 1[0.57 1 0.50 l 0.55 0.30 l 0.31 1 0.35 l 0.381 0.02 0.01 1 0.02 l 0.02 0.04 1 0.03 l 0.05 SAChE ACTIVITY Day Old 183.58!69-89*!15.02!18-78 !20.35i25-U2*!41.07 139.53 71.34 160.14*113.67 !18.10*174.28 170.05* 169-29 182.29 One month 124.28123.43 125.94127.88 139.45139.11 138.46 118.42 49.76 118.47*115.54 110.70*167.45 123-51* 120.99 118.98 Two month 123.86116.46*132.60133.06 143.32172.90*118.30 132.14 | 9.88 110.94 l 9.36 115.04*118.62 133-236*110.22 [23-24 Three months 117-20117-85 125-82127-16 131-21129-07 120.79 117-00 , 10.19 l 9.58 114.56 110.31*126.78 124.70 110.08 i 9-43 Four months l 5.3U 6.04 l 7.33! 7-74 119-24121.01 1 5.80 1 8.52 , 9.56 1 8.31 ! 5.28 l 5.22 l18.99 117-66 [ 7.51 1 9-99 S.E.M 1 1.77 l 4.01, l 2.59 l 2.87 2.90 [ 0.75 l 3-95 l 4.42 * paired values (male/female) under a brain region and * in an age (in the same horizontal column) group bearing the sign * are significantly different (P<0.05). UNIVERSITY OF IBADAN LIBRARY 5^ TABLE 3.10 AChE ACTIVITY. PROTEIN CONTENT AND SAChE ACTIVITY IN THE PORCINE BRAIN AT DIFFERENT AGES (MEAN + S.E.M.) iPons !Cerebellum !Amygdala !Hippocampus Hypothalamus !Cerebral Cortex! Mid Brain !Medulla Oblongata AChE ATIVITY 1 1 Day old !j 7.38+0.143 b| d b 1 a c ! b i 1.76+0.18 2.02+0.09 1 3.81+0.14 I 5.92+0.16 1.37+0.02 6.37+0.12 8.94+0.32a c b a a 11 One month J 4.47+0.09 3.69+0.27 4.79+0.13 1 3.90+0.21 15.62+0.19 1.90+0.05 7.26+0.32a. b a 1 C " G b 1 7.67+0.15 1 Two months j 4.29+0.23 3.19+0.10 j 7.70+0.40 1 4.52+0.23 ;3.83+0.17 1.38+0.02 j 6.11+0.17 1 4.78+0.17° Three months J 4.25+0.17 3.14+0.22aj 7.11+0.17 11 4.68+0.15a t4.15+0.02b° 1.57+0.05 j 6.02+0.151 1 5.48*0.17° c c a b b - ' b b 11 Four months ! 3.08*0.05 ! 2.43+0.15 ! 8.50+0.28 ! 3-60+0.07 14.61+0.20 1.58+0.03 5.92*0.26 1 5.48+0.16 Values in the same vertical column differenttly £ superscripted significantly differ (P<0.05) PROTEIN d o d Day old jO.09+0.0004 jO.10+0.01 jO.09+0.001 j0.09+0.002 0.09+0.002 0.09+0.00g d 0.09+0.02 0.13+0.06 One month 0.19+0.005 0 .17+0.01 0.13+0.011 ]0.16+0.02° 0.20+0.003 be 0.23+0.17° 0.21“+0 .01°h 0.41+0.06° Two months 0.21+0.001' 0.10±0.01 0.14+0.001° Jo.19+0.01° |0.37tP.010° 0.12+0.01° d b b 0.27+0.02 0.33+0.01 Three months 0.25t0.01 0.12+0.01C 0.24+0.01 J0.26+0.02 '0.42+0.01 Lbe a 0.13+0.005° a 0.24+0.01 0.55+0.01 Four month;s ;0.55t0.006 !0.32+0.01 i0u.4+3pt*u0.u0ni a !r0.52+0.03a 10.52+0.03s 0.31+0.023 * values in the same vertical column diff0.05 S.E. standard Error of Estimate UNIVERSITY OF IBADAN LIBRARY 55 Not surprisingly therefore, highly significant and negative correlation coefficients were recorded for the pons, hippocampus, hypothalamus, cerebral cortex, midbrain, medulla oblongata and very low and insignificant negative correlation coefficients recorded for the cerebellum and amygdala (figure 3.6). The cerebral cortex as earlier reported recorded a rather low value of 15.89 at day old which was significantly superior (PC0.05) to the value recorded at one month. The activity recorded at one month was however similar to values recorded at two and three months but significantly lower than the value recorded at four months of age. * Significant sex effects were observed in the pons, amygdala, hypothalamus, cerebral cortex and midbrain. Males had higher levels than females at one and three months of age in the pons while the exact opposite was observed in the amygdala. In the hypothalamus, cerebral cortex and mid brain SAChE levels were higher in the males at day old and one month of age while at two months of age, the trend was reversed in the cerebral cortex and midbrain. MINERAL PROFILE Tables 3.1.2 and 3-1.3 summarize the influence of age and sex on the mineral content of the various brain regions. The regression curves are displayed in figures 3.9 to 3*15. Highly significant age influence was observed in the cation content of the various brain regions. UNIVERSITY OF IBADAN LIBRARY 57 CALCIUM With Calcium, slight but significant increases were observed in concentration with age in the Pons, cerebellum, amygdala and midbrain. Table 3.1.4 shows the correlation coefficients of the cation distribution in the various brain regions with age. However significant negative correlations were observed in the hippocampus, hypothalamus and cerebral cortex. A non-significant correlation was observed in the medulla oblongata. This is due to the non-consistent nature of calcium concentration in the medulla oblongata with varying age. With the exception of the cerebral cortex that shows a significant increase in calcium content of the male brain at four months of age over the female brain, no significant sex influence was discovered. MAGNESIUM Slight but significant increases were observed in the pons, cerebellum and midbrain evidenced by the strong and positive correlation coefficients in table 3.1.4 and the regression curves in figures 3.1.1. On the other hand, significant negative decline in magnesium concentration was observed in the hippocampus and cerebral cortex. The amygdala and hypothalamus displayed non-significant negative correlation coefficients while the medulla oblongata showed a rather weak and positive correlation coefficient. Significant sex influences were observed only in the pons where the females had higher values than the males at day old and four UNIVERSITY OF IBADAN LIBRARY 56 months of age and also in the hippocampus where the same trend was observed at day old and two months of age but the trend was reversed at three and four months of age. UNIVERSITY OF IBADAN LIBRARY - 59 - _ L ) ■:s' \\ 7.60 - > 4-80 H-----1-----1-----•- 1 .0 1 .8 2.6 . 3.̂ 0.05 UNIVERSITY OF IBADAN LIBRARY 68 POTASSIUM Potassium increased significantly with age in the pons, cerebellum, amygdala, hypothalamus, mid brain and medulla oblongata. The hippocampus showed a non-significant positive correlation coefficient while the cerebral cortex displayed a non-significant negative correlation. Significant age/sex interaction was observed only in the hypothalamus with the female being superior to the males at two and four months of age. SODIUM Sodium increased significantly in the pons, cerebellum, hypothalamus, midbrain, medulla oblongata (as shown by the positive correlation in table 3.14) and non significantly in the amygdala, hippocampus and cerebral cortex. No significant sex influence was observed except in the hypothalamus where the females were superior to the male at three and four months of age. The male however displayed a higher sodium content at day old than the female. UNIVERSITY OF IBADAN LIBRARY 69 COPPER Generally copper increased significantly with age from day old to four months of age. The only exception was the hypothalamus which had a non-significant correlation of 0.283(P>0.05). (Table 1.14 and figure 3.1.4). No significant sex influences were observed. ZINC Data in table 3.1.4 Indicate that only the pons, cerebellum, hypothalamus, midbrain and medulla oblongata displayed significant increases in zinc concentration with age. The amygdala and hippocampus displayed a non- significant negative correlation with age while the cerebral cortex on the other hand had a non-significant increase with age. Significant age sex interactions were observed only in the pons and midbrain where the females had slightly higher levels than the males at one three and four months of age. UNIVERSITY OF IBADAN LIBRARY 70 3.1.1.3 THE HYPOPHYSES Tables 3.1.5 and 3.1.6 show the influence of age and sex on AChE activity, protein content and SAChE activity in the hypophyses. The regression curves are also displayed in figures 3.7. and 3.8. AChE ACTIVITY In both sexes, AChE activity was higher in the adenohypophysis than the neurohypophysis. Both the adenohypophysis and the neurohypophysis showed a significant decrease in AChE activity with age (r=-0.99 and 0.84, respectively (P<0.001; table 3.1.9). Significant sex difference was observed only in the adenohypophysis with the female being superior to the male at day old. No significant sex effect was observed in the neurohypophysis. PROTEIN CONCENTRATION Protein concentration increased significantly with age in both the adenohypophysis and the neurohypophysis as shown by the highly significant and positive correlation coefficients in table 3.1.7. No sex influence was observed. SAChE ACTIVITY SAChE activity declined significantly with age in the hypophyses. In the adenohypophysis, the decline was fairly steady whereas in the neurohypophysis there was a sharp drop from 20.03 Umole/g protein/min at day old to 7.65 at one month followed by a very steady decline to 2.88 at four months. The only significant sex influences was observed at day old with the female being superior to the male. UNIVERSITY OF IBADAN LIBRARY 71 3.1.1.4 MINERAL PROFILE ADENOHYPOPHYSIS Calcium, Sodium and copper increased linearly with age evidenced by the positive and significant correlations recorded for these cations (table 3.1.8). Magnesium and potassiun did not show a consistent trend resulting in non-significant negative correlations. Zinc content also declined with age but not at a significant rate (P>0.05). The only significant sex difference was observed at one month of age when the female piglets had higher sodium content than the male (PC0.05). NEUROHYPOPHYSIS Zinc and potassium displayed an initial rise in levels from day old to between one and two months of age followed by a decline which was significant in potassium (r=-0.66; P<0.01, table 3.1.9). Magnesium and calcium also displayed non-significant negative correlations with age but sodium recorded a highly significant and positive rise with age (r=0.86; P<0.001). Figure 3.1.6 illustrate the various regression curves obtained. Significant sex differences were only recorded in calcium, magnesium and Sodium levels, (table 3.1.8). The males were superior to the females at Day old and two months of age with respect to Calcium levels while the reverse was observed with sodium level. Magnesium content in the neurohypophysis of females was also higher than in the males at Day old and One month of age only. UNIVERSITY OF IBADAN LIBRARY 12 Table 3-1.5 EFFECT OF SEX ON THE DEVELOPMENT OF AChE ACTIVITY, PROTEIN AND SAChE CONTENTS IN THE HYPOPHYSES WITH INCREASING AGE. ! AChE ACTIVITY |PROTEIN CONCENTRATE SAChE ACTIVITY jMale |Female Male !Female Male !Female ADENOHYPOPHYSIS OD I naey molodn th 22.00 2 .J20. ' 0.09 20.36*|29.49»Two months 1.1 l!l§ 00..1226 1? :Il FTohS.u rre e mmoonntthhs s 1.0. _ 0.47 , 0.4 00..745E.M '■'l 5 o'M ijo'M 0.056 O . W 0.563 DNaEyU RoOOne ml HdY POPHYSIS onth 21..1096 1 1 1 1I 00..1083 I1 0.0Two months 0.08 I 1I 0.15 8 i 183*.29 1 0.20 6.0 FTohurre em omnotnhtsh s 0.T9 1 Q-21 0.11 0.20 S.E.M 0.. 0009.23 gPraoiurpe d bevaarliunegs t(hmea le* /fseimganl es)i gnini fithcea ntslaym e dhifofreirz on(tPaCl0 .0c5o)lumn and age Table 3-1.6 CHANGES IN AChE, PROTEIN AND SAChE CONTENT THE HYPOPHYSES WITH INCREASING AGE ! ADENOHYPOPHYSIS | NEUROHYPOPHYSIS ADCahE ACTIVITY 1 y old 2.37+0.086 a 11 i1 1.69-+0 .070 a One month i 2.07tP.0116 11 1.07+0.021 c Two months c b! 11.17+0.071 1c 1 0.99+0.103Three months 1.06+0.028 i1 1.05*0.024 b Four months d! 0.47+0.023 \ 1 d 1 0.61*0.019 DPaRyO ToElIdN CONTENT i 1 0.09+0.002d 11 0.08*0.00 "1“d’J One month 0. d 1 c12+0.010c 1 0.14+0.007 Two months 0.28+0.020 111 0.20+ —0 .010ab Three months 0.42+0.034b 11 ba 1 0.19*0.003Four months ! 0. 172+0.036 11 0.21+0.004 a SDAaCyh Eo ldACTIVITY i 1 a 24.93+1.458a 1l1 20.03+0.618 One month 17.48*0.312 1c 11 7.65+0.366Two months 4.33+0.344 1 c1 5.02+0.515c Three months 2.64+0.129 1e 1 5.56+0.098Four months 1 d! 0.78+0.123 l1 2.87*0.038 vsaulpueerssc riinp ttehde ssaimgen ifhoirciaznotnltya ld ifcfoelru(mPn< 0d.i0f5f)erently UNIVERSITY OF IBADAN LIBRARY 73 Table 3.1.7. CHANGES IN THE CATIONS CONTENT OF PORCINE HYPOPHYSIS WITH INCREASING AGE ADENOHYPOPHYSIS NEUROHYPOPHYSIS CDAaLyC oIlUdM 2.01+0.010e | 2.11+0.040 be One month 2.55+0.032c(j ! 2.38+0.035^ Two months 2.39+0.084 ! 2.15+0.029° Three months c2.85*0.013a 2.04+0.037Four months be3.52+0.067 2.08*0.045 MDAaGy NEolSdIUM 2.06+0.012be ca 1.92+0.043One month a2.20+0.012 2.42+0.024 Two months 2.07+0.040be 2.05*0.000b Three months 2.02+0.012c 2.02+0.012b Four months 2.09+0.014b 2.03*0.012b DPaOyT AoSlSdIUM b19-49+0.308a 15.99+0.266 d One month "" a31.11-+0 .460 c 19.45+0.221Two months 16.88+0.272 bb 17.22■+"0 .337 Three months 19.84+0.806 c 16.19+0.739 Four months b e20.02+0.881 10.94+0.235 SDOaDyI oUlMd b d539.50+1.329 One month ab 527.50+~ 1.586 a 541.25~+3 .34 ab 531.25+0.851Two months 542.62+0.966a 527.37+0.746bThree months 547.37+1.225b 539.63t1.676Four months 538.75+1.650 550.63+1.974a DCaOyP PoElRd 0.20+0.001 b 0.20+0.002e One monthr'"J-* ' Oh 20*~0 .001b 0.21+0.004d - Two months 0.16*0.006°b 0.28+0.1053 Three months b0.22*0.005 0.23*0.005 Four months 0.28*0.018a 0.21*0.003° ZDIaNyC old b • c0.39"+*•0 .017 0.42+0.008 One month 0.50+' 0.012 a ~ a c 0.62*0.005 Two months 0.24+0.00•6 c c 0.41+0.007 Three months 0.25*0.011 c d 0.42—+0 .009 b Four months 0.21*0.003 0.52*0.008 vsaulpueerssc riinp ttehde ssaimgen ivfeirctainctally dcioflfuemrn d(iPf JO ASaHOHYPOl'BJi. «*nlllOS»“OPK*S!S . w » . - O.JU 9 ) >OJL 8 0.60I 9 I •& 0.80 a T 0 .0 0 0.40 0.00 t. -4 — I .0 1.8 1 .0 1 .8 *.6 6 .8 5.0 F ig . 3 . 7 . R e i j i u , h i p Betw een Ag an a a ..x . ^na T o t a l P r o t e in in t h e i-/*. -joh. e? . UNIVERSITY F IBADAN LIBRARY 26.00 ADENOHIPQPHT S IS / .1.U ■"ig.3.8. Relationship Between Age and SAChE Activities in the Hypophyses. UNIVERSITY OF IBADAN LIBRARY 77 ACC (MONTHS) Fig 5.9. Relationship Between £ £ § and Calcium . Concentraticn £ 2 in the Different Brain Regions. [ UNIVERSITY OF IBADAN LIBRARY 78 age ( ko;:t h s) Fig 3«9. (Continued) t UNIVERSITY OF IBADAN LIBRARY 79 Fig. Relationship Be•-t-we11en Age and Magnesium Concentrations in the Different Brain Regions. UN MAGNESIUIM V(PPM) E .. ' MAGNESIUM (PPM)RSITY OF IBADAN LIBRARY 80 as Fig. 3.1*1 (Continued) 1 UNIVERSITY OF IBADAN LIBRARY 81 Aq** (-ionths) Plii. 3.1.2. Relationship Between. £ ga and Potassium Co He an trail' o n in the Different Brain Regions. UNIVERSITY OF IBADAN LIBRARY 82 Fig. 3*1*2. (Continued) UNIVERSITY OF IBADAN LIBRARY 83 AG; (tSOIfVHS) F ig . 3»1»3» Relationship Between Age and Potassium Concentrations in the Different Brain Regions. UNIVERSITY OF IBADAN LIBRARY - 8 4 - Fig. 3«1«3« (Continued) UN SODIUM (ppa) SODIUM (pp.)IVERSITY OF IBADAN LIBRARY 85 ID E (HCKTHS) *Bt (MONTHS) 1. 00- 74 HIPPOCAHPDS 0.80- 0.60- 0.40- 0. 20- - T . 0.106 ♦ O.OOBi 0.00 J---- 1-----H --- h--- 1.0 1.8 2.6 3.6 6.2 AGS (MON1H5) AGS (MONTHS) Fig. 3*1.4* Relationship Between Age and Copper Concentrations in the Different Brain Regions. UNIVERSITY OF IBADAN LIBRARY 86 AGE (MONTHS) Fig.3.1.*+. (Continued) UNIV COPPER (pp«)ERSITY OF IBADAN LIBRARY - 8? - AGE (MONTHS) AGS (MONIrJS) AGS (MONTHS) Fig.3.1.5. Relationship Between Age and Zinc Concentrations in the Different Brain Regions. UNI ZIHC (PP«) Zinc (ppa)VERSITY OF IBADAN LIBRARY - 88 - Fig, 3*1*5* (Ccmtimued) UNIV ZINC (ppm)ERSITY OF IBADAN LIBRARY - 89 - Fig. 3.1.6. Relationship Between Age and Minerals Concentrations in the Hypophyses. UN CALCIUM (pp«) CALCIUM (pp«>IVERSITY OF IBADAN LIBRARY 90 Fig. 3*1*6. (Continued; UNIVERSITY OF IBADAN LIBRARY 91 1.00 1.00.80 90t i METO OHtPOPHTSIS NEOHOHTPOPHTSIS 0.80-. 0.80. 0.60. 0.60. t . 6.1,8 - 8.0«&T 0.40. to .40_ _ I . 0.417 ♦ O.OOJx 0.20 0.20 0.00. 0.00.H .o i;8 216 jfcS 47i FTc1 .0 1 .8 2 .6 3 .4 4-.2- --5l.0 AGC (MONTHS) *02 (HONTHS) Fig 3.1.6. (COmtinued) / UNICOPPES (ppn) ' c o m ® (pp«)VERSITY OF IBADAN LIBRARY 92 TABLE 3.1.9 REGRESSION TABLE OF AChE, PROTEIN AND SAChE CONTENT OF THE HYPOPHYSIS ON AGE Y |Age X! PEQRUEADTIICOTNION r j PROBABILITYi S, E i 1 AACdehEno hAyCpToIpVhITYNeurohypophyyssiiss ! jI yy==21..8783--00..4282xx --00..8947 !I PPC<00..i S : U ! 000011 APdReOnToEhIyNp oCpOhNyCsEiNTRATION Neurohypophysiss i! jj yy==00..0047++00..1003xx 00..5868 | P<0.01 i 8 :A ? I 1 P<0.001 SAAdCenhoEh yApCoTpIhVyIsTYNeurohypophysiiss i m --00..983 1 P<0.001 I ! m i t t : ® 3 ! p 1C 5 nuclei of the hypophyseal secretory cells. Another interesting point to note is the concomitant decrease in AChE and SAChE activities in the hypothalamus coupled with sex-differences. Thus the hypothalamic-hypophyseal axis may be implicated in the low enzyme activities observed in these organs. The highly significant positive correlations observed in calcium, sodium and copper concentrations in the adenohypophyses with age and the lack of a consistent trend in magnesium, potassium and Zinc levels indicate that the minerals that are more essential to growth correlated well with age. The exception is zinc which cannot be readily explained. The result may also be explained by the fact that calcium and sodium tend to antagonize magnesium and potassium by competing with them for active sites on enzyme systems (Dixon and Webb, 1961) although the reasons for this are not very clear. Potassium showed a negative and significant correlation while sodium displayed a positive and significant correlation with age in the neurohypophysis. This may also be explained by the sodium-potassium interrelationship or reciprocity. All the other minerals showed non- significant correlations. UNIVERSITY OF IBADAN LIBRARY - 105 - CHAPTER IV EFFECT OF AGE AT ORCHIDECTOMY AND TESTOSTERONE ON PORCINE BEHAVIOUR AND BRAIN AND HYPOPHYSEAL PHYSIOLOGY UNIVERSITY OF IBADAN LIBRARY - 107 - 4.1.1 INTRODUCTION Among other functions, the testes secrete testosterone, which is known to have diverse influences on the male vertebrate: viz:- The action of androgens (e.g. testosterone) is evident on the sexual characteristics of the male. Androgens also possess the following biological activities in the male: 1) They stimulate spermatogenesis in the hypophysectomized animal and hasten the onset of spermatogenesis in the seasonal breeders. 2) They prolong the life span of epididymal sperm. Sperm motility lasts for approximately thirty days in the guinea pig following castration whereas androgen treatment will increase sperm viability to the normal period of seventy days. 3) They promote growth, development and secretory activities of the accessory sexual organs such as the prostate, vesicular glands, bulbo urethral gland, vas diferens, cowper's gland, penis and scrotum. 4) They stimulate sexual behaviour and libido in the male. 5) They induce nitrogen retention distinct from its action on the reproductive tract. In other words, testosterone possesses protein anabolic activity which involves the total organism. Androgens stimulate growth and have also been associated with nitrogen balance by facilitating nitrogen retention which is vital for protein anabolism (Kochakian, 1946). 6) They also influence the intensity and frequency of sexual bahaviour and aggression in adult male vertebrates (Guhl, 1961, Hart, 1974 and Young, 1961). UNIVERSITY OF IBADAN LIBRARY - 108 ' Because they are sensitive to androgens, these behaviours are co­ ordinated to some degree with each other, with related physiological and morphological changes and with developmental secretion. Hence, this study was designed to evaluate the effects of castration at various ages with or without restosterone replacement in boars. Sexual differentiation is a phenomenon among mammals and birds for which distinct critical periods exist in early brain development. There also seems to be a link between sex differences in brain structure and sex differences in neuroendocrine function and behaviour (McEwen, 1980). A possible reason for sexual dimorphism in the brain may also be due to sex differences in gene products subject to genomic regulation by steriod- receptor complexes e.g. the same enzyme may be regulated at a different rate in say the male hypothalamus and the female hypothalamus. Another reason may be due to differences in the inducibility of a gene product such as occurs in estrogen induction of progestin receptors (Moguilevsky and Raynard, 1979). 4.1.2 LITERATURE REVIEW The age-dependence of the hypothalamic pituitary-gonadal axis has been well established (Masafumi et al., 1981). Also well known is the age- dependence of steriod hormone receptors in the brain (Cidlowski and Muldoon, 1976). This is partly why most of the conflicting reports on the effects of castration depend on the age at which the operation was carried; whether pre-pubertally or post pubertally. Thus Yahr and Coquelin (1980), while not being able to distinctly explain why pre- versus post-pubertal castration produced different effects on aggression in male gerbils, discovered that differences in age at UNIVERSITY OF IBADAN LIBRARY - 109 - castration can help to explain why prepubertally-castrated males and intact females were about twice as aggressive as post-pubertally castrated males. Anisko et al (1973) also observed increased aggressiveness of pre-pubertal gerbil castrates over the normal males. Androgens act on the hypothalamus and the pituitary gland to exert a feed-back system of gonadotropin secretion. In recent years, receptors for sex steriod hormones have been found in the brain and the pituitary glands of rats suggesting that they perform important role in this feed-back control system (Cidlowski and Muldoon, 1976). Thus castration increases metabolism at the hypophyses and hyhothalamus (Roy and Laumas, 1969). The interaction between hormone synthesis and protein anabolism is made more evident by Martini (1973) who discovered that inhibition of protein synthesis interferes with the synthesis of releasing factors in the rat hypothalamus. He further found that the cerebral cortex and amygdala are not androgen-sensitive whereas the hypothalamus (particularly the median eminence) and the adenohypophysis are androgen-sensitive. Although not much work has been done on dose-response relationships between testosterone administration and sexual bahaviour, Yahr et al̂ (1979) found that the castration-eliminated sexual activity in male gerbils was prevented by large injections of testosterone propionate. Mating behaviour, indexed by mounting, intromission and ejaculation, was reinstated. Sodersten et al (1980) also confirmed earlier reports that sexual behaviour persists for some time after abrupt testosterone withdrawal i.e. castration. They also reported that blood titre levels of testosterone in male rats is normally higher than that needed for maintenance of sexual behaviour. Davidson (1977) remarked that castration of sexually UNIVERSITY OF IBADAN LIBRARY - 110 - experienced rats is usually followed by a gradual decline in sexual bahaviour whereas castration increased plasma Luteinizing hormone (LH) within 12 hours (Nansel et a_l, 1969) followed by a further progressive increase over a 3 week period (Damassa et al., 1976; Gay and Haugen, 1977). The prolonged response of the brain mechanisms controlling sexual behaviour and possibly LH secretion to androgen withdrawal is at variance with the prompt response of the accessory sexual glands which are androgen- sensitive. Both the central and the peripheral responses can be prevented by testosterone treatment (Sodersten et al.., 1980). It has also been suggested that castration results in a gradual desensitization of the brain and that as a consequence, more testosterone is needed for restoration of the behaviour in long-term castrates than acutely castrated rats (Davidson, 1972). Furthermore, the level of testosterone stimulation needed for induction of the behaviour in castrated sexually inactive rats over a brief period of time is lower than the serum testosterone present in normal males. The specificity of testosterone for maintenance of sexual behaviour was also confirmed by Sodersten (1973) and Paup et al (1974) who discovered that subcutaneous injections of estradiol benzoate stimulated the display of mounts and intromissions by castrated rats but failed to induce ejacula­ tions partly due to the inability of the estradiol benzoate to stimulate the peripheral androgen-sensitive sexual organs, particularly the penis. The importance of adequate androgenic stimulation of the penis for sexual behaviour was demonstrated by Beach and Levinson (1950). Meyerson (1964) postulated that brain monoamines mediate the effects of gonadal hormones on sexual behaviour in the female rat. Brain monoamines inhibition facilitate the induction of sexual behaviour by UNIVERSITY OF IBADAN LIBRARY - 111 - testosterone in castrated rats (Mayerson, 1964; Malmenas, 1973). Christensen and Clemens (1974) established that testosterone acts on the hypothalamus to induce sexual behaviour. The hippocampus and septum have also been suggested to be target areas for testosterone (Fuxe et al., 1978 Kohler et al., 1978). Other results indicate that differences in the behaviour of adult rats are related to differences in the perinatal milieu (Donler and Wuttke, 1965; Pang et_ al., 1979; Goldman, 1978). Hece the presence of testicuar secretions during th first ten days of life facili­ tates the display of ejaculations by male rats in adulthood and inhibits display of female behaviour. Hupp et al. (1961) had long ago observed that castration however produced some changes that are not reversible by testosterone administration alone except when combined with adequate doses of estrogen. They therefore suggested a synergistic effect of testosterone and estrogen on the secretory activity of the various accessory sex glands of the boar. However, Booth (1980) found that prepubertally-castrated boars receiving testosterone injection had increased weights of accessory glands, increased zinc and fructose levels in the seminal vescles and raised plasma androgens with full restoration of mating behvaiour. It has also been established that apart from having receptors for testosterone, the brain also contains enzyme, responsible for the conver­ sion of testostorone to estradiol. Halasz (1969) also suggested that in the male, the hypothalamus is sufficient to maintain near-normal testicular activity without afferent input. In a more interesting experiment, Kang et al. (1970) found that stimulation of the genital afferent nerves and injections of gonadal hormones into adult rhesus monkey reulted in electrical responses in the UNIVERSITY OF IBADAN LIBRARY - 112 - hippocampus and limbic structures which are involved in satiety and "need" state of sex drive. A direct evidence of the presence of receptor systems was established by Lisk (1962), Michael and Scot (1964) and Harris and Michael (1969) who placed testoterone in the brain of a castrated male rat and found it sufficient to stimulate mating behaviour. According to Eisenfeld and Axelrod (1965, 1966), Xato and Villee (1967b, McGuire and Lisk (1968) and McEwen and Pfaff (1970), testosterone- 3H also tends to be retained by cells of limbic hypothalamus system which includes the septum, amygdala, hippocampus, with a limited retention capacity by the medial preoptic area. Pfaff and Pfaffmann (1969a) also added that testosterone injected into castrated male rats influences the spontaneous activity and the responses to peripheral stimili of individual neurons in the preoptic area, olfactory bulb and mesencephalic reticular formation. Also reported is the fact that the absolute magnitude of a neuron's respnse to an individual odour is androgen-sensitive but the relative magnitude of a neuron's responses to different odours (measured by a different trial response analysis) is not androgen-sensitive (Pfaff and Pfaffman, 1969b, Pfaff and Gregory, 1971a). Castration has been known to increase monoamine (Norepinephrine) levels in the anterior pituitary (Stefano et al., 1965). These levels are decreased again following hormonal theraphy. Some other workers (Wurtman, 1968) found that castration increased the whole brain turnover rates of norepinephrine even through there were no large changes in norepinephrine concentration. Fuxe and Hokfelt (1969a, b) found increased in the utiliza­ tion of dopamine in the median eminence after low doses of pituitary qr testosterone in castrated rats. Moguilevsky et al., (1966) observed changes UNIVERSITY OF IBADAN LIBRARY - 113 - in hypothalamic oxidative and anaerobic metabolism following sex hormone variations. Sutherland and Gorski (1970) also found that drugs which inhibit and rogenization at the level of the brain such as barbiturates induce lower enzymes capable of inactivating plasma steroids. This supposes that androgens suppress liver enzymes capable of inactivating plasma steroids and suggests an elevation of plasma steroids on androgen treatment. Moguilevky et al., (1966) further observed that endogenous oxygen uptake and anaerobic metabolism in the hypothalamus was significantly depressed in male rats by castration. Androgens may also prevent development of a serotogenic system in the brain or induce the development of another system antogonistic to the serotogenic system (Ladosky and Gaziri, 1970). Gorski, (1973) also advanced the view that androgens alter fundamental neurochemical processes, perhaps protein synthesis within the suprachiasmatic region and this in some way prevents the cyclic release of Gonadotropin (GTH) in the adult. Further, androgen deficiency during a critical hypothalamic differentiation phase results in predominantly female organization of the brain (Dorner, 1969). Thus male homosexuality could be completely and permanently prevented by a single androgen injection administered during the hypothalamic differentiation phase. This indicates an androgen prophylaxis of neuroendocrine-conditioned male sexuality. Dorner (1973) concluded by suggesting that changes in the androgen and/or estrogen levels during the hypothalamic organization phase may results in permanent disorders of gonadal function and/or sexual behaviour during the post-pubertal hypothalamic functional phase. Therefore in gonadal males, androgen UNIVERSITY OF IBADAN LIBRARY c - 114 - deficiency during the hypothalamic differentiation period leads to post- pubertal hypo- or even homosexuality. It has been shown by several authors that neurons in the anterior preoptic area involved in LH release may pass through the basal hypothalamus (Eveett, 1964); Halasz and Gorski, 1967). They also found that a gross lesion of the median eminence and the adjacent area of the tuberal regions results in gonadal atrophy and a reduction of pituitary LH synthesis. Green et al (1957) also induced hypersexuality in the monkey and cat with ablations of the pyriform cortex or amygdala while MacLean (1970a, b) reported intensive grooming and penile erection following the after­ discharges induced by electrical stimulation of the hippocampus in the rat, cat, and monkey. The limbic system has also been established to influence gonadotropin secretion, the stimuli arriving from the amydgala being facilitatory and those of the hippocampus inhibitory (Koikegami et al., 1954; Bunn and Everett, 1957; Velase and Taleisnik 1969a, b). Although contradictory results exist in literature concerning the influence of the mid brain on gonadrotropin secretion, a dual role of the midbrain has been described for the control of corticotropin hormone release (Mangili et al., 1966, Fortier 1966), but even in that case the lack of specific organized systems is evident. Injection of androgens into castrated mammals prevents the atrophy of penis, scrotum, prostrate, seminal vesicles, epididymis and bulbo urethral glands which normally follows removal of the testes, and the increase in weight of the prostrate of the castrated rat after androgen treatment is the basis of a method of testosterone bioassay. UNIVERSITY OF IBADAN LIBRARY - 115 - 4.1.3 PORCINE PLASMA TESTOTERONE Although dose-response relationship is not well established several workers had determined the testosterone levels in the boar. Andresen (1976) observed an increase in plasma testosterone level from 2.5 ng/ml plasma at 100 days of age to 5.5 ng/ml plasma at 156 days of age followed by a fall of 2.7 ng/ml at 213 to 226 days of age. These determinations were based on the fact that boars reach adolscence between 110 to 125 days old and become sexually mature between six and seven months. Gray et al., (1971) similarly established a rise in the testosterone level in boars from 15.9 ug/100 ml bolld at three months of age to 27.0 lug/100 ml blood at six months of age followed by a drop to 11.6 ug/100 ml at nine months of age. More recently Booth and Baldwim (1980) observed average testosterone levels of 1.27-6.28 ug/ml in boars. Various dosages of testosterone have been adminstered to castrated boars. Joshi and Raeside (1973) administered a weekly im injection of 37.5 mg/boar followed by 75 mg for another six weeks. Hupp et al (1961), administering deep intramuscular injection of testosterone propionate in sesame oil to 24 months old boars gave dose leves of 7.5, 37.5 and 187.5 ug/lb body weight for three separate periods. The hormone was aministered in each period three times weekly. After castration, each boar received an initial dose of 7.5 ug/lb body weight to be increased to the next level if the castrate failed to serve during a two week interval at this level. Bidner et al (1973) deviated from this mode of administration by including 2.2 mg methyl-testosterone per kg of ration fed to pigs lbetween 23 and 90 kg body weight. In all these instances it could be seen that all dosages were far higher than the physiologic level of testosterone in the boar blood. UNIVERSITY OF IBADAN LIBRARY - 116- 4.1.4 POSSIBLE MECHANISMS OF HORMONAL ACTIONS Several mechanisms for hormone action have been postulated. It has been demonstrated that a number of different hormones act on the active transport across cell membrane. This has been shown for insulin with regard to the transport of glucose into the cell and STH and androgens with regard to the transport of amino acids. A current hypothesis is that the hormones act as gene-activators leading to the production of messenger- RNA and induced enzyme synthesis (Karlson and Sekeris, 1966). The general effects produced by the action of hormones in the living organisms may be classified as follows: (1) Morphogenesis, (2) Maintennce of physilogical events. The somatotrophic (STH) hormone is an outstanding hormone with regards to morphogenetic action where the overall growth of the organism reflects the action of this hormone. The morphogenetic action of steroid hormones on the reproductive tract is seen in the growth of the uterus following treatment with estradiol and of the prostate following treatment with testosterone. The main argument is that hormones exert a large portion oft heir actions through the activation or inhibition of enzyme systems. There is evidence that many hormones bring about their intracellular effects through the mediation of a second messenger that has been shown to be 3',5' cyclic adenosine monophosphate (cylic AMP) (Sutherland et al., 1968). Through the action of adenyl cyclase, adenosine triphosphate (ATP) is converted to cyclic AMP, which then acts within the effector cell to produce the appropriate hormonal response. Cyclic AMP has now been established as a second messenger that mediates some of the effects of UNIVERSITY OF IBADAN LIBRARY - 117 - quite a number of hormones: ACTH on the adrenal cortex, TSH on the thyroid, vasopressin on the kidney, epinerphrine and glucagon on the liver, LH on bovine corpus luteum and Interstitial cells stimulating hormone (ICSH) on the testis which directly stimulates testosterone production. Hormones may also alter the permeability of the cell membrane or the membranes of intracellular organelles. In this way, the hormone could influence the movement of materials into the cells or between subsellular structures and thus condition the rate of some biochemical sequence (Levine and Goldstein, 1955). The cell membrane acts as a barrier and prevents the free entry of some materials but the hormone such as steroids that promotes the synthesis of proteins act at this cell surface to facilitate the entrance of amino acids into the intracellular pool. There is a growing interest in the possibility that some hormones activate specific genes (DNA), thus promoting the transcription of new kinds of M-RNA which then code for the synthesis of specific proteins at the ribosomal level. Hormones in circulation are frequently bound to specific carrier proteins and are continuously subjected to enzymatic inactivation or destruction in such organs as the liver and kidneys. 4.1.5 THE RECEPTOR MECHANISMS A lot of mention has been made about steroid receptors in the brain and the gonads. It is therefore pertinent to review the concept of hormonal receptors. The advent of hormone labelling has amply demonstrated that particular hormones are selectively concentrated by specific target cells and tissues; for example, estrogens by the uterus, androgens by the male UNIVERSITY OF IBADAN LIBRARY accessory sex glands and FSH and LH by the gonads. In general, the tissues that respond most profoundly show the highest uptake of the hormone and retain it longest. The mechanism of action of a hormone involves its interaction with receptor sites of the target cells and the chain of intracellular events that eventually leads to the organismal adjustments generally regarded as the effects of the hormone. The receptor hypothesis therefore states that every target cell has specific sites that bind particular hormones and some progress has been made in identifying and characterizing the macromolecules which serve the cells in this capacity. There seems to be two major types of hormone receptor sites: those on the cell surface associated with plasma membrane and others within the cell either as part of the internal membrane structure of present in the cytosol itself. It has also been found that in many cases, the receptor is or involves a protein (Sutherland, 1972). 4.1.6 THE ROLE OF MINERALS ON TESTOSTERONE METABOLISM The possible role of minerals and vitamins in reproduction has been the object of studies by some researchers in recent years. Calcium has for a long time been linked with protein synthesis (Kunerth and Pitman, 1939) and the growth and parathyroid hormones have also been implicated in the increase observed in the absorption of calcium ions in the young amimal. In addition, a high level of potassium ions is associated with a high rate of ATP depletion in conditions of high metabolic rate because ATP is the metabolic substrate for the energy-dependent transmembrane transport of potassium and sodium ions. UNIVERSITY OF IBADAN LIBRARY - 119 - The role of zinc in protein synthesis has also been well established (Lieberman et al., 1963). Zinc also plays a significant role in sexual maturation (Sandstead et al., 1973) and is important for the function of many enzymes (Mikac-Devic, 1970). Prasad and Oberleas (1974) found that since deficiency in rats depresses zinc content of the testis, hones, muscles, oesophagus and kidney. Dietery zinc deficiency has also been known to result in decreases in most organ weights such as liver, lungs, testis whereas the kidneys and adrenal glands increase in weight (Prasad and Oberleas, 1974). The condition also depresses the activities of many zinc-dependent enzymes. Since one of the most prominent effects of zinc deficiency is growth retardation, one may conclude that the protein content of the cell is being decreased concomittantly. Thus Fullis (1958), Millar et al (1958) and Barney et al (1968) observed marked atrophy of the testis in zinc deficient animals. Zinc is also involved in carbohydrate and lipid metabolism. Potassium, along with sodium, plays a major role in electroylte balance. While sodium functions extracellularly, potassium functions intracellularly. Their deficiency retards growth and lowers the utiliza­ tion of digested proteins. Sodium is also involved in fat deposition and testicular development (Orent-Keiles et al., 1937). Low potassium in diets causes slow growth and sterility (Orent-Keiles and McCollum, 1942). Sodium and potassium are also antagonistic to calcium and magnesium. Thus high levels of potassium disturb metabolism of magnesium. Testosterone improves calcium and phosphorus balance (Reifenstein and Albright, 1947). UNIVERSITY OF IBADAN LIBRARY - 120 - Magnesium is an enzyme activator for many enzyme systems involved in the metabolism of carbohydrates, proteins and fats. Copper plays a vital role in the synthesis and proper functioning of several oxidative enzymes necessary for normal metabolism. It's deficiency leads to lesions in the brain stem and spinal cord and poor growth. 4.1.7 ROLE OF THE HYPOPHYSEAL - ADRENAL - THYROID - GONADAL AXIS IN ANDROGEN METABLOSM The importance of the hypothalamus in the maintenance of pituitary functions has already been discussed. In recent years, much evidence has accumulated to show that impairment of pituitary, thyroid and adrenal functions have serious effects on normal reproductive behaviour in mammals. The thyroid hormone influences reproduction and fertility by helping to maintain the pituitary-hypophyseal-gondal relationship and also by affecting the metabolic pool of nitrogen and available energy, thus allowing the proper growth of tissues of the reproductive system and the growing embryo. Thus hypothyroidism impairs testicular development in young animals and delays sexual maturity and in certain cases abolishes testosterone production. Some studies (Samperex et̂ al., 1969) have indicated that the \ hypophyses have a much higher capacity to bind androgen molecules than the hypothalamus and that castration considerably activates the transformation of testosterone into dihydrotestosterone (DHT) in the hypophyses and that the rate of conversion increases with the length of time after castration (Martini, 197 3). UNIVERSITY OF IBADAN LIBRARY - 121 - This conversion is also activated by castration at the hypothalamic level. This suggests that testoterone must be converted into "active" metabolite before initiating the feedback responses mentined earlier and also reinforces the conclusion that both the anterior pituitary and hypothalamus are the sites on which androgen exerts its feedback effect on gonadotropin secretion. It is therefore probable that the pituitary plays a more important role in this process than the hypothalamus. It is interesting to note that following castration, a higher uptake of radioactive testosterone has been reported to occur both in the anterior pituitary and in the hypothalamus (Roy and Laumans, 1969). In addition, among the large number of steroids found in the adrenal cortex are sex hormones which include androgens, estrogens and progre- sterone-like substances. The amount of androgens produced by the andrenal cortex has been known to increase after castration, but the level is not sufficient to restore normal reproductive functions. 4.1.8 MATERIALS AND METHODS Fifty-one male Large White pigs housed, fed and provided water as described earlier were used. They were randomly divided into four groups as follows according to the age at which castration was done: Group 1: Orchidectomy as performed at one week of age. Group 2: Orchidectomy as performed at 3-4 months of age. Group 3: Orchidectomy as performed at 5-6 months of age. Group 4: Orchidectomy as performed at 7-8 months of age. Orchidectomy was performed by open surgery according to the method of Berge and Westhues (1966). UNIVERSITY OF IBADAN LIBRARY I - 122- Eac.h group therefore comprised (a) intact control boards (IC), (b) orchidectomized boars treated with 1 ml of corn oil containing 25 mg of testosterone enanthate intramuscularly (equivalent to 18.0 mg testosterone) (OT) and (c) orchidectomized control boars (OC) which were orchidectomizedand given intramuscularly 1 ml of corn oil. The injections were administered at 0900-1000 hours every Monday for five weeks. 24 hours after the last injection all the animals were slaughtered and their brains and hypophyses quickly removed, disseted and processed as described earlier. 4.1.9 BEHAVIOURAL TESTS 24 hours after the second testosterone administration, all animals in grups 2, 3 and 4 were regularly introduced to cycling gilts and the following parameters studied and respectively scored as follows: (a) SEXUAL BEHVIOUR Parameter Scores (i) Courtship 1 (ii) Mounting 2 (iii) Intromission 3 (iv) Ejaculation 4 AGONISTIC BEHAVIOUR Parameter Scores Investigative behviour 1 Strutting 2 Restless/Slashing 3 Violent/Biting 4 UNIVERSITY OF IBADAN LIBRARY - 123 - Scoring was based on the behaviour of each animal within a five-minute period. STATISTICAL ANALYSIS The data were subjected to multifactor analyses of variance and the treatment means compared by the least significant difference method of Steel and Torrie (1960). 4 . 2.0 EFFECT OF PRE-WEANING CASTRATION WITH OR WITHOUT TESTOSTERONE ON PORCINE BRAIN AND HYPOPHYSEAL AChE ACTIVITY TOTAL PROTEIN AND SAChE ACTIVITY The results are summarised in Table 4.1.1. Preweaning castration without testosterone replacement depressed AChE levels (P<0.05) in the amygdala, hypothalamus, midbrain and medulla oblongata while testosterone therapy restored AChE activity to normal levels in only the amygdala. The AChE content in the pons of OC and IC animals were similarly inferior to that of OT animals (P<0.05). AChE activity in the cerebellum and hippocampus of OT animals was higher than in OC animals but not IC animals which however did not differ significantly from the other controls. AChE in the cerebral cortex and hypothalamus was depressed by orchidectomy with or without testosterone while in the mesencephalon and medulla, the enzyme was partially restored and enhanced, respectively. In the adenohypophysis AChE activity was similar in the OT and IC but inferior to that in the OC animals while in the neurohypophysis, the testosterone therapy elevated the enzyme level beyond that of the IC but not that of the OC. The control animals were however similar. UNIVERSITY OF IBADAN LIBRARY 1 2 4 TABLE <1.1.1. THE EFFECT OF PREWEANING CASTRATION WITH OR WITHOUT. TESTOSTERONE ON 'ACETYLCHOLINESTERASE ACTIVITY IN THE PIG. ANIMAL GROUPS BRAIN REGIONS 0RCHIDECT0MI2ED a) ‘Acetylcholinesterase Kith testosterone Without testosterone Intact control ^ u m o i W g / m i n ) ^ Pons <4. 8 7 << + 0.270a 0.258 + 0.311b 0.180 + 0 .1 2 3 b —~_ Cerebellum 3.280 + 0.187a 2.513 + 0.0O2b 2.970 + 0.08lab Amygdala 5.899 ’+ 0.337a 3.590 + 0 .1 8 0 b 6.053 + 0.062a Hippocampus 0.979 + 0.226a 0.092 + 0.OlOb 0.091 + O.37oab Hypothalamus 0.153 + 0.135b 0.572 + 0.357b 5.737 + 0.172a Cerebral cortex 1 . ' 2 8 6 + 0.032b 1.398 + 0.021b 1.723 + 0.107a Mid brain 7.493 + 0.273b 0.070 + 0.211c 8.370 + 0.235a Medulla oblongata 7.079 + 0.353a ■ 0.601 + 0.150c 6.999 + 0.090 Adenohypophysis 0.561 + 0.053b 1 . 1 1 6 + 0.070 a 0.356xf 0.023b Neurohypophsis 1.120 + 0.063a 0.890 + 0.030ab 0.515 + 0.002b Grand mean 0 . 1 1 3 + 2.523 3.150 + 1 . 5 2 0 7.101 + 2.731 b) “ Frotein concentration ^QRCHIDECTOMIZED 1 ( g / 1 0 0 m l ' ) With testosterone Without testosterone Intact; control Pons 1.360 + _0 .035a 0 . 0 0 9 + 0.010b 1.390 + 0 . 0 8 6 a Cerebellum 1 . 2 0 6 + 0.000a 0.157 + 0 .0 0 8 c O.930 Amygdala 1.128 + 0 .0 0 5 a 0.206 0.007c .0.610 + 0 .0 3 3 b ‘ Hippocampus '1.058 + 0.028a G. 173 + 0.007c 0.791 + 0 .0 3 7 b Hypothalamus 1.101 + 0.028a 0.773 + 0.010b 0.989 + 0.050a Cerebral 1.090 + 0.000a 0.139 + 0.010b 0.966 + 0.027a Mid brain 1.260 + 0.o80a 0.068 + 0.029b 1.091 + 0.020a Medulla oblongata 1.510 + B.100a 0.020 + 0.009c 1.166 + 0.028a Adenohypophysis 1.0.59 + 0 .0 3 1 a 0.239 + 0.008b 1.033 + 0.051a Neurohypophysis. 1.096 + 0.058 a 0.250 + 0.025b * 1.037 + 0.028a Grand mean 1.188 + 0 . 1 5 0 0.331 + 0.198 1.005 + 0.209 c) ‘“ Specific acetylcho­ linesterase activity ;Tf7i;I,7.Fn (fimnl e / g p r o t e i n / m i n Vlth testosterone Without testosterone Intact control Pons ' 3 . 5 7 6 + 0.577b 9.490 + 0.657a 3.051 + 0.212b Cerebellum 2 . 7 3 9 + 0.203c 15.974 + 2.424a 3.248 + 0.282b Amydgala < 5 . 3 1 3 + 0.450c 14.690 + 0.869a 10.047 + 0.557b Hippocampus 4 . 7 1 8 •f 0.121b 23.736 + 2.470a 5.734 + 0 . 5 6 lb Hypothalamus 3 . 7 7 0 0.097c 25.218 + 1 .9 8 8 a ‘ 5.845 + 0 .2 6 2 b Cerebral cortex 1 . 0 9 4 + 0.100b 10.370 + 0.985a 1.732 + 0.097b Mid brain 6.020 + 0.394b 9.732 + 0.679a 7.678 + 0.227b Medulla Oblongata 4.930 + 0.199b 10.934 + 0.546a 6.012 + 0.114b Adenohypophysis 0.537 0.063b 4.694 + 0.358a 0 . 7 4 8 '-f 0.071b ”1? 11 v 0 h y p 0 P h y lz 1.036 ■f 0 .0 8 lb 3.667 4 0.305a 0 . 4 9 8 + 0.01 in UNIVERSITY OF IBADAN LIBRARY Jr-OH VO 0 0 +1 - 125 - Castration without testosterone significantly depressed protein levels in all brain regions. In addition, protein levels in the OT animals and the IC were similar in the pons, hypothalamus, cerebral cortex and midbrain while in the cerebellum, amygdala, hippocampus and medulla oblongata of the OT animals, protein levels were significantly enhanced. Protein concentration in both hypophyses of OT and IC animals were similar and significantly superior to the OC animals (P<0.05). SAChE activity in the OT and IC animals was similar and significantly inferior (P<0.05) to that in the OC animals in the pons, cerebellum, hippocampus, cerebral cortex, midbrain and medulla. SAChE activity was enhanced in the amygdala and hypothalamus of the OC animals than the IC which in turn had higher levels than the OT animals. Both the OT and IC animals have similar hypophyseal SAChE activity which were significantly lower than the OC animals. 4.2.1 MINERAL PROFILE IN THE BRAIN AND HYPOPHYSES The effects of the various treatments are summarized in Table 4.1.2. CALCIUM Calcium levels were similar in the OT and OC animals and superior to those in the IC animals in the pons and cerebral cortex while the reverse occurred in the hippocampus, hypothalamus and medulla oblongata. Calcium was much enhanced in the cerebellum, amygdala and mid-brain of the OC animals while much lower levels were observed in the IC and OT animals (P<0.05). In the adenohypophysis, the OC and the IC animals had similar calcium levels but inferior to the OT animals. In the neurohypophysis, the UNIVERSITY OF IBADAN LIBRARY - 126 - IC and OC animals had similar calcium levels which were superior to the OT animals. MAGNESIUM In the different brain regions, magnesium levels in the OT and OC animals were similar and superior to the IC animals in the pons and amygdala. In the medulla oblongata, the OC animals and the IC had similar magnesium levels which were superior to their OT counterparts. In the cerebral cortex, the OT animals and control had similar magnesium levels significantly (P<0.05) higher than the OC animals. The other brain regions viz: the hypothalamus, hippocampus and the cerebral cortex did not show a particularly consistent trend. In both hypophyses, the OC animals had significantly higher magnesium levels than the other two groups (P<0.05) which were similar to each other. ZINC Zinc was depressed in the hippocampus, hypothalamus and medulla oblongata of the OC animals while it was considerably enhanced and moderate in the OT and IC animals, respectively. Highest zinc levels were recorded in the cerebral cortex and midbrain of the OC animals, medium in the OT and least in the IC groups. Zinc was depressed in the cerebellum and amygdala of the IC animals, moderate in the OC animals and considerably enhanced in the OT animals. UNIVERSITY OF IBADAN LIBRARY «' 127 ' TABLE 4.1.2 FEPFC"’ OF PRK-KFANIMO CASTRATC ?N AND TE8TC PTERCIIE ON T:‘E * MINERAL PROFILE IN THE BRAIN AND HYPOPHYSES OF PIGS. ANTMA! 1R0U?s ORCHIDECTOMi: ED a) CALCIUM With testosterone Without testosterone Intact control Pons 1.639 + 0.175 a 1.796 + 0.058 a 1.374 + 0.021 b Cerebellum 2 . 0 6 1 + 0.025 a 2.172 4 0.059 ab . 1.883 + 0 . 0 5 0 .b Amygdala 1.53^ + 0.035 b 2.214 + 0 . 0 2 8 a 1.760 + 0.033 b Hippocampus 2.670 + 0.051 b 1.474 + 0.031 b 2.053 + 0.032 a Hypothalamus 1.480 + 0.030 b 1.679 + 0 . 1 0 8 b 2.180 j: 0.031 a t Cerebral cortex 1.772 + 0.034aa 1.630 + 0.022 a 1.406 + 0.004 b Mid brain 1.264 + 0.031 c 1.690 + 0 . 0 3 8 b 2.246 + 0.062 a Medulla oblongata 1.638 + 0.059 b 1.645 + 0.034 b 2.530 + 0.025 a Adenohypophysis 2.350 + 0.045 a 2. C39 + ■C.010 b 2.053 + 0.030 b Neurohypophysis 2.070 + 0.019 b 2.420 + 0.067 a 2.516 + 0.098 a Grand mean ■>: 1.753 + 0.323 1.906 + 0.303 2.001 + 0.403 b) ’MAGNESIUM ORCHIDECTOMIZED With testosterone Without testosterone Intact control Pons 1 . 3 6 8 + 0.020 a 1.362 + 0 . 0 3 4 a 1.258 +^0.017 b Cerebellum 1.341 + 0.013 ab 1.406 + 0 . 0 1 3 a 1.305 + 0.013 b Amygdala 1 . 3 6 1 + 0.029 a 1.342 + 0 . 0 0 7 a 1.196 + 0.004 b Hippocampus 1.270 + 0 . 0 0 8 c 1.330 + 0 . 0 0 9 “ 1.490 + 0.043 af Hypothalamus 1070 + 0.021 a 1.227 + 0 . 0 3 t9 "l.OlO + 0.058 c Cerebral cortex 1.542 + 0 . 0 6 5 a 1.484 + 0 . 0 2 0 at 1.422 + O.O6 3 b Mid brain 1.326 + 0.045 a 1.183 + 0 . 0 2 7 b 1.397 + 0.005 a Medulla oblongata 1.390 + 0.045 b - 1.491 + 0 . 0 6 8 a - L.53S + 0 . 0 0 8 - Adenohypophysis ’***'“" 1.257 + 0.008 b 1.528 + 0.004 a 1.325 + 0.015 b NeurohyooDhysis - 0.901 +■ 0.060 b 1 . 1 8 9 + 0.005 a ’ - -_• -1-.0-05 + 0.035 b Grand mean 1.313 + 0.164 1.360 + 0.125 1.295 + 0.183 c) ZINC 0RCKIDECT0MI ZED ̂ With testosterone Without testosterone Intact control Pons 0 . 3 2 6 4- Q.0.0.6 c 0.332 + 0.004 b 0 . 4 3 7 + O'. 003 a Cerebellum 0 . 4 7 3 + Q. 0 1 0 a ' 0.373 + 0.072 b 0 . 3 1 3 + 0 . 0 0 6 c Amygdala 0 . 5 8 1 *-0.012 a 0.-10 + 0.006 0 0 . 3 0 8 + 0.003 b Hippocampus 0 . 4 7 2 + 0.007 a 0.262 + C.006 c 0 . 3 0 3 + 0 . 0 0 3 b Hypothalamus 0.464 +. 0 . 0 0 6 a 0.287 + 0.004 c 0 . 3 5 0 + 0.003 b Cerebral cortex 0.319 + 0.001 b 0.364 + 0 . 0 0 3 a 0 . 2 5 1 + 0.003 c Mid brain 0 . 3 0 2 + 0.002 b 0.416 + 0.006 a 0.242 + 0.005 c Medulla oblongata 0.482 + 0.005 a 0.336 + 0 . 0 0 8 c 0.402 + 0.005 b Adenohypophysis 0.454 + 0.002 b 0.307 + 0.003 a 0.520 0.003 a Neurohypophysis 0.409 + 0.002 b 0.273 + C.005 c 0.47 4 + 0.003 a Grand mean 0.428 + 0.039 0.359 + O.O8 3 0.371 + 0.093 Values in the same horizontal column differently superscripted differ significantly {?< 0.05) Values are means + S.E.M. * Values In parts per million (ppm). UNIVERSITY OF IBADAN LIBRARY 1 2 8 TABLE 4.1.2 Continued BRAT" REGIONS ANIMAL 1 ROUPS OFCHIDECfOMTZED r) POTASSIUM With tes:tosterone Without testosterone Intact control Pens 22.800 + 0.860 a 25.415 + 1.187 a 22.592 0.094 a Cerebellum 25. ̂55 + 1.645 a 25.400 + 1.596 a 22.5 5 + 0.779 a' Amygdala 2 7 . 2 0 0 + 2.482 a 1 8 . 1 0 0 + 2.190 b 21.000 •i- 1.129 b Hippocampus 18.750 + 2.46 b 24.700 + 0.952 a 21.000 + 1.346 a Hypothalamus 2 3 . 0 0 0 + 1.658 a 22.070 + 0.398 a 22.204 + 0.647 a Cerebral cortex 27.946 + 1.416 a 27.465 + 1.377 a 27.540 + 1.418 a Mid brain 21.710 + 1.514 a 21.445 + 0 . 8 9 6 a 21.976 + 0.664 a " ... Medulla oblongata 22.000 + 1.458 a 25.929 + 0.839 a 25.868 + 1 . 2 3 2 a Adenohypophysis 27.550 + 3 . 1 8 6 ab 29.123 + 1;220 a 24.803 + 1.996 h Neurohypophysis 20.915' + 0-. 551 ab 1 8 . 0 4 6 + 0 . 9 7 3 b ' 24.130 + 1.183 a Grand mean 23.733 + 3.136 23.769 + 3.748 24.377 + 3.092 e) SODIUM ".... ORCKIDECTOMIZED With testesterene Without 'testost;one Intact control Pons 528,9.60 + 2,430 a- 530,650 + 0.970 a 5 1 8 . 0 0 + 1.176 a Cerebellum 531.000 + 4.848 a 528.350 + 1 . 2 8 8 a 525.000 + 3.688 a Amygdala 515. + 5.701 a 531.200 + 1.268 a 522.750 + 2.694 a Hippocampus 5 3 2 . 0 0 + ‘).062 a 531.900 + 1 . 2 0 8 a 523.000 + 6 . 8 7 9 a Hypothalamus -- - 5 2 9 . 0 0 0 + 5.788 a 534.700 + 2.502 a 5 2 0 ^ 6 0 0 + 1.517 a Cerebral cortex 518.700 + 1.364 a 521.355 + 0.847 a 514.200 + 3.397 a Mid brain 514.32Q + 3.747 a 521.450 + 2.488 a 532.400 + 2.502 a Medulla oblongata 523v200 + 0.231 a 521.620 + 1.071 a 5 7 2 . 8 0 0 + 3.007 a Adenohypophysis 536.900 + 1.8Q6 a 512,000 + 7.314 a 5 3 1 . 0 0 0 + 2.214 ■a Neurohypophysis 521.800..+ 4.934 a 539.400 + 2.132 a 520.400 + 3.010 a Grand mean 524.gg2 + 7.512 528.462 + 6.443 523.553 + 6.152 a COPPER -0 —RC H' IVD ECT0MIZ■ -E D • - - •• - 1. :■ With testosterone Without stestosterone Intact control Pons 0.120 + •0.001 b 0 . 1 2 6 + 0 . 0 0 6 b 0.153 + 0.007 a Cerebellum 0.154 + 0.003 a 0.153 + 0.008 a 0.163 + 0.003 a Amygdala r _ 0.170 + 0.001 a 0.149 + 0.007 b 0.093 + 0.004 c • Hippocampus . D.,124 + 0.003 C 0.152 + 0.Q05 b- ■*— * 0.17J. + G.A0 3 a Hypothalamus 0.152 + 0.010 b 0.145 + 0.008 c 0.191 + 0.007 a Cerebral cortex 0 . 1 6 0 + 0.QQ2 a 0.122 + 0.003 b 0.172 + 0.005 h Mid brain *. O.litil + 0.00.8 b 0 . 1 2 7 + 0.Q02 c 0.205 + 0.007 a Medulla oblongata 0.148 + 0.QQ8 b 0.123 + 0.00.1 c 0.170 + 0.004 a Adenohypophysis 0 , 2 6 0 + 0.0C3 c 0.280 + 0 . 0 0 7 b 0.302 + 0.010 a Neurohypophysi s 0,268 + "0.00 4 a 0.145 + 0 .OO.3 b 0.265 + 0.012 a Grand mean 0.171 + 0.051 0.153 + 0.046 0.189 + 0.057 Values In the same horizontal column differently superscripted differ significantly (p< 0.C5) Values are means + S.E.M. UNIVERSITY OF IBADAN LIBRARY - 129 - In the adenohypophysis, both the OC and IC groups had similar levels which were higher than the OT groups (P<0.05). In the neurohypophysis, highest levels were observed in the IC animals with the OC animals recording the lowest level while the OT animals showed a slightly higher value (P<0.05). POTASSIUM Potassium levels in the three treatment groups were similar in all the brain regions except in the amygdala where potassium was significantly depressed in the OC animals and in the hippocampus where the OC animals had higher levels than the other two groups. In the adenohypophysis, both the OT and OC animals had similar potassium content but potassium is considerably more enhanced (P<0.05) in the OC than the IC animals. In the neurohypophysis, the OC animals had similar level with the OT animals but inferior to the IC animals. The IC group was however similar to their OT counterparts. SODIUM There was no significant (P>0.05) treatment effect on both brain and hypophyseal sodium levels. COPPER Copper was significantly depressed in the medulla, midbrain, cerebral cortex, hypothalamus and pons of the OC animals. No significant changes were observed in the cerebellum but in the hippocampus, the OT animals had depressed copper levels while moderate and considerably enhanced levels were observed in the OC and the IC animals, respectively. UNIVERSITY OF IBADAN LIBRARY - 130 - Copper was lowest in the amygdala of the IC animals, medium in the OC animals and highest in the OT group. In the adenohypophysis, the highest copper level was recorded in the IC animals (P<0.05) followed by the OC group and least in the OT group. In the neurohypophyses, testosterone therapy restored copper levels to the same level as found in the intact control while orchidectomy significantly depressed it. 4.2.2 EFFECT OF PRE- VERSOS POST PUBERTAL CASTRATION WITH OR WITHOUT TESTOSTERONE THERAPY ON SEXUAL BAHAVIOUR IN MALE PIGS Each group of experimental animals was tested with intact and mature sows and the response noted. Table 4.1.2. CASTRATED PIGS WITHOUT REPLACEMENT THERAPHY The prepubertally castrated pigs, i.e. those castrated at three months of age were very timid and lacked the urge to go for the females. It was also observed that when the females discovered this docility in the pigs, they became very aggressive and chased the barrows all over the pen, biting and slashing at them all the way. After about 3 weeks of constant exposure to the females, about 60% of them became fairly aggressive and repelled the attack of the females. However, only about 20% attempted mating while the remaining 80% showed apparent disinterest in the coutship process. The pigs castrated between 5-6 months of age and 7-8 months of age were fairly aggressive towards the female within 10 seconds of exposure but still lacked of interest in mating. They were however more interested in UNIVERSITY OF IBADAN IBRARY •31 Table 4.1.2.1 PARAMETERS OF AGGRESSION AND SEXUAL BEHAVIOUR DISLAYED BY PRE-VERSUS POST-PUBERTALLY CASTRTED MALE PIGS COMPARED TO GONADALLY INTACT MALES Pre­ Post- pubertal ly pubertal ly Castrates Castrated Castrated Intact Treated with Males Males Males Testosterone ALL SUBJECTS 12 24 36 36 (Total number tested) Number of animals observed to bes Investigative 4 2 30 18 Strutting 2 4 15 15 Restless 7 5 32 26 Violent 1 13 25 23 Aggression index 2.36 + 0.97 3.21 + 1.02 2.50 + 1.16 2.66 + 1.11 Main parameter Strutting/ Restless/ Strutting/ Strutting/ exhibited Restless Violent Violent Restless SEXUAL BEHAVIOUR Number of animals displayed: Courtship 3 18 34 20 Mounting 2 4 32 18 Intromission - - 5 - Ejaculation - - 5 - Behaviour index 1.40 + 0.49 1.18 + 0.38 1.75 + 0.85 1.47 + 0.50 Main behaviour Courtship/ Courtship/ Courtship/ Courtship/ exhibited Mounting Mounting Mounting Mounting UNIVERSITY OF IBADAN LIBRARY - 132 - agon i s t i c behaviour such as strutting, slashing and biting (see Table 4.1.2). CASTRATED PIGS WITH TESTOSTERONE REPLACEMENT THERAPY All the three treated groups showed marked interest in the female whether on heat or not by the third week of testosterone therapy. However, the interest was more aggressive initially without about 65% of them becoming very aggressive within 10 seconds of contact with the females and displayed characteristic agonistic behaviour. By the fourth week of treatment about 50% were attempting mating within 30 seconds of contact with the female while over 80% of them still displayed aggressive behaviour by the end of the fifth week with 60% attempting mating. INTACT BOARS Above 90% of the intact controls attempted to mate when exposed to females. They displayed characteristic boar sexual behaviour by sniffing the urinogenital area of the females and following them around. In some cases when the females urinated, the male would sniff the urine perhaps trying to discern a particular odour which may indicate receptivity by the sow or not, depending on her estrus status. This courtship period normally lasted for a few seconds and the boars attempted to mount within 5 seconds of entry into the female pens. Females that were not on heat and which restricted mounting were vigorously pursued and attacked. They too fought back. It should also be noted that the sows accepted the intact boars more readily than they did the castrates. UNIVERSITY OF IBADAN LIBRARY - 1 3 3 - 4 . 2.3 EFFECT OF ORCHIDECTOMY AT 3-4 MONTHS OF AGE AND TESTOSTERONE ON THE PIG BRAIN AND HYPOPHYSEAL PHYSIOLOGY Table 4.1.3 summarizes the effects of orchidectomy with and without testosterone on the acetylcholinesterase activity, specific acetylchol- insterase activity, and protein content of the porcine brain and hypophyses. Castration without testosterone significantly depressed AChE in the pons, cerebellum, hippocampus, hypothalamus and medulla oblongata while replacement therapy boosted activity to above normal levels in the same regions except the hypothalamus. The cerebral cortex, midbrain and amygdala were not significantly affected by orchidectomy but the OT animals had considerably enhanced activity in the amygdala and midbrain, cerebellum, pons, amygdala, hippocampus, hypothalamus and medulla oblongata than the IC animals. AChE activity in the adenohypophysis of the OC and IC animals were similar and inferior to the OT group. However, no significant differences (P>0.05) were observed between the OT and the OC animals. In, addition no significant differences were observed in the neurohypophysis. Castration without replacement therapy similarly depressed protein levels in the pons, cerebellum and medulla oblongata (P<0.05) while in the OT animals it was elevated more than the IC animals In the pons, cerebellum, amygdala, hippocampus and midbrain. No significant effects were observed in the hypophyses. SAChE activity was significantly depressed in the amygdala, hippocampus, hypothalamus, cerebral cortex and midbrain of the OT animals and elevated in the OC and IC animals. SAChE activities in the hippocampus, cerebral cortex and midbrain of both the OC and IC were similar. UNIVERSITY OF IBADAN LIBRARY 134 TABLE 11.1.3 '.'HE EFFECT OF ORCHIDECTOMIZED WITH OR WITHOUT TESTOSTERONE AT 3-# MONTHS OF AGE ON FROCINE BRAIN AND HYPOPHYSEAL PHYSIOLOGY. BRAIN REGIONS ANIMAL GROUPS a) •ACETYLCHOLINESTERASE ORCHIDECTOMIZED ACTIVITY With testosterone Without testosterone Intact; control Pons 7.310 + 0 . 3 6 3 a 2.965 + 0.256 C 9.217 + 0 . 1 7 0 b Cerebellum A . 3 0 0 + 0 . 1 8 0 a 2.209 + 0.0S5 c 2 . 9 8 1 + 0 . 1 0 3 b Amygdala 5.960 + 0.300 a 5.022 + 0.190 b 9.793 4 0.119 b Hippocampus 6.1468 + 0 . 1 7 6 a 3.395 + 0.o59 c 9.565 4_ 0.286 b i Hypothalamus 6.059 + 0.196 a 9.138 + 0.379 b 5.826 + 0 . 1 6 1 a Cerebral cortex 1.8h3 + 0 . 0 8 6 a 1.690 + 0.057 a 1.779 + 0.037 a Mid brain 9.663 + 0.956 a 7.960 + 0.113 b 7.222 + 0.111 b Medulla oblongata 9.516 + 0.295 a 5.257 + 0.283 c 6.193 + 0.327 b Adenohypophysis 1.855 + 0.023 a 1.918 + 0.032 ab 0 . 8 6 1 4 0.059 b Neurohypophysis 1.196 + 0.099 a 1.075 + 0.025 a 1.151 + 0.020 a Grand mean 5.1*12 + 3.076 3.908 +. 2.050 3.955 + 2.199 b) **PROTEIN CONCENTRATION ORCHIDECTOMIZED With testosterone Without testosterone • Intact control Pons 1.021 + 0 . 0 8 6 a 0.572 + 0 . 1 0 9 0.965 + 0.157 ab ( Cerebellum 0.799 + 0.070 a 0.328 + 0.090 c 0.560 + 0.087 b Amygdala 0.962 + 0.056 a 0.127 + 0.003 b 0 . 1 6 8 + 0.0C8 b Hippocampus 0.907 + 0.088 a 0.177 + - . 0 3 2 b 0.198 + 0.012 b Hypothalamus "0.909' + 0.093 a 0.l97n+ 0 . 0 0 5 b 0 . 2 6 3 + *0.019 ab Cerebral cortex 0 . 2 3 6 + 0.015 a 0.097 + 0 . 0 0 6 a 0.136 4 0.013 a Mid brain 0.932 + 0.109 a 0.279 + 0 . 0 2 5 b , 0.273 + 0 . 0 3 8 b Medulla oblongata 0.979 + 0 . 0 2 3 a 0.257 + 0.020 b 57968 + 0.025 a Adenohypophysis 0.117 + 0.003 a 0.175 + 0.010 a 0 . 1 8 3 + 0.020 a Neurohypophysis 0.109 + 0.009 a 0 .1 8 ^ + 0.005 a 0.088 + 0.005 a C-rand mean 0.596 + 0.396 0.239 + 0 . 1 3 8 0.330 4 q. 2 6 7 c) SPECIFIC ACETYLCHOLINE­ ORCHIDECTOMIZED STERASE ACTIVITY Pons — 7.276 + 0.990 a 9.732 + 0.897 a — . 9.653 4 ,9 . 6 0 5 a Cerebellum 5.599 + 0.686 a 7.151 + \1 . 2 1 9 a 5.787 T 1 . 1 2 6 a Amygdala 13.686 + 2.279 C 90.903 + 9 . 3 8 1 a 28.639 4 1 . 5 2 5 b Hippocampus 7.27& + 0 . 5 2 0 b 20.799 + 3 . 3 8 6 a 23.390 + 2.037 a Hypothalamus 15.967 + 2.029 0 28.067 + 2.002 a 22.999 + 1 . 3 2 5 b Cerebral cortex 7.891 + 0.198 b 16.979 + 0.726 a 13.5*7 4 1 . 3 9 3 a Mid brain 10.597 •+ 1.122 b 27.770 4 2.177 a 27.991 4 3 . 6 0 1 a Medulla oblongata 20.197 + 0.839 a 20.557 + 0.815 a 13.315 4 0 . 8 3 5 b Adenohypophysis 15.933 + 0.646 a 8 . 2 0 3 + 0.651 b 5.762 4 1 . 3 1 8 b Neurohypophysis 11.102 + 0.289 a 5.691 + 0 . 0 7 0 b 13.191 4 0 . 3 7 6 a Grand mean 11.983 + 9.720 18.029 + 11.809 15.862 4 9. 37 5 Values In the same horizontal column different superscripted differ significantly (P< 0.05) Values are means + S.E.M. « SChE act!vtty-in pmole/g/minc; ** Total protein lr. g/100 ml *** f.ACM- activity in umole/g/protein/min. UNI ERSITY OF IBADAN LIBRARY - 1 3 5 - The OC animals however had higher activities than the IC in the amygdala and hypothalamus. The pons and cerebellum were not significantly affected (P>0.05) and SAChE activity in the OC and OT animals were similar and superior to the IC. In the adenohypophyses, SAChE activity was significantly higher (P<0.05) in the OT animals than either the OC and IC groups which were similar. SAChe activity in the neurohypophyses of both the OT and IC were similar and superior to the OC animals. 4.2.4 MINERAL PROFILE IN THE BRAIN AND HYPOPHYSIS Table 4.1.4 summarizes the effects of the treatments on the brain and hypophyseal mineral content. Castration without testosterone significantly depressed calcium levels in the pons, cerebellum, amygdala, hypothalamus and midbrain (P<0.05). Higher calcium levels were observed in the medulla oblongata, midbrain, cerebral cortex and hippocampus in the IC than in the OT group while a reverse of this trend was observed in the pons. Calcium levels in the cerebellum and hypothalamus of both the IC and OT animals were similar. Calcium was significantly depressed in the adenohypophysis of the OC animals and partially restored in the OT group. In the neurohypophysis, similar calcium levels were observed in the OC and OT animals but the OC animals had significantly lower level than the IC animals (P<0.05). UNIVERSITY OF IBADAN LIBRARY - 1 3 6 - MAGNESIUM Magnesium levels were relatively stable in the cerebellum and hypothalamus. In the medulla oblongata and amygdala, the OC animals had significantly lower (PC0.05) levels than the IC and OT animals whereas in the hippocampus and cerebral cortex, the untreated castrates had higher magnesium levels than either of the other two groups. Magnesium was highest in the adenohypophysis of the OT group, medium in the OC and low in the IC animals. In the neurohypophysis, the OT and IC had similar levels which were superior to that in the OC group. ZINC Castration without testosterone significantly reduced zinc level in all brain regions except the midbrain where no significant treatment effects were observed. Testosterone partially restored zinc level in the cerebellum, amygdala, hippocampus, hypothalamus, cerebral cortex and medulla oblongata. Zinc level in the pons of OC and OT animals were similar and significantly higher than the IC animals. POTASSIUM Castration without testosterone significantly depressed potassium level in all brain regions while testosterone partially restored it. The IC animals also had higher levels than the OT animals in the medulla oblongata, cerebral cortex, hypothalamus and pons while similar levels were observed in the amygdala and midbrain. Castration without testosterone significantly depressed potassium level in both hypophyses and testosterone partially enhanced it in the adenohypophsis but not in the neurohypophysis. UNIVERSITY OF IBADAN LIBRARY 137 . 'ABLE 9.1.9; EFFECT OF ORCHIDECTCMY AT 3-4 MONTHS OF AGS AND TESTOSTERONE ON THE *MINERAL PROFILE OF THE PORCINE BRAIN AND HYPOPHYSIS, BRAIN REGIONS ANIMAL GROUPS • ORCHIDEI'TCMIZED a) CALCIUM With' testotercne Without testosterone Intact control Pons 2.200 + 0.091 a 0.925 + 0.093 C 1.375 + 0.025 b Cerebellum 2.175 + 0.085 a ■ 1.000 + 0.071 b 2.325 + 0.085 a Amygdala 0.995 + 0.067 b 0.900 + 0.091 c 1.385 + 0.075' a Hippocampus _1.125 + 0.025 b 1 . 0 2 5 + 0.075 b 2.925 + 0.133 a Hypothalamus 1.525 + 0.063 a 1.100 + 0.082 b 1.362 + 0.029 a Cerebral cortex 1.025 + 0.025 b 1.100 + 0.025 ■C 2.225 + 0.335 a Mid brain 1.575 + 0.085 b 0.825' +'0.025 cc 2.125 + 0.098 a Medulla oblongata . 1.062 + 0.025 b 1.000 + 0.091 b 2.975 + 0.199 a Adenohypophysis 1 . 8 9 0 + 0.012 b 0 . 8 9 5 + 0.005 -C 2.605 + 0 . 0 6 6 a Neurohypophysis .1.287 + 0.031 ab 1 . 1 2 5 + 0.098 b 1.375 + 0.025 a Grand mean ~ 1 .1186'+ 0.097 0.939 + 0.213 2 . 0 6 8 + 0.655 b) 'MAGNESIUM ORCHIDECTOMIZED With testosterone Without; testosterone Intact control Pons 2.375 + 0.025 a 2.137 + 0.029 b 1 . 8 8 7 £*0.012 c / Cerebellum 2.000 + 0.025 a 2.037 + 0 . 0 2 8 a . 2 . 0 2 7 + 0.029 a Amygdala 2.305 +0.051 b 1.099 + 0.073 c 2 . 5 5 0 + 0.096 a Hippocampus 1 . 1 8 0 + 0.029 b 1.782 + 0.069 a f 1 . 2 1 2 + 0.093 b Hypothalamus 2.375' + 0.098 a 2.265 + 0.057 a 2 . 3 6 2 + 0.037 a Cerebral cortex 1.725 + 0.183 b 2.195 + 0.098 a 1 . 1 8 7 + 0.031 c Mid brain 1.895 +.0.021 a 1.555 + 0.012 a 1 . 2 8 2 + 0.022 b Medulla oblongata 2.'385 + 0.053 a ' 1.975 + 0.085 b 2.907 + 0.067 a Adenohypophysis 2 . 0 3 0 + 0.029 b 1.730 + 0.093 b 1.525 + 0.032 c Neurohypophysis 1.725 + 0.091 b 2.075 + 0.025 a 1.830 + 0.029 b • Grand mean • 2.002 + 0.369 1.879 + 0.351 1.827 + 0.513 c) ZINC ORCHIDECTOMIZED", Pons 0.538 + 0.023 a 0 . 6 0 2 + 0.022 a 0.288 + 0.007 b Cerebellum 0.1(62 + 0 . 0 1 8 a 0.258 + 0.020 b 0.530 + 0.S09 a Amygdala 0.301) + 0.009 a 0.190 + 0.005 b 8.311 + 0.007 a Hippocampus ^ 0.360 + 0 . 0 2 6 b 0.156 + 0.017 C _ 0.505 —+ .0.090 a * Hypothalamus 0.256 + 0.005 b 0.139 + 0 . 0 0 6 C ' 0 . 5 2 0 + 0.031 a Cerebral cortex 0 . 2 5 0 + 0 . 0 1 8 b 0.231 + 0.095 b 0.513 + 0.052 a Mid train 0.192 + 0.019 a 0.162 + 0.025 a 0.220 + 0.011 a Medulla oblongata 0.259 + 0.020 b 0.198 + 0.016 c 0.625 + 0 . 0 5 8 a Adenohypophysis 0.239 + 0.012 b 0 . 1 1 3 + 0.009 c 0.692 + 0.090 a fieurohypophysis 0.229 + 0 . 0 1 1 & 0.168 + 0.001 a 0.220 + 0 . 0 0 8 a Grand mean 0,308 + 0 . 1 1 3 0.217 + 0.192 0.992 + 0.169 Values in the same horizontal column differently superscripted differ significantly (P <0.05) Values are means +_ 3.E.M. •Values In parts per million (ppm) UNIVERSITY OF IBADAN LIBRARY 1 3 8 TABLE 4.1.4 (CONTINUED) BRAIN REGIONS ANIMAL GROUPS a) POTASSIUM OR CH ID EC TOM 11 ED With testosterone Without testosterone Intact control Pons 8.3(30 + 0.644 b 6.QOO 4 0 . 6 3 7 b 2 7 . 2 7 5 4 1 . 0 5 1 a Cerebellum 22.375 + 1.028 b 1*1.250 + 0 . 7 5 0 c 3 0 . 3 7 5 4 1 . 0 6 8 a Amygdala 20.625 + 1 . 3 0 7 a 8.375 4 0 . 5 9 1 b 20.125 4 0 . 8 7 6 a Hippocampus 17.750 + 1 . 0 2 8 a • 5.625 + 0 . 6 2 5 c 11.500 4 0 . 6 1 2 b Hypothalamus 21.250 + 0.722 b 1 .625 4 1.068 c 31.750 4 1 . 1 8 1 a Cerebral cortex 7.625 + 0.31*1 c 11.875 4 1.181 b 22.250 4 1 . 0 3 1 a Mid brain 1*1.000 + 0.657 a 7.375 + 1.021 b' 16.875 4 0 . 7 1 8 a Medulla oblongata 17.500 + 1.021 b 10.500 + 1.028 c 28.375 4 0 . 4 7 9 a | Adenohypophysis 9.625 + 0.375 b . 5.875 + 0.975 c 25.875 4 1 .8o3 a I Neurohypophysis 9.125 + 0.987 b 7.625 + 1.028 b 1*1.375 4 0.473 a Grand mean 1*1.817 + 5.795 8.512 4 2.8*12 22.877 4- 6.986 e) SODIUM 0RCHIDECT0MIZED With testosterone Without testosterone Intact control Pons 520.500 + 2.1(32 a **53.750 + 5.5*13 b 5 2 0 . 0 0 0 4 4.082 a Cerebellum 528.750 + *1.732 a 316.250 + 2.39*1 b . '526.250 4 5.543 a Amygdala 516.250 +_ 3.1**6-a 51*1.250 + *1.0*19 a 5 0 8 . 0 0 0 4 1.779 a Hippocampus 538.125 + 1.779 a 522.500 + 1.031 a 5 *1 5 . 0 0 0 4 4.083 al Hypothalamus 235.000 + 2Q.1Q*1 b 3 0.3. Q0.CL + 12.069 t 536.250 4 3.750 a Cerebral cortex 526'.250 + 6.575 a 5*10.0.0.0 + 3-535 a 536.875 4 5-340 a Mid brain 5*H'.815 + 1.197 a 5 0 6 .7 5 0 :+ 2.360 b 551.750 4 3.119 a Medulla oblongata 093.750 + 7.065 b 537.500 + **.330 a 522.500 4 4.787 ab Adenohypophysis 5*16.750 + 1.97*1 a 2 7 6 . 1 2 5 + 5.35** b 55*1.250 4 2.834 a Neurohypophysis 2 6 5 .OOO + 17.100 b 523.750 + 3.750 a 517.375 4 2.626 a / Grand mean 071.220 + 117.153 *1*1 9 . 3 8 7 ~~53l.8l9 4 15.47 e) COPPER ORCHIDECTOKIZED With testosterone Without testosterone Intact control Pons 0.262 + 0.Q05 a 0.139 + 0.070 a 0.112 4 0.011 a Cerebellum 0.155 + 0.0.Q5 a 0.113 + 0.003 a 0.095 1 0.005 a Amygdala 0.367 + 0.013 b . 0.179 + 0 . 0 0 8 b 0 . 6 0 2 4 0.0.51 a Hippocampus 0.100 + 0 . 0 0 0 a Q. Oh 8 + O.Oil 0.179, 4 0.005 a Hypothalamus 0 . 0 8 0 + 0.003 a 0.050 + 0.002 i - 0 .08*1 4 0.003. a Cerebral cortex 0.157 + 0.025 a O.O6 3 + 0.0Q3 a 0.212 4 0.014 a Mid brain 0.060 + 0.00.5 a 0 . 0 7 0 0.001 a 0.251 4 0.010 a Medulla oblongata 0.080 + 0.002 a 0 .0*16 + 0.005 a 0 . 1 1 8 4 0 . 0 0 6 a Adenohypophysis 0.087 + 0.Q05 a 0.120 + 0.076 a 0.105 4 0.003 a Neurohypophy s i s 0.090 + 0.00.6 a 0.200 + 0.0]*t a 0.162 4 0.005 a Grand mean Q.1*1*1 + 0.035 0.103 4 0.056 0.192 4 0.154 Values in ,the same horizontal column differently superscripted .differ significantly (P < 0.05) Values are means + S.E.M. UNIVERSITY OF IBADAN LIBRARY - 1 3 9 - SODIUM The OC animals had significantly depressed sodium levels in the pons, cerebellum, hypothalamus and midbrain while testosterone restored the level to normal. The other brain regions were not significantly affected (P>0.05). Castration without testosterone significantly depressed sodium level in the adenohypophysis compared with the IC and OT animals which were however similar. COPPER No significant effects were observed in the pons, cerebellum, hippocampus, hypothalamus, cerebral cortex and medulla. In the midbrain and amygdala, the OC and OT animals were similar and inferior to the IC animals (P<0.05). No significant effects were observed in both hypophyses. 4.3.0 EFFECT OF ORCHIDECTOMY AT 5-6 MONTHS OF AGE WITH OR WITHOUT TESTOSTERONE ON THE PORCINE BRAIN AND HYPOPHYSEAL ACETYL­ CHOLINESTERASE LEVELS Table 4.1.5 summarizes lthe effect of orchidectomy at 5-6 months of age with or without testosterone on the porcine brain and hypophyseal acetylcholinesterase activity. AChE activity is depressed in the pons, cerebellum, amygdala, hypothalamus and medulla oblongata of the OC animals while testosterone considerably enhanced it in the amygdala, hippocampus, hypothalamus and medulla oblongata. The cerebral cortex and the hypophyses were unaffected. UNIVERSITY OF IBADAN LIBRARY 1 ZfO. TABLE 11.1.5: THE EFFECT OF ORCHIDECTOMY AT 5-6 MONTHS CE AGE AN? TESTOSTERONE ON FROCINE BRAIN AND HYPOPHYSEAL PHYSIOLOGY BRAIN REGIONS A M MAI. GROUPS ft) •ACETYLCHOLINESTERASE ORCHir‘KCTOMI "EDACTIVITY With testosterone Without tes:tcsterone Intact control Pons 3.1*29 + 0.106 a. 3.325 + 0.0S4 a 4.914 + 0.112 a Cerebellum. 2.805 + 0 .0 7 S a 2.037 + 0 . 0 2 8 b 2.700 + 0.227 a Amygdala 6.057 + 0 . 0 7 9 a 3.54S + 0.379 c 4.571 + 0.128 b Hippocampus 5 . 1 7 4 + 0.226 a 3.519 + 0.276 b 3.638 + 0.143 b Hypothalamus. 7.051 + O.UOl) a 3.220 + 0.329 c 3 . 8 0 0 + 0.209 b Cerebral cortex 1.9^ *4 + 0.030 a 1.456 + 0.339 a 1 . 8 2 6 + 0.055 a Mid brain ,6 .1)16 + 0.336 a 6.677 + 0.305 a 5.353 + 0.301 b Medulla oblongata 5.158 + 0.421 a 4.152 + 0.248 b 4.552 + 0.374 ab Adenohypophysis 1.1)78 + 0.090 a 1.205" + 0.350 a 1 . 0 9 2 + 0.026 a Neurohypophysis 1.144 + 0.041 a 1.2cl 0.021 a 0.971 + 0 . 0 2 8 a Grand mean i). 069 + 2.178 3.012 + 1.670 3.342 + 1 . 6 0 6 b) ••PROTEIN CONCENTRATION ORCHIDECT0MI2;sd Pons 1 . 3 0 1 + 0.014 b 0 . 4 9 4 + 0 .C0 6 a ^ C. 4.88 + 0.012 a Cerebellum 0.1)11 + 0.059 a 0 . 1 1 7 + 0.004 b 0.336 + 0.012 a Amygdala 0.290- + 0.027 a 0 . 1 1 6 + 0.C03 b 0.194 + 0.012 ab f Hippocampus 0.31)3 + 0.04Q a 0 . 1 7 9 + O.C0 8 b‘ ___ 0 . 2 2 6 + 0 . 0 0 6 ab Hypothalamus ''0.1)90’ + 0.036 a 0 . 2 5 3 + 0.020 * 0.294 + 0.007 b Cerebral corbex 0.2i)9 + 0 . 0 2 8 a 0.156 + 0.005 ab 0 . 1 1 8 + 0.002 b Mid brain 0.621 + 0.002 a 0 . 1 8 5 + 0 . 0 0 8 b 0.618, + 0 . 0 3 8 a Medulla oblongata 0.872 + 0.021 b 0.287 + 0.013 c 1.403 + 0 . 2 1 8 a Adenohypophysis 0.225 + 0.011 a 0.155 + 0.001 a 0 . 1 6 6 + 0 . 0 0 9 a Neurohypophysis 0.146 + 0 . 0 0 9 a 0.097 + 0.003 a> — — 0.102 + 0.004 a Grand mean 0.1)95 + 0 . 3 5 5 0.204 + 0.113 0.394 + 0.390 % % X c) SPECIFIC ACETYLCHOLINE­ 0RCHIDECT0MIZED STERASE ACTIVITY Pons 2.635 + 0 . 0 7 0 c 6.732 + 0.158 b 10.073 + 0 . 1 3 8 a Cerebellum 7.253 + 1 . 0 8 1 b 17.515 + 0.810 a 8.403 + 1.260 b Amygdala 21.698 + 0 . 6 8 2 t 27.005 4.860 a 23.972 + 2.054 ab Hippocampus 15.238 + 0 . 9 0 8 b 19.695 + 1.155 a 16.135 + 0.974 Hypothalamus li).61(7 + 1.414 a 12.762 + 1.675 a 1 2 . 8 9 6 . + 0.496 a Cerebral xcortex 8.052 + 0.838 b 9.335 + 0.414 b 15.482 + 0.552 a Kid brain 10.350 + 0.646 b 36.297 + 1.950 a 8.844 4- 1.027 b Medulla oblongata" 5.957 + 0 . 0 3 1 b 14.663 + 1.413 a 3.430 + 0.437 b Adenohypophysis 6.553 + 0.201 a 7.799 + 0.233 a 6.645 + 0.436 a Neurohypophysis 7.8D8 + 0.2*15 13.001 + 0.411 9.527 + 0.497 Grand mean 10.023 + 5 . 6 2 2 16.481 -f•9.233 11.541 5.832 Values In the same horizontal column differently superscripted differ significantly (P < 0.05) Value:-, are means + S.E.M. * AChE actlvlty~lri urnoLe/g/mln ** Total protein in g/100 ml *** SAChE activity In pmole/y prot.e1n/min UNIVERSITY OF IBADAN LIBRARY - 1 4 1 - Orchidectomy significantly reduced protein levels in the cerebellum, midbrain and medulla oblongata with testosterone therapy significantly enhancing protein levels in all the brain regions. No significant differences were observed between the IC and the OC animals in the pons, amygdala, hippocampus, hypothalamus and cerebral cortex. No significant differences were observed in the hypophyses. SAChE activities were highest in the cerebellum, hippocampus, midbrain and medulla oblongata of the OC animals but similar in both the IC and OT animals. The hypothalamus was however not affected. Castration with or without testosterone significantly depressed SAChE activity in the pons and cerebral cortex. No significant effects were observed in the adenohypophysis while in the neurohypophysis, the IC and OT animals had similar levels which were inferior to the OC animals. 4.3.1 MINERAL PROFILE IN THE BRAIN AND HYPOPHYSES CALCIUM Calcium is significantly depressed in the OC animals in the amygdala, medulla, hippocampus and hypothalamus and restored by testosterone in the OT animals. (Table 4.1.6). The midbrain, cerebral cortex, pons and cerebellum were not significantly affected (P>0.05). No significant effects were observed in the adenohypophysis while in the neurohypophysis the OC animals had depressed calcium level which was partially restored by testosterone treatment in the OT animals. UNIVERSITY OF IBADAN LIBRARY TABLE. A. 1.6: EFFECT OF ORCH1DECTOMY AT 6-6 MONTHS OF AGE AND TESTOSTERONE ON THE "MINERAL PROFILE OF THE PORCINE BRAIN AND HYPOPHYSES. i BRAIN REGIONS ANIMAL GROUPS a) CALCIUM ORCHTDECTOMIZED With testosterone Without testosterone Intact control Pons 1.027 j_ 0.024 a 1.050 + 0.029 a 2.007 + 0.005 a Cerebellum 0.890 + 0.024 a 1.237' + 0.024 a 1.740 + 0.048 a Amygdala 1 . 2 3 0 + 0.012 ab 0.992 + 0.064 b 2 . 0 0 8 + 0.403 a Hippocampus 1.512 + 0.031 ab ,1.150 + 0.096 b 2.325 + 0.071 a Hypothalamus 2.150 + 0 . 0 3 1 a 0.8477 + 0.050 b 2.716 + 0.041 a Cerebral cortex 1.587 + 0.031 a 1.075 + 0.050 a 1.6 7 + 0.020 a Mid brain 1.026 + 0.015 a. 1 . 3 2 0 + 0.012 a 1.692 + 0.0.40. a Medulla oblongata 2 . 2 9 2 + Q.0 8 A a 0.826 + 0.244 b 3.062 + 0.242 a Adenohypophysis 1.792 + 0.015 a 2.075 + 0.'050 a 2.630 + 0.051 a Neurohypophysis ' 2.33Q + 0.047 at 1.837 + 0.024 b 2.887 + 0 . 0 8 8 a .Grand mean "1.587 + 0.537 1.241 + 0.411 2.272 + 0.524 b) MAGNESIUM ORCHIDE. TOMIZED With testosterone Without testosterone In/tact control Pons 1 . 2 8 2 + 0.048 c 2.393 +0.025 a 1.605 + 0.021 b Cerebellum I.I8 0 +0.027 c 2.485 + 0 . 0 6 2 a 2.051 + 0.030 b Amygdala .— 1.275 + 0.027 c 2.425 + 0.033 a ! 2.068 + 0.021 b Hippocampus 11551 + 0 .Q1 8 b 2.480 + 0.063 a 1.485 + 0.023 b Hypothalamus 1 . 8 1 0 + 0.077 b 1.435 + 0.050 c 2.014 + 0.003 a Cerebral cortex 1.249 + 0.056 c 2.372 + 0.070 a 1.612 + 0.041 b Mid brain 1.460 + 0.029 b 2.220 + 0.039 a 1.250 + 0.025 c Medulla oblongata 1.582 + 0.Q14 c 2.117 + 0.046.-4 1.935 + 0.025 b Adenohypophysis 1.871 + O.Q55 a 1.625 + 0.085 '> l.J^950 + 0.029 a Neurohypophysis . 1.674 + 0.097 = 2 . 3 6 7 + 0.035 a 1.956 + 0.037 b Grand mean 1.473 + 0.247 -^192 + 0.369 1.793 + 0.283 c) ZINC ORCHTDECTOMIZED With testosterone Without testosterone Intact control Pons 0.264 + 0.005 b 0.198 + O.OOS-c- 0.355 + JI.010 a Cerebellum 0.164 + Q.005 0 0.402 + 0.00^ a’ 0.336 + 0.014 b . Amygdala 0 . 2 1 6 + 0 . 0 0 3 b 0.190 + 0.007a 0.162 + 0.015 a Hippocampus 0 . 3 3 5 + 0 . 0 0 3 b 0.407 + 0.005 a 0.332 + 0.025 a Hypothalamus 0.301 + 0.004 b 0.148 + 0.004 c 0.541 + 0.015 a Cerebral cortex " 0.189 + 0.009 c 0.309 + 0.005 a 0.242 + 0.001 b Mid brain 0.417'-+ 0.002 b 0.249 + 0.005 c 0.543 + 0.020 a Medulla oblongata 0.347 + 0.144 b 0 . 2 5 0 + 0 . 0 0 6 c 0.578 + 0 . 0 1 6 a Adenohypophysis 0.363 +0.005 b 0 . 2 6 2 0.012 c 0.429 + 0.012 a Neurohypophysis 0.256 + 0.008 b 0.187' t 0.012 c 0 . 3 0 1 + 0.00? a. Grand mean 0.276 + 0.094 0.260 + 0.089 0.382 + 0.138 Values in the same horiz intal column differently. uperscripted differ significantly (P< 0.05) Values are means +_ SVE.M • *Va 1 ues 1 n parts per’ m:i 1lion (ppm). UNIVERSITY OF IBADAN LIBRARY TABLE it,1.5 (CONTINUED) - 143 BRAIN REGIONS AMIMAt, GROUPS d) POTASSIUM ORCHIDECTOMIZED With testosterone Without testosterone Intact control Pons 30.250 + 1 . 1 8 1 a IS . 5 0 0 + 0.515. c 25.625 + 1 . 0 2 8 b 1 Cerebellum 2 5 . 5 0 0 •K 0.500 a • 19.875 + 0 . 9 2 1 b 24,625 ♦ 0 . 8 6 6 a Amygdala 2 1 . 0 6 2 + 0 . h 7 2 a 15.625 ■f 0.625 h 20.625 0.851 a V Hippocampus 27.500 0.353 b 21.000 + 0.613 c 31.000 0.577 a Hypothalamus 26.880 + 0.375 b 20.625 ■f 1.197 c 35.500 + 0.500 a 1' Cerebral cortex 25.937 + 0.926 a 11.430 + 0.743 b 28.875 + 0.479 a i Mid brain 32.7QQ + 1.051 a 17.300 + 0.829 b 34.500 + 0.500 3a ’• Medulla oblongata 25.375 + 0.378 a 15.375. + 0.944 c 22.625 + O . 6 2 5 b Adenohypophysis 22.625 + Q.462 b 1 1 . 5 0 0 + 0.645 c 2 5 . 0 0 0 + O . 8 1 6 Neurohypophysis 21.750 + Q.tt79 a 11.425 + 0.472 b 2 1 . 5 0 0 + 0.645 a r Grand mean 25.952 + 3.667 16.265 + 3.819 2 6 . 9 8 7 + 5.266 e) SODIUM ORCHIDECTOMIZED r With testosterone Without testosterone Intact control ■ - Pons ’ 543.000 + 1.205 a 506.250 + 5.543 b 536.750 + 2.609 a Cerebellum 536.875 + 1.541 a 191.250 + 10.282 b 534.250 + 4.049 a Amygdala 529.250 + 2.689 a 331.250 + 11.250 b ^31.250 + 1.250 a Hippocampus 532.750 + 1.031 a 269.375 + 8.315 b 541.750 + 1 . 1 8 1 a Hypothalamus 537.500 + 5.204 a 270.500 + 13.823 b 545.500 +_ 4.406 a Cerebral cortex 524.250 + 5.313 a 242.500 + 5.95 b 53§.750 + 2.839 a ■ Mid brain q 5441750 + 5.186 a 537.500 + 3-227 a 539.250 + 1.49 3 a Medulla oblongata 5 3 0 . 0 0 0 + 2.041 ab 515.000 + 4.082 b 543.000 + 1.225 a Adenohypophysis 530.000 + 1.041 a 303.750 + 4.732 5' 529.250 + 1.493 Neurohypophysis - 432.500 + 1.041 a 523.000 + 6.178 a '53r.T50 + 1 . 1 8 1 a Grand mean 534.'087 + 6.416 3 6 9 ^ 0 3 7 + 135.470 537.150 + 5.446 d COPPER ORCHIDECTOMIZED V/ith testosterone Without testosterone Intact control s Pons 0.098 + 0.001 a 0 . 0 5 7 + 0.002 a 0.149 + 0.007 a Cerebellum 0.282 + 0.002 a 0.195 + 0.003 ab 0.109 + 0.003 b Amygdala 0.103 + 0.004 ab 0.085 + 0.003 b1 0 . 2 1 8 + 0.009 a Hippocampus 0.094 '+ 0.006 a 0.049 + 0.003 a 0 . 1 5 1 + 0.010 a / Hypothalamus 0.087 + 0.002 a 0.131 + 0.006 a 0.099 + 0.003 a Cerebral cortex 0.068 + 0.002 a 0.055 + 0.005 a 0.101 +0.004 a Mid brain 0.068 + 0.007 a 0.035 + 0.003 a 0.116 + 0.004 a Medulla oblongata 0.127 + 0 . 0 0 8 ab 0.060 + 0.004 b 0 . 2 0 3 + 0 . 0 0 2 a i ■ ? Adenohypophysis 0 . 0 7 7 + 0 . 0 0 6 a 0.154 + 0 . 0 0 8 a 0.155 + 0.402 a Heurohypophysis 0.101 + 0.001 a6 0.048 + 0.003 b 0.175 + 0 . 0 0 6 a Grand mean 0 . 1 1 0 + 0 . 0 6 3 0 . 0 8 7 + 0.05-4 0.148 + 0.042 Values In the same horizontal column differently superscripted differ 3 lgnlflcantly (P< 0.05) Values are means + UNIVERSITY OF IBADAN LIBRARY - 1 4 5 - MAGNESIUM Magnesium levels were highest in the pons, cerebellum, amygdala, hippocampus, cerebral cortex, midbrain and medulla oblongata of the OC animals, moderate and lowest in the IC and OT animals respectively. However, in the hypothalamus castration depressed magnesium level and was only partially restored in the OT animals. Magnesium levels in the adenohypophysis of the IC and OT animals were similar and superior to the OC animals. In the neurohypophysis, the OC animals had considerably higher magnesium level than either the IC or OT groups- ZINC Castration significantly depressed zinc levels in the pons, hypothalamus, midbrain and medulla oblongata and was partially enhanced by testosterone treatment. However OC animals had higher zinc levels in the cerebellum, and cerebral cortex than either the IC or OT groups. The IC animals in turn had higher levels than the other two groups in all the brain regions except the hippocampus. Castration depressed zinc content in both hypophyses and was partially restored by testosterone treatment. POTASSIUM Potassium was significantly reduced by castration in all the brain regions (PC0.05). However the OT animals had higher levels than the IC animals in the pons and medulla oblongata while the reverse occurred in the hippocampus and hypothalamus. Similar levels were recorded in the UNIVERSITY F IBADAN LIBRARY - 1 4 6 - cerebellum, amygdala, cerebral cortex and midbrain of both the IC and OT animals. SODIUM The IC and OT animals were similar and significantly superior to the OC animals in all the brain regions except the midbrain where no significant differences were observed. No significant differences were observed in the neurohypophysis while in the adenohypophysis, castration depressed sodium content and was restored by testosterone therapy. COPPER No significant differences (P<0.05) were observed in the pons, hippocampus, hypothalamus, cerebral cortex and midbrain. In the amygdala and medulla oblongata, the OC animals were similar to the OT animals but significantly inferior to the IC group (P>0.05). No significant differences were recorded in the adenohypophysis but in the neurohypophysis, the OC animals were inferior to the IC group but similar to the OT animals. 4.4.0 EFFECT OF ORCHIDECTOMY AT 7-8 MONTHS OF AGE AND TESTOSTERONE ON PROCINE BRAIN AND HYPOPHYSES Table 4.1.7 summarizes the effect of orchidectomy at 7-8 months of age and testosterone on porcine brain and hypophyseal physiology. No significant differences were observed in AChE activity in the cerebral cortex and neurohypophysis. AChE activities in the amygdala, hypothalamus and adenohypophysis of the IC and OC animals were similar and UNIVERSITY OF IBADAN LIBRARY 1 w? TABLE * .1 .7: THE .EFFECT OF ORCHIDECTOMY AT 7-8 MONTHS OF AGE AND TESTOSTERONE ON ?ROC:\E BRAIN AND HYPOPHYSEAL PHYSIOLOGY, BRAIN- REGIONS ANIMAL GOUP3 _____ ____________ •ACETYLCHOLINESTERASE fmCHinFCTOMIZED ACTIVITY With testosterone Without testosterone Intact contr.-l Pons 3.360 + 0.129 a 9.163 + 0.133 ab 3.050 + O.lE r Cerebellum 2.970 + 0.G19 a 2.233 + 0.333 b 3.021 + 0.Pit a Amygdala 5.992 + 0.176 a 5.201 + Q. 215 b 5.393 + 0.515 ; Nippocampus it.779 + 0.239 b 6.981 + 0 .9 3 7 - a 5.123 ± 0.9:1 t Hypothalamus 5.097 + 0.051 a 9.163 + 0.296 b 9.965 + C.6:I i • Cerebral cortex 1.761 + 0.009 a 1.711 + 0.026 a 1 . 6 6 9 + 0.C" , Mid brain 8.992 + 0.996 b 9.972 + 0.662 a 9.826 + C.520 a Medulla oblongata 7.902 + 0.163 b 6.705 + 0.351 c 9.635 + 0.912 a 'Adenohypophysis 1.599 + 0.192 a 1.138 + 0.136 h 1 . 0 0 8 + 0.0’7 t Neurohypophysis 1.099 + 0.079 a 1.939 + 0.057 a 1.398 + 0.063 a Grand mean 9 . 2 9 5 +2 . 6 0 8 9.270 + 2.739 9.393 + 2.937 b) ** PROTEIN CONCENTRATION ORCHIDECTOMIZED With testosterone Without testosteron/e Intact control Pons 0.695 + 0 . 0 1 6 a 0.586 + 0 . 0 3 2 b 0.597 + 0.0<1 i Cerebellum 0 . 1 8 6 + 0 . 0 1 0 a 0.099 + 0.009.- b 0 . 1 3 5 + 0 . 0 1 0 t Amygdala 0 . 8 0 8 + 0.031 a 0 . 1 3 5 + 0.011 b— 0.157 + 0.011 t Hippocampus 0.918 +. 0 . 0 3 8 a 0.191 + 0.008 c 0.365 + 0.021 b Hypothalamus 0.312 + 0.009 a 0.122 + 0.003 o' 0.296 + 0.C05 b . Cerebral cortex 0.220 + 0.007 a 0.079 + 0.009 c Q.128 + 0.031 t Mid brain 0.289 + 0.009 a 0.199 + 0.009 c 0.236 + 0.033 i Medulla oblongata 0.937 + 0.005 a 0.179 + O'. 009 c 0.338 + 0.029 t Adenohypophysis 0 . 2 3 1 + 0.006 a 0.260 + 0.00fl~ da'---- 0.9l8 + 0.013 a Neurohypophysis 0.399 + 0.Q2S a 0.089 + 0.005 c 0.158 + 0.037 t Grand mean 0.399 + 0.207 0.183 + 0.150 0.276 + 0.133 c) •’’SPECIFIC ACETYLCHO-" ORCHIDECTOMOHIZED LINESTERASEAACTIVITY With testosterone Without testosterone Intact, contrrl Pons 9,892 + 0.191 a 7.171 + Q.9Q6 a 5.622 + 0.3-5 a Cerebellum 16.197 + 1 . 2 8 6 a 20.357 + 1.911 a 22.807 + 1.762 a Amygdala 7.958 + 0.393 b 39.193 + 3.199 a 3 5 . 1 2 6 + 3.51-3 a Hippocampus 11.755 + 1.202 b 96.330 + 3.962 a 11.993 + 1.237 b Hypothalamus 16.299 + 0,591 b 39.013 + 1.592 a 18.198 + 0.939 b Cerebral cortex 7.978 + 0,989 b 23.212 + 1.155 a 13.081 + O.lci b Mid brain 29.930 + 0.611 b 98.762 + 1.583 a 91.837 + 2.9l£ a Medulla oblongata 18.097 + 0.990 b 37.916 + 0.719 a 25.819 + 1.516 t Adenohypopphysis 6.928 + 0.9c9 a 9.377 + 0.231 a 2.919 + 0.059 a Neurohypophysis 2.999 + 0.111 b 16.169 + 0.650 a 7.203 + 0.2+2 b Grand mean 12.251 + 8 . 8 0 8 27.700 + 15.739 18.909 + 12.936 Values in the same horizontal column differently superscripted differ signi ficantly (? < C.0G7>)) Values art -meanr. __+"l.El11. * ACHE activity In umole/g/min. ’'Total protein- in g/10Q r.l *** SAChE activity in umole/protcin/rain. UNIV RSITY OF IBADAN LIBRARY - 1 4 3 - inferior to the OT animals. Contrariwise AChE activites in the midbrain of the OC and IC animals were similar and superior to the OT animals. Castration significantly depressed AChE activity in the medulla oblongata and cerebellum and was partially restored by testosterone. However in the pons and hippocampus, AChE activites in the OT and IC animals were similar and inferior to the OC animals. Castration significantly depressed protein levels in the hippocampus, hypothalamus, cerebral cortex, midbrain and medulla oblongata while testosterone considerably enhanced it in OT above IC animals in all the brain regions. However protein levels in the pons, cerebellum and amygdala of the OC animals were similar to the IC but inferior to the OT groups• Castration also significantly depressed protein content in both hypophyses but was considerably enhanced by testosterone in the neurohypophysis of OT animals. No significant differences were observed in the SAChE activites in the pons and cerebellum, whereas in the amygdala and midbrain the OC animals were similar to the IC group but superior to the OT group. SAChE activites in the hippocampus, hypothalamus, cerebral cortex and medulla oblongata of the IC animals were similar to the OT animals but inferior to the OC group. No significant differences were obseved in the adenohypophysis (P>0.05). In the neurohypophysis however, the OC animals had higher levels than either the OT or IC groups which were similar. UNIVERSITY OF IBADAN LIBRARY 4 . 4 . 1 M I N E R A L P R O F I L E I N T H E B R A I N A N D H Y P O P H Y S E S The results are displayed in Table 4.1.8. CALCIUM The cerebral cortex, midbrain and medulla oblongata were not significantly affected (P>0.05). Calcium levels in the pons, cerebellum, hippocampus and hypothalamus of the OC and OT groups were similar and significantly inferior to the IC animals (P<0.05). In the amygdala, calcium was depressed by castration and partially resotred by testosterone. Calcium levels were depressed by orchidectomy in the hypophyses but partially restored by testosterone in the neurohypophysis. MAGNESIUM Results obtained on magnesium concentration did not follow any consistent pattern. OC animals were higher than OT animals but inferior to the IC in the amygdala and hippocampus while the control was inferior to the OC group in the hypothalamus. In the cerebellum, midbrain and medulla oblongata, magnesium levels in the OC and OT animals were similar but inferior to the IC (P<0.05). In the pons and the cerebral cortex, the OC animals were similar to the IC but superior to the OT animals (P<0.05). In the adenohypophysis, both the IC and OC animals were similar and superior to the OT animals. In the neurohypophysis, the highest level was recorded in the OT animals while the OC and IC were moderate and low respectively. UNIVERSITY OF IBADAN LIBRARY 150 £ l $j i TABLE 4.1.8: EFFECT OF ORCHTDECTOKV AT '-3 ffOMTlS OF AGE AN? TESTOSTERONE ON THE «MINEFA PROFILE OF THE PR0CIN3 B R A I N AND HiPOPHVSES L f F R A I N R E G I O N S A N I M A L G R O U P S a - C A L C I U M O R C K I D E C T C M I ZED With testosterone Without testosterone Intact control Fens 1.292 + ?. 015 't> 1.C50 4 0.025 b . 2.705 4 0.095 a Cerebellum 0 . 9 2 0 + C.071 b 0.832 4 0.031 b 1.592 4 0.052 a Amygdala 1.492 + 0.022 b ' 0. 997 + 0.002 c 2.945 4 0.214 a Hippocampus 1 . 2 6 0 + 0.029 b 0 . 9 7 5 + 0.025 c 3.044 + 0; 367 •a Hypothalamus 1.076 + C.025 b 1 . 2 2 7 + 0.477 b 2.148 4 0.090 a Cerebelum cortex 1 . 1 9 1 + 0 . 0 2 8 a 1.037 4 0.043 a 1.219 4 0.050 a Mid brain ' 1.442 + C.067 a 1.154 + 0 . 0 3 8 a 1.405 + 0.06S a Medulla oblongata 1.027' + c.027 a i.'ojo + 0.03? a 1.230 + 0.092 a Adenohypophys i s 1 . 9 7 2 + C.O. 3 2 b 2. 042 + 0.026 b 3.531 + 0.670 a Neurohypophysis 1.750 + C. 020 b 1.137 +_ 0 . 0 6 2 c 2.580 + 0.053 a Grand mean 1.342 + C.329 . 1.160 + 0.329 2.240 4 0.839 b) MAGNESIUM ORCHIDECTOMIZED With testosterone Without testosterone Intact control Pons 1 .3 S5 + 0 . 0 3 1 S 2 . 1 3 0 + 0.077 a 2.131 + 0.084 a Cerebellum 1.397' + 0.027 b 1 . 3 3 0 4 0.037 b 2.105 + 0.061 a Amygdala 1.560 + 0.089 c 1 . 8 3 5 + 0.338 t/ 2.523 + 0.036 a Hippocampus J..5Q5 + 0 . 0 1 3 e 1 . 9 3 0 + 0.054 b 2.272 + 0.130 a Hypothalamus- 1.621 + 0.046 b 2 . 3 0 8 4 0.106 a 1.279 + 0.096 a Cerebral cortex 1.815 +0.050 5 2.os8 + 0.146 ab 2.170 + 0 . 0 8 8 a Mid brain 1.795 + 0.083 t* 1 . 9 2 2 + 0.034 b * * 2.277 + 0 .0 6 6 a Medulla oblongata 1 . 8 2 0 + 0.067 b 1 . 9 1 0 + 0.165 b 2.405 + 0.142 a Adenohypophysis 1.745 + 0.Q26 b 2.241 + 0.149 x_" 2.290 + 0.105 a Neurohypophysis 1 . 8 3 0 + 0.030. a 1. 4?8 + 0.069 b 1.073 + 0.044 c Grand mean 1.647 + 0.177 r^l.9:5 +0 . 3 1 6 2.053 + 0.481 c) ZINC ......OTCBIDFCTOKIZSD Vfith testosterone Without testosterone Intact contro Pons Q.350 + 0.019 b 0.165 + 0.127 c 0.460 + 0.051 a Cerebellum 0.242 + 0.025 a 0.222 + 0.015 a 0.183 + 0.013 a Amygdala 0.392 + 0.015 t 0.305 + 0.910 b 0.401 4 0 . 0 0 8 a 1 ! Hippocampus 1.267' + 0.027 a 0.220 + 0.522 c 0.511 4 0.039 b Hypothalamus 0.161 + 0.012 b Q. 245 + 0.C21 a 0.292 4 0.014 a Cerebral cortex 0.251 + 0.012 b 0 .1 9 ? + 0.C10 b 0.390 4 0.012 a Mid brain - 0.297 + 0.019 a 0.164 + 0.C13 b 0.352 4 0.205 a Medulla oblongata 0.125 + 0.904 c 0 . 2 2 5 + 0.C11 b 0.309 4 0.034 a Adenohypophysis 0 . 2 2 3 + 0.012 c 0.28? + 0. C5W b 0.410 + 0.027 a Neurohypophysis 0.202 + 0.908 a 0.15? + 0.C11 ab 0 . 1 3 2 + 0.007 b Grand pie an 0.351 + 0.33? 0.21? + 0.C51 0.364 + 0.128 Values in the same horizontal column differently superscripted dl ffer significantly (P < 0.05) Values are means + JValues In parts per million UNIVERSITY OF IBADAN LIBRARY ■TABLE it. 1.8 (CONTINED) 151 BRAIN REGIONS ANIHAL GROUPS d) P O T A S S I U M ORC HIDECTOM12 ED With testosterone Without testosterone Intact control i; Pons 37.375 4 1 . 6 5 0 a 10.312 4 0 . 5 7 1 C 15.500 4 2 . 1 0 2 b Cerebellum 29.500 4 1 . 6 5 8 a * 6.322 4 1.265 c 36.625 4 1 . 5 5 9 b Amygdala 19.625 + 2.3*10 b 7.6112 4 0.657 c 211.500 4 0 . 8 6 6 a Hippocampus - 31.250 4 3 .1*16 b 11.737 4 0.69*1 c 37.000 4 1 . 8 6 0 • Hypothalamus 2U.875 + 0.921 b 10.500 4 1.0*11 c 30.375 4 *1.100 a '• ' Cerebral cortex 2 0.o5). UNIVERSITY OF IBADAN LIBRARY - 1 5 3 - In the pons and midbrain, both the IC and OT were similar and superior to the OC while in the hippocampus and hypothalamus, the OC and IC were similar and significanlty inferior to the OT animals (P<0.05). Castration also significantly depressed copper levels in both hypophyses (P<0.05) and testosterone therapy failed to exert a significant restorative effect. DISCUSSION 4.5.0 ORCHIDECTOMY AND TESTOSTERONE THERAPY ON SEXUAL BEHAVIOUR IN BOARS The apparent lack of interest of the orchidectomized boars in mating behaviour suggests that orchidectomy must have largely diminished sexual response in such boars. This confirms earlier reports by Davidson (1972) that castation usually abolishes sexual behaviour. The fact that a few percentage of the boars still attempted mating also suggests that sexual behaviour still persists for some time after castration probably due to increased plasma luteinizing hormone secretion (Nansel et al., 1979). The increased aggressive behaviour observed in boars orchidectomized post pubertally over the pre-pubertal castrates tend to oppose the report by Anisko et al. (1977) that castration increases aggression in pre-pubertal male gerbils. However they performed their experiment on pre-pubertal males whereas in this study, the post-pubertal castrates were more aggressive than the pre-pubertal castrates. This study also conflicts with the research by Yahr and Coquelin (1980) that pre­ pubertal males were more aggressive than post-pubertal males. These differences might be due to specie differences because it is known that copulation in the pigs may be more violent than in some other species of UNIVERSITY OF IBADAN LIBRARY - 1 5 4 - animals particularly the gerbils which were used for the Yahr and Coquelin experiments. This study therefore suggests that post-pubertally castrated boars might be able to withstand the responses due to androgen withdrawal (e.g. atrophy of the secondary sexual organs) more than pre-pubertal castrates. It also indicates that the age at castration affects the behaviour of the animal. The ability of testosterone to partially restore sexual behaviour lends credence to the view that androgen therapy if started early enough restores sexual behaviour in castrated males. This is more probably achieved through the growth-promoting functions of androgen in stimulating the development of the accessory sexual organs and restoration of sexual behaviour and libido in the male. 4.5.1 AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY IN THE BRAIN REGIONS The results indicate that AChE activities are highly depressed by castration in most of the brain regions of the four groups of animals used- and restored by replacement therapy. Of particular interest is the fact that AChE activities were depressed constantly in the hypothalamus, midbrain, medulla oblongata and pons in all the age groups except the pons which was unaffected in the pons of preweaning castrates and the hypothalamus in the animals castrated at 7-8 months of age. These results thus support the view that the hypothalamic-pituitary gonadal axis may be age-dependent (Masafumi et al., 1981). UNIVERSITY OF IBADAN LIBRARY - 1 5 5 - The resistance of the hypothalamus to testoterone withdrawal at puberty may also be due to the fact that the hypothalamus through aromatization may induce the conversion of estradiol to testosterone (McEwen et a_l 1980). This suggestion is further strengthened by the report of McEwen and Pfaff (1970) that estradiol is concentrated in the limbic hypothalamic system and tends to be retained by cells in the same regions as testosterone -jH. It is also noteworthy that AChE activities are depressed by castration in androgen-sensitive areas of the hippocampus, hypothalamus, and midbrain and relatively unaffected in the cerebral cortex of all the age groups studied. This discovery is of particular interest since according to Martini (1973), the cerebral cortex is not androgen-sensitive. It is pertinent to note that the functions performed by the cerebral cortex involve higher mental activities not necessarily induced by sexual stimuli. The fairly higher levels of AChE activities in the testerone- treated groups above the control also suggests that the amount of testosterone administered may be slightly more than normally occurs in the serum of normal males. This may therefore increase brain-induced sexual behaviour and activity of the androgen-receptor cells in the androgen- sensitive areas of the brain. These actions would definitely be reflected by more normal activity culminating in increased release of AChE. The decline in the protein concentration by castration in all the brain regions of the pre-weaning castrates may be a result of the absence of the protein anabolic activity of testosterone. Also since testosterone is implicated in the release of somatotrophic hormone and therefore functions in morphogenesis, it is not surprising that testosterone deprivation at a very early and critical period of growth may lead to UNIVERSITY OF IBADAN LIBRARY - 1 5 6 - impaired protein synthesis and possible inactivation of enzyme systems involved with growth (Sutherland et al., 1968). In the other age groups, the pons, cerebellum, medulla oblongata, midbrain and hypothalamus had their protein levels significantly reduced by castration and this may also be due to increased nitrogen excretion after castration (Roy and Laumas, 1969). Gorski, ( 1973) also advanced the view that androgen withdrawal may alter fundamental neurochemical processes such as protein synthesis within the hypothalamus and this may in some way prevent the cyclic release of the gonadotrophins in the adult. The observed suppression of protein synthesis in parts of the brain by testosterone withdrawal confirms this hypothesis. Bass et al., (1970) provided a link between protein synthesis and brain development when they discovered that pigs maintained post-natally on protein restricted diet had severe growth retardation and imparied brain development. The cerebral cortex was however relatively unaffected. « The present study also indicates that the cerebral cortex and the hippocampus except at 5-6 months were also relatively unaffected. This apart from being due to reasons connected with the functional capacity of the cerebral cortex may also be due to the fact that the cortex has been established as the most metabolically active region in the animal brain and is directly related to ACh synthesis. Such a vital role may make it relatively resistant to abuse and thus helps the animal to maintain its coordination and retain its higher mental functions in the face of stress. The fairly high specific acetylcholinesterase activities recorded in most brain regions of the testostrone-deprived animals may be directly linked to the very low protein concentrations of those regions which normally results in high SAChE activity. The repercussion is that higher UNIVERSITY OF IBADAN LIBRARY - 1 5 7 - than normal activity occurs in the neurons in an attempt to maintain the normal physiologic functions of the animal. When this is viewed against the very low protein content and AChE activities of the regions, neural fatigue may easily occur in such animals. This would readily reduce major enzyme activities and results in poor body growth. Although Williamson and Payne (1975) reported that castration of pigs facilitates management and prevents indiscriminate mating, no effects were observed on flavour, odour or tenderness of the meat. On the other hand, they found intact boars to be better converters of food than castrates. 4.5.2 MINERAL PROFILE IN THE BRAIN REGIONS With the exception of the pre-weaners where calcium levels were not consistent in the brain regions of treated and untreated castrates versus the control, calcium levels were generally depressed by castration and restored by replacement therapy in the pons, cerebellum, amygdala, hypothalamus, hippocampus and midbrain (3 months castrate) and medulla oblongata (5 months castrates). The cerebral cortex was unaffected. This trend could be explained by the decline in rate of protein synthesis caused by castration and which invariable slows down the rate of calcium absorption from diet. This would also lead to decreased calcium retention in the brain. The results thus confirms the work of Kunerth and Pitman (1939) who have linked calcium ions retention with protein synthesis. Also Collier and Ilson (1971) report that calcium ions are necessary for the release of ACh at neuromuscular junctions and their necessity for the transmission of nerve impulses also lends evidence to the UNIVERSITY OF IBADAN LIBRARY - 1 5 8 - depletion of calcium from the critical brain regions involved with active behaviour and growth. This also explains the steady concentrations observed in the cerebral cortex since AChE activities in the cortex were unaffected by testosterone withdrawal. A fairly inconsistent trend was observed with magnesium content of the brain regions in the various age groups. The pre-weaning castrates did not exhibit any consistency in magnesium concentration compared to the controls or the T-injected groups, and like the calcium levels may be due to the fact that these piglets were still actively growing and as such their brains would be very active. This would imply increase in metabolic turn-over rates of major metabolites in the brain and may account for the inconsistency in the mineral concentra­ tions of the brain regions of the animals. The rise in magnesium levels in the cerebral cortex and midbrain of the animals castrated at 3 months of age and the amygdala, hippocampus, pons, cerebral cortex, cerebellum, midbrain and medulla oblongata of those castrated at 5-6 months of age indicates a possible compensation for the calcium depletion observed in most of the brain regions mentioned. This view is the fact supported by report that alkaline earth metals often compete with one another and in particular, a number of magnesium-activated enzymes are inhibited by calcium ions while the calcium-activated myosin adenosine triphosphate is inhibited by magnesium ions (Dixon and Webb, 1961 ). UNIVERSITY OF IBADAN LIBRARY - 1 5 9 - In the animals orchidectomized at 7-8 months animals, the cerebral cortex was unaffected by testosterone withdrawal which is in line with earlier reports on the stability of the metabolic pathways in the cerebral cortex. The depression of sodium and potassium levels by castration and their subsequent restoration by T-treatment is an indication of their vital roles in neuromuscular functions and utilization of proteins and energy. It also supports the view that sodium concentrations are affected by the level of potassium in the medium because the two act in concert to maintain electrolyte balance. Hence a disturbance of the membrane permeability of the brain which may be brought about by impaired protein synthesis as a consequence of testosterone withdrawal (Levine and Goldstein, 1955) would definitely result in disturbed movement of materials (particularly ions and amino acids) in and out of the cells and the sub cellular structures and thus impair the rate of biochemical sequence-through the blood-brain barrier. This argument is further supported by the depression of endogenous oxygen uptake and oxidative and anaerobic metabolism of the hypothalamus following castration observed by (Mogvilevsky et al., 1966) which would interfere with normal functioning of the brain. With the exception of the 3-4 months group that could not show any consistent trend, castration also depressed copper levels in brain regions such as midbrain, hypothalamus, pons and medulla oblongata which are areas concerned with sexual development and active behaviour. The lack of effect of T-withdrawal on cerebral cortex, amygdala and cerebellum of the 7-8 months group may stem from the earlier report that the amygdala and cerebral cortex are not testosternone-sensitive and are UNIVERSITY OF IBADAN LIBRARY - 1 6 0 - thus relatively stable to testosterone-withdrawal especially when castration is performed post-pubertally. The depression of zinc levels by castration in the hippocampus, hypothalamus and medulla oblongata and its elevation in the cerebral cortex (except inthe 7-8 months castrate) indicates that the depression occurs in regions that control growth which probably depend on zinc-dependent enzyme systems while the elevation in the cortex is an attempt to prevent brain malformations and abnomalities usually associated with a deficiency of zinc. Although there is no direct evidence that zinc is an activator of the AChE hydrolytic systems, the metal is known to be a component of several enzyme systems and plays a role in sexual maturity. The report of Fullis (1958) indicates that zinc deficiency brings about atrophy of the testis which resembles the testicular atrophy associated with castration. It should also be mentioned that in many of these brain regions, and with most of the minerals studied, T-injection on castrates while elevating the mineral levels significantly above the levels observed in the untreated castrates was not quite able to bring the levels above those observed in the control animals. The reason for this is not very clear and may suggest that other metabolites apart from testosterone in the intact pig contribute to mineral metabolism in the brain. It is therefore clear from all the foregoing that castration renders the brain to a lot of abuse and disrupts the normal functioning of the brain. Other probable deleterious effects of castration are impaired brain protein synthesis and electrolyte balance. UNIVERSITY OF IBADAN LIBRARY - 1 6 1 - Si nee the brain controls body coordination and growth processes, any condition which disturbs the metabolic pathways of the various enzymatic processes in the brain should be avoided. It is thus clear, that castration whether performed pre or post - pubertally has very little if any advantages on the animal. On the other hand, it lowers the performance, stress adaptability and impairs endocrine balance of the animal. The only consolation appears to be the striking and welcome stability and resistance of the cerebral cortex which is the most active region of the brain to hormol abuse. 4.5.3 AChJB ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY HYPOPHYSES The inability of castration to significantly influence AChE activities in the hypophyses may be due to the feed back response of the hypophyses to lowered serum testosterone brought about by castration. In addition, studies have indicated that the hypophyses have a very high capacity to bind adrogen molecules (Samperez et al 1969) and castration considerably activates the transformation of testosterone into dihydrotestosterone (DHT) in the hypophyses (Martini, 1973). McEwen (1978a,b) has also shown that 5 DHT is one of the major brain metabolites involved in brain AChE sexual differentiation. Hence, it is very probable that castration and its attendant elevation of LH production by the hypophyses results in increased activity of the hypophyseal gonadotrophs which helps in maintaining AChE activity at near normal levels. Although a number of workers have deomostrated that brain AChE activity is associated with overall metabolic condition or resting metabolic rate (RMR) (Ling, 1970) and that AChE activity was lowered in the UNIVERSITY OF IBADAN LIBRARY - 1 6 2 - hypothalamus of thyroid-deficient animals (Geel and Timirasm 1967), the present study failed to show any significant influence of castration on AChE activity in the Pituitary gland. The reason for this may be that the absence of testosterone in protein anabolism and the growth promoting metabolic pathways may enchance augmented secretion of TSH by the pituitary through the feed-back systems which would maintain normal secretion of the thyroid gland hormones and thus sustain the calorigenic and protein anabolic activities of the thyroid gland. The decline in the protein concentrations in the hypophyses following castration and the restoration effect of testosterone shows that castration or testosterone withdrawal has an effect on the hypothalamic- hypophyseal axis. Thus the increased nitrogen excretion and disturbance of nitrogen balance associated with testosterone withdrawal may have profound effects on the amino-acid metabolism at the cellular level in the pituitary gland. The increase in SAChE acitivities in the hypophyses of castrated pigs is a direct reflection of the decreased protein levels in those organs which ultimately results in higher than normal SAChE activities and which may lead to cellular fatigue. 4.5.4 MINERAL PROFILE IN THE HYPOPHYSES The depression of calcium in the adenohypophysis and neurohypophysis of the castrated animals and inability of testosterone therapy to fully restore calcium levels to normal may be linked to the decline in the protein concentrations of the hypophyses of the castrated animals. ► UNIVERSITY OF IBADAN LIBRARY - 1 6 3 - Although growth-rate studies were not carried out on the animals, it is not unlikely that the impaired protein synthesis and consequent disturbed absorption of calcium may have serious implications on growth. As was mentioned earlier, the result that testosterone therapy is unable to completely restore the mineral levels suggests that the testis apart from producing testosterone probably produced some other metabolities which enhance clacium and other mineral balance in the brain. Another attractive hypothesis is that the products of the testis may function to maintain normal endocrine status of the animal which testosterone injections alone would not do. The elevation of magnesium by castration particularly in the neurohypophysis and the adenohypophysis (the pre-weaning castrates) is a further proof of magnesium acting to compensate for calcium depletion or decreased absorption by the adenohypophysis. The elevation of magnesium by castration may also be due to the increased activity of the hypophyses of the castrated animal which would invariably require magnesium ions as a co-enzyme of the phosphorylated transferase concerned with metabolic processes which may be facilitated to corpensate for the effects of castration. Although sodium levels in the pre-weaners were not consistent and did not show any defined pattern irrespective of treatment, generally, castration depressed sodium and potassium levels in both hypophyses and this may be linked to the synergistic nature of the two minerals in maintaining electrolyte balance. It is also not surprising that a condition that leads to protein depletion is attended by lowered sodium and potassium levels. UNIVERSITY OF IBADAN LIBRARY - 1 6 4 - Copper and zinc ions were similarly depressed by castration although the zinc levels of the neurohypophyses were relatively stable especially in the older animals. In both cases too, testosterone therapy restored copper and zinc levels but not to the same levels as the controls. This further supports the suggestion of certain extra testosterone metabolites in the testis contributing to the mineral metabolism of the hypophyses. The decline in zinc and copper levels by castration may be related to their roles in body growth and sexual maturation. It is interesting to note that in most of the cases where depression of mineral levels were observed, the adenohypophysis is invariably involved. This may be due to the fact that the adenohyphysis is more associated with the hypothalamus since it receives most of its gonadotrophin-release-inhibiting factors through the hypothalamic portal vein. It is therefore probable that conditions which disturb the normal functioning of the hypothalamus also have some effect on the hypophyses. UNIVERSITY OF IBADAN LIBRARY - 1 6 5 - CHAPTER FIVE EFFECT OF OVERIECTOMY WITH ESTRADIOL OR PROGESTERONE ON PORCINE BRAIN AND HYPOPHYSEAL PHYSIOLOGY UNIVERSITY OF IBADAN LIBRARY - 1 6 6 - INTRODUCTION 5.1.0 SECRETIONS OF THE OVARY The ovary possesses two functions: The production of ova and the production of hormones. These hormones are estrogens, progestogens and relaxin. The first two are the female sex hormones and are steroids in nature. Relaxin is a polypetide and is active towards the end of gestation. The two estrogenic steroids secreted by the ovary are estradiol and estrone. Like androgens, estrogens are not stored in the body but are removed through inactivation by the liver and elimination in both urine and faeces. Approximately 10% of blood estrogens are eliminated and the remainder inactivated. Stimulation of estrogen release from the ovary is under the control of the gonadotropins from the anterior pituitary gland. Hence, hypophysectomy of the female is followed by atrophy of the reproductive tract and all structures dependent on the presence of estrogens. According to current concepts, a feedback mechanism operates whereby the level of FSH and LH is controlled by the concentration of estradiol and progesterone in the blood. Very low levels of estradiol appear to stimulate FSH release which in conjunction with LH causes marked increase in estradiol release. An increase in the blood estradiol level eventually acts back on the pituitary inhibiting further, the release of FSH and hence, a drop in estradiol occurs. Thus excessive quantities of FSH are produced following ovariectomy and decrease in FSH following estradiol injection. UNIVERSITY OF IBADAN LIBRARY - 1 6 7 - 5.1.1 FUNCTIONS OF ESTROGENS (1) The hormone stimulates growth and secretory activity of structures receptive to the hormone and acts in concert with other hormones especially progesterone and relaxin to elicit normal reproductive functions. (2) Estrogens stimulate marked growth of the uterus resulting in increase in the mass of both the endometrium and myometrium. Estrogen administration to ovariectomized females has striking effects on the uterus. The effect on uterine growth is preceded by various alterations in tissue composition and enzyme activity (turner and Bagnara, 1975). An early change occuring in the uterus within an hour or so after estrogen administration is an increased blood supply associated with increased permeability of the uterine capillaries (Szego and Sloan, 1961). This is accompanied by an uptake of water and electrolytes by the uterine tissue and within four hours both aerobic and anaerobic glycolyses are elevated. The inhibition of water by uterine tissues results in marked increase in uterine weight and accelerated incorporation of C'‘ amino acids into the uterine tissues followed by cellular proliferation (Szego and Lawson, 1964). Other effects include increase in the RNA content, rates of respiration and glycolysis in the uterus. (3) Estrogens stimulate uterine contractility by increasing both the amplitude and rate of contration. (4) Estrogens also stimulate growth and muscular activity of the oviducts. In addition, they stimulate growth and development of the mammary duct system in all species and both the duct and alveolar systems in some species. (5) They also stimulate loosening of the Pubic symphysis and the increase in size of the interpubic ligament. UNIVERSITY OF IBADAN LIBRARY - 1 6 3 - (6) Although ovulation is a consequence of the effects of pituitary gonadotropins, heat, sexual receptivity and other psychic manifestations are probably brought about by the ovarian hormones acting through the central nervous system. (7) Although estrogen injections alone may induce sexual receptivity in the rat, full mating behaviour generally depends upon both estrogens and progesterone. However, progesterone is usually required in small amounts. Thus Timiras (1971) proposed that estrogens act as "organizer" of behavioural activity during critical periods of brain development because the hormone increases RNA synthesis in emrbryonic neural tissue. Vernadakis (1973) also observed increased RNA synthesis, increased cellular activity and increase in cell number, specifically of glial cells, by estradiol. Estrogen also induce enlargement (hypertrophy) of the adenohypophysis (Schreiber, 1973). The mode of action is supposed to be direct (Lisk, 1967b) because the adenohypophysis behaves like a target tissue for estrogens (Eisenfeld and Axelrod, 1966). They also induce thyrozine binding by adenophypohyseal proteins (Schreiber et al, 1970 a,b). The above effects may be reversed by simultaneous treatment of the animal with estrogens and thyroxine, a classic feed back effect. PROGESTOGENS Progesterone is the most prevalent, naturally occuring progestogen and is secreted mainly by the lutein cells of the corpus Luteium. This hormone is also secreted by the placenta while some amounts have been isolated from the adrenal gland. U IVERSITY OF IBADAN LIBRARY - 1 6 9 - Like other steroids, progesterone is not stored in the body. It is either rapidly utilized or excreted and so is present in low concentrations in the body tissues. FUNCTIONS: The action of progesterone is difficult to separate from that of other hormones particularly estrogens- This is due to the fact that progesterone normally acts in conjunction with estrogens and other steroids and produces few specific effects when active alone. Generally estrogens primarily promote growth processes whereas progestogens encourage tissue differentiation. Other major functions include: (1) The induction of the formation of a secretory endometrium in a uterus previously sensitized by estrogens. This is characterized by increases in mucosal thickness, increased coiling of the glands, edema of the stroma and presence of glycogen droplets in the glandular cells. (2) Progesterone inhibits spontaneous uterine motility and the response of the myometrium to oxytocin. (3) Progesterone in conjunction with estrogen induces growth of the lobule alveolar system of the mammary gland. (4) Progesterone is necessary for the maintenance of gestation. (5) Progesterone acts synergistica1ly with estrogen to induce behavioural estrus in the female. (6) Progesterone induces ovulation in the cow, bird, rat and rabbit but also inhibits ovulation when given chronically. UNIVERSITY OF IBADAN LIBRARY — IV 0 — 5.1.1 LITERATURE REVIEW The nervous system of mammals possesses groups of cells which release hormones. For some time now, the output mechanisms of these neuroedocrine cells have been under considerable attention. The cells are able to respond to circulating steroids by either a decrease or an increase of the membrane potential (Schade' and Van Wilgenburg, 1970). The involvement of the nervous system and the hypophyseal gonadal axis in reproduction has been extensively reviewed in previous chapters with more emphasis on the male animal. The main role in the regulation of anterior pituitary function is played by the hypothalamic-hypophysisotrophic factors or hormones (Guillemin, 1964, 1967, Schally et al, 1968, Campbell, 1970). These factors are either releasing factors or inhibiting factors whose primary effect is the release or reduction of anterior pituitary hormones into the blood, i.e. their secretion and not their increased synthesis. At the moment, it is now well established that the hypothalamic-hypophysiotrophic hormones directly influence biosynthesis of the anterior pituitary hormones (Schally et al, 1977). A number of brain regions have been implicated in gonadal function and sexual behaviour. Kluver and Bucy (1939) and Green et al (1969), induced hypersexaulity in monkeys and cats with ablations of the pyriform cortex or amygdala. In addition, electrical stimulation of the amygdala led to ovulation in the rabbit (Koikegami et al, 1953) and in the light- induced constant estrous rat (Bunn and Everett, 1957), while stimulation of the hippocampus blocked spontaneous ovulation in the rat (Velasco and Taleinsnik, 1969) and slightly facilitated the induction of ovulation in UNIVERSITY OF IBADAN LIBRARY - 1 7 1 - the rabbit (Xawakarai et al, 1966a, Kawakami et al, 1967). Bilateral lesions of the hippocampus or amygdala in the adult rat altered the estrus cycle (Koikegami et al 1960, Koikegami, 1964) while the destruction of the amygdala in the immature rat induced precocious puberty (Elwers and Critchlow, 1960, 1961). On the other hand, destruction of the hippocampus delayed the onset of puberty (Riss, 1958, Riss et al 1963). In 1970, Koves and Halasz demonstrated that the neural trigger for ovulation is located in the medial preoptic area. Kawakami et al (1970b) found that the effect of sex steroids on the electrical activity of the hypothalamus differed depending on the timing of administration of steroids either in castrated or cyclic rats. Injection of estrogen caused a rise in the multiple unit acitivity in the dorsal hippocampus, amygdala and hypothalamus. Administration of progesterone did not show particularly striking results. Kawakami et al (1973) also found that stimulation of medial preoptic area, amygdala and hippocampus resulted in increased FSH and LH release by the adenohypophysis in prepuberal rats. A lesion of the anterior hypothalamus has been made to induce vaginal estrus (Dey 1941, Flerko and Bardos, 1960). This proves that the cyclic secretion of gonadotropic hormones is controlled by the anterior and preoptic area of the hypothalamus. In the anteior preoptic area, a feed back centre for ovarian steroids exists controlling the cyclic discharge of gonadotropic hormones (Flerko and Bardos, 1961, Terasawa and Sawyer, 1970). While stimulation of medial preoptic area in mature rats induced increased level of serum FSH and LH in the blood, stimiulation of the hippocampus alone inhibited the elevation of serum LH level. This fact suggested that the hippocampus UNIVERSITY OF IBADAN LIBRARY - 1 7 2 - stimulation inhibited the release of FSH. Thus the hippocampus may participate in the gonadotropin secretion dominating the hypothalamus. Also, stimulation of the midbrain can evoke both facilitatory and inhibitory influences on gonadotropin secretion. Thus, stimulation of the midbrain inhibits some neurons and activates those of the hypothalamic ventromedial nucleus (Tsubokawa and Sutin, 1963) and the posterior hypothalamus. Stimulation of the amygdala also facilitates gonadotropin secretion. It has also been found that the midbrain projects into the amygdala (Machne and Segundo, 1950). Anatomical stuidies have demonstrated that the midbrain is linked with the hippocampus by a pathway which permits reciprocal influences between these two structures thus forming a limbic forebrain-midbrain circuit (Nauta, 1958). This circuit therefore explains the involvement of hippocampus in the inbhibition of gonadotropin secretion after midbrain stimulation (Velasco and Taleisnik, 1969b). These two antagonistic systems subserve the transmission from the periphery to the hypothalamus of most of the sensory stimuli affecting gonadotropin secretion. The midbrain thus acts as a central station for the distribution of flow of afferent impulses to the hypothalamus or to the limbic system but also modulates the magnitude of sensory imputs according to the information received from upper neural structures and the prevailing hormonal background. It has already been mentioned in preceeding chapters that during the sex differentiation of the brain, the production by the neonatal testes of androgen is the factor which determines the course of brain development, suggesting that the neonatal is more sensitive to androgen than ovarian steroids. The sex differntiation of the brain is made more evident by the UNIVERSITY OF IBADAN LIBRARY - 1 7 3 - fact that the male animal will most usually desplay sexual behaviour when presented with a receptive female whereas reproductive activity of the female is cyclic. Ovulation which is the key to the estrous cycle of the female is brought about by the neural activation of the pituitary gland in response to circulating levels of ovarian estrogen. Because of the dependence of this brain function upon estrogen, one might assume that the sex difference in reproductive activity is due to the presence of a functional ovary in the female and its absence in the male. However, when an ovarian graft is transplanted into the castrated rat, ovulation does not occur. Even in the presence of ovarian steroids, the brain of the male rat cannot bring about ovulation. With respect to the pattern of pituitary gland secretion, the ability of the brain of the female rat to regulate the cyclic surge of gonadotropic hormone responsible for ovulation accounts for this fundamental sex difference. It has already been established (Gorski, 1971a) that neurons within the medial basal hypothalamus and the arcuate nucleus are responsible for follicular growth and estrogen secretion but cannot independently bring about ovulation. In the female ovulation is regulated by the preoptic anterior nypothalamus (Halasz and Gorski, 1967, Koves and Halasz, 1970) and Studies have indicated that this region binds to labelled estrogen (Pfaff, 1968, Zigmond and McEwen, 1970). Thus intraphypothalamic infusion of estradiol benzoate (EB) induces anovulatory persistent estrus in the rat (Wagner et al 1966, Sutherland and Gorski, 1970). It is already known that one major functional result of sex steroid effect on brain tissue is the alteration of pituitary gonadotropin release and investigation show that radioactive estradio1-17B is highly UNIVERSITY OF IBADAN LIBRARY - 1 7 4 - concentrated in the ventromedial hypothalamus, preoptic area, ainydgala and septum and less concentrated in other brain areas such as cortex. This regional distribution has been determined both from scintillation counting of dissected brain regions (Eisenfeld and Axelrod, 1965, Green et al 1969, Kato and Villee, 1967a, McEwen and Pfaff, 1970) and from autoradiographic description of estrogen concentration following systemic injection (Michael, 1965, Pfaff, 1968a). Estradiol concentrations have also been established in the limbic hypothalamic system which also includes the hippocampus. Estradiol-3H tended to be retained by cells in the same region as testosterone-3H (McEwen and Pfaff, 1970). They also observed low estradiol uptake capacity in many structures outside the limbic hypothalamic system. According to Moguilevsky and Raynard, (1979), a possible reason for sexual dimorphism in the brain may also be due to differences in the inducibility of a gene product such as occurs in the estrogen induction of progestin receptors. A similar situation is in the estrogen induction of choline acetyltransferase in the preoptic area (Luine et al, 1975). As is already known, changes involved in sexual differntiation are supposed to be located in the hypothalamus and the preoptic area (Ifft, 1972). In addition, study by Kato et a_l (1968) indicates an increase in the hypothalamic estrogen receptor concentrations during sexual maturation. There are also indications that estrogens and androgens act on the hypothalamus and pituitary gland to exert a feed back system of gonadotropin secretion. Baum and Schietlen (1979) observed highest estradiol binding in the pituitary followied by the hypothalamus, midbrain, amygdala and cerebral cortex. Although not much data is available on dose-response relationship, UNIVERSITY OF IBADAN LIBRARY - 1 7 5 - Will s o n et al (1979) recorded estrogen concenstration in gilts of 6.8 to 10.2 ng/ml plasma. Average plasma volumes of progesterone were recorded at 2 ng/ml in non pregnant gilts and between 7 to 25 ng/ml during pregnancy. Estrogen level during pregnancy was somewhat stable at 7 ng/ml (George et al, 1978). 5.1.2 EFFECT OF OVARIAN STEROIDS ON OVAIECTOMIZED GILTS Immediately after ovariectomy, serum LH increased significantly while FSH and prolactin did not show any appreciable change. Progesterone administered to ewes ovariectomized during pregnancy showed increased volume of allantoic fluid whereas estradiol benzoate prevented excessive accummulation of allantoic fluid. Sensitivity of the brain to ovarian steroids depends on the neonatal treatment. For example, McEwen (1980) observed that neonatal treatment of female rats with testosterone reduces adult sensitivity to estradiol with respect to a variety of estrogen- dependent neuroendocrine and behavioural parameters. A similar observation was recorded for adult sensititivity to progesterone. After ovariectomy, the oviduct, uterus, vagina and mammary glands atrophy and may be largely revived through adequate estrogen therapy. Even in the adult human female, the menstrual cycle is interrupted in the absence of estrogenic hormones. Estrogen administration on ovariectomized rats induced the inhibition of hydrocortisone-mediated suppression of the compensatory ACTH secretion and may be interpreted as an interference of the two steroids on common receptive sites at both the hypothalamic and the pituitary levels (Tallian, 1973). UNIVERSITY OF IBADAN LIBRARY - 1 7 6 - Also changes in corticotropin releasing factor content of the hypothalamus was observed. This suggests an increased FSH and TSH secretion which plays a role in the increase of the pituitary-adrenal axis function which is mediated through an increase in plasma corticosterone binding capacity (Fortier et al., 1970). Ross et ad 1971) demonstrated that progesterone rapidly facilitates lordosis behaviour in ovariectomized rats primed with estradiol benzoate in the mesencephalic reticular formation. Kawakami et al (1970a,b) in a pioneering work described sudden increases in multi-unit activity in the medial basal hypothalamus on the afternoon of proestrus which are susceptible to change by ovariectomy and estrogen or progesterone administration. BarraClough and Cross (1963) had earlier described the effects of injected progesterone on responses of individual hypothalamic neurons to peripheral stimuli. Tach et al (1972) found that estradiol injection of ovariectomized rats increased mitotic division and RNA synathesis in the rat uterus whereas progesterone injection tended to block these estradiol-dependent effects. Effects of ovarian steroids on hypothalamic monoamine levels have also been reported. Norepinephrine levels increase in the anterior hypothalamus after ovariectomy (Stefano et al 1965) and are decreased again following estrogen and progesterone treatment (Donoso and Stefano, 1967). In the normal female rat, they are minimum at estrus after a peak at proestrus. These changes in norephinephrine levels complement the results of Kobayashi et a_l (1963) who found that hypothalamic monoamine oxidase (MAO) activity increased after ovariectomy, decreased again after treatment with estrogen and was highest in proestrus. After ovariectomy, hypothalamic choline acetylase changes were the UNIVERSITY OF IBADAN LIBRARY - 1 7 7 - reciprocal of MAO changes (Kobayashi et al, 1963). The above findings were more or less confirmed by Zolovick et al (1963). Anton-Tay and Wurtman (1968) found that ovariectomy increased the whole brain turnover rates of norephinepherine even though there were no drastic changes in norephinephrine concentration. Since it is known that drugs which deplete the brain of monoamines (in particular of norepinephrine) block ovulation and that sex steroids alter monoamine levels (Coppola et al, 1965), it is possible that estradiol and progesterone regulate ovulation through the alteration of the levels of norephinephrine and other monoamines. Gonadal hormones have also been known to play an important role in behavioural changes which are in part mediated through cholinergic elements of the central nervous system (Oliverio et al 1973). Iramain et al (1979) discovered that orchidectomy plus estradiol administration decreased AChE activity in the rat cerebral cortex and mesencephalon while it was increased in the amygdala. These workers also observed diminished AChE activity in the adenohypophysis of orchidectomized rats. Nayeemunisa (1976) reported that the hormone may influence protein synthesis as manifested in decreased protein content and AChE levels in the rat brain on in vivo administration of progesterone. Moudgil and Kanungo, ( 1973) reported that 17-B-estradiol induced AChE activity in rat brain while ovariectomy decreased AChE activity in the cerebral hemisphere. The increase in AChE acitivity after estradiol administration is supposed to be due to an increase in the transcription of M-RNA which increases the synthesis of the enzyme. For example, Hansel (1959) showed a decrease in the level of estrogen in women with old age and this may affect the level of AChE activity of the brain and thereby alter the behavioural pattern of the female. UNIVERSITY OF IBADAN LIBRARY - 1 7 8 - AChE is involved in the feed back control of Luteinizing hormone secretion (Florindo and Martini, 1975) and behavioural changes which are in part mediated through cholinergic central mechanisms and are influenced by steroid hormones (Olivero et al., 1973, Lindstrom, 1975). 5.1.3 SOME METABOLIC EFFECTS OF OVARIAN STEROIDS ON MINERAL METABOLISM Not much information is available on the interaction between ovarian steroids and mineral metabolism in the animal body. However, since estrogens are involved in protein synthesis and a deficiency of some cations such as zinc results in decreased DNA synthesis (which reduces protein syntesis), it is not too presumptious to assume that the mineral may also enhance the normal functioning of the enzyme. Estrogens also stimulate retention of water, sodium, calcium and nitrogen. Estrogen increases zinc accumulation (Gunn and Gould, 1955) and induces increased zinc incorporation in the dorsoolateral lobe of castrate rats (Muntzing et al, 1977). 5.1.4 MATERIALS AND METHODS Twelve Large White gilts housed, fed and provided water as described earlier were used. They were randomly assigned to 3 equal groups: Ovariectomized and treated with estradiol (OE), ovariectomized and treated with progesterone (OP) and sham operated (SO). OE and OP gilts were bilaterally ovariectomized while SO gilts were sham operated and all the animals were allowed to recover for four weeks. OE gilts were thereafter given 5 daily intramuscular injections each of 3 mg of estradiol valerate (E2) in 1 ml of corn oil between 0900-1000 hours. OP gilts were similarly treated with 20 mg of hydroxyprogesterone caproate in 1 ml of UNIVERSITY OF IBADAN LIBRARY - 1 7 9 - corn oil while the sham operated gilts received the corn oil only. Ovariectomy was performed through a mid-ventral incision according to the method of Berge and Westhues, (1966). 24 hours after the last injection, all the animals were slaughtered and their brains and hypophyses quickly removed, dissected and processed as described earlier. 5.1.5 STATISTICAL ANALYSES The Data were subjected to multifactor analyses of variance as described earlier. RESULTS 5.2.0 EFFECT OF OVARIECTOMY WITH ESTRADIOL OR PROGEESTERONE ON BRAIN AND HYPOPHYSEAL AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY The effects of the treatments on AChE activity, total protein and SAChE activity are summarized in Table 5.1 AChE acitivity was significantly and similarly depressed (P>0.05) in the cerebellum, amygdala and hippocampus by estrogen or progesterone treatment alone. In the midbrain, however, progesterone was more potent in depressing the enzyme activity. On the other hand, estrogen was potent in enhancing the AChE activity (P<0.05) in the pons, cerebral cortex and medulla oblongata. In the cerebral cortex, AChE was depressed by progesterone alone but unaffected by estrogen. AChE activity in the adenohypophysis were similar in both the sham operated and the OE gilts but inferior to the OP animals. No significant differences were observed in UNIVERSITY OF IBADAN LIBRARY 180 TABLE 5.1.1: EFFECT OP OVARIECTOMY WITH ESTRADIOL OR PROGESTERONE 1IHERAPY ON PROCINE BRAIN AND hyp ophys eal PHYSIOLOGY. i s BRAIN REGIONS ANIMAL GROUPS __________________i t a ) ACETYLCHOLINESTERASE Sham operated OVARIKCTOMIZED ACTIVITY* With estradiol With progesterone Pons 3.052 + 0.284 c 4.623 + 0.053 a 3.503 + l.OSl b Cerebellum <1.553 + 0.080 a 2.878 + 0.160 b 2.918 + 0.070 b Amygdala 9.11 <4 + 0.492 a 5.447 + 0.203 b 5.060 + 0.73 b Hippocampus 4.326 + 0.245 a 3.421 + 0.185 b 3.123 + 0.049 b “Hypothalamus 4.469 + 0.071 a 4.423 + 0.093 a 3-914 + 0.043 b Cerebral cortex 1.614 + 0.089 a 1.292 + 0 . 0 1 6 ab 1.007 + 0.014 b Mid brain 7.. 530 + 0.259 a 5.550 + 0.276 b 4.335 + 0.467 c Medulla oblongata 5.447 + 0.163 a 5.043 + 0.102 a 3.842 + 0.134 b Adenohypophysis 0.509 + 0.057 b 0.548 + 0.046 b 1.005 + 0.049 a Neurohypophysis 1.059 + 0.027 a 0.931 + 0.007 a 1.041 + jo . 024 a 1' f Grand mean 4.167 + 2.758 3.416 + 1.915 2.975 + 1.447 Sham operated OVARIECTQMIZED b) PROTEIN CONCENTRA- • TION * * With estradiol x With progestorcne - ' Pons " 11 V0.571 + 0.024 a 0.512 + 0.017 b 0.548 + 0.014 ab Cerebellum 0.363- + 0.0'23 a 0 . 2 6 1 + 0.013 C / : 0.311 + 0.017 b Amygdala '0.356 + 0.013 a 0.220 + 0.005 b 0.170 + 0 . 0 1 9 c Hippocampus O . 3 2 9 + 0.007 b 0.414 + 0.021 a 0.267 + C.007 c Hypothalamus 0.382 + 0.015 a 0.304 + 0 . 0 3 3 b 0.231 1 0.042 c Cerebral cortex 0.356 + 0.020 a 0. 230 + 0 . 0 0 2 b 0.296 + 0.009 ab Mid brain 0.338 + 0.004 a 0.253 + 0 . 0 1 0 b 0.239 + 0.010 b Medulla oblongata 0.573 . + 0.003 c 0.698 + 0 . 0 0 9 ^____ 0.624 + 0 . 0 1 8 b Adenohypophysis 0.319 + 0 . 0 0 6 a 0.244 + 0 . 0 0 7 b 0.179 + 0.006 c Neurohypophysis 0.222 + 0.010 a ^jD . 2 2 7 + 0 . 0 1 5 a 0.172 + 0.004 b 1 Grand mean 0.379 + 0.110 0. 341 + 0 . 1 5 6 0.304 + 0.158 c) SPECIFIC ACETYL­ OVARIETCTOMIZED CHOLINESTERASE Stam operated ACTIVITY*** With estradiol With progesterone Pons 5 . 3 8 5 + 0.637 b 9 . 0 5 1 + 0.315 a 7.145 + c . 1 9 9 ab Cerebellum 1 2 . 7 1 9 + 0.922 a 1 1 . 2 1 3 + 1.192 a 9.466 + 0 . 5 6 2 a Amygdala 2 5 . 7 8 1 + 1.591 b 24.811 + 1.137 b 30.808 + 3.387 a Hippocampus 1 3 . 1 3 3 + 0.626 a 8.322 + 0.543 b 11.714 + 0 . 5 0 1 a Hypothalamus 1 1 . 7 5 6 + 0.503 c 15.202 + 2 . 0 1 6 b 19.401 + 4 . 6 5 1 a Cerebral cortex 4 . 8 0 7 + 0.149 a 4.621 + 0.070 a 3.245 + c . 1 2 3 a Mid brain 2 2 . 2 9 0 + 0.7 48 a 22.060 + 1.386 a 18.101 + 1 . 6 0 8 Medulla oblongata 9.514 + 0.318 a 7.232 + 0.168 ab 6 . 1 6 6 + G . 3 2 2 b Adenohypophysis 1.559 + 0.169 b 2.239 + .0 . 1 3 2 b 5.611 + 0 . 1 7 0 a Neurohypophysis 4.786 + 0 . 1 9 0 a 4.153 + 0.317 a 6.057 + 0 . 1 0 8 a Grand mean 11.173 + 7.842 10.390 + 7.602 11.771 4- 8 . 5 7 7 --- Values in the same horizontal column bearing different superscripts differ significantly (P< 0.05') Values are means + S.E.M. * AChE-act1vity in m.ole/g/min ** Total protein In r/IOO ml LAC I v i t . y i n i i:nole/g p r o t e i n / m i n . UNIVERSITY OF IBADAN LIBRARY - 1 8 1 - the neurohypophysis (P>0.05). Total protein was significantly depressed (P<0.05) by estrogen and progesterone in the cerebellum, amygdala, hypothalamus and midbrain. However, estrogen was more potent in depressing total protein in the cerebellum while progesterone was more potent in depressing total protein in the amygdala, hippocampus, hypothalamus, and midbrain. In the cerebral cortex and pons, total protein was depressed by estrogen but unaffected by progesterone. In the medulla oblongata, both estrogen and progesterone considerably enhanced total protein content with estrogen being superior to progesterone. In the adenohypophysis, total protein was significatnly depressed by estrogen and progesterone alone (P<0.05) with estrogen being superior to progesterone. In the neurohypophysis, total protein in the SO and OE animals were similar and superior to the OP animals. No significant differences were observed in the SAChE activities in the cerebellum, cerebral cortex and nuerophypophysis (P>0.05). In the pons, SAChE acitivity was enhanced by estradiol but unaffected by progesterone while in the adenohypophysis and amygdala, SAChE activities were similar in the SO and OE animals but inferior to the OP animals. In the midbrain and medulla oblongata SAChE acitivity was depressed by progesterone (P<0.05) but unaffected by estrogen, while in the hippocampus SAChE activity was depressed by estrogen but unaffected by progesterone. Both estrogen and progesterone significantly enhanced SAChE activities in the hypothalamus but progesterone was more significantly potent than estrogen. UNIVERSITY OF IBADAN LIBRARY - 1 8 2 - 5.2.1 EFFECT OF OVARIECTOMY WITH ESTRADIOL OR PROGESTERONE ON THE MINERAL PROFILE OF THE PORCINE BRAIN AND HYPOPHYSES CALCIUM The results are summarized in Tables 5.1.2 and 5.1.3 Calcium in the pons and adenohypophysis of OE and OP animals were similar and superior to the sham operated animals. Similarly, both estrogen and progesterone significantly elevated calcium content in the cerebellum but estradiol was more potent than progesterone. In the amygdala and hippocampus, calcium levels in the OE and OP animals were similar but inferior to the SO animals. Calcium was enhanced by progesterone in the hypothalamus and midbrain but unaffected by estrogen. Estrogen and Progesterone significantly enhanced calcium levels in the neurohypophysis with progesterone being more potent than estrogen (P<0.05). No significant changes were observed in the medulla oblongata (P>0.05). MAGNESIUM Both estradiol and progesterone significantly enhanced magnesium levels (PC0.05) in the pons, medulla oblongata, adenohypophysis and neurohypophysis. However, in each case, estradiol was significantly more potent than progesterone (P<0.05). In the Amygdala and midbrain, progesterone significanlty elevated magnesium levels but was unaffected by estrogen. In the hippocampus and cerebral cortex, magnesium was depressed by estrogen but unaffected by progesterone. In the cerebellum and hypothalamus, magnesium was significantly depressed by estradiol and enhanced by progesterone. UNIVERSITY OF IBADAN LIBRARY 183 TABLE' ̂ .1.2: EFFECT OF OVARIECTOMY WITH ESTRADTOI OR PROTESTOR!ME THERAPY ON THE ‘CALCIUM, HAOENSSIUH AND ZINC LEVELS IN CHE PRC-CINE BRAIN AND HYPOFHYSIS. A> TMAT CROUPS BRAIN REGIONS OVARII CTOMIZED a) CALCIUM Sham operated With estradiol With progesterone Pons 1.005 + 0.021 b 1.4 06 4 O.OCl a 1 . 2 2 9 4 0.010 a Cerebellum 0.952 + 0.022 c 1.662 4 0.012 a 1 . 9 7 4 4 0.010 b Amygdala 1.5^5 + 0 . 0 2 6 a 1.200 4 0 . 0 5 7 b 1 . 1 0 5 4 0.069 b Hippocampus 1.312 + 0.031 a 1.024 4 0 . 0 1 5 b 1 . 1 1 5 4 0.066 b Hypothalamus 1.400 + 0.074 b ' 1.562 4 0 .0 S5 ab 1 . 6 3 9 4 0.052 a Cerebral cortex 1.306 + 0.059 a 1.337 + 0 . 0 5 9 a 1 . 1 0 2 4 0.071 b Mid brain 1 .2 5 0 '+ 0.064 b 1.200 + 0 .0 S2 b 1 . 5 0 0 4 0.079 a Medulla oblongata 0.937 + 0.047 a 1.100 + 0 . 0 5 7 a 1.124 4 0.076 a I Adenohypophysis 2.04? + 0.478 a 2.0S7 4 0.043 a 1.626 4 0.053 b \ Neurohypophysis 1.631 + 0.02S c 2.055 + 0.025 b 2.220 4 0.092 a £ Grand mean ~~ 1.339 + 0.328 1.459 + 6.369 1.413 4 0.358 1 ___Il b) MAGNESIUM Sham operated OVRAIECTOMIZED f With estradiol Wi£h progesterone Poyis 1 . 2 9 5 + 0.015 c 1.471 4 0.013 b 1 . 7 5 1 4 0.002 a i Cerebellum 1 . 4 9 3 + 0.136 b 1.397 + 0.003 e 2 . 0 0 1 4 0.003 a 1 Amygdala 1.431 + 0.003 b 1.500 4 9.002 2 . 0 2 1 4 0.009 a b f 1 Hippocampus 1.914 + 0.013 a 1.580 + 0.005 b --- 2 . 0 5 9 4 0.020 a Hypothalamus 1. 540 + 0.005 b 1.4 8 3 + 0.007 c 2.046 4 0.029 a r Cerebral cortex 1'; 546 + 0.003 a 1.423 + 0.003 a 1.536 4 0.035 a Mid brain 1 . 5 2 6 + 0.003 b_ 1.482 + 0.003 b 2.133 4 0.029 a Medulla oblongata 1.396 + 0.004 c 1.571 4> 0.010 b 1.912 4 0.029 a Adenohypophysi s 1.476 + 0.003 c 1.914 + o.o4r b . ̂ 2.282 4 0.007 a f Neurohypophysis." l-S1̂ 4 0 . 0 1 7 C 1.712 + 0.020 b 2.124 4 0.031 a £ t. Grand mean 1.501 + 0 . 1 6 8 1̂.-553' 4 0 . 1 5 5 1.937 4 0.211 — r OVARIECTOMOZED c) ZINC Sham'operated With estradiol With 'progesterone i Pons 0-.9S7 + 0 . 0 5 2 a 0 . 6 6 8 + 0.022 b 0 . 5 4 9 4 0.020 c t Cerebellum 0.961 +- 0.012 b 0 . 6 9 7 + 0~002 c 1.039 4 0.010 a > Amygdala 0.529 + 0.010 c 0 . 6 0 2 + 0 . 0 1 5 b 0.952 4 0 . 0 6 0 a Hippocampus 0.24? + 0.017 b Q &. 7 0 2 4 0.014 a 0.748 4 0.010 a Hypothalamus 0.687 + 0.036 b 0 . 7 9 7 4 0.012 a 0.779 4 0.029 a | Cerebral cortex 0 . 9 2 2 + 0.005 a 0 . 7 3 4 4 0 . 0 3 0 b 0.592 4 0 . 0 0 8 c Mid brain 1.047 4 0 . 0 0 6 a 0 . 5 7 6 4 0.007 b 0.604 4 0 . 0 0 8 b 1 Medulla oblongata 1.037 + 0.011 a 0 . 6 7 7 4 0.014 C 0.796 4 0.019 b Adenohypophysis 0.457 + 0.022 c 0 . 8 1 3 4 0.006 b 1.169 4 0.003 a Neurohypophys i s 0.797 + 0.038 b 1 . 1 1 0 4 P.003 a 1.1-6 4 0.009 a : Grand mean 0.756 j_ 0.278 0.73 s 4 0 . 1 5 0 0.84? 4 0.239 . Values in the same hori zontal column bearing different superscr ptr- Jiffer significantly (*>'. 5.U5T - t Values are means +_ S.E. M. •Values are in parts.pe r million (ppm). 1 ' UNIVERSITY OF IBADAN LIBRARY I8if TABLE S t . 3: EFFECT OF OVARIECTOMY WITH SSTRAPIOL OR FROGEATORONE THERAPY ON THE * POTASSIUM SODIUM AND COFFER LEVELS IX PROCI.' S BRATS AND HYPOPHYSES. BRAIN REGIONS ANIMAL GROUPS A ) POTASSIUM Sham operated ' • 0VARX ECTOMIZED . . . . Vt’ith estradiol Kith progester one Pons 37.980 + 2.05? a 27.461 + 1.465 b 23.025 + 0.842 c Cerebellum 33.293 i 1.137 a 25.663 + 0.546 b 2 6 . 8 6 1 + 0 . 6 2 7 b Amygdala A2 .1 7 5 ' + 0 . 9 3 4 a 27.S07 + 0.476 b 2 5 . 0 0 0 + 0 .8 ' 6 b Hippocampus 33.637 + 1.492 a 26.901 + 0.689 b 2 5 . 0 0 0 + 0.4S6 b - Hypothalamus 25.186 + 1.381 a 26.187 + 0.357 a 20.609 + 0.963 b Cerebral cortex 32.325 + 2.QS: a 23.61? + 1 . 3 1 0 a 27.890 + 0.656 a Mid brain 33.476 + Q.42T a 3 0 .500 + 0 . 6 1 2 a 31.317 + 1.807 a Medulla oblongata 32.300 4 0.39" a 30.375 + 0 . 8 9 8 a 25.250 ++1.031 b Adenohypophysis 23.050 + O . 7 3 3 b 32.250 + 0.722. a 35.025 + 0.953 a Neurohypophysis 22.027 + 0.711 b 28.250 + 0.616 a 17.394 + 1.066 c Grand mean 32.01)5' + 5.765 ,28.401 + 2 . 0 8 3 25.737 + 5.032 e) SODIUM Sham operated ' ■4'" 0YARIECT0HIZED With estradiol ^ With progesterone Pons 5GS.750' + i).2 7 a a 5 3 5 .paa + 4.as2 a 5 2 8 . 2 5 0 + 3 . 3 7 0 a Cerebellum 527.875 + 1.-443 a 525.000 + 5.400 L 5 2 9 . 2 5 0 + 1 . 0 5 1 a Amygdala 539.375 + 2.135 a 529.750: + 1.051 a 5 2 7 . 6 2 5 + 7 . 7 3 3 a- Hippocampus - 537.500 + 3.227 531.250 + 2.39.4 a 5 3 2 . 1 1 2 + 0 . 6 1 6 a Hypothalamus 537.750 + 1.531 a 530.125 + 0.935 a 5 3 6 . 3 7 5 + 2 . 2 8 6 a Cerebral cortex 51)1.875' + 2‘,391'a 5 2 6 . 8 7 5 + 9 . 0 9 3 a - • 5 3 2 .5 0 0 : + 8 . 2 9 2 a ‘ Mid brain 541.375 + 1.25C a 526.250'+ 4.270 a 5 4 5 . 0 0 0 + 3 . 5 3 6 a Medulla oblongata 542.000' +■ 3.227 a 529.375 + 2.095''a* 5 1 7 . 5 0 0 + 8 . 7 8 0 a Adenohypophysis 517.375 + 2.996 a 533.375 I . 0 6 1 a 545.000 + 2.041 a Neurohypophysis 533.750 + 4.27C a ^532.850'+_ 6 . 6 3 1 a 542.625 + 2.500 a Grand mean 533.262 + 1 1 . 5 6 7 529.835 + 3.278 533.624 + 8.787 f) COPPER Sham operated OVAPJ ECTOHIZED* With estradiol Kith precesterone Pons 0.191 + 0.004 a 0.037 + 0’.005 b 0 . 0 8 0 + 0.007 b Cerebellum 0.122 + 0 . 0 0 3 t 0.130 + 0.009 b 0.202 + 0 . 0 0 8 a Amygdala 0.120 + 0.008 a 0.076 + 0.004 b 0.087 + 0.004 b Hippocampus 0.136 + 0.002 t 0.156 + 0.009 a. 0.084 + 0.001 c Hypothalamus 0.142 + 0.004 a 0.135 + 0.008 a 0.096 + 0.002 b Cerebral cortex 0.147 + 0.003 a 0 . 1 0 1 + 0 . 0 0 9 b 0 . 0 8 2 + 0 . 0 0 1 b Mid brain 0.126 + 0.002 a 0 .1 : 9 + 0.001 a 0 . 0 9 6 + 0 . 0 0 3 b Medulla oblongata 0.124 + 0.003 2 0.092 + 0.002 b 0.085 + 0.003 b Adenohypophysis 0.173 + 0.006 a 0.057 + 0.004 c 0 . 1 3 9 + 0 . 0 1 0 b Neurohypophysis 0.126 + 0.012 a 0.104 + 0.004 b . 0.074 + 0.402 c Grand mean 0.141 + 0.024 0.109 + 0.026 0.102 + 0.039 Values in the same hori '■ ontal col un r; b c a r 1 r. * diTt crcnfc s u p e r scr i p t s -ti i V r et s i g n i f i c a n t l y v F < 0.05, Va 1 ue s a r** me a ns + 3 . F...M. * V a l u e r arn in part..-, pe.r m i l l i o n (rip-,). UNIVERSITY OF IBADAN LIBRARY - 1 8 5 - ZINC Estrogen and progesterone significantly depressed zinc levels in the pons and cerebral cortex. However, in each case, progresterone was more potent in depressing zinc than estrogen. Zinc levels in the OE and OP animals were similar and superior to the sham operated animals in the neurohypophysis, hippocampus and hypothalamus. In the amygdala and adenohypophysis, zinc levels were significantly enhanced by estradiol and progesterone but the OP animals were superior to the OE animals. Estrogen and progesterone depressed zinc levels in the midbrain and medulla oblongata, however, the medulla of OE animals was inferior to the OP animals. In the cerebellum, estradiol significantly depressed zinc level (P<0.05) but was considerably enhanced by progesterone. POTASSIUM Both estrogen or progesterone treatment alone depressed potassium level in the pons (P<0.05) but the OP animals were inferior to the OE animals. In the cerebellum, Amygdala and hippocampus, potassium levels in the OE and OP animals were similar and inferior to the SO animals (P<0.05). In the hypothalamus and medulla oblongata, the SO animals and OE animals were similar and superior to the SO animals (?<0.05). In the neurohypophysis, estrogen injection along significantly enhanced potassium level (P<0.05) while progesterone depressed it. No significant changes were observed in the cerebral cortex. UNIVERSITY OF IBADAN LIBRARY - 1 86- SODIUM No significant differences were observed in the brain and hypophyseal sodium levels (P>0.05). COPPER Copper levels were significantly depressed by estrogen or progesterone treatment alone in the pons, amygdala, cerebral cortex and medulla oblongata (P<0.05). In the hypothalamus and midbrain, SO and OE animals were similar and superior to progesterone-treated animals. In the cerebellum, the SO and OE animals were similar and inferior to OP animals; while in the hippocampus, copper levels were significantly enhanced and depressed by estradiol and progesterone respectively (P<0.05). In the hypophyses, estrogen or progesterone treatment alone significantly depressed copper levels (P<0.05) but in the adenohypophysis, the OE animals were inferior to the OP animals whereas in the neurohypophysis, the OE animals were superior to the OP animals. 5.3.0 DISCUSSION The depression of AChE activities in the pons, cerebellum, amygdala, hippocampus and midbrain of bilaterally ovariectomized gilts irrespective of steroid treatment is in line with the reports of Pfaff (1968), Eisenfeld and Axelrod, (1975) who have linked these brain regions with steroid metabolism by neural cells. It also lends credence to the observation of Iramain et al (1979) that orchidectomy plus estradiol administration decreased AChE activity in the rat cerebral cortex. Moudgil and Kanungo (1973) also observed a UNIVERSITY OF IBADAN LIBRARY - 1 3 7 - decrease in the AChE activity of the rat cerebral hemisphere following ovariectomy. The rise in AChE activity in the pons, hypothalamus, cerebral cortex, midbrain and medulla oblongata of the Estradiol-treated gilts over the progesterone treated animals implies that estrogen has a more facilitatory role on nervous transmission than progesterone. Since AChE activity has been known to enhance adaptability, Estradiol may also be more useful in allowing the animal to adjust favourably to changes brought about by gonadal steroids withdrawal in the female. The decline in AChE activity upon gonadal steroids withdrawal in the female also emphasizes the necessity of these hormones in maintaining t normal metabolic functions of the brain. Hence ovariectomy may possibly lead to impairment of brain activities. The ability of the hypothalamus, midbrain, pons and medulla oblongata to respond more to estradiol treatment than progesterone also supports the report of Kawakami et al (1970b) that estrogens cause a rise in the multiple unit activity in the dorsal hippocampus, amygdala and hypothalamus while progesterone did not show appreciable results. However, it must be mentioned that in the x^resent study, progesterone and estradiol injections had similar effects in the cerebellum, amygdala and hipx^ocampus. The reason for this may be due to the dual purpose function of the midbrain which is known to exert both facilitatory and inhibitory effects on gonadotrophin secretion. In addition, stimulation of the midbrain may activate the neurons of the hypothalamic ventromedial nucleus (Tsubokawa and Sutin, 1963) and UNIVERSITY OF IBADAN LIBRARY - 1 3 3 - posterior hypothalamus while at the same time inhibiting the hippocampus and amygdala in gonadotrophin secretion (Velasco and Taleisnik, 1969b). This reciprocity of the midbrain in gonadotrophin release may thus explain the higher AChE activity induced by Estradiol in the midbrain and hypothalamus over the amygdala and hippocampus. While it is known that the hypothalamus is responsible for follicular growth and estrogen secretion and also regulates ovulation (Halasz and Gorski., 1967, Koves and Halasz, 1970). Other studies have shown that stimulation of the hippocampus may in fact block ovulation in the rat (Velasco and Taleisnik 1969). A further evidence of the fairly antagonistic nature of the hippocampal neurons to the hypothalamus comes from the report of Terasawa and Sawyer (1970) and Flerko, and Bardos, (1961) that while stimulation of the medial preoptic area of the hypothalamus in the rat increased serum FSH and LH levels in the blood, stimulation of the hippocampus alone inhibited the elevation ofserum LH level and suggests that the hippocampus stimulation inhibited the neural surge of the ovulatory hormone and facilitated the release of FSH. This is not to say that the hippocampus is antagonistic to behavioural estrus or gonadal function in the female because Riss et a_l (1963) have demonstrated that destruction of the hippocampus delayed onset of puberty in the rat. It has also been shown by Nauta, (1958) that the anatomical connections of the brain regions contribute largely to these striking and related differences in their functions. Thus it has been established that the neurons of the midbrain projects to the amygdala and are linked with the hippocampus by a bi-directional medial fore-brain bundle which permits reciprocal influences between the regions with the midbrain acting as a UNIVERSITY OF IBADAN LIBRARY - 1 3 9 - central station for the distribution of flow of afferent impulses to the hypothalamus or to the limbic system and modulates the magnitude of sensory inputs according to the information received from upper neural structures and the prevailing hormonal background. The lack of difference in AChE activity in the cerbral cortex and medulla oblongata of the sham ovariectomized and Estradiol treated gilts is a reflection of the steroid receptivity and sensitivity of these structures. Thus while it is known that estradiol is highly concentrated in the hypothalamus, preoptic area, amygdala and septum (Pfaff, 1968), very low concentrations have been found in the cortex. Another possible reason for the AChE activity induction by estradiol is the discovery that estrogen induces choline acetyltransferase in the preoptic area (Luine et aJL, 1975) and AChE is also known to be involved in the feedback control of LH secretion which is elevated in the serum after ovariectomy and depressed by estradiol administration. The apparent potency of estradiol over progesterone may be due to the fact that estradiol when administered alone to ovariectomized animals may be able to restore near normal gonadal functions probably due to its ability to induce progestin receptors or to make use of the small amounts of progesterone secreted by the adrenal gland to elicit the appropriate responses. Progesterone on the other hand when administered alone tends to behave as an anti-estrogen unless the target tissues are previously sensitized by estrogens. The higher protein concentrations observed in the sham ovariectomized gilts than the other bilaterally ovariectomized gilts in the cerebellum, amygdala, hypothalamus and midbrain supports the earlier suggestion that these regions play profound roles in sexual development and UNIVERSITY OF IBADAN LIBRARY - 1 9 0 - maturation of the animal and hence require the two major female gonadal steroids estrogen and progesterone working together synergistically to maintain sexual behaviour. The higher protein concentrations observed in the estradiol-treated castrates over their progesterone treated counterparts in the amygdala, hippocampus, hypothalamus and medulla oblongata implies higher protein synthesis induced by estradiol since it is known that estradiol promotes body tissue growth presumably due to an increase in the transcription of M- RNA. Chan and O'Malley (1978) opined that exogenous estradiol causes functional changes in target-tissues involving an initial hormone specific receptor interaction ultimately leading to enhanced protein synthesis and increase in the activities of specific enzymes. Progesterone contrariwise is reported to facilitate protein catabolism. Hence since the hypothalamus, amygdala and hippocampus have been identified as receptor sites of estradiol, it is hardly surprising that increased protein synthesis is enhanced in the regions. Another possible evidence is the increase in blood flow (hypermia) which follows estrogen administration after ovariectomy. Concurrent with this is an increased permeability of the capillaries (Szego and Sloan, 1961). Although these changes take place in the uterus, it is not unlikely that they are manifested in the brain too. Thus an increased permeability of the capillaries would allow more metabolites particularly enzymes and amino acids plus the necessary ions to escape through the blood-brain barrier into the brain cell membranes which is the primary site of protein synthesis. The similarly in protein concentration of the cerebral cortex and midbrain of both groups may arise from the earlier suggestion that the UNIVERSITY OF IBADAN LIBRARY - 1 9 1 - cortex is not a major site of estradiol metabolism while the midbrain is known to have a dual "carriageway" link with the limbic system and the hypothalamus. In addition, the cortex is less associated with sexual behaviour and more with learning and memory functions. The reason for the higher protein concentration induced by progesterone in the cerebellum is not very clear and may need further investigation. The higher SAChE activities observed in the brain regions of progesterone treated castrates is a reflection of the depressed protein concentration of the regions and their lowered AChE activities. These depressions would ultimately result in elevated SAChE activities implying a compensatory attempt by the neural cells to offset the metabolic inbalance brought about by changes accompanying ovariectomy and progesterone administration. 5.3.1 MINERAL PROFILE IN THE BRAIN The results indicate that ovariectomy depressed calcium levels in the amygdala and hippocampus irrespective of hormonal treatment while it elevated calcium in the pons and cerebellum. Progesterone treatment alone elevated calcium in the midbrain and hypothalamus and depressed it in the cortex. This trend closely reflects the inhibitory role of the midbrain on the hippocampus and amygdala. The mechanism whereby calcium is depressed by ovariectomy and either progesterone or estradiol administration cannot be readily explained in the light of available information but a likely possibility is an interference with some enzyme activated systems in the brain. It is well established that the ouput mechanisms of neuroednocrine cells respond to circulating steroids by either a decrease or an increase UNIVERSITY OF IBADAN LIBRARY - 1 9 2 - in the membrane potential of the cells which are principally determined by the electrolyte balance between sodium and potassium- It is therefore possible that the lack of effect of steroid withdrawal on sodium concentration of the brain regions is a result of an increase in turnover rates of sodium to maintain fairly normal concentrations of the ions- However, the results show that while estradiol facilitated potassium ions retention in the medulla oblongata, pons and hypothalamus than progesterone administration, similar levels were observed in the cerebellum, amygdala, hippocampus, midbrain and cerebral cortex. This suggests that estradiol facilitates mineral retention by the brain cells in response to their role in protein synthesis. It also shows that estradiol has a higher capacity to increase cell permeability through increased blood flow than progesterone. The elevated magnesium level induced by progesterone administration on ovariectomized gilts in most of the brain regions is indicative of the compensatory role magnesium tends to play during calcium depletion. It also suggest possible progesterone induction of magnesium which may be required in the several magnesium activated enzyme metabolic pathways required for maintenance of neuromuscular activities. It also suggests possile antagonism between magnesium and calcium. The depressed copper levels in the ovariectomized gilts is also indicative of the impaired mineral retention of the brain cells consequent upon ovarian steroids withdrawal. The slightly higher copper levels in the estradiol-treated castrates suggests increased blood flow to the affected brain regions resulting in passage of more metabolites across the blood- brain terrier to the brain cells. The effect of ovariectomy and ovarian steroids therapy on brain UNIVERSITY OF IBADAN LIBRARY - 1 9 3 - zinc levels were not very consistent and further study is recommended to further elucidate the role of ovarian steroids metabolism on zinc retention. Suffice to say however, that estradiol and progesterone treated castrates had higher zinc levels than the sham ovariectomized gilts in the hippocampus and hypothalamus which is in agreement with the reports that estrogens increases zinc accumulation (Gunn and Gould, 1956) and induced increased zinc incorporation in the dorsoventral lobe of the castrate rats (Muntzing et al, 1977). 5.3.2 AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY IN THE HYPOPHYSES No treatment effects were observed in the AChE activity of the neurophypophysis which suggest a considerable amount of tolerance to ovarian steroids withdrawal. It also indicates that the positive feed back mechanisms that normally follow ovariectomy such as LH and FSH surge did not influence the activity of the gland. However, the adenohypophysis which is the site of FSH and LH release showed increased AChE activity in progesterone-treated castrates. The restorative effect of estradiol in AChE activity in the adenohypophysis of the ovariectomized gilts confirms the observation that the LH and FSH surge in the serum following ovariectomy can be diminished largely by estradiol administration while progesterone has a very limited effect. Thus the adenohypophysis of the estradiol-treated ovariectomized gilt is able to respond to the negativefeed back effect of exogenous estradiol administration thereby allowing the gland to function normally. This is also another evidence of the sensitivity of the hypothalamic- UNIVERSITY OF IBADAN LIBRARY - 1 9 4 - hypophyseal-gonadal axis to estrogen administration. The decreased protein concentrations of the hypophyses of the progesterone-treated, ovariectomized gilts and the conconmitant restorative effect of estradiol on protein synthesis lends credence to the presumption that progesterone is protein catabolic while estrogen is protein anabolic. While the SAChE activities in the neurohypophysis were unaffected by the treatments, the adenohypophysis of the progesterone-treated ovariectomized gilts recorded higher levels than the other two groups which attracts the earlier explanation of an attempt to compensatte for decreased protein synthesis resulting in increased SAChE activity. 5.3.3 MINERAL PROFILE IN THE HYPOPHYSES The inability of progesterone to maintain normal calcium levels in the adenohypophysis of the ovariectormized gilts suggests that the protein catabolic action of progesterone also facilitates decreased calcium retention. The restoration of calcium level to normal by estradiol in the ovariectomized gilts may be linked to the role of estradiol (Florindo and Martini, 1975), and calcium ions in the release of ACh at neuromuscular junctions (Rahamimmof, 1976). In addition, increased calcium absorption is known to favour increased protein synthesis and the utilization of amino acids. The increased retention of calcium in the neurohypophysis of the progesterone-treated ovariectomized gilts may probably have a link with the accumulation of fluids observed in the uterus of progesterone-treated, ovariectomized sows during pregnancy and the inhibition of the oxytocic effect of neurohypophysis. (Alexander and Williams, 1968). Magnesium as usual is induced by progesterone treatment and lowest UNIVERSITY OF IBADAN LIBRARY - 1 9 5 - in the hypophyses of the sham ovariectomized gilts. This again suggests a possible role for progesterone in magnesium metabolism which has to be investigated further. Potassium is highest in the adenohypophysis of the ovariectomized groups of animals than in the sham ovariectomized group while in the neurohypophyses, the progesterone injected group had the least concentra­ tion. Possible reason for this may be due to cations interaction and possible antagonism between potassium, calcium and magnesium. Sodium on the other hand remained relatively stable indicating its role in electrolyte balance and osmotic regulation of the body fluids of the animal. The lowering of copper levels by ovariectomy and the fairly restorative effects of estradiol confirms the role of copper in red blood cells formation and the synthesis and activation of several oxidative enzymes necessary for the normal metabolism of the pig. Complement to this is the increased blood flow associated with estrogens which may readily result in increased copper retention by the hypophyses. The higher levels of zinc in the hypophyses of the ovarian steroids treated ovariectomized gilts may be due to the regenerative action of zinc on body tissues and a compensatory increase of the mineral in an attempt to reduce the degenerative changes characterizing ovariectomy. Another possible evidence for this function is the known fact that zinc deficiency or impaired zinc metabolism in the rat body results in atropy of the sex organs and brain malformations (Fullis, 1958, Hurley, 1974) which are effects also manifested by gonadal steroid withdrawal. Thus the elevation of zinc in the hypohyses of the ovariectomized ovarian steroids-treated gilts may be a feed back response to attenuate the effects of ovariectomy. UNIVERSITY OF IBADAN LIBRARY C H A P T E R S I X ' EFFECTS OF TESTOSTERONE INJECTION ON THE AChE ACTIVITY AND CATIONS IN THE BRAIN AND HYPOPHYSES OF GILTS UNIVERSITY OF IBADAN LIBRARY - 1 9 6 - CHAPTER SIX 6.1 EFFECTS OF TESTOSTERONE INJECTION ON THE AChE ACTIVITY AND CATIONS IN THE BRAIN AND HYPOPHYSES OF GILTS 6.1.1 INTRODUCTION Preceeding chapters have dealt rather exhaustively on the role of the gonadal hormones in reproductive behaviour. How this behaviour is mediated by the nervous system has also received considerable attention. Evidences abound that the presence or absence of perinatal androgens appears to determine irreversibly the nature of adult sexual behaviour shown by rats and other rodents whereas perinatal ovarian secretions do not appear to play such a role (Gorski, 1973, Arnold, 1980). Thus genetic females injected with a single dose of androgen around the time of birth can be permanently masculinized behaviourally as well as defeminized. That is, such a genetic female will show typical male behaviours more frequently and typical female behaviours less frequently at adulthood compared to a normal female. Similarly, males deprived of androgens by castration at birth will be defeminized and demasculinized (Arnold, 1980). It therefore appears that androgens, when administered to female animals act as anti-estrogens. 6.1.2 LITERATURE REVIEW The concept of organizational effects of hormones suggests that Sex differences exist in the brain and workers have shown differential receptivity to gonadal steroids by certain brain regions. That is, some UNIVERSITY OF IBADAN LIBRARY - 1 9 7 - regions in the male brain are different in their functions, steroid receptivity and probably structure from the same brain regions of female animals (Nottebohn and Arnold, 1976). Thus Pfaff (1970) reported that the effect of testosterone on male behaviour in male rats can be mimicked using testosterone injections in females. McEwen, (1970) noted that neonatal treatment of female rats with testosterone reduces adult sensitivity to estradiol with respect to a variety of estrogen-dependent neuroendocrine and behavioural parameters. Indications also show that neonatal androgenization may influence adult sensitivity to progesterone as some male rats exhibit somewhat the same reduced hormone sensitivity as neonatally androgenized females. Testosterone injected into gilts in the early and middle of the luteal phase of the estrous cycle blocked ovulation but did not at the end of the phase. Also, the treated gilts were in estrus longer (4.0 to 6.8 days) than the control (2 days) (Ciro and Torres, 1974). The work of Clemens et al. (1969, 1970) shows that while a normal female rat shows lordosis behaviour after ovariectomy and priming with estradiol benzoate and progesterone, lordosis behaviour in the androgenized female is suppressed. Further evidence is provided by Gorski (1968) who reported that when as little as 10 ug testosterone propionate is administered to the one or two-day-old female rat ovulation is prevented but when such females are tested for lordosis behaviour after ovariectomy and replacement therapy with both estradiol benzoate and progesterone, they display normal levels of female behaviour (Clemens et al 1969). Flerko and Mess (1968), McGuire and bisk (1968) observed that testosterone administration on intact females decreases estradiol uptake by the hypothalamus. However, information is lacking on dose-response ■ UNIVERSITY OF IBADAN LIBRARY - 1 9 8 - relationship on androgenization of the female and induced male behaviour. A striking anomaly observed by Gorski (1973) was the fact that androgenized female rats displayed significantly higher lordosis quotient following estradiol benzoate treatment than control females. However, Gorski was quick to add that the dose of testosterone propionate, the age at injection, probably the age at testing, the hormonal replacement and conditions under which the tests are conducted are critical factors which affect the results obtained. Thus 10 ug testosterone propionate given to the 6-day-old female was much less effective in inducing anovulatory persistent estrus than the same dose given on day 4. A critical question on the mechanism of androgenization is whether the androgen merely prevents the maturation or development of the fully functional cyclic regulatory system or it induces changes which actually masculinize the brain. Ladosky and Gaziri (1970) reported that brain levels of serotonin increases significantly in the female and that testosterone antagonizes the serotonergic system. Another evidence was provided by Dorner (1971) that an absolute independence of the "Sex- specific" brain differentiation from the genetic sex exists. Thus, a complete inversion of sexual behaviour after post pubertal androgen activation was observed between male and female rats following androgen deficiency in the males and androgen overdosage in the females during the hypothalamic differentiation period. Dorner also observed that prepubertal administration of androgen to female rats resulted in a normal or approximately normal cyclic ovarian function after puberty. However, these females displayed predominantly heretotypical (i.e. homosexual) behaviour. This suggests a partial chronological dissociation in the differentiation of hypothalamic sex and mating centres On the other hand, such UNIVERSITY OF IBADAN LIBRARY - 1 9 9 - testosterone treatment during the hypothalamic organization phase results in a predominantly male differentiation of these centres and acyclic pituitary gonadotropin secretion coupled with hypo or homo-sexuality in post pubertal life. Another example of the anti-estrogenic effect of androgen is its inhibition of the estrogen-induced hypertrophy of the anterior pituitary and also the inhibition of the thyroxine-binding capacity of anterior pituitary proteins (Schreiber, 1973). It is also suggested that estrogens and androgens compete for identical protein binding sites in the anterior pituitary. This suggestion is confirmed by the discovery by Gorski et al (1968) of a morphological sex difference in the suprachiasmatic nuclei of the hypothalamus (SCN) and the findings of Moore (1978) that neonatal testosterone treatment of females disrupts the periodic release of LH in the same way lesions of the SCN do. The similarities between the effects of perinatal testosterone treatment and SCN lesions on the disruption of LH release in female led to the speculation that testosterone treatment may affect the function of the SCN and other brain areas that affect female sexual behaviour such as the frontal cortex, hippocampus and Septum (Sodersten et al, 1980). Emmens and Miller (1969) therefore advanced the hypothesis that testosterone belongs to the anti-estrogens which do not inhibit or reduce estrogen concentration in the target tissues, but exert their influence in some unknown way. UNIVERSITY OF IBADAN LIBRARY - 200- 6.2.1 MATERIALS AND METHODS Ten 4-month-old Large White gilts raised and maintained at the physiology unit of the university of Ibadan farm were sacrificed for this study. The animals were housed and managed as described in preceeding chapters. The gilts (28 to 35 kg body weight) were injected intramuscularly with 1 ml of corn oil containing 25mg of Testosterone enanthate (equivalent to 18.0mg testosterone). The injections were administered every Wednesday at 0900-1000 hours and repeated every Wednesday for five weeks. The other group of gilts (CG) received the oil vehicle only at the same time and rate as TE gilts. 24 hours after the fifth and last injection, all the gilts were sacrificed and their brains and hypohyses quickly removed and processed as described earlier. 6.2.2 STATISTICAL ANALYSIS All results were subjected to statistical analyses as described in preceeding chapters. RESULTS 6.3.1 EFFECT OF TESTOSTERONE INJECTION ON THE AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY OF THE BRAIN AND HYPOPHYSES OF GILTS. The results are summarized in Table 6.1. Testosterone injection significantly depressed AChE activity in all the brain regions (P<0.05). However, no significant differences were observed in the hypophyses (P>0.05) UNIVERSITY OF IBADAN LIBRARY 2 0 1 TABLE 6.1. EFFECT OF TESTOSTERONE INJECTION ON THE AChE ACTIVITY, TOTAL FROTEIJ .• s AND SAChE ACTIVITY IS THE BFAIN AND HYPOPHYSES CF GILTS. * * * (a) *AthE ACTIVITY ANIKAL GROUPS BRAIN REGIONS TESTCSTERC*J£«■INJECTED GILTS CONTROL g :[LTS Pons 2.370 1 0.139b 4.868 t 0.155® Cerebellum 2.424 i 0.193b 3-829 i 0.1653 Amygdala 2.385 ± 0.177b 4.079 1 0 .0 6 2 ® Hippocampus 2.519 t 0.213b 6.153 ± 0.227a Hypothalamus 3.658 * 0 . 1 6 3 6 4.895 + 0 .2 1 2 a Cerebral cortex 1.343 + O.C59b +1 . 8 6 1 0.0?0a Mid Brain 5.760 + 0.231b 10.130 ± 0.363® Medulla Oblongate ♦6 . 0 6 1 t 0.127b 6 . 9 2 1 0.124® "Adenohypophysis 0.445 ± *0.020a 0.710 + 0.031® Neurohypophysis 1.292 t 0.0502 1.455 + 0.031® GRAND MEAN , . 2.876 + 0.735 -....... 4.490 + 1.130® (b) “ TOTAL PROTEIN ANIMAL . . .. GROUPS BRAIN REGIONS. TESTCSTERC: ■INJECTED GILTS ’ CONTROL GILTS Pons 0.464 ± 0.019a f 0.340 ± 0.023b Cerebellum 0.264 + 0.015b 0.638 + 0.071a Amygdala 0 . 3 0 0 ± 0.006a +0 . 3 2 2 0.007® Hippocampus 0.252 + 0.011a 0.331 + 0.007® Hypothalamus 0.346 ± 0.025b . „ 0:767 + o.o3oa Cerebral cortex 0.188 ± 0.004b 0.332 ± 0.004® Mid Brain 0 . 6 0 1 ± 0.0045 — 0.602 £ 0.054a Medulla Oblongata 0.543 + 0.022^ ‘ 1.037 ± 0.030® Adenohypophysis 0.169 ± 0.004a 0.179 £ 0.003® NeOrohypophysis Chi 2 6 + 0.009a 0.076 ± 0.004® GRAND MEAN 0.325 £ 0.065b 0.462 ± 0.117a (c) ♦♦♦SAChE ACTIVITY ANIMAL" GROUPS BRAIN REGIONS. TESTOSTERONE-INJECTED GILTS CONTROL (GILTS Pons 6.185 ± C.1 2 6 b 14.477 ± 0.560® Cerebellum 9.414 ± 1 .2 8 6 a 6.359 + 0.974b Amygdala 7.995 ± 0.703b 12.696 ± 0.316® Hippocampus 10.147 + 1.096b 18.667 £ 0.936® Hypothalamus 1 0 . 8 0 0 i 0.089a 6.725 + 0.912b Cerebral cortex 7 . 1 8 2 ± 0 .3 6 0 ® 5.613 ± 0.252® Mid Brain 9.597 ♦ 0.4l6b 17.763 ± 2.177® Medulla Oblongata 11.248 ± 0.513® 6.706 ± 0.289b Adenohypophysis 3-527 0.100a 3.979 ± 0.222a Neurohypophysis 10. 390 + 0.680b 19.312 ± 0 .6 9 5 ® GRAND MEAN 2.648 ± 0.971® 11.230 + 2.-02b Values in the same horizontal column differently sup cripted differ significantly 'P>0.-5;. •AchE Activity in pmole/g/itiB- “ Total Protein fn. g/100 ml. ♦“ AchE Activity in umole/E prtte lr./r.in. •♦“ Values are means l stanic-.rc error of the mean. UNIVERSITY OF IBADAN LIBRARY - 202- Testosterone injection further depressed total protein levels in the cerebellum, hypothalamus, cerebral cortex, medulla oblongata, and elevated it in the pons (P<0.05). No significant differences were observed in the amygdala, hippocampos, midbrain, adenohypophysis and the neurohypo­ physis, (P>0.05). The control gilts (CG) had significantly higher SAChE activities than the TE gilts in the pons, amygdala, hippocampus, mid brain neurohypo­ physis and inferior to the TE gilts in the Cerebellum, hypothalamus and medulla oblongata (P<0.05). No significant differences were observed in the Cerebral cortex and the Adenohypophysis (P>0.05). 6,3,2 EFFECT OF TESTOSTERONE INJECTION ON THE MINERAL PROFILE IN THE BRAIN AND HYPOPHYSES OF GILTS. The results are summarized in Tables 6.2 and 6.3. CALCIUM Testosterone injected gilts exhibited depressed Calcium levels in all the brain regions and the hypophyses (P<0.05). MAGNESIUM Unlike the trend in calcium, testosterone administration on gilts significantly elevated magnesium levels in all the brain regions and hypophyses (P<0.05). ZINC In a similar trend with magnesium levels, testosterone administration also raised magnesium levels in intact gilts above the controls (P<0.05) in all the brain regions and the hypophyses. UNIVERSITY OF IBADAN LIBRARY TABLE 6.2: EFFECT OF TESTOSTERONE INJECTION ON THE ‘CALCIUM, AJNESIUM AND ZINC LEVELS IN THE BRAIN AND HYPOPHYSES OF GILTS'* (a) CALCIUM ANIMAL GROUPS BRAIN REGIONS. TESTOTERONE--INJETTED GILTS CONTROL GILTS Pons 0.880 ± 0.015 b 2.640 4 0.075a Cerebellum 0.826 + ’ 0.01Gb 3.040 1 0.08la Amygdala 0.900 4 0.0l6b 2.980 ± 0.222a Hippocampus 1.680' + 0.025& 3.090 4- 0.l8la Hypothalamus 0.901 ± 0.0l£b 1.860 ± 0.108a Cerebral cortex 0.911 ± o'.oisb 2.020 ± 0 .0 5 8 a Mid Brain 1.081 ± 0.02# 2 . 9 2 0 i 0.159a Medulla Oblongata 1 . 2 8 1 0.03# 2.580 + 0.1l6a Adenohypophysis 1.340 + 0.08# 2.160 i 0.070a Neurohypophysis 2.260 ± 0 .0 6 # 3.220- 4 0.080a GRAND MEAN '1.206 + 0 .1 8 # 2.651 f 0 . 1 9 4 / (b) MAGNESIUM ANIMAL GROUPS BRAIN REGIONS. TESTOSTERONE-INJECTED GILTS CONTROL GILTS Pons •2.625 ± 0.08# / 2^270 ± 0.040b Cerebellum . 2.534 ± 0.040* 2.292 ± 0.004b -Amygdala 2.655 + 0.07# 2.312 ± 0.040b Hippocampus 2.753 + 0.40# 2. 200 ± 0.040b Hypothalamus 2.737 ± 0.03# 2.129 4 0.080b Cerebral cortex 2.705 + 0.080B 2.281 4 0.022b Mid Brain 2.590 + 0.1472 2~-lOk 4 0 .3 0 b Medulla Oblongata 2 . 8 1 8 + 0.100s 2.256 + 0.090b Adenohypophysis 2.536 + 0.070E 2.297 ± 0 .0 3 0 b Neurohypophysis - 2.246 4 0.063a 2. 3 6 6 ± 0 .0 3 0 a GRAND MEAN 2.620 ± 0.064a 2.271 t 0,026b (c) ZINC ANIMAL . GROUPS -BRAIN REGIONS . . TESTOSTERONE-INJECTED C-KtfS CONTROL .0XLTS, Pons 1.396 ± 0.0l8a 1.098 4 0 .040b Cerebellum 1.211 4 0.003a 1.136 ± 0 .0 5 0 a Amygdala 0.869 4 0.020a 0.614 4 0. 04b Hippocampus 0.903 + 0.004a 0 . 5 2 8 4 0.020b Hypothalamus 0.896 4 0.206a 0.619 ± 0.107b Cerebral cortex 1.270 4 0.004a 1.101 4 0.608b Mid Brain 0.956 t 0 .0 3 1 a 0.608 4 0.020b Medulla Oblongata 1 . 2 6 1 4- 0 ,040a 1 . 0 0 6 ± 0.020b Adenohypophysis 0.931 4 0 .0 5 0 a 0.678 ± 0.030b Neurohypophysis 1.458 t 0.0403 1.127 + 0.0l8b GRAND MEAN 1.115 i 0.091a 0.851 k 0.104b Values on the same horizontal line bearing different superscript;) differ significantly (P>0.05). 'Values are in pacts per million (ppm). "Values are means t standard error of the isean. UNIVERSITY OF IBADAN LIBRARY 20k TABLE 6.j: EFFECT OF TESTOSTERONE INJECTION OK 7:-: •' I "'., SODIUM AND COPPER LEVELS IN THE BRAIN AND Esi-.-HYSES OF GILTS* * (d) POTASSIUM ANIMAL .GBOUFS BRAIN REGIONS. TESTOSTERONE--INJECTED GILTS CONTROL OH Pons 3.900 ♦ 1 . 9 5 0 ® 19.000 ± l.stt® Cerebellum - . 13.500 i 1.498® 9 . SCO ± 1.297' Azygda'la 20.100 + 0 .9 0 0® 7 . a o + O.SlTh Hippocampus a . 6 o o ± 1.976® 13.87t i 1.136b tfy-pothalanus 23.260 ± 0.350® 9 . 9 7 0 i 0.t22h Cerebral cortex 20.000 + 1.581® 1 0 . 2 5 0 + 0 .2 6 8b Mid Brain . 23.500 ± 1.207® - 1 2 . 5 0 0 ± 0.805b Medulla Oblongata 12. tOO ± 0.796® 7 . t o o ± 0.331b Adenohypophysis 19.960 i 0.98t® 5 . 2 0 0 + 0 .8 6 0b NeUrohypophysis 16.900 + 1.536® 1 2 . 0 0 0 ± 0.7tlb GRAND MEAN. 19.71 ± 1.733® 1 0 . 7 2 0 ± 1.570b (e) SODIUM ANIMAL •fflOBPS / BRAIN REGIONS TESTOSTERONE-INJECTED GUTS CONTROL GILTS Pons 5t9-000 + 2.tt9a /' 553.000 ± 3.391® Cerebellum 539.000 ± 2.tt9® 5t3.200 + 3.2t6® Amygdala 5t7.000 ± 2.5t92 5 1 2 . 0 0 0 + t.062b Hippocampus 525.000 + 3.536® 5 3 3 . 0 0 0 + 2.000® Hypothalamus 529.000 ± 5.100b 5t7.000 ± 5.148® Cerebral cortex 533.000 ± 5.6l2b 5t8.000 i 3.391® Mid Brain 531. t o o + 2.821b ^ '553.000 + 3.742® Medulla Oblongata 520.500 + 1.531® 509.000 ± 3.317b Adenohypophysis 5t3. 00 ± t.0 7 0 a S t o . o o o + 3-162® Neurohypophisis 532.000 ± 6 . O t 2® 5 2 8 . 0 0 0 + 2.5t9a GRAND MEAN 53t.900 + 3.752® 5 3 6 . 6 2 0 ± 6.373® (f) COPPER ANIMAL GROUPS BRAIN REGIONS . - TESTOSTEROi-IE-INJECTED OiLiS CONTROL GUILTS 1 Pons 0.168 + 0.007s 0.100 ± 0.003®- Cdrebellum o . n o + 0 . c o t ® O.lOt + 0 . 0 0 5 ® Amygdala 0.162 ± 0.008* 0.l6t + o . o o t ® Hippocampus 0.13 t ± 0 . 0 0 2 ® 0.187 + o . o o t ® Hypothalamus 0.190 ♦ 0 . 0 03* 0.102 * o . o c t a Cerebral Cortex 0.l8t ♦ 0 . 0 0 7 ® O.lOt + 0 . 0 0 2 ® Mid Brain 0 . 1 2 8 ♦ 0 . 0 5 6 ® 0 .1 2 6 ± 0.006® Medulla Cblongala 0.160 ♦ 0.008* 0.126 + o . o o t ® Adenohypchysis 0.12t i o . o o t b 0.6t0 * o . o o t ® Nevrohypophysis 0.137 i 0 .0 0 3 1’ 0.631 ’ t 0 .0 0 3 ® GRAND MEAN. 0.157 i O .C iO b 0 . 2 2 8 ± 0-.086® Values in the same horizontal line bearlag different superscripts differ significantly (P>0.0;,;. 'Values are in parts per million (ppm) "Values are means t standard Error of' trie neaa. UNIVERSITY OF IBADAN LIBRARY - 2 0 5 - POTASSIUM Potassium levels in the testosterone-injected gilts were significantly superior to their control counterparts in all the brain regions and hypophyses (P<0.05). SODIUM Testosterone administration caused a significant rise (P<0.05) in the sodium levels observed in the medulla oblongata, amygdala and a significant decrease in the sodium levels in the cerebral cortex, mid-brain and hypothalamus (P<0.05). No significant changes were observed in the pons, cerebellum, hippocampus, adenohypophysis and neurohypophysis (P>0.05). COPPER No significant treatment effects were observed in all the brain regions (P>0.05). However, the copper levels of the hypophyses of the testosterone-injected gilts were inferior to the intact controls (P<0.05). 6.4.1 DISCUSSION That androgens behave as anti-estrogens in the female has been well established (Arnold, 1980) and their role in the inversion of female behaviour when administered during the hypothalamic differentiation period has been well described by Dorner (1971). Thus the depression of AChE activities in the brain regions of testosterone-treated gilts confirms the inhibitory role of testosterone on the development of the cholinergic system in the female. UNIVERSITY OF IBADAN LIBRARY - 2 0 6 - Ladosky and Gaziri (1970) had already established that testosterone inhibits the increase in brain levels of serotonin in females. The present study thus lends credence to the view that testosterone may be involved in some or the whole family of neurohumoral transmitters including acetylcholine, norepinephrine, dopamine and Serotonin. The anti-estrogenic effect of testosterone is further supported by the discovery by schreiber (1973) that estrogens and androgens compete for identical protein binding sites in the anterior pituitary. The report of Moore (1978) that perinatal treatment of females with testosterone mimmicks the effects of lesions of the suprachiasmatic nuclei of the hypothalamus characterized by a disruption of the LH release enhances the speculation that testosterone affects other brain areas such as the frontal and entorhinal cortex, hippocampus and septum. The present study also confirms this speculation. It is therefore not surprising that testosterone also blocked ovulation in gilts (Ciro and Torres, 1974) and decreased estradiol uptake by the hypothalamus (Flerko and Mess, 1969, McGuire and Lisk, 1969). The foregoing argument may be extended to explain the depression of protein levels in the Cerebellum, Cerebral cortex, hypothalamus and medulla oblongata of testosterone injected gilts. Such result is a reflection of the fact that testosterone injection into the female reduces the sensitivity of the hypothalamus to estradiol and disturbs the family of estrogen-dependent neuroendocrine and behavioural parameters such as M-RNA and DNA Synthesis, respiratory and glycolytic rate of the brain and the enzyme-synthesis pathways UNIVERSITY OF IBADAN LIBRARY - 2 0 7 - The lowering of SAChE activities of testosterone injected gilts also suggests the inability of the brain cells to compensate for the decline in AChE activity and protein concentrations through increased metabolic and turnover rates. This indicates that testosterone may, apart from blocking the uptake of estradiol by brain cells, actually destroy or seriously impair the cellular integrity of the cells thereby reducing the functional capacity of the brain. The consequences of such a condition in the animal would be very grave and would probably include a very slow or complete destruction of the ability to respond to appropriate stimuli, slow growth and cessation of reproductive functions and a possible lack of co-ordination. 6.4.2 MINERAL PROFILE IN THE BRAIN REGIONS The depression of calcium levels in Testosterone injected gilts suggest a possible disruption of enzyme systems necessary for transmission of impulses and which are calcium-dependent. The lowered calcium levels would also impair the release of ACh as observed in the study and may have serious implications for skeletal development and utilization of dietary amino acids. It thus appears that the depression of calcium levels partly contributed to the lowered protein concentrations observed in the brain regions. This condition may therefore very easily put the animal in a negative nitrogen balance, reduced feed intake and depressed feed conversion efficiency UNIVERSITY OF IBADAN LIBRARY - 2 0 8 - Paradoxically, magnesium levels were elevated in the brain regions of the testosterone-treated gilts and probably indicates an attempt to replace or compensate for the depletion of calcium ions especially in the maintenance of the magnesium-dependent metabolic processes involving fats, proteins and carbohydrates. It is however very doubtful if magnesium ions can functionally replace calcium despite the fact that they are both divalent. On the other hand, they may even compete with each other and the magnesium ion-activated enzyme systems may in fact be inhibited by calcium (Dixon and Webb, 1961). Thus the high levels of magnesium observed in the brain of the testosterone-injected gilts may be due to the inability of the brain cells to efficiently utilize the mineral. The relative stability of the brain sodium and copper levels to testosterone injection supports the importance of sodium in maintenance of electrolyte balance and osmotic regulation of the body fluids and copper in the synthesis of red blood cells and iron mobilization. This implies that the brain tries to resist the probable degenerative changes induced by testosterone treatment by using the extra cellular sodium to act as a buffer system and the copper in maintaining oxidative enzymes necessary for the metabolism of the animal. The elevated potassium and zinc levels may not be easily explained in the light of the preceeding discussion because according to Muntzing et al., (1977) estrogens induce the increased zinc incorporation in the dorsolateral lobe of castrate rats and by its anti-estrogenic activity, testosterone would be expected to depress zinc levels in the brain. However a possible explanation of this anomaly is that the brain being a very sensitive and well protected organ may posses certain mechanisms which tend to protect it from excessive abuse by a disturbance of the metabolic UNIVERSITY OF IBADAN LIBRARY - 2 0 9 - systems. It is therefore probable that zinc, by virtue of its vital role in metabolism, sexual development and protein synthesis, is retained by the brain cells or allowed to pass in through the blood-brain barrier. It is however also possible that the disruption of the membrane permeability of the brain cells and the blood-brain barrier contribute to these anomalies. 6.4.3 AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY IN THE HYPOPHYSES The possible influence of testosterone administration on the hypophyses of intact gilts is described by Dorner (1971) that testosterone treatment during the hypothalamic organization phase results in a predominantly male differentiation of these centres and acyclic pituitary gonadotrophin secretion coupled with hypo- or homosexuality in post pubertal life. Schreiber, (1973) also reported that testosterone inhibits the estrogen-induced hypertrophy of the thyroxine-binding capacity of the anterior pituitary proteins. There is still a further suggestion of identical protein binding sites in the anterior pituitary for estrogens and androgens. The study therefore suggests that testosterone-injected gilts would exhibit impaired sexual behaviour. However in the present study, the above suggestion could not be confirmed as testosterone injection had no effect on the AChE activity, protein concentration and SAChE activity of the adenohypophyses. Likewise in the neurohypophysis, AChE activity, and protein concentration were unaffected by testosterone injection but SAChE activity was depressed in the neurohypophysis of the testosterone injected gilts UNIVERSITY OF IBADAN LIBRARY - 210- The present results imply that the disturbance of the estradiol binding sites of the hypothalamus by testosterone would interfere with the release of FSH and LH by the pituitary. However, the lack of effect on the pituitary AChE activity and protein levels suggest that the target cells of testosterone in the female hypothalamus are the only ovarian-steriods receptive areas. This presumably allows the other areas involved in the secretion of releasing and inhibiting factors (the hypophysiotrophic neurohormones) which regulate the release of other hypophyseal hormones to function normally. This view is supported by Martini (1973) who implied in his brilliant work that the hypothalamus contains specific neurons and sites for the production of specific hypophysiotrophic neurohormones. Thus while testosterone injection of gilts may interfere with the functioning of the hypophysial gonadrotrophs, other cells such as the somatotrophs, lactotrophs, corticotrophs and the thyrotrophs may continue to function at least for sometime. 6.4.4 MINERAL PROFILE IN THE HYPOPHYSES The depression of calcium and copper levels with the concomitant elevation of magnesium, potassium and zinc in the adenohypophysis may be due to a possible interference in the cellular activity of the gonadotrophs resulting in decreased retention of calcium and copper ions presumably resulting from changes in the enzyme systems involved in oxidative metabolism and transmission of nervous impluses. The same trend was observed in the neurohypophysis except that magnesium and sodium levels were not affected by testosterone treatment. UNIVERSITY OF IBADAN LIBRARY - 211- This trend may also be due to changes in the transmission of nervous impulses and the calcium activated release of ACh at nerve endings in view of the anatomical connection of the neurohypophysis with the hypothalamus. The foregoing therefore indicates that testosterone administration on gilts has potentially unfavourable consequences on the animal by its ability to interfere with endocrine functions and enzyme metabolic pathways and has no discernable advantages at the moment. However further studies are necessary to elucidate the direct mechanism by which testosterone exhibits these manifestations in the female animal. UNIVERSITY OF IBADAN LIBRARY - 212- CHAPTER SEVEN INFLUENCE OF HEAT STRESS AND WATER DEPRIVATION ON PORCINE BRAIN AND HYPOPHYSEAL ACETYLCHOLINESTERASE AND CATIONS UNIVERSITY OF IBADAN LIBRARY - 2 1 3 - INTRODUCTION 7.1.1 STRESS Psychologically, stres implies a number of factors, viz: (1) Some external object which excites it. (2) Specific feelings characteristic of particular emotions and (3) The emotion tends to find expression in some characteristic action. Accompanying the feelings and the motor activities there are (4) certain physiological states in which the autonomic nervous and the endocrine systems play an important part and finally there is often (5) a pre-existing physiological state which is necessary if the appetite or emotional needs is to be experienced. This is most obvious in the case of hunger, thirst and sexual impulse. 7.1.2 HEAT STRESS: ITS EFFECT ON BODY METABOLISM After prolonged exertion in hot surroundings, the normal rise in body temperature with profuse sweating can be followed by rapid onset of coma, convultions and finally death. Areas affected usually include the floor of the third ventricle or the pons. The hypothalamus also affects heat regulation, peripheral vasoconstriction, vasodilation and sweating (Grossman, 1960, 1962). The ability to lose heat by sweating decreases in the following order: Man, horse, camel, cattle, sheep, goat, pig, cats and chicken. Conversely, the ability to lose heat by panting increases in roughly the same order. UNIVERSITY OF IBADAN LIBRARY - 2 1 4 - 7 .1.3 WATER DEPRIVATION: EFFECTS ON BODY METABOLISM Shortage of water causes more immediate and more intolerable distress than shortage of food. Thirst creates a 'dry-sensation' of the mouth and craving for fluid rapidly becomes compelling. As time goes on, the dryness of the mouth increases, production of saliva decreases and finally ceases. Swallowing of food becomes impossible. This is finally followed by delirium and death within a day or two in a dry climate or a little longer in a moist environment (Bell et al., 1972). Normally, the intake of water in food and drink is so regulated that it balances the loss of water in urine, faeces, sweat and breadth. The body weight and amount of water in the body therefore remains constant. When the rate of water loss is greater than the rate of water replacement, dehydration of the body results and there is a reduction in physical efficiency. LITERATURE REVIEW 7.1.4 HEAT STRESS Heat stress is generally a combined effects of temperature and humidity. Air humidity is important because evaporation is inhibited by the reduction in water vapor pressure gradient between the lungs and the air. Studies of the effect of humidity level on the well being of the pig indicate that body heat loss by ventilation becomes increasingly difficult as the humidity level is raised (Ingram, 1965). It must also be noted that in carrying out its varied functions, blood must maintain homeostasis and homoiothermy in the organism. UNIVERSITY OF IBADAN LIBRARY - 2 1 5 - A shift in the temperature of the environment beyond a certain range can be expected to bring about gradual quantitative and qualitative changes in certain blood constituents. The reactions which can be observed vary according to the nature, duration and intensity of the climatic stimulus and follow the pattern of the general adaptation syndrome i.e. shock, adpatation and exhaustion. In the pig, at times of exogenous physical stress e.g. hyperthermy, blood volume increases owing to mobilization of reserves (Steinhardt and Studzinski, 1967). Heat stress is also usually accompanied by a decline in red blood cell count. However, long term heat stress at a low or moderate level seems to lead to haemoconcentration as a result of fluid loss. The pig is at particular disadvantage under short-term heat stress. Although it can achieve an increase in circulating blood volume and thus lose heat by vasodilation, its weak cardiovascular system is not able under continued stress to circulate the enlarged blood quantity quickely enough thereby ensuring the transport of oxygen and carbon-dioxide. A rise in ambient temperature leads to increased respiratory activity and a condition of respiratory alkalosis. An increase in metabolic activity causes a rise in the presence of acids or acidosis from which the organism must be protected. An increase in physical stress creates an acid surplus due to formation of lactate, metabolic and respiratory acidosis which may exhaust the bicarbonate buffer system of the blood resulting in a fall of pH. The detrimental effect of high ambient temperatures on the growth processes of pigs or sexual development, spermatogenesis and embryonic survival is based on a number of physiological interactions which animal production research has been trying to unravel for many years. UNIVERSITY OF IBADAN LIBRARY - 2 1 6 - Heat stress during gestation is also known to reduce birth weights in rats and sheep (Cartwright and Thwaites, 1967, Benson and Morsis, 1971, Brown et al., 1977). Heat stress during gestation also reduced placental weight (Alexander and Williams, 1971) and uterine blood flow (Oakes et al., 1976). Heat stress in swine reduces food intake (Nichols et al., 1980) while sprinkling with water increased food intake and average daily gain. Heat stress exerts a myriad of effects on body functions viz, reproduction, metabolism etc. Hence heat stress affects protein concentration in the blood of the pig via interaction with blood volume. An increase in blood volume arising from vasodilation leads to a drop in total protein concen­ tration as a consequence of haemodilution (Yanga, 1972). Heat stress also disturbs body metabolism by increasing blood suger levels (Tewes et al., 1981). Heat stress is also known to disturb the activity of some endocrine glands chief among which are the thyroid, hypophyses and the adrenal glands. Thus Brooks et al., (1962) and Yousef et al.., (1967) observed a reduction in thyroid function in heat stressed cattle and sheep and an impairment of endocrine dynamics in cattle (Collier et al., 1982). Acclimatization to raise environmental temperatures is accompanied by a decrease in the basal metabolism (Bedrak et al̂ , 197 1) and a lowered concentration of thyroxine and thyrotrophin (Tal and Sulman, 1973) while TRH content of the hypothalamus stays constant (Bedrak et al., 1980). While Bedrak et al., (1980) observed an increase in the activity of enzymes associated with steriod metabolism with increasing temperature of incubation, they recorded concomitant decline in serum testosterone in heat acclimatized rats. Their explanation is based on the assumption that heat UNIVERSITY OF IBADAN LIBRARY - 2 1 7 - stress presumably brings about an increase in the rate of androgen catabolism by the liver and kidney. The same workers also observed decreased sperm production and impaired integrity of sertoli cells in heat exposed rats. The results were confirmed by Egbunike and Dede, (1980) who observed that short-term exposure of boars to tropical sunlight resulted in a drop in sperm production and increased sperm abnormalities. The works of Bedrak et al., (1971), Sod-Moriah and Bedrak, (1976) further suggest that the lowered basal metabolism and increased urinary excretion of steroids induced by heat stress are due to lowered thyroidal function coupled with an increased secretion of ACTH and an increase in the rate of steroid inactivation. In spite of the relatively scanty informa­ tion on the effect of heat stress on pigs, the impairment of thyroid function seems to be the area that is continuously attracting attention. The exposure of warm blooded animals to cold environments increase thyroid function. The stress-induced inhibition of TSH secretion and elevated ACTH level could arise as a result of a competition between the corticotropin­ releasing factor (CRF) and Thyrotropin releasing factor (TRF) at the hypothalamic level so that enhanced release of one principle would necessarily depress the release of the other (Fortier, 1973). Another possibility could involve a competition of a similar type at the pituitary level between ACTH and TSH secretion, the two processes being inversely related, so that stimulation or inhibition of one would have the opposite effect on the other. It is also presumed that the dorsal hippocampus exerts opposite influences on TSH and ACTH secretions and may be involved, in association with other components of the limbic system, in the stress-induced shift of these secretory activities. This is due to the inhibitory nature of the UNIVERSITY OF IBADAN LIBRARY - 2 1 8 - hippocampus on the pituitary-adrenocortical system (Endoczi and Lissak 1962; Kawakami et al^, 1968). Thus according to de Wied (1973) the pituitary-adrenal axis is the system "par excellence" of homeostasis and is responsible for the relative freedom which higher organisms exhibit in a constantly changing environ­ ment. Stress not only results in the discharge of ACTH from the adenohypophysis but also in the release of vasopressin from the posterior hypophyses. This close relationship between the vasopressor and adrenocorticotropic response to stress led to the hypothesis that vasopressin may be responsible for the release of ACTH. Another striking evidence is the efficacy with which vasopressin induces the release of ACTH, and the inhibution of ACTH release in animals with extensive lesions in the median eminence of the hypothalamus which at the same time causes diabetes insipidus as the result of the destruction of the hypothalamic-neurohypophyseal connections (McCann, 1957, de Wied et al., 1958). 7.1.5 WATER DEPRIVATION The sensation of thirst is presumably produced by an increased osmotic pressure of the fluid in cells and calculations based on experiments in man with hypertonic saline show that thirst is produced when about 1 percent of the intracellular water of the body has passed into the extracellular space. The osmoregulator mechanism for antidiuretic hormone (ADH) release and the central thirst mechanism may not be identical. The drinking effect may thus be due to a stimulation of nervous elements specifically sensitive to an elevated sodium chloride concentration of the UNIVERSITY OF IBADAN LIBRARY - 2 1 9 - internal evironment. The thirst mechanism is therefore probably stituated more posteriorly than the osmoregulators. Other symptoms of water deprivation as described by Harper, (1981) include nausea, vomiting, a hot and dry body, a dry tongue, loss of co­ ordination and a oncentrated urine of small volume. Death usually occurs when the body loses 10 to 20 percent of its water content. 7.1.6 HORMONES AND ELECTROLYTES IN WATER METABOLISM The brain, the hypophyses, the adrenal glands and the thyroid gland are all involved in the maintenance of water balance in the body. Any attempt to deprive the animal of water therefore results in serious imbalance in the level of circulating hormones and electrolytes. Under normal circumstances, the osmotic pressure of the plasma varies only slightly despite wide variations in the intake of fluid and solutes. The neurohypophysis releases into the blood an antidiuretic hormone (ADH) or vasopressin, the chief action of which is to increase water reabsorption in the distal tubules and collecting ducts. This eanables the kidney to defend the osmolarity of the plasma. Water starvation therefore leads to elevated ADH secretion and the produc­ tion of a concentrated urine. This results in an increase in osmotic pressure and an elevation of the sodium ions in the blood due to the fact that the rate of water loss is usually greater than the rate of electrolyte loss. Experiments by Verney (1958) revealed that osmoreceptors are present in the hypothalamus and stimulation of the hypothalamus stimulates drinking while its destruction abolishes the sense of thirst. UNIVERSITY OF IBADAN LIBRARY - 220- In another experiment, Grossman (1960, 1962a) found that injection of cholingergic stimulants (such as carbachal) into the hypothalamus enhanced water intake in both satiated and water-deprived rats. Contrari­ wise, injection of cholingergic blocking agents reduced water consumption (Grossman, 1962b). Also Leibowitz (1970) reported that blocking of cholinergic system in the rat hypothalamus increases the thirst sensation and decreases hunger. 7.2.1 MATERIALS AND METHODS Three experiments were carried out. All the animals used for the experiment were boars farrowed and reared in the physiology unit of the University of Ibadan Farm as earlier described. Experiment !_ Eight Large White boars weighing between 38 and 48 kg and aged between 6 and 8 months were used. They were randomly assigned to two experimental groups of 4 boars each. One group (ES) eas exposed to direct sunlight without water or shade for one hour everyday between 1200 to 1300 hours for five days. The other group (CS) were used as controls and kept in the pen throughout the experimental period. Both the ES and CS groups were slaughtered immediately after the last and final exposure hour. Experimental 2 Twelve Large White boars (6-8 months old) divided into 3 groups of four boars each were used. In the first group, the four boars were exposed to direct sunlight everyday for one hour between 1200 and 1300 hours for a UNIVERSITY OF IBADAN LIBRARY - 221- 3-day period (ES3). In group two, all the boars were challenged with the same exposure treatment but this time for an extended period of six days (ES6). In group three (CS) all the four boars were used as controls and kept indoors throughout the duration of the experiment. In groups one and two, the animals were slaughtered immediately after the last and final exposure hour. The controls were slaughted at the end of the exposure of the ES6 animals. Experiment _3 Twelve Large White boars made up of four boars for the control and four in each of the two experimental groups were used. In group one, all the four boars were deprived of drinking water for twenty-four hours with free access to food. In group two, all the four boars were deprived of drinking water for 48 hours also with uninhibited access to food. The control group was allowed ad lib feeding and drinking. Immediately after the water deprivation period, experimental group of animals were slaughtered. The controls were slaughtered at the end of the 48 hour water-deprivation of the group two animals. Immediately after slaughter, the individual brain samples and hypophyses were dissected out and processed as described earlier. 7.2.2 PHYSIOLOGICAL AND CLIMATIC MEASUREMENTS During exposure, the rectal temperature and respiratory rate of all boars were recorded every fifteen minutes. Also the dry- and wet-bulb temperatures in the open and in the pen were recorded every fifteen minutes using a zeal (Z.H. Zeal, London), Mason's type wet - and dry-bulb UNIVERSITY OF IBADAN LIBRARY - 222- hygrometer and the relative humidy calculated therefrom. The physio­ logically effective temperature (PET) was calculated by respectively weighting the dry- and wet-bulb temperatures by 0.6 and 0.4 (Ingram, 1965; Steinbach, 1971). 7.2.3. STATISTICAL ANALYSES The data were subjected to statistical analyses as described in preceeding chapters. RESULTS TABLE 7.1.1 CHANGES IN MEAN TEMPERATURE, RELATIVE HUMIDITY, RESPIRTORY RATE, RECTAL TEMPERATURE AND PHYSIOLOGICALLY-EFFECTIVE TEMPERATURE (PET) DURING THE EXPOSURE OF BOARS TO DIRECT SUNSHINE (Means + S.E.M.) LEVEL OF DIFFE­ IN THE OPEN INSIDE THE PEN RENCE BETWEEN (a) (b) (a) and (b) Air Temperature (°C) 35.255 + 0.545 30.175 + 0.476 PC0.001 Relative Humidity % 50.325 + 0.131 67.850 + 0.476 P<0.001 P.E.T. (°C) 32.00 + 0.245 29.3 + 0.114 PC0.0001 Respiratory Rates 122.2 + 3.419 48.1 + 2.084 PC0.001 (breaths per minute) Rectal Temperature 41.637 + 0.056 39.475 + 0.035 P<0.0001 (°C) Table 7.1.1 shows the mean temperatures, relative humidities, physio- logically-effective temperature (PET), respiratory rates and rectal temperatures in the open and inside the pen. These results indicate that the above indices were significantly different with respect to the location of the animals at the time the readings were taken. animals in the open displayed considerable signs of hyperthermia exhibited by very erratic movement, profuse salivation and flushing of the face UNIVERSITY OF IBADAN LIB ARY - 2 2 3 - 7 .3.1 INFLUENCE OF SHORT-TERM EXPOSURE TO TROPICAL SUNLIGHT ON AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY IN THE PORCINE 3RAIN AND HYPOPHYSES The results are displayed in Table 7.1.2. Heat stress sharply elivated AChE activity (P<0.05) in the pons, cerebellum, amygdala, hippocampus, midbrain, and medulla oblongata while AChE activity in the hypothalamus of heat-stressed animals was inferior to the control. However, no significant changes were observed in the cerebral cortex, adenohypophysis and neurohypophysis (P>0.05). Total protein levels in heat stressed animals were generally inferior to the control group in the pons, cerebellum, hippocampus, hypothalamus, midbrain, and medulla oblongata (PC0.05). No significant changes were observed in the amygdala, cerebral cortex, adenohypophysis and the neurohypophysis (P>0.05). The results also indicate that heat stress significantly elevated SAChE activities in all regions except in the cereberal cortex where no significant effect was observed. 7.3.2 INFLUENCE OF SHORT-TERM EXPOSURE TO TROPICAL SUNLIGHT ON THE CARTIONS CONCENTRATION IN THE PORCINE BRAIN AND HYPOPHYSES The results are summarized in Tables 7.1.3 and 7.1.4. CALCIUM Calcium levels were higher in the pons, hippocampus, hypothalamus, midbrain, adenohypophysis and neurohypophysis (P<0.05) of the heat stressed animals than the control. Heat stress however depressed calcium level in the amygdala and cerebral cortex (P<0.05 while no significant change was observed in the medulla oblongata (P>0.05). UNIVERSI Y OF IBADAN LIBRARY 22Zf . ' .TABLE 7.1.2: ' INFLUENCE OF SPORT-TERM EXPOSURE TO TROPICAL 57NLII-T . :-w ACTIVITY, TOTAL PROTEIN AND SAChE ’ACTIVITY IN. THE PORCIN:' BRA I - I . .POPHYSES. a) AChE ACTIVITY ANIMAL GROUPS BRAIN REGIONS Heat stressed Control Pons 8 .694 + 3.531 a i.9*45 + 0 .6 * 1 2 b Cerebellum 10.211) + 1.15*1 a f.6 *)l + 0 .1)*)*) b Amygdala 10.365 + 3.588 a ‘ 7.178 + 0.*)?*) b Hippocampus 7.156 + 3.D0O a 5.103 + 0.519 b Hypothalamus 7.301 + 0.880 b 9.699 + 0 . 6 1 6 a Cerebral cortex 3.050 + 3.153 a 3.992 + 0.938 a Mid brain 1*1.795 + 3.866 a 7-956 +_0 . 909 b . Medulla oblongata 15.388 + 1.266 a 1C.892 + 0.766 b Adenohypophysis 0 .6 6 *) + 3.093 a 1.716 + 0.129 a Heurohypophysis 0.528 + 3.028 a 1.578 + 0 . 3 1 6 a GRAND MEAN 7.386 + 2.127 5 .9 2 0 ' + 1 . 2 6 0 b) TOTAL PROTEIN ANIMAL GROUPS BRAIN REGIONS Heat stressed Control s'" Pons 0.990 + 0.070 b 1 . 1 7 8 + 0 . 0 2 a Cerebellum 0 . 6 1 2 + 0 . 0 6 6 b 1.269 + 0 .0 6 T a Amygdala 0.3*48 + 0 . 0 2 8 a 0.992 + 0 . 0 1 6 a- Hippocampus 0.563 + 0.029 b . 1.089 + 0 . 1 2 1 a Hypothalamus 0.35& +0.023 b 0.573 + 0 . 0 1 8 a Cerebral cortex " 0 . 5 2 6 + 0 . 0 8 2 g. 0.501 + 0.019 a _ Mid brain 0.207 + 0.00*) b 0.619 + 0.010 "a Medulla oblongata' 0.*)32 + 3.015 b 3.580 + O'. 036. a Adenohypophysis • 0.299 + 0.03*) a 0.361 + 0.029'a ’ Neurohypophysis 0.126 + 0 . 0 0 8 a C.195 + 0.009 a GRAND MEAN 0 .*) 46 + 0.098 C.672' + 0.199 c) SAChE ACTIVITY ANIMAL GROUPS BRAN REGIONS Heat stressted Control Pons '9.020 + 1.202 a 9 . 3 8 5 + 0.897 a Cerebellum 17 .038 + 1.872 a 5 . 3 0 0 + 0.380 b Amygdala 29.986 + 1.033 a 16.017 + 0.993 b Hippocampus 12.912 + 1. * ) 1 6 a -.739 + 0.298 b Hypothalamus 20.963 + 3.061 a 16.869 + 1.027 a Cerebral cortex 6.198 + 0.938 a 6.952 + 0.807 a Mid brain 71.236 + 6 . . 0 6 1 a 12.992 + 1.916 b Medulla oblongata 36.65*) + 1.968 a 15.855 + 1.119 b Adenohypophysis 2.921 + 0.*)i)6 a +.793 + 0.390 a * Neurohypophysis *4.286 + 0.507 a 1C.809 + 1.911 b GRAND MEAN 21.077 + 3.351 11.165 + 2.265 Valuer. In the samt- horizontal column bear I n r different superscripts differ sign! f icantly fP < 0 . 0 5 ) *AChE activity uniole/g/raln,, ** Total protein in g/100 ml ***"ACh.% activity in Mmole/g protein/mir.’. Values are means. + S.E.M. UNIVERSITY OF IBADAN LIBRARY 2 2 5 TABLE 7.1.3: INFLUENCE OF SHORT-TERM EXPOSURE TO TROPICAL SUNLIGHT K THE ■CALC1 . MAGNESIUM AND ZINC LEVELS IN THE PROCISS BRAIN AND HYPOPHYSES. a ) CALCIUM ANIMAL GROUPS BRAIN REGIONS Heat stressed Control Pons , 1.691 + 0.059 a 1 . 5 0 0 + 0 . 0 7 0 b Cerebellum 1.565 + 0.012 a 1.976 + 0.019 a Amygdala 1.11)3 + 0.002 b 1.987 + 0.029 a Hipocampus 1.939 + 0.055 a 1.399 + 0 . 0 1 6 b Hypothalamus 2.01)1 + 0.01)0 a 1.631 + 0.031 b Cerebral cortex 1.707 + Q.0.16 b 1.915 + 0.013 a Mid brain 2.019 + O.Q53 a 1.390 + 0.012 b i Medulla oblongata 1 .5 0 0 + 0.020 a 1.580 + 0.013 a Adenohypophysis 3.01!) + 0.555 a 2.329 + 0.Q02 b Neurohypophysis 2.171 + 0.033 a 1.369 + 0.027 b GRAND MEAN 1.871) + 0.201 1.602 + 0.129 b) MAGNESIUM ANIMAL GROUPS BRAIN REGIONS Heat stressed Control , Pons 1.081)' + Q. 0.09 b 1.20,3 + 0.-003 a Cerebellum 1 . 2 7 0 + 0.00,3 a 1.200 + 0.017 a Amygdala Q . 9 3 6 + 0 . 0 1 1 b ,1.369|+- 0.021 a Hippocampus 0.689 + 0.Q21 a 0.707 + O.Q25 a Hypothalamus Q . 8 7 0 + 0.021 a 0 . 6 7 a + Q.025 a Cerebral cortex Q.815 + 0.00.9 b 1.395 + 0 . 0 0 8 a Mid brain 1 . 1 1 6 + 0.113 b 1.303 + 0.027 a . Medulla oblongata 1 . 0 1 3 + 0 .0 0,1) h 1 .1 8 9 ; + Q. 0.01 a Adenohypophysis Q . 9 3 9 + 0 . 0 1 2 a 17022 + 0.028 a Neurohypophysis . 1.311 + 0.009 a 0 .9 5 0 V'Crroc b GRAND MEAN 1.109 + 0 . 0 7 8 I . 0 5 1 + 0.131 c) ZINC ANIMAL GROUPS BRAIN REGIONS Heat stressed Control Pons a . 3 9 8 + 0 .Q0 9 a 0.366 + 0.019 a Cerebellum 0 . 3 8 0 + 0.016 a £.102 + 0.019 a Amygdala 0 . 2 8 1 + 0.003 a b.315~ + 0.010 a Hippocampus Q . 3 8 6 + 0 . 0 1 2 a 0.253 + 0.013 b Hypothalamus 0.310 + Q.005 a 0.289, + 0.039 a Cerebral cortex 0.220 + 0.00,7 b 0.369 + 0.005 a Mid brain 0 .30, 8 + 0.003 a 0 . 3 2 2 + 0.005 a Medulla oblongata . 0 . 3 1 8 + 0.00,3 a Q . 3 0 8 + 0.002 a Adenohypophysis 0 . 2 7 0 + 0.002 b 0,379 + 0.015 a Neurohypophysis 0.269 + 0.QU5 b 0 . 3 1 6 + 0.007 a GRAND MEAN 0.313 + 0.023 0.329 + 0.017 Values in the same horizontal column hearing different superscripts significantly (P < 0.05) Values are in parts per million, (ppm). UNIVERSITY OF IBADAN LIBRARY 2 2 6 TABLE 7.1. It: INFLUENCE OK SHORT-TERM EXPOSURE TO TROPICAL SUNLIGHT ON Ti-S ‘POTASSIUM SODIUM AND COPPER LEVELS IN THE FROCINEL BRAIN AND HYPOPHYSES (Mean + SiE.M). i ) POTASSIUM ANIMAL GROUPS BRAIN REGIONS Heat stressed Control Pons 12.511 + 0.501 a 13.290 + 0.464 a Cerebellum 20.865 +0.479 a 21.656 + 1.136 a Amygdala 6.262 + 0 . 3 2 6 b 18.489 + 0.330 a Hippocampus 15.512 + 0.208 a 10.063 + 0.739 b Hypothalamus 15.522 + 0.836 a 11.265 + 0.4-9 b 1 Cerebral cortex 12.964 + 0.829 a 9.732 + 0.510 b Mid brain 15.190 + 0.103 b 25.314 + 0 .8 - 3 a Medulla oblongata A.9 2 6 ' + 0.1115 b . 17.756 + 1.052 a Adenohypophysis 7 . 5 1 1 2 + 0 . 2 7 8 a 8.511 + 0.345 a Neurohypophysis ' - 8.1129 + 0.504 a 5.337 + 0.261 b GRAND MEAN ^ 11.717 + 1.969 14.14? + 2 . 5 5 6 ] e) SODIUM ANIMAL GROUPS BRAIN REGIONS Heat stressted Control __ Pons 536.312 + 3.146 a 520.125 + 8.502 b Cerebellum 528 + 1.554 a 536.250 + 2.529 a Amygdala 513.720 + 4.270' b 534/556 + 2.527 a Hippocampus 515.000 + 6.319 b 532.250 + 2.730 a Hypothalamus 538.500 + 6 . 3 8 3 a 539.50D + 2.954 a Cerebral cortex 5 2 6 . 0 0 0 + 3 . 1 8 8 a 529.250 + 4.916 a . Mid brain 525.25U + 2.639 b 539.125 + 1.433 a Medulla oblongata 171.75Q' + 7.836 b 537.250 +2.287 a Adenohypophysis 258.000 +5-593 b 54l.«2'5a + 2.353 a Neurohypophysis 540.000' + 3.535 a 540.00 + 4.213 a GRAND MEAN 465.319 + 53.533, 534.956 + 2.565 f) COPPER ANIMAL GROUPS BRAIN REGIONS Heat stressed Control Pons 0.230 + 0.003 a 0. 0 8 8 + 0.031 a Cerebellum 0.113 + 0.004 a 0 . 0 9 1 + 0.001 a ) — Amygdala 0.073 + 0.006 a 0.159 + 0.004 a ' Hippocampus 0.123 + 0.021 a 0.082 + 0.033 a Hypothalamus 0.070 + 0.002 a 0.134 + 0.033 a Cerebral cortex 0 . 0 6 0 + 0 . 0 0 8 a 0 . 1 1 6 + 0.020 a Mid brain 0,070 + 0.002 a 0.0.43 + 0.002 a Medulla oblongata 0.079 + 0.003 a 0 . 0 5 4 + 0.002 a Adenohypophysi s 0.100 + 0.004 a 0.149 + 0.001 a : Neurohypophysis 0.140 + 0.002 a 0.060 + 0.033 a GRAND MEAN 0,106 + 0.020 0 . 0 9 5 + 0 . 0 1 5 - ----U Values In the same horizontal column bearing, different superscripts differ significant ly (P< 0.05) •Values are in parts per mill!on (ppm). UNIVERSITY OF IBADAN LIBRARY - 2 2 7 - MAGNESIUM Heat stress depressed magnesium level in the pons, amygdala, cerebral cortex, mid brain and medulla oblongata and elevated it in the neurohypophysis (P<0.05). No significant changes were observed in the cerebellum, hippocampus, hypothalamus and adenohypophysis (P>0.05). ZINC Heat stress significantly depressed zinc level in both hypophyses and the cerebral cortex and elevated it in the hippocampus (P<0.05). No significant changes were observed in the pons, cerebellum, amygdala, hypothalamus, midbrain and medulla oblongata (P>0.05). POTASSIUM Heat stress significantly elevated potassium level in the hippocampus, hypothalamus, cerebral cortex, neurohypophysis (P<0.05) and depressed it in the amygdala, midbrain and medulla oblongata (P<0.05). No significant changes were observed in the pons, cerebellum, and adenohypophysis (P>0.05). SODIUM Sodium level in the amygdala, hippocampus, midbrain, medulla oblongata and adenohyphysis of the heat stressed group were inferior to the control (P<0.05). The pons of the heat stressed boars however had a higher sodium level than the control. No significant differences were observed in the cerebellum, hypothalamus, cerebral cortex and neurohypophysis (P>0.05). UNIVERSITY OF IBADAN LIBRARY - 2 2 8 - COPPER No significant differences were observed in all the brain regions and the hypophyses (P>0.05). 7.4.1 EFFECT OF ACUTE AND PROLONGED HEAT STRESS ON THE AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY OF THE PORCINE BRAIN AND HYPOPHYSES The results are summarized in Table 7.1.5. Animals heat stressed for either 3 days or 6 days had similar AChE activities but were significantly higher than the controls in the cerebellum, hypothalamus, cerebral cortex and medulla oblongata. No significant differences (P>0.05) were observed in the amygdala and the hypophyses. However in the pons and hippocampus, the ES3 animals had higher AChE activity levels than the controls while no significant differences were observed between the ES6 group and the control group. Contrariwise, in the midbrain, the ES6 group was superior to the ES3 group which in turn was superior to the control group. Total protein levels were unaffected by heat stress (P>0.05) in the pons, amygdala, hippocampus, cerebral cortex and adenohypohysis and the neurohypophysis. In the hypothalamus, medulla oblongata and midbrain the ES6 group had higher protein level than the control groups which were similar to the ES3 groups. Surprisingly however, protein level was least in the cerebellum of the ES3 animals, medium in the control group and highest in the ES6 group. SAChE activities were not affected by heat stress (P>0.05) in the pons, cerebral cortex and neurohypophysis. UNIVERSITY OF IBADAN LIBRARY 22 9 TABLE 7.1.R: EFFECT OP ACUTE AND PROLONGS Kr A? STRESS ON THE AChE ACTIVITY, TOTAL PROTEIN AND 13AChE ACTIVITY OF THE FORCIN ' BRAIN AND HYFDPHY3E3 (Means.+ S.E.M,) a) * AChE ACTIVITY ANIMAL GROUPS BRAIN REGIONS HEAT STRESSED . ___■CONTROI.S 3 DAYS 6 DAYS Pons 8.8l8 + 0.158 a* 8.282 + 0.107 ab 3.786 4 0.115 b Cerebellum 3 . 6 8 9 + 0.165 a 3.851 + 0.138 a 2.888 4 0.062 b Amygdala 7.276 4 O . 8 7 8 a 7.025 + 0.135 a " 7.235 4 0.205 a Hippoeaunpus 5.278 + 0.077 a 8 . 1 6 6 + 0.178 £ 3.883 4 0.093 b Hypothalamus 8.083 + 0.088 a . 8.067 4 0.056 a 2.582 4 0.023 b Cerebral cortex 6.857 4 0.279 b 8.375 + 0.335 a 8.632 + 0.213 c Mid brain 6.857 4 0.279 b 8.375 + 0.335 a 8 . 6 3 2 + 0.213 c Medulla oblongata 5.162 4 0.171 a 5.768 + 0 . 0 8 6 2 3.586 + 0 . 1 1 8 b Adenohypophysis 0.651 + 0.032 a 0.826 + 0.088 a 0.865 + 0.009 a Neurohypophysis 0.892 + 0.012 a 1.117 + 0.036 a O . 9 8 9 + 0.088 a GRAND MEAN - 3.960 + 1.130 8 . 0 9 6 + 1 . 2 8 6 3.170 + 0.930 — ------------------1 b)**T0TAL PROTEIN ANIMAL GROUPS BRAIN REGIONS HEAT STRESSED CONTROLS 3 DAYS 6 DAYS — Pons 0.611 + 0.028 a 0.698 4 0.012 a o'568 + 0.019 a Cerebellum 0 . 0 9 6 + 0.005 c 0 . 3 7 0 4 0.017 a 0 .2 5 7 ' + 0 . 0 1 8 b Amygdala 0.177 + 0.006 a 0.257 + 0.008 0 . 2 8 9 + 0 .0 0 S a 2 f Hippocampus 0.202 4 0 . 0 1 8 a 0 . 2 5 8 4 0.006 — 0 . 1 5 8 + 0 . 0 0 8 a Hypothalamus 0.131 4 0.008 b 0.283 + 0.010 a 0.198 + 0.009 ab Cerebral cortex Ci.212 4 0.022 a 0 . 2 3 0 4 0.006 a 0 . 2 9 8 + 0.009 ab Mid brain '' 0.298 4 0 . 0 0 6 CALCIUM ANIMAL GROUPS BRAIN REGIONS HEAT STRESSED CONTROLS 3 DAYS _____________ 6 DAYS Pons . 0.95^ 4 0.031 b 1.907 4 0.019 a 1.969 4 0.076 a Cerebellum 0.977 4 0.017 b 2.970 4 0.031 a 1.001 4 0.013 b Amygdala 0.795 4 0.095 c 1.380 4 0.006 a 1.110 4 0.031 b Hippocampus 1.205 + 0.082 b 2.091 + 0.09? a 0.877 4 0.035 c Hypothalalmus 1.533 + 0 . 0 8 6 b . 1 . 8 6 0 4 0.055 a 1.139 4 0.070 c Cerebral cortex 0.720 + 0.008 c 2.019 + 0.022 a 1.171 f 0.035 b Mid brain 1.129 + 0.010 b 1.905 + 0.097 a 1.190 4 O.O6 3 b Medulla oblongata 1.275 4 0.019 b 2 . 1 6 2 4 0.038 a 0 . 8 6 7 4 0.090 c Adenohypophysis 1.792 + 0.019 b 2.032 + 0.023 a 1.297 4 0.051 c Neurohypophysis 1.903 + 0 . 0 3 1 a 1.932 4 0 . 0 3 8 a 1.306 + 0.072 b GRAND MEAN 1.219 + 0.201 1.921 + 0.162 1.137 + 0.092 b) KAONESSIUM ANIMAL GROUPS BRAIN REGIONS HEAT STRESSED CONTROLS 3 DAYS' . 6 DAY Pons 0.831 + 0 . 0 1 7 a 0.699 + 0.0S8 b Q. 566 4 0.062 b Cerebellum 0 . 8 5 2 + 0 . 0 5 9 a 0 i911 + 0.105 a • 0.720 4 0.090 a Amygdala 0.930 4 0 . 0 3 6 a 1 . 0 5 0 4 0.099 a 0.562 4 0.085 b f Hipoocambus 0.701 + 0 . 6 2 2 a 0.911 + 0 . 0 7 0 a __0.511 4 0.093 c Hypothalamus '-0.895 + 0 . 0 2 2 a 0.795 + 0 . 0 8 6 a 0.239 4 0.071 b Cerebral cortex .1 . 0 5 8 + 0 . 0 2 6 a 0.761 + 0 . 0 9 6 b ' 0.501., 4 0.022 c Mid brain 0.695 + 0 . 0 1 0 a 0.931 + 0 . 0 6 5 b 0.391 4 0.027 b Medulla oblongata 0.7 62 + 0 . 0 1 9 b 1.217 + 0 . 0 3 6 a 0 . 2 6 9 4 0.036 c Adenohypophysis 1 . 5 2 6 + 0 . 0 9 1 a 1.125 + 0.1 li b 0.617 4 0.029 c Neurohypophysis 1.900 + 0.071 a 1 . 0 6 2 + 0.129 bx > __ 0^961 4 0.055 b GRAND MEAN 0.969 + 0.192 0.386 + 0.121 0 . 5 2 8 4 0 . 1 0 8 c) ZINC ANIMAL CKUl/PS BRAIN REGIONS HEAT STRESSED 3 DAYS ' ‘ ' 6 DAYS C O lITRQLS Pons 0 . 0 2 6 + 0.002 b 0 . 0 3 2 ++0.002 b 0.201 4 0.005 a Cerebellum 0.097 + 0 . 0 0 3 b 0 . 0 1 9 + 0.002 b 0.106 4 0.002 a Amygdala 0.901 + 0 . 0 3 2 a 0 . 2 5 9 + 0.029 b 0.151 4 0.001 c Hippocampus 0.201 4 0 . 0 1 0 a 0 . 0 6 7 + 0 . 0 0 8 c 0.113- 4 0.001 b Hypothalamus 0.073 \± 0.009 a 0 . 0 2 0 + 0.002 b 0 . 0 7 0 4 0.009 a Cerebral cortex 0.326 4 0.019 a 0.099 + 0.002 c 0.109 4 0.003 b Mid brain 0,079 4 0.009 a 0.016 + 0.002 b 0.079 4 0.002 a Medulla oblongata 0.021 4 0.009 b 0.069 + 0.009 a 0.078 4 0.001 a Adenohypophysis 0.9 9“ 4 0.025 b 0.706 4 0.030 a 0.377 4 0.020 c Neurohypophysis 0 . 5 0 2 4 0.021 a 0.980 + 0.029 a 0.189 4 0 . 0 0 8 b GRAND MEAN ‘ 0.216 4 Q.099 0.171 + 0.129 0.197 4 0.096 Values In the same horizontal lino'tearing different superscripts differ significantly (P < 0.05) ‘Values are in parts per million (ppm) UNIVERSITY OF IBADAN LIBRARY 232 Table 7.1.7 EFFECT OF ACUTE, AND PROLONGED KEAT STRESS CN THE POTASSIUM, SODIUM J AND COPPER LEVELS. IN THE PORCINE BRAIN AND HYPOPHYSES (Means « S.E.M.) POTASSIUM ANIMAL C-ROUPS B^AIN REGIONS ' HEAT STRESSED 3 days 6 days CONTROL Pons 16.050 + 1.415a . 12.875 + 0.314b 12.500 + 1.085b Cerebellum 15.500 + 0 .6 1 2 a 14 .-875 + 0.125a 11.200 + 0.-515b Amygdala 16.875 + 0.875a 17.375 + 0.375a • 10.350 + 0 .6 5 6 b Hippocampus - 16.000 + 0.718a 13.750 + 0.595b 10.000 + 0.722c Hypothalamus 15.625 + 0.289a 14.625 + 0.68Sa 6.725 + 0.395b Cerebra Cortex 16.375 + 0.554a 14.625 + 0.55^3-b 12.837 + 0.827b Mid Brain , ' U.125 + 0.826a 10.500 + 0.645a 8.300 + 0.212b Medulla Oblongata 12.750 + 0.520a 1 6 . 0 0 0 + 0.473b 6.680 + 0.289c Adenohyp ophy sis 11.750 + 0.433b 11.375. + 0.746b 15.250 + 0.898a Netirohypophysis 19.125 + 0.427a 19.500 + 0.645a 13.212 + 0.635b GRAND MEAN "15.117 + 1.24 14/.550 + 1.340 . 10.755 + 1.427 SODIUM BRAIN REGIONS 3 days 6 days CONTROL Pons 290.000 + 0.607a 270.025 + 4.516a 292.000 + 9.120a Cerebellum 269.750 + 8.370b 271.500 + 7.963b 301.500 + 8.694a Amygdala ._-- 288.500 + 10.905a 296.000 + 3.674a ! _ 2 .8 3 . 2 5 0 + 15.146a Hippocampus 29^.350 + 8.602a 252.250 + 8.066b 3 0 0 . 7 5 0 + 8.835a Hypothalamus 292.000 + 8.042ab 275.750 + 5.138b 3 0 4 . 5 0 0 + 10.380a Cerebral Cortex /275.750 + 4.564b 265.000 + 6.455b 3 0 0 . 0 0 0 + 5:401a Mid Brain 280.500 + 4.113b 314.750 + 5.252a 3 1 6 . 7 5 0 + 11.346a Medulla Oblongata 293.000 + 5.972a 297.500 + 10.49-la 3 0 .7 - . 5 0 0 + 5.010a Adenohypophysis 312.500 + 8.540a 285.000 + 6.455b >1-29-6.. 970 + 2.926ab Neurohypophysis 322.500 + 4.790a 322.500 + 2.500a 296.500 + 5.694a GRAND MEAN 291.885 + 7.931 28^027 + 11.221 299.972 + 4.490 COPPER BRAIN REGIONS 3 days 6 days CONTROL Pons 0.134 + 0.004a. • 0.171 + 0.002a • 0.167 + 0.003a Cerebellum 0.137 + 0.003a 0.159 + 0.003a 0.167 + 0.002a Amygdala 0 . 1 2 5 + 0.003a 0.151 + 0.002a’ 0.129 + 0 .0 0 6 3 . Hippocampus 0 . 1 6 6 + 0.005a 0.125 + 0.003b 0.169 + 0.005a Hypothalamus 0 . 1 8 2 + 0 .0 0 6 a 0 . 1 6 6 + 0.008a 0.167 + 0.009a Cerebral Cortex 0.142 + 0.010a 0.159 + 0.004a 0.175 + 0.008a Mid Brain " 0.144 +• 0.005a 0.112 + 0.001a 0 . 1 2 6 + 0.009a Medulla Oblongata 0 . 1 6 2 + 0.005a 0.106 + 0.001b 0.150 + 0.017a Adenohypophysis 0.150 + 0.002a 0 . 1 6 2 + 0.006a 0.147 + 0.007a Neurohypophysis O.lol + 0.001$ 0.150 + 0 .0 0 3 a 0 . 1 6 6 + 0.004a GRAND MEAN 0.150 + 0.009 0.146 + 0.012 0.156 + 0.009 Values in the same horisontal line bearing different superscripts significantly differ (P<0.05). ___V a J ju -o q . - r,. r̂. ; M on ■ UNIVERSITY OF IBADAN LIBRARY - 2 3 3 - Magnesium level in the amygdala and hypothalamus of the heat stressed groups were similar and superior to the control (P<0.05). In the cerebral cortex and adenohypophysis, magnesium level was highest in the ES3 group, medium in the ES6 group and least in the CS group. In the hippocampus and medulla oblongata, the ES6 group were superior to the ES3 group whch in turn were superior to the CS group. No significant effects were observed in the cerebellum (P>0.05). ZINC Heat stress reulted in a significant delcine in zinc level in the pons and cerebellum while the reverse occured in the neurohypophysis. Results in other brain regions indicate that heat stress for 3 days was more potent in elevating zinc level in the amygdala, hypothalamus and midbrain than heat stress for a 6-day period. However, in the adenohypophysis the six-day heat stress period had a more potent effect in elevating zinc level than the 3-day stress period. In the cerebral cortex and hippocampus, zinc was highest in the ES6 group while in the medulla oblongata, zinc level in both the ES6 and CS animals were similar and inferior to the ES3 group (P<0.05). POTASSIUM The results indicate that heat stress significantly elevated potassium levels in the cerebellum, amygdala, hypothalamus, midbrain and neurohypophysis (P<0.05) whereas in the cerebral cortex and pons, only the ES3 animals had significantly higher level than the other two groups. UNIVERSITY OF IBADAN LIBRARY - 2 3 4 - In the medulla oblongata, and hippocampus, potassium was highest in the ES3 group, medium in the ES6 and least in the CS groups. In the adenohypophysis, heat stress significantly depressed potassium level (P<0.05). SODIUM While no significant treatment effects were observed in the pons, amygdala, medulla oblongata, neurohypophysis (P>Q.05), other brain regions were appreciably affected. Thus in the cerebellum and cerebral cortex, the heat stressed groups had significantly depressed sodium levels than the control (P<0.05). In the hippocampus and adenohypophysis, sodium levels in the ES3 and the CS goups were similar and superior to the ES6 groups. Heat stress for 3 days significantly depressed sodium levels in the midbrain (P<0.05) while the 6- day stress period had no significant effect (P>0.05). In the hypothalamus, the six-day stress period was more potent in depressing sodium levels than the 3-day stress period. COPPER Only the six-day heat stress period significantly depressed copper levels in the medulla oblongata and hippocampus. (P<0.05). No significant effects were observed in the other regions (P>0.05). 7.5.1 EFFECT OF ACUTE WATER DEPRIVATION ON THE AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY IN THE PORCINE BRAIN AND HYPOPHYSES The results are summarized in Table 7.1.8. While no treatment effects were observed in the cerebral cortex, adenohypophysis and the neurohypophysis UNIVERSITY OF IBADAN LIBRARY - 2 3 5 - (P>0.05), appreciable results were obtained in other brain regions. Thus in the amygdala, medulla oblongata and hypothalamus, water deprivation significantly depressed AChE actitivy but to varying extents depending on the duration of water deprivation. For instance, in the amygdala, water deprivation for 24 hours significantly depressed AChE activity (P<0.05) while it was partially restored after 48 hours of water deprivation. In the hippocampus, water deprivation for 24 hours had no significant effect (P>0.05) while the 48-hour deprivation depressed AChE activity (P<0.05). In the medulla oblongata and pons, water deprivation for 24 hours depressed AChE activity and was restored after 48 hours. In the midbrain, animals water-deprived for 24 hours had signifi­ cantly depressed AChE activity (PC0.05) much more than the 48 hour water deprivation did. In the cerebellum, water deprivation for 24 hours elevated SAChE activity (P<0.05) but was depressed after water deprivation for 48 hours. Generally, water deprivation caused a delcine in protein levels. More specifically, water deprivation for 24 hours was more potent in depressing total protein in the pons and hypothalamus than the 48-hour period (P<0.05). In the cerebellum, total protein was only depressed in the group deprived of water for 24 hours. The other group was unaffected (P>0.05)> In the amygdala, water deprivation caused a rise in total protein level but only the 24-hour water-deprived group displayed a significant effect. No particularly consistent trend was observed in the cerebral cortex where as shown, the animals water-deprived for 24 hours were superior to the control whereas the 48-hour-water-deprived group were inferior to the control. UNIVERSITY OF IBADAN LIBRARY 236 TA3LE 7.1.3 EFFECT OF ACUTE WATER DEPRIVATION OH THE AChE ACTIVITY, TOTAL PROTEIN AND SAChE ACTIVITY IN THE PORCINE BRAIN AND HYPOPHYSES (Mean » S.E.M.' AChE ACTIVITY ANIMAL. GROUPS BRAIN REGIONS WATER DEPRIVED 24 hours A8 hours CON'rROL Pons 3. *436 + 0.923c 6.058 + 0 .0 3 7 a A. 37A + 0.224b Cerebellum A.601 + 0.297a 2.610 + 0 .2 6 7 c 3.386 + 0.196b Amygdala. A. 1.89 + 0.3A0c 6.833 + O.AA6b 8 . 0 8 1 + 0.044a Hippocampus A.010 t 0.230a 3.332 + O.A89b A.252 + 0.256a Hypothalamus - A.9 2 A + 0.150b 5.073 + 0.13Ab 6.666 + 0 .06-.a Cerebral Cortex 1.A89 ♦ 0.110a 1.7A8 + 0.055a 1.893 + 0.05ia Mid-Brain 7.006 + 0.073a 6.339 + 0.424b 5.614 + 0.286c Medulla Oblongata 3.866 + 0.199b 5.317 + 0.072a 4.847 + 211a Adenohypophysis 0.511 + 0 .0 2 5 a 0 .3 SA. + 0 .0 1 6 a 0.865 + 0.009a Neurohypophysis 0.770 + 0 .0 3 0 a 1 . 0 1 8 + 0.232a 0.949 + 0.044a GRAND MEAN - -3.480 + 1.012 3.871 + 1.177 4.093 + 1.192 TOTAL PROTEIN BRAIN REGIONS 24 hours - 48 hours CONTROL Pons 0 . 3 7 2 + 0.021c 0 . 5 2 2 + 0.020b 0.670 + 0.019a Cerebellum 0 .1 A9 + 0.010b 0 . 2 1 9 + 0.026a OY239 + 0.010a Amygdala 0 . 2 5 7 + 0.008a 0 . 2 2 5 + O.OlOab ' 0.198 + 0.007b Hippocampus 0 . 2 5 5 + 0.013b 0 . 1 5 3 + 0.008c r 0.398 + 0.022a Hypothalamus 0.121 + 0.009c 0 . 1 7 9 + 0.017b -0.258 + 0.014a Cerebral Cortex 0 . 3 3 1 + 0.015a 0 . 1 9 2 + 0.002c 0.256 + 0.008b Mid Brain 0 . 3 2 5 + 0.017b 0 . 2 1 3 + 0.082c ’0.490 + 0.025a Medulla Oblongata -0 . 6 2 5 + O.OAOb 0 .5 2 A + 0 .0 3 6 c 0.679 + O .020a Adenohypophysis 0 . 1 5 1 + 0.008b 0 .A6 8 + 0.026a 0.170 + 0.007b Neurohypophysis 0 . 1 7 5 + 0.009b 0 .3 2 6 .+ O.0A3a'' 0.202 + 0.00 3b GRAND MEAN. ' 0 . 2 7 6 + 0.075 0 . 3 0 2 + 0.07A x -— rrr356 + 0.097 SAChE ACTIVITY BRAIN REGIONS 2A hours / 48 hours CONTROL Pons 9.165 + 0.359ab 1 1 .6 A7 + 0.551a 6.571 + 0.493b •Cerebellum 31.578 + A.A55a 1 1 . 9 7 1 + 0.197b 14.240 + 1.146b Amygdala 1 6 .2 A7 + 0.887c 3 0 . 2 5 2 + 0.825b 40.766 + 1.535a Hippocampus 15.732 + 0.lA2b 2 2 .2 7 A —+ 4.500a 13.354 + 0.426ct Hypothalamus Ad. 8 6 1 + 1.753a 2 9 .1 1 A + 3.725b 25.980 + 1.067b Cerebral Cortex A.A86 + 0.12b 9 .0. 80 + 0.195a 7.413 + 0.207ab Mid Brain 21.699 * 1.390b 3 1 . 6 0 1 + 8 .2 8 2 a 16.7.71 + 0.532c Medulla Oblongata 6.193 + 0.078b 10.2A2 + 0.8l3a 7.174 + 0.45 lab Adenohypophysis 3.A23 + 0.3l6ab 0.821 + 0.010b 5.119 + 0.190a Neurohypophysis A.A3 2 + 0.386a 3.2A2 + 1.012a 4.642 + 0 .1 6 3 a GRAND MEAN 15.382 + 6 . 3 6 2 ~ 1 6 .0 2 A + 5.69A 14.203 + 5.723 Values in the game horizontal line differently superscripted differ significantly (F<0 .05). •AChE activity In N mole/g/min ••Total protein in g/lOOnl ••SAChE activity In H mole/g proteiri/mln UNIVERSITY OF IBADAN LIBRARY - 2 3 7 - In the hypophyses however, water-deprivation for 48 hours signifi­ cantly raised protein level (P<0.05) while the other two groups were unaffected (P>0.05). Water deprivation generally elevated SAChE activities in the various brain regions to varying extents (P<0.05) except in the neurohypophysis where no significant changes were observed (P>0.Q5), and in the amygdala where the control group recorded the highest SAChE activity levels. The 24-hour water deprivation period was more potent in elevating SAChE activity above normal than the 48-hour water deprivation period in the cerebellum, hypothalamus, and adenohypophysis (PC0.05), while the reverse was observed in the pons, hippocampus, medulla oblongata and the midbrain. No particularly consistent trend was observed in the cerebral cortex. 7.5.2 EFFECT OF ACUTE WATER DEPRIVATION ON THE M INERAL PROFILE IN THE PORCINE BRAIN AND HYPOPHYSES I The results are summarized in Tables 7.1.9. and 7.2.0. CALCIUM While water deprivation for 24-hours did not significantly affect the cerebellum and cerebral cortex (P>0.05), the 48-hour water deprivation significatly lowered calcium levels below normal (P<0.05). In the amygdala, midbrain and hippocampus, calcium levels were highest in the 24-hours water-deprived group, medium in the controls and least in the 48-hours water-deprived group. UNIVERSITY OF IBADAN LIBRARY 238 TABLE 7.1.9 EFFECT OF ACUTE WATER DEPRIVATION ON THE CALCIUM, MAGNESIUM AND ZINC LEVELS IN THE PORCINE BRAIN AND HYPOPHYSES (Mean ♦ S.E.M). CALCIUM ANIMAL GROUPS BRAIN REGIONS . WATER DEPRIVED CONTROL 29 hours 93 hours Pons 1 . 3 0 1 + 0.022b 1.199 + o.oo3c 1.969 + 0.076a Cerebellum 1.097 + 0.019a 0.801 + 0.027b 1.011 + 0 .0 1 8 a Amygdala• 1.267 + 0.098a 0.809 + 0.033c 1.110 + 0.031b Hippocampus 1.991 + 0.099a 0.752 + 0.033c 0.877 + 0.035b Hypothalamus 1.985 + 0.391a 1.295 + 0.097b 1.139 + 0.070b Cerebral Cortex 1.158 + 0.092a 0.899 + 0.035b 1.171 + 0.085a Mid Brain • ~ 1.378 + 0.119a 0.921 + 0.081c 1.190 + 0.063b Medulla Oblongata 1.980 + 0.088a 0.761 + 0 .0 3 0 b 0.867 + 0.0 9 0b Adenohypophysis 1.797 + 0.017a ‘ 1.296 + 0 .0 3 0 b 1.297 + 0.051b Neurohypophysis 1.850 + 0.098a 1.979 + 0.010b lr.306 + 0.072c GRAND MEAN 1.975 4- 0.155 1.010 + 0.133 1.138 + 0.092 MAGNESIUM BRAIN REGIONS 29 hours 98 hours CONTROL Pons 1.252 + 0.130a O . 7 8 9 + 0.0l9c 0.969 + 0.051b — Cerebellum 0.992 + 0.095a 0.796 + 0 .0 7 8 b 1.032 + 0.071AT Amygdala 1.015 + 0 .0 2 8 a 0.917 + 0.082a 0.579 + 0.100b Hippocampus 1^059 + 0.135a 0.752 + 0 .0 5 6 b 0.697 + 0i096b Hypothalamus 1.113 + 0.068a 0.797 + 0.032b 0.800 + 0 .06'9b Cerebral Cortex 1.255 ' + 0.023a • 0.529 + 0.071c 0 . 7 6 1 + 0.053b Mid Brain 1.255 + 0.09la • 0.866 + 0.09lb 0.750 + 0.096b Medulla Oblongata 1.989 + 0.089a 0.750 + 0.096b 0.550 + 0 .0 6 8 c Adenohypophysis 1.322 + 0.032a 0 . 8 6 2 + 0.012b 0.617 + 0.029c Neurohypophysis 1.259 + 0.013a 1.109 + 0.085ab 0.961 '+ 0.055b GRAND MEAN 1.200 + 0.077 0 . 8 1 1 + 0.073 0.771 + otSSs'-’ ZINC 29 hours 98 hours CONTROL BRAIN REGIONS Pons 0.377 + 0.015a 0.085 + 0.007b 0.126 + 0.019b Cerebellum 0.295 + 0.009a 0.106 + 0.009b 0.115 + 0.005b Amygdala 0.189 + 0.007a 0.103 + 0.002b 0.167 + 0.009a Hippocampus 0.927 + 0.010a 0.106 + 0.006b a. 0.00 8b Hypothalamus 0.999 + 0.012a 0 . 1 8 1 + 0.019c 0 .3 3 ^ 0.028b Cerebral Cortex 0.899 + 0.009a 0.109 + 0.009b 0.129 + 0 .0 0 6 b Mid Brain 0.896 + 0 .0 2 8 a 0.100 + 0.002b 3.108 + 0.007b Medulla Oblongata 0.967 + 0.031a 0.077 + 0.007c 3.139 + 0.005b Adenohypophysis 0.995 + 0.015b 0.526 + 0.035a 3.038 + 0 .0 0 6 c Neurohypophysis 0.592 + 0.089b 0.673 + 0.013a 3.932 + 0.008c GRAND MEAN 0.993 + 0.119 0 . 2 0 6 + 0.106 3.178 + 0.057 Values In the 3ame horizontal line differently superscripted differ significantly (P<0.05). •Values are in parts per million (ppm) UNIVERSITY OF IBADAN LIBRARY 23 9 TABLE 7.2.0 EFFECT OF ACUTE WATER DEPRIVATION ON THE POTASSIUM, SODIUM AND COFFER LEVELS IN THE PORCINE BRAIN AND HYPOPHYSES (Mean + S.E..M.) POTASSIUM - ANIMAL GROUPS BRAIN REGIONS WATER DEPRIVED CONTROL 24 hours 43 hours Pons lh. 3 0 0 + 1.328a 15.775 + 1.212a 15.625 + 0.796a Cerebellum 17.000 + 0.115b 15.000 + 0.393b 21.225 + 1.314a Amygdala 17.012 + 3.165a 17.625 + 2.048a 11.550 + 0.368b Hippocampus 13.112 + 0.517a 15.125 + 1 .1 6 7 b 16.775 + 0 .6 3 9 ab Hypothalamus 15.912 + 1.444a 14.775 + 1.24la 11.500 + 0.408b Cerebral Cortex 19.750 + 0 .8 8 2 a 14.200 + 0.536b 14.425 + 0.285b Mid Brain 12.300 + 0.892b 13.750 + 1.453b 17.125 + 1.083a Medulla Oblongata 1H.450 + 1.097a 12.450 + 1 .3 1 2 a 14.275 + 0.714a Adenohypophysis 25.175 + 0.788a 11.750 + 1.302b 1 2 . 3 0 2 + 1.045b Neurohypophysis 26.750 + 1.202a 13.375 + 0.607b 13.212 + 0.635b GRAND MEAN 18.076 + 2.334 14.432 + 0.831 - 14.701 + 1.575 SODIUM BRAIN REGIONS 24 hours 48 hours CONTROL Pons 292.500 + 15.595a 2 9 5 . 0 0 0 + 11.547a 273.750 + 6,129a Cerebellum 290.500 + 4.333a 2 9 3 . 7 5 0 + 9 .2 8 0 a 301.250 + 5.154a Amygdala '302.500 + 14.530a 2 9 2 . 5 0 0 + 14.530a 305.500, + 11.449a Hippocampus 297.750 + 21.657a 2 6 7 . 5 0 0 + 7 .6 3 8 b 291.750 + J.929ab Hypothalamus 308.250 + llt667a 3 0 1 . 7 5 0 + 14.450a 305.750 + 6.169a Cerebral Cortex 314.750 + 10 .,171a 3 0 8 . 0 0 0 + 8.718a 3 0 8 . 0 0 0 + 7 .6 8 1 a Mid Brain 277.250 + 4.702b 3 0 0 . 5 0 0 + l4.622ab 306.250 + 11.6l4a Medulla Oblongata 254.500 + 9.939b 2 8 0 . 2 5 0 + 3.480ab 30C.250 + 3.391a Adenohypophysis 281.500 + 21.940a 2 7 3 . 7 5 0 + 7.638a 296.972 + 2 .9 2 6 a Neurohypophysis' 285.750 + 11.591a 2 9 2 . 5 0 0 + 8.570a 296.500- ±—5^-694a GRAND MEAN 290.525 + 8.626 2 9 0 . 5 5 0 + 6.425 299.197 + 4.285 COPPER BRAIN REGIONS 24 hours 48 hours CONTROL Pons 0.149 + 0.008a 0.086 + 0.012b 0.167 + 0 .0 0 3 a Cerebellum 0.160 + 0 .0 0 6 a 0.113 + 0.008b 0.158 + 0.002a Amygdala 0.146 + 0.002a 0,075 + 0.005b - -0.129 + 0 .0 0 6 a Hippocampus 0 . 1 6 2 + 0 .0 0 3 a 0.070 + 0.005b 0.'l59 + 0.005a Hypothalamus 0.159 + 0.007a 0.058 + 0.002b 0.157 + 0.009a Cerebral Cortex 0.115 + 0.004b 0 . 1 0 6 + 0.010b 0.175 + 0.008a Mid Brain 0.114 + 0.006a 0.078 + 0.003b 0 . 1 2 6 + 0.009a Medulla Oblongata 0."220 + 0.013a 0 . 0 6 0 + 0.004c 0.150 + 0.017b Adenohypophysis 0.322 + 0 .0 2 3 a 0 . 1 8 1 + 0.011c 0.147 + 0.007b Neurohypophysis 0 . 2 8 6 + 0.011a 0.199 + 0.0C2b 0 . 1 6 6 + 0.004c GRAND MEAN 0.183 + 0.035 0.103 + 0.025 0.156 + 0.009 Values in the 3ame horizontal line differently suoerscripted differ significantly (P<0.05). •Values ar.e in parts per million (ppm).. UNIVERSITY OF IBADAN LIBRARY - 2 4 0 - In the hypothalamus, medulla oblongataland adenohypophysis, the 48- hours lwater deprived gorup and the controls were similar and inferior to the 24-hours water-deprived group (PC0.05). In the ons, calcium was highest in the controls, medium and least in the 24-hours and 48-hours water-deprived groups respectively (P<0.05), while in the neurohypophysis, calcium was highest in the 24-hours water- de£>rived group, medium in the 48-nours water-deprived group and least in the controls. MAGNESIUM The forty-eight hours water deprived and control groups were similar and inferior to the twenty-four hour water deprived group in the hippocampus, hypothalamus and midbrain (P<0.05). In the medulla oblongata and adenohypophysis, magnesium was highest in the 24-hours water deprived group, medium and least in the 48 hours water-deprived and control groups respectively (P<0.05). Water deprivation also elevated magnesium level in the amygdala (P<0.05) while in the cerebellum the 48-hours water deprived group was inferior to the other two groups, which were similar. In the pons and cerebral cortex, water deprivation for 24-hours significantly elevated magnesium level above normal (P<0.05) while the 48-hours water deprived group had significantly depressed magnesium level. The 24 hours water deprived group was also superior to the control in the neurohypophysis (P<0.05) UNIVERSITY OF IBADAN LIBRARY - 2 4 1 - ZINC Zinc levels in the 48-hours water-deprived group and the control group were similar and inferior to the 24-hours water deprived group in the pons, cerebellum, cerebral cortex, midbrain and hippocampus (PC0.05). In the hypothalamus medulla oblongata however, water deprivation for 24-hours significantly elevated zinc levels while the 48-hours water-deprivation period depressed it (P<0.05). Water deprivation for 48 hours also significantly depressed zinc level in the amygdala (P<0.05). In the hypophyses, zinc was highest in the 48-hours water-deprived group, medium and least in the 24-hours water-deprived and the control groups respectively (P<0.0 5). POTASSIUM Water deprivation significantly elevated potassium levels in the amygdala and hypothalamus (P<0.05) while the reverse ocurred in the cerebellum and midbrain. In the cerebral cortex, hippocampus and the hypophyses, the 24-hour deprivation period caused a significant increase in potassium level over the other two treatments while no significant effects were observed in the pons and medulla oblongata (P>0.05). SODIUM In the midbrain and medulla oblongata, water-deprivation for the 24-hour period caused a significant decline (?<0.05), in sodium levels while the other group was unaffected (P>0.05). In the hippocampus, the decline in sodium level was noticed only in the group water-deprived for 48 hours. T h e o t h e r r e g i o n s w e r e n o t s i g n i f i c a n t l y a f f e c t e d ( P > 0.05). UNIVERSITY OF IBADAN LIBRARY - 2 4 2 - COPPER In the pons, cerebellum, amygdala, hippocampus, hypothalamus and midbrain, copper levels were significantly depresed by the 48-hour water- deprivation treatment (P<0.05) whereas the 24-hour water-deprivation treatment had no significant effect (P>0.05). In the medulla oblongata and adenohypophysis, water-deprivation for 24 hours resulted in an increase in coper levels (P<0.05), while the 48 hours water deprivation treatment caused a significant decline (P<0.05). Water deprivation aso resulted in significant lowering of copper levels (P<0.05) in the cerebral cortex and in the neurohypophysis, copper was highest in the 24-hour water-deprived group, medium and least in the 48-hour water-deprived and control groups respectively (PC0.05). DISCUSSION 7.6.1 Heat Stress Heat stress is perhaps the most important environmental factor affecting livestock production in the tropics. Heat stress like exogenous hormonal treatment disturbs endocrine dynamics and elicits very active responses in the animal. The elevation of AChE activity in the pons, cerebellum, amygdala, hippocampus, midbrain and medulla oblongata of heat stressed animals is indicative of increased activity of the neural cells which may induce the marked neuromuscular activity and muscular movements characteristic of heat-stressed animals. It should be mentioned that during heat stress, the animals were very restless, agitated, very aggressive and many of them even attempted to jump the fence restraining them. Such gross muscular activity is indicative of nervous reaction to heat stress and is in line with UNIVERSITY OF IBADAN LIBRARY •2 4 3 - reports of early researchers that heat stress induces convulsions, coma and affects the floor of the third ventricle and pons. The increase in AChE activity may also be linked with the increase in blood volume, respiratory and metabolic activities accompanying hyperthermy. As a rise in AChE activity directly reflects increased secretion of ACh at synaptic junctions, the rise in ACh would need to be quickly removed to prevent the animal from being in a state of continous excitation and convulsive muscular activity which would utlimately lead to exhaustion and collapse. The rapid removal of ACh by AChE also has a heat regulatory action becuase ACh has been known to cause excitation of the heat production pathway (Findlay and Thompson, 1968). Bedrak et al«, (1980) also observed an increase in the activity of enzymes associated with steroid metabolism (in vitro) with increasing temperature of incubation and a concomitant decline in serum testosterone in heat acclimatized rats. Thus the increased AChE activity not withstanding, heat stress also brings about increased androgen catabolism by the liver and kidney (Bedrak et al 1980) and impaired integrity of sertoli cells. This may explain the lowered AChE activity observed in the hypothalamus of heat stressed pigs. This result is particularly striking in view of the multiple role of the hypothalamus in both androgen production and heat regulation. The rise in AChE acitivity in the hippocampus concomitant with a decrease in the hypothalamus also lends support to the inhibitory role of the hippocampus on the hypothalamic steroid-metabolic dynamics. The hippocampus is also known to inhibit the pituitary-adrenocortical system which responds directly to hypothalamic stimulation (Kawakarai et al̂ , 1968). UNIVERSITY OF IBADAN LIBRARY - 2 4 4 - Heat stress directly lowers thyroxine and TSH release which may also indirectly lower the release of Thyrotropin releasing hormone (TRH) by the hypothalamus of heat stressed animals and may therefore explain the sharp drop in testicular and reproductive capacity of boars exposed to heat stress• The relative tolerance of the cerebral cortex to heat stress implies that learning and memory functions are not impaired by heat stress and lends support to the view that the cortex seems to possess special metabolic mechanisms protecting it from abuse by stressors. From the second experiment, it appears that the 3-day heat stress period did not evoke as drastic a response as the 5 and 6-day heat stress did. This may be due to the fact that the animals were still able to recover fairly quickly after the heat stress period. The inhibitory effect of thermal stress on Basal metabolic rate (BMR) and a lowered concentration of thyroxine and thyrotropin (Tal and Sulman, 1973) coupled with decreased serum testosterone level suggests an impairment of adenohypophyseal functions. This is evidenced by the observed decline in AChE activity and protein concentrations of the hypophyses. The decreased protein level is reflective of the increased steroid inactivation induced by thermal stress. The vasodilatory effect of thermal stress also implies decreased secretion of vasopressin by the neurohypophysis. The depression of protein concentraions of the pons, cerebellum, hippocampus, hypothalamus, midbrain and medulla oblongata of heat stressed animals confirms reports that the increase inblood volume arising from vasodilation leads to a drop in total protein concentration as a result of haemodilution (Yanga, 1972). In addition, the lowered basal metabolic rate UNIVERSITY OF IBADAN LIBRARY - 2 4 5 - and thyroid gland functions are reflective of decreased protein synthesis. The cortex, through a not very clear mechanism was able to maintain normal protein synthesis which may still be resolved by its role in higher mental functions. It is therefore clear that heat stress when prolonged would eventually lead to brain injury. Although many of the exotic breeds of pigs reared in the tropics are heat-acclimatized, studies by Bedrak et al (1980), Egbunike and Dede (1980) reveal that such animals, when exposed to thermal stress even of short duration evoked changes in enzyme metabolism and reproductive behaviour. The sharp rise in the SAChE activity of the brain regions of heat stressed boars over the control also simulates the rise observed in AChE activity and attracts the same reasons advanced for the increase. 7.6.2 MINERAL PROFILE IN THE BRAIN AND HYPOPHYSES The rise in calcium concentration of several brain regions of heat stressed boars suggest increased calcium absorption at the neural centres in response to increased ACh and AChE activity. This supports the report that calcium ions facilitate the release of ACh at neuromuscular junction and neurotransmitter release. The same trend was observed for potassium and indicates increased potassium retention in response to the increase in membrane excitability. The increase in membrane excitability would depolarise the membranes and trigger of the sodium-pump mechanisms resulting in more potassium ions in the extracellular fluid and sodium ions replacing the potassium ions. Although the concentrations of the minerals were not determined at the cellular level, it is presumed that this action may in part explain the decreased sodium concentrations induced by heat UNIVERSITY OF IBADAN LIBRARY - 2 4 6 - stress. Zinc levels were also similarly reduced by heat stress especially after 6 days in some of the regions with the amygdala and hippocampus being exceptions. This reduction in zinc levels may have a link with the concomitant decline of AChE activity in the hypothalamus and the presumed UNIVERSITY OF IBADAN LIBRARY - 2 4 6 - stress. Zinc levels were also similarly reduced by heat stress especially after 6 days in some of the regions with the amygdala and hippocampus being exceptions. This reduction in zinc levels may have a link with the concomitant decline of AChE activity in the hypothalamus and the presumed impairement of reproductive function. The similarities of copper content of the brain regions of both heat stressed and control boars may result from the fact that the increased blood volume by vasodilation induced by heat stress is accompanied by fluid loss and the resultant decrease in red blood cells is offset by the haemoconcentration of the blood. The lack of a consistent trend in magnesium concentrations in the brain regions of both heat stressed and control boars relate to earlier observation in preceeding chapters that magnesium seems to be antagonistic to calcium and its mode of action is not very clear. In addition, it appears as if the mineral does not play a very clear role in adaptation to environmental and hormonal stress. While heat stress did not affect copper level in the hypophyses, calcium and magnesium were elevated which is indicative of a shift in the electrolyte balance of the gland. The some argument can be advanced for the lowering of potassium and sodium levels on the adenohypophysis of heat stressed animals. The mechanism by which this shift is brought about is not yet determied. 7.6.3 WATER DEPRIVATION The importance of the brain in osmotic balance of body fluids and regulation of water intake has been established for quite some time but reports have been inconsistent. UNIVERSITY OF IBADAN LIBRARY - 2 4 7 - Grossraan (1960, 1962a) discovered that the cholinergic system of the hypothalamus enhances water intake in both satiated and water-deprived rats while Leibowitz (1970) reported that blocking of the cholinergic system in the rat hypothalamus increased the thirst sensation and decreased hunger. The present study showed a decrease in AChE activity of water deprived pigs in the pons, amygdala, hippocampus, hypothalamus and medulla oblongata. The study further indicated that water deprivation for 24 hours caused a higher decrease in the affected regions than water deprivation for 48 hours. Contrariwise, AChE activity was elevated in the midbrain of the same pigs over the control. The decrease in AChE activity of some regions particularly the amygdala, hippocampus and hypothalamus is suggestive of the fact that these regions are involved in the thirst sensation. The classical experiments of Verney (1958) revealed that osmoreceptors are present in the hypothalamus and stimulation of the hypothalamus stimulates drinking while its destruction abolishes the thirst sensation. The thirst mechanism is presumably due to a stimulation of nervous elements specifically sensitive to an elevated sodium chloride concentration of the internal environment. Thus a lowering of AChE activity in the hypothalamus of water-deprived boars may be a direct response to acute sensation of thirst. This response may bring about a suppression of the thirst mechanism. The rise in AChE activities in the pons (after 48 hours of water deprivation), midbrain and the restoration of activity in the medulla oblongata after the 48-hour water deprivation period is indicative of an UNIVERSITY OF IBADAN LIBRARY - 248- attempt to reduce the stress of thirst sensation through a concomitant decrease of the hunger sensation. As the tropical environment is essentially stressful, a combination of thermal and water-deprivation stress have very serious concsequences for the animal. Stimulation of the midbrain has been known to suppress the hunger sensation (Adamek, 1976) and the pons co-ordinates reflexes concerned with swallowing, vomitting and cardiovascular controls such as blood pressure and salivation. It is thus possible that the increase in AChE activity in the pons may enhance the water-conservation mechanism of the brain. A possible role may also be a regulation and perhaps stimulation of increased synthesis of ADH by the neurohyphysis in a bid to concerve water loss. The slightly more pronounced effects of water deprivation for 24 hours compared to 48 hours indicates that the reaction of the animal to water deprivation during the first 24 hours involves more physiologic and metabolic activities than in the second day. It is also possible that during the second day of water-deprivation, the animal had achieved its optimal water conservation mechanism or status. This suggestion agrees with observation by earlier workers that the onset of thirst creates a dry sensation of the mouth and craving for fluid rapidly becomes compelling but as time goes on, the dryness of the mouth increases, production of saliva decreases, finally ceases and food intake stopped. The cessation of good intake would therefore minimise metabolic activities and put the animal in another physiologic status. Although the physiological interactions elicited by water- deprivation have not been clearly unravelled, the hypothalamic-hypophyseal adrenocortical axis is presumed to play a major role. This is borne out by UNIVERSITY OF IBADAN LIBRARY - 249- the fact that workers have been able to produce experimental diabetes insipidus and inhibition of ACTH release by destroying the hypothalamic- hypophyseal connections (de Wied et al, 1958). It is therefore presumed that water-starvation causes increased release of the vasopressor ADH which is also thought to facilitate ACTH release by the adenohypophysis. The stimulation of ACTH exerts an inhibitory effect on TSH release (Fortier, 1973), thereby diminishing the activity of the thyroid hormone. The present study confirms these views. The failure of water deprivation to influence AChE activity of the hypophyses eliminates the possible impairment of hypophyseal function by the treatment. If further enhances the view that both the adenohypophysis and the neurohypophysis possess mechanisms of ensuring osmoneutrality in the animal. The decrease in the protein concentration of the hypothalamus, hippocampus, medulla oblongata, midbrain and pons of the water-deprived animals implies decreased protein synthesis presumably due to a decrease in feed intake and the utilization of ingested amino acids. It may also be a result of a decrease in the metabolic activity of the body. The increased protein levels in the hypophyses of the water- deprived animals after 48 hours suggests increase in the turn-over rate of enzymatic activities responsible for the increased synthesis of ACTH and ADH. This increase in protein turn-over rate of the adenohypophyses may contribute to the decline in SAChE activity observed in the adenohypophyses of water-deprived boars. The result also indicates that boars water- deprived for 48 hours had adjusted to the effects of thirst sensation more than those deprived of water for 24 hours. UNIVERSITY OF IBADAN LIBRARY - 2 5 0 In the same vein, the increase in the SAChE activities of the brain regions of water-deprived boars is reflective of the decreased total protein and AChE activity observed in these regions. This presumably leads to higher SAChE acttivities in an attempt to maintain the enzyme turnover rate. Of particular interest is the relative stability ofthe cerebral cortex to water-deprivation which is reflected in its role in learning and memory activities which are not immediately impaired by environmental stress. 7.6.4 MINERAL PROFILE IN THE BRAIN AND HYPOPHYSES The depression of calcium levels in the brain regions of water- deprived boars suggests a reduction of neuromuscular transmission and is in line with the decline in AChE activity of the brain regions concerned with osmoregulation. It also reflects the lack of efficient incorporation of amino acids into proteins evidenced by lowered protein content of the brain regions of these animals. The elevation of magnesium in the medulla oblongata, midbrain, hypothalamus, hippocampus and amygdala of water-deprived boars may be a compensatory attempt for the depletion of calcium in the affected regions. In addition, the magnesium may be functioning to prevent electrolyte imbalance that may result in more interference with the metabolic activities of the animals. It is also well known that magnesium is a co­ enzyme of many metabolic pathways, thus the mineral may be helping in sustaining the vital physiologic processes of the water-deprived boars. The decline in magnesium content of the pons, cerebellum and cerebral cortex may be an evidence of differential metabolic rates of differnt brain UNIVERSITY OF IBADAN LIBRARY - 2 5 1 - regions whereby the highly active areas during water-deprivation retain magnesium more than the less active regions. Although no substantial changes were observed in the potassium content of the hippocampus and medulla oblongata of water-deprived boars, the rise observed in the concentrations of the mineral in the cerebral cortex, amygdala and hypothalamus of the same group of animals coupled with a decline in potassium content of the cerebellum and midbrain also reflects differences in the metbolic rates of the mineral in the brain regions during water-deprivation. Thus the brain areas that exhibited an increase in potassium concentrations over the controls may be more involved in osmoregulation and may be more osmoreceptive than other groups. The failure of water-deprivation to significantly influence sodium levels in the brain regions save for a decline in the midbrain and medulla oblongata suggests that the brain possessed some mechnanism for maintaining osmoneutrality and homeostasis. The elevation of zinc in several brain regions may enhance the stimulation of enzyme-metabolic pathways it activates to compensate for the decline in the protein turnover rate of the brain regions. The elevation of hypophyseal levels of magnesium and zinc by water deprivation may be due to the increased secretion of ACTH and the concomitant release of the Adrenal cortex hormones namely the mineralocorticoids (e.g. aldosterone) and the gluccerticcids. The release of the adrenocorticoids would therefore enhance the retention of the minerals in a bid to maintain osmotic balance both intracellularly and extra-cellularly UNIVERSITY OF IBADAN LIBRARY - 2 5 2 - An interesting trend is displayed by copper, calcium, potassium and magnesium where water-deprivation for 24 hours resulted in a rise in the levels of the minerals in the hypophyses followed by a reduction after 48 hours. A possible explanation for this is that the surge of ACTH released in direct response to the onset of thirst is gradually diminished as the animal adapts to the new condition. This further confirms the hypothesis that the animals that were water-deprived for 48 hours showed higher adaptability to water-deprivation than those water-deprived for 24 hours. As suggested earlier, the manifestations of such adaptation would include decreased urine production, decreased salivation, decreased or cessation of feed intake and decreased locomotory activities. 7.6.5 SUMMARY These results have conclusively shown that the pig suffers from serious brain impairment and possible interference with endocrine dynamics during thermal stress. They also indicate that the present design of the pig pens in use at the university farm protects the animals from the thermal stress of the humid tropical climate and provide optimum temperature and relative humidity conducive to normal development of the animal. These studies further reveal that water deprivation even for a short period like 24 hours results impairs nerous transmission in the brain with possible interference with normal brain activity. They also show the relative ability of the hypophyses to withstand water-deprivation stress presumably through feed-back mechanisms UNIVERSITY OF IBADAN LIBRARY C H A P T E R E I G H T C O N C L U S I O N S UNIVERSITY OF IBADAN LIBRARY - 2 5 4 - CHAPTER EIGHT CONCLUSIONS 8.1.0 The studies reported herein have not only provided adequate details about the role of sex steroids and enzymes in the maintenance of the physiological integrity of the animal particularly at the central nervous system level but have also elucidated to a large extent, the importance of a physiological balance between the circulating hormones inside the animal and the external environmental factors. The studies also highlight the critical role of acetyl- cholinestrease enzyme (AChE) activity as a major link between the endocrine and nervous system. The ontogenic studies indicate that AChE activity can be used as an index of development in the fetal brain and correlated positively with gestation length. They further suggest that brain development is not necessarily accompanied by a rise in total protein content of the brain. The increase in calcium content of the fetal brain with gestation length is directly related to its vital requirement for bone development while the decline in copper and zinc levels may be responsible for the piglet anaemia normally associated with piglets shortly after birth. Postnatally, the studi.es also indicate that the brain regions could be classified rnto 'high', 'medium' and 'low' activity regions depending on the level of AChE activity obtained in such regions. Thus the 'high activity' areas are the amygdala, midbrain and medulla oblongata which recorded the highest AChE activities. This may be related to the fact that these are areas that are involved in highly active, quick and short-termed UNIVERSITY OF IBADAN LIBRARY - 2 5 5 - responses such as aggressive sexual behaviour and predatory actions as described by Kang et al., ( 1970). These are actions characterized by high nervous stimulation and manifested by considerable muscular activities. The hypothalamus, hippocampus and pons which are concerned with basic reflexes such as respirattion, swallowing and normal hormonal actions and co-ordinated responses thus naturally fall into the 'medium activity' class whereas tne constant low activity observed in the cerebral context and cerebellum reflects on the fact that these regions control higher and lower intellectual functions which are acquired by the body over time and retained for a long period of time. As animals do not posses the ability to regnerate neurons after birth, it is not surprising therefore that a decline in AChE activities after birth with age was observed in the pig brain. Thss further confirms that the pig brain had reached its peak of neuronal development by the time of birth and very little changes in quality of the cellular constituents of the neurons take place. The higher activities observed in some brain regions of the male pigs over the female pigs could be responsible for the more aggressive nature of the male animal over the female. The increase in total protein concentrations of the pig brain with age relates to the growth requirements of the young animal which include increased rate of RNA Synthesis, protein turnover and tissue deposition. It is also noteworthy that positive correlations were observed in the calcium, magnesium, potassium, sodium, copper and zinc contents of the pons, cerebellum, medulla oblongata and midbrain which are areas involved in growth and protein synthesis. The results of the experiments on castration also tend to confirm the notion that castration abolishes sexual behavior. The studies also UNIVERSITY OF IBADAN LIBRARY - 2 5 6 - reveal that the age at castration has an influence on the effects of castration in the animal. Thus post-pubertally castrated male boars appeared to tolerate the responses due to androgen withdrawal more than pre-pubertal castrates. The studies further show that trestosterane enhances AChE activity in the central nervous system of the boars while castration interferes with protein synthesis. The decline in che concentrations of some cations such as calcium, sodium, potassium, copper and zinc in castrated boars is indicative of a possible interference with cation absorption from the diet. This closely agrees with the observation of Kunerth and Pitman (1939) who related calcium ions retention to protein synthesis and according to Fullis (1958), zinc deficiency also brings about testicular atrophy associated with castration. The striking ability of the cerebral cortex to maintain normal levels of AChE activity in castrated pigs supports the view that the cortex is involved in high mental activities which are not immediately impaired by short-term hormonal inbalance. It is also probable that the failure of castration to significantly influence AChE activity in the hypophyses is presumably due to the feed-back response of the hypophyses to a lowered serum testosterone level brought about by castration. On the effects of ovariectomy on brain physiology, it is safe to conclude that both estrogens and progesterones are necessary for normal functioning of the nervous .system and the nervous system responds to estrogens much more than it does to progresterone UNIVERSITY OF IBADAN LIBRARY - 2 5 7 - On the role of testosterone as an antl-estrogen in intact gilts, the results indicate that testosterone administration to intact gilts interfers with AChE activity and mineral profile in the brain and has no distinct advantages at the moment. It is therefore clear that androgens cannot serve as primers in the female animal body. Apart from endocrine factors, environmental factors also play a part in the physiological well being of the animal. Studies carried out on thermal stress and water deprivation have conclusively shown that the pig suffers from endocrine malfunction during thermal stress. The vulner­ ability of the pig to high temperatures indicate that the pig cannot be successfully reared on an extensive system of management. However, the results show that the present design of the pig pens at the University farm adequately protects the animals from thermal stress during the day and provides tolerable temperature and relative humidity conducive to the normal functioning of the animal. The study further indicate that water deprivation even for a short period of 24 hours impairs nervous transmission. It also shows that the pig responds immediately to short-term water deprivation of 24 hours and adjusts progressively as the period of water deprivation increases which is an evidence of adaptability. As acetylcholinesterase is known to influence adaptive behaviour, the ability of the animal to regain fairly normal AChE activity levels in some brain regions after 48 hours of water deprivation may partly explain why water deprivation for 48 hours elicited less reponse than water deprivation for 24 hours. The study further confirms the cerebral cortex as an area tnat tolerates to a large extent, short-term inbalances in endocrine and environmental factors thereby allowing it to effectively maintain its vital role in learning and high mental activities. UNIVERSITY OF IBADAN LIBRARY - 2 5 8 - The hypophyses too are presumably able to tolerate the effects of stressors through feed-back mechanisms. In summary, for optimum productivity of the animal, the basic requirements of energy, protein, vitamins, minerals and managemental procedures should be strictly adhered to. 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VALUE P MEAN SQUARES f 299 _ r t X 1 (ICD f t l ct x H fD T3 “ O" P 0) c5 <* o m3 s ^ 3 * £03 o > fDct" ft *• ,-r - I t ' T— .**«.-> VU£>) HVJ1 xr M HKj XrVO ${ • M . 1 ro VO VJI G . o o o o o o o o o o o o o o o o o o o o o o u i o o ^ h o o n r y o v p* H U 1 o H H H o 8 VO O Mp u- o^_&__8_ co vo On rv) - __8_ _^__ Xr M CTvH OOU) oo co ro o ro ctvm xr ro p » vo o \vji co £ ft & & X X X X X X° . ° . S P* M P* P» M P*®i CO u> CO CO °,cic?‘?c? r xjrr ccoo HooV Oov ro xr xr ro H W -J X Xr OO <^3 CO M CD XT * * * 3 * 3 * * * co 3 ? * •cd cn * CD o o o o o o o O O O O O O O o o o o o o o o o o o o o o • • • • • • • O P O O fM O t T ' O O O Q O U U Q S pH W C7NC0 O go l\> O M O M O —J WOOOP OCO OWV XJrI OXrV fO\J —O3C O* VOJNI VJ1 OO Ol\JVO HVJ»X CT\̂ VO-O -O>oI 3o —§4 -§4 8 §a\cgo §—3 X X X * X X p- 8 P* P* P » Q M° l C.O co LO S S S iS ° lCO co XT M CO ot u i 00 h-* VJ1 M COCO VJI o M O ->J P» 10-4 H —4 x r CO— 1 rroo -v-j3i _o_OXM CD o o • fO VJI VO vo o OrvV_VoOO > vo p* ror O VJI k ) --4 ro Xr ON x r OCO >->3 a\ ro -> i v o -o co 1 * * Dc * 3• 3 » 3 * 3 ■a * * * • * • 3 ? * * « * CO CD * CD CD U * • CD § o o o o o o o o o o o o o o o o o o o o o 0 0 3 0 - ^ Vjf K ) VJI Oo rXoT oO oO xxrr or\j -rog ON p* — 4 rV) Xr Q Xr IV3 rv> o co oo-4 to CO O O P* OO O CO OOfUHVOW w o CO O Xr VJI VJI c o a\ VJI vo COOVO VJI co X 'X x X X X X X X X X X M & M M M P» P* M Q P* p. 1 8 ° . 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X cf ?J CO CO CO CO Xr 8* , f o \ M -Mg vPJ VJIM o -a o o M O LOOD HVO P ----— CTV a\H vo vo o VO OtH o * VcOo Xr XrVO xr CD XrVO P* O -̂ 1 xr s OO OOTO M OO 3 ? * 3 3 3 3 3 3 3 3 >K 3 CO CO * * c•o co a CD 01 a co ca Dc CD o o o o o o o O O O O O O O Ho COxiO oO rWu uXrj Kh) Oo O O O O O P» O o\ oo ro ro ro ov ro W O V H H W ^ O J o M vji vo vo co co * QP.*I CO i w CO ON M• O• •O •OO o o coco ovco CO CO -4 --J x r xr p VJI VO-̂ J -Cr ro ro VJI CT\ 3 3 * * ♦ 3 * * ? ? co a CO bi • a . o o o o o o o o o o o o o o 8 c o c o b ro o o vju i o v o w o a O 8vni oroo rxor oro po wav - 4 ro x r H P Xr p w VJI ro x r X X X X X X X x o o P* M M PJ M I I o o o o o CO CO X rcov jl CO Xr ro vo VJI H O \ P* VJI VO O 5> co oo ro O O 1^ -3 Xr CO "0 -a -4 O VO—3 PC O - 1̂ o \ VO Xr 3 m 3 * • co CD l ■ •r., UNIVERSITY F IBADAN LIBRARY 026 039 300 TENDIX analysis of variance (aov) WITH and without androgen therapy tables for effect of pre-weaning castration PROTEIN SAO-E ACir.TF: !ACRE ACTIVITY KAN SQUARES P. VALUE p JRCE OF VARIATION DF MEAN SQUARES F. VALOE P MEAN SQUARES F. VALUE P ** 1.072 s.s14 14.681 0.028 1.061 n.s 2.cions 250 ..521874 106.630 H I .pies 12 640.741 **» 0.330 12.378 0.027 2.347 i-or A 48 *«» 11.317 1477.454 ftftft 1138.016 409-571 IH H I eatments 2 56.039 **« 0.115 H.851 ftftft 73-975 26.62316.859 . ppr.ictlon 2410{4 0.0212140.171 3&‘i&7 .a,*. 19̂ MAGNESIUM POTASSIUM 0.296 n.s pill cations 0.03? 1.079 n«.*s• 0.003 0.555 n#.#s 3.858 # 82.900 . 6.368 > HI rples 0.794 21.973 0.257 52.592 13.018 -ror A 3b 0.036 0.005 n.s2 0.778 23.870 §** 0.056 9-710 ## 7.965 0.828 11 êatments #** 0.061 10.547 *** 44.698 4.654 -.teraction 18 0.499 1̂ -319 9.607 rror B 8Q 0 0.006 149 0..013436 0 . 0 2 8 . 18.921:tal POPPER ZINC n.s 14 279.606 0.394 n.s 0.1421 x 10-3 2.667 0.258 x 10"3 1.639 *«» rp11cations 0.032 , 201.576 Q.053 3 3 14 .7 8 6 inples 9 750.307 1.057 n.s oll57 x 10-3 rror A 36 709.402 0.158 x 10"3 103-862 ftftft 0.067 371.763 »** reatments 2 1553.119 2.469 n.s 0.017 25.822 ftftft 0.032 o 179.578 »** -teraction 18 1019.700 1.621 n.s . 0.166 x 10“3 0.182 x 10" 3 rrar B 80 629.003 0.003 0.0Q8 otal 1H9 705.978 * = P<0.05 4 : ** = P<0.01 ■ f -t* = p<0.001 Vfp>n __ 4 4-.. ̂-PPENDIX 3 ANALYSIS OF VARIANCE (AOV) TABLES OF EFFECT OF CASTRATION AT 3-4 FDNfflS OF AGE WITH AND WITHOUT ANDROGEN A fl__________THERAPY ACHE ACTIVITY PROTEIN_________SACbB ACTIVITY SOURCE OF VARIATION DF MEAN SQUARES F. VALUE P MEAN SQUARES F. VALUE P MEAN SQUARES F. VALUE P . Replications 0.698 n.s Samples 3 0 153 0.027 1.806 n.s 0.702 i 12 82.520 0.079 n.s |378.183 «»* 0.542 111 Trror A 36.493 763.887 86.116' ### 36 0.219 0.015 8.870 Treatments 2 — 35.805 182.655 111 0.817 *«* 18.247 #«« Interaction 24 52.477 168.8433.109 15.863 111 0.110 7.092 111 Trror B 78 0.196 162.533 17.565 ##» / 0.015Total 0.081 9-253155 7.485 93.215 • CALCIUM MAGNESIUM POTASSIUM plications 3 0.05 1.868 n.s 0.008 0.342 ri.'sTanples h i 0.527 0.144 n.s9 0.913 31.137 1.067 42.093 111 l83.HO 50.038 «§#Trror A 27 0.029 Treatments **» 0.0252 fit# 3.65912.733 443.845 111 Interaction 18 0.928 111 0.315 21.397 2073-799 560.41432.351 0.612 41.533. 111 89.367 24.150 •••Trror B 60 0.029 Total 0.015 . 3.700 119 0.446 0.192 64.929 POPPER ZINC -cpli cations 3 962.680 1.111 n.s 1.486linples 0.020 n.s 0.006 n.fe9 37664.265 43.483 »## . «# 2.5970.085 6.188 ftftft Trror A 0.092 37.52527 866.171 0.014 0.002 reatments 2 80960.352 89.460 • #»» 0.085 - , 5.054 # , 0.516 ### ■"Traction < 240.39818 36768.398 40.628 «## 0.039 ■ 2.247 # ## . ror B 60 0.077 36.137 otal 904.979 0.017 0.002 119 10447-930 0.026 0.029 * = P<0.05 a«*' •* = P<0.01 •i' 1 1 ** =* P<0.001 .s = not significant (P>0.05) •I J UNIVERSITY OF IBADAN LIBRARY • ANALYSIS OP VARIANCE (AOV) TABLES OP EFFECT OF CASTRATION AT 5-6 PC-. TBS OF AGE WITH AND WITHOUT ANDROGEN THERAPY' ACNE ACTIVITY ' ■ PROTEIN SAChE ACTIVITY r.CE OF VARIATION DF MEAN SQUARES F. VALUE P MEAN SQUARES F, VALUE MEAN SQUARES F. VALUE P 0.167 1.522 n.s 0.002 0.356 n.s 3.954 0.653 n.s 3 •»« 117.532 495.382 81.908 ««»12 49 445.692 0.706 36 0 -.016190 0.006 6.048 i t * * 2 7.751 47*715 **««** 0.815 129.235 486.388 99.339 24 2.226 13.701 0.154 24.339 *t* 82.912 16.933 HI 78 0.162 0.006 4.896 155 4.354 0.094 61.411 CALCIUM MAGNESIUM POTASSIUM 3 , 0 . 5 0 8 1.026 n.s 0.010 0.846 n.s 1.124 0.414 n.s 1.746 11 0.134 11.055 •*« 160.083 59.106 «i« 9 3.527 27 0.495 0.012 2.708 2 11.486 682.426 **• 23.936 111 5.183 779-370 1353.965 18 0.777 1.620 n.s 0.488 73-403 111 30.631 15.438 «** 60 0.480 0.007 1.984 119 '-0.809 0.177 4•.1 ,-i.1 3." 9- sodium COPPER ' rt-~, ZINC ■eclicatlons 3 69.114 0.922 n.s 0.004 0.744 n.s 0.37 x 10-3 0.843 Arples 9 25550.263 340.874 \ *** 0.015 2.848 * 0.071 - 162.072 27 74.955 0.005 0.439 x 10-3 Tror A ««« « 0.183 420.263 Treatments 2 370084.627 2857-152 »## 0.037 4.239 24070.777 185.832 0.009 1.113 n.s '0.037 86.369_:eraction 18 0.436 x 10-3 Irror B , 60 219.529 4. 0.009 /Trtal 119 11877.298 ; 0.009 0.014 ri •• r !/ - ANALYSIS OF VARIANCE (AOV) TABLES OF EFFECT OF CASTRATION AT 7-8 MONTHS OF AGE WITH AND WITHOUT ANDROGEN THERAPY , ACNE ACTIVITY | , PROTEUS'—— SAChE ACTIVITY IOURCE OF VARIATION DF MEAN SQUARES F. VALUE P MEAN SQUARES F. VALUE P MEAN SQUARES F. VALUE P Replications 3 0.110 1.244 n.sCamples >.001 n.s12 98.951 1114.416 *** 2.285 *** 2.4900.194 0.069 n.s Irror A _ 281.845 1 1 1 36 45.180 Treatments 0.089 0.689 X 10~3 1627.369 2 0.161 *** 36.0192.134 n.s Interaction 24. «** 0.468 541.361 2518.862 80.331 1 1 1, 1.107 14.632 1 1 1Irror B 0.053 60.82078 10-3 22.2.753 7.104 H I 0.076 Total 0.865 X 31.356 155 7-895 Tr- . * ?.. 0.030 217.175 CALCIUM j\: MAGNESITM POTASSIUM RTeempplliecsations 3 0.035 0.693 n*.«s* 0.015 1 1.152 n.s 14.682? 2.268_ 44.510 2.317 n.s Irror A 0.531 40.720 t * * 293.508 46.332 H I 27 0.051 0.013 Treatments 2 13.363 334.808 111 *«« 6.335 Interaction *## 1.7050.706 71-3130.460 3672.363 340.107 H I 18 Irror B 17.685 19.252 1 1 1 60 0.040 168.039 15.562 **» 0.024 Total 10.798119 0.535 0.154 116.588 ffc., - SODIUM « OOPPER ZINC replications fanples 3 82.119 0.383 n».«s« 0.178 X 10" 3 1.068 n.s 1.4829 28520.213 133.074 0.534 X 10"3 0.003 n.s 3.204 1 1 Irror A 0.22627 10-3 114.143 H I 214.317 0.166 X Treatments 0.0022 343806.338 1460.249 111 0.014 69.456 1 1 1 0.261 #**Interaction 18 27403-078 116.389 *## 0.002 »*« 125.4:4 9.877 0.145 69.723 *Error B 60 235.443 0.205 x 10"̂ 0.002 UNIVERSITY OF IBADAN LIBRARY 302 APPENDIX ANALYSIS OF VARIANCE (AOV) FOR EFFECT OF OVARIECTOMY AMD HORfCMAL REPLACEMENT THERAPY 1 AChE ACTIVITY PROTEIN SAChE ACTIVITY SOURCE OF VARIATION DF MEAN SQUARES F. VALUE P MEAN SQUARES VALUE p MEAN SQUARES F. VALUE P Replications Sanples 3 0.007 0.051 -«n».«s 0.575 X 10"3 0.840 n«.«s0.244 « 2.519 0.438 n.s 12 Error A 57.689 392.774 356.670 694.41436 0.147 0.684 x 10-3 ftftft 120.945 «•« Treatments 123.298 5.7412 12.618 155.250 H I 0.120 20.704 ft Interaction 2*) 11.1662.480 Error B 30.519 0.011 3.913H I «• 78 0.081 • 0.978 X 10-3 18.342 3-467 Total 5.290155 5.088 0.023 60.913 CALCIUM MAGNESIUM POTASSIUM Replications 3 0.039 3-195 ft 10-3Sanples 0.388 X9 1.124 0.407 n.s 1.488 0.449 n.s Error A 90.271 ftftft 0.192 10-3 201.896 ««» 114.083 34.443 »«• 0.012 Treatments 272 0.211 11.216 •»« 0.952 X 3-312 Interaction ftftft 0.353 2291.930 387.977 70.332 »«• 18 0.229 12.222 0.098 78.634 ««« 680.403 12.400 «•«Error B 60 0.019 0.001 Total 119 ^,0.136 5.5150.078 29.066 ^ SODIUM 0QPPER ZINC * Replications 3 874.226 0.845 n.s 0.878 X 10-3Sanples 0.-692 n.s 0.960 x 10-3 n.s9 1044.866 1.010 n.s 0.0035 28.302 ««« 0.554 «§« Error A 0.18427 1034.117 0.127 x 10-J 106.407 Treatments 0.0022 IO89.986 1.423 n.s «»* siftInteraction 0.017 144.826 56.76418 1681.422 ft 0.C04 0.117 ««« Errpr B 2.19560 765.752 0.117 x 10-3 32.238 /' H.217 105.180 Total 0.002119 994.439 0.001 0.050 » * P<0.05 K- ** » P<0.01 !- *** = P<0.001 - ■ ' n.s = not significant (CP>0.05) • X \ . APPENDIX ; ANALYSIS OF VARIANCE TABLES FOR EFFECT OF ANDROGIEN AIKENISTRALION ON GILTS - AChE ACTIVITY PROTEIN ' ----. SAChE ACTIVITY SOURCE OF VARIATION DF MEAN SQUARES F. VALUE 1 < P MEAN SQUARES F. VALUE P MEAN SQUARES F. VALUE P Replications 3 5-356 2.690 n.s /-0.029 4.203 ft 0.372 n.sSanples 9 134.318 5.731 67.467 0.697 99-491 ftftft Error A 1201.371 78.108 ««« 27 1.991 0.007 15.381 Treatments 1 75.695 ' 49.712 ftftft 1.042 ftftft 2380.540 183.982 Interaction 9 18.495 12.146 ftftft 0.102 95-103 9-313 ftftft 670.588 «««Error B 51.82730 Total 1.523 0.011 12.939 79 19.829 0.112 253.782 CALCIUM~ru'; ;i’ y MAGNESIUM POTASSIUM Replications 3 0.005 Sanples 1.064 0.719 n«.««s 0.0013 ' 0.537 n.s 4.648 3-687 ft 9 159-868 0.281 Error A 0.002 129.693 ftftft 168.023 133.313 «»« 27 0.007 1.260 Treatments 1 1.478 201.745 ««« 0.043 9.125 ««« ftftft 94.124 ««« Interaction 72.3319 0.334 45.592 0.306 64.525 97.664 75.570 ftftii Error B 30 0.007 0.005 1.292 Total 79 0.183 0.070 32.558 SODIUM • COPER ZINC Replications 3 134.038Sanples 1.737 n«.«s« 0.011 0.767 n.s 0.118 x 10-3 0.536 n.s9 34822.486 451.266 0.015 1.050 n.s 0.009 *«ftError A 41.13577.166 0.014 0.220 x 10-3 Treatments 271 Interaction 97125.017 1472.267 «*» ««« 0.019 1.339 n.s 0.005 9.3699 37002.862 560.906 0.020 1.384 n.s 0.012 23-253 ftftiiError B 30 65.970 0.014 0.503 x 10-3 Total 79 9468.581 0.015 0.0C3 * - P<0.05 «* - P<0.01 *** = P<0.001 n.s = not significant (P>0.05) UNIVERSITY OF IBADAN LIBRARY ’PENDIX 6 . ANALYSIS OF VARIANCE TABLES FOR EFFECT OF ACUTE HEAT EXPOSURE OF PIGS AChE ACTIVITY PROTEIN SAChE ACTIVITY OURCE OF VARIATION DF MEAN SQUARES F. VALUE P MEAN SQUARES F. VALUE P MEAN SQUARES F. VALUE P Replications 4 0.010 0.065 n.s 0.009 2.465 n.s 2.446 0.570 n.s anples 9 51.873 334.281 «** 0.443 117.188 ftftft 128.576 29-963 rror A 36 0.155 reatments 1 65.172 485.051 t** 0.004 ftftft 4.291 *«* nter̂ atlon 9 ~ 4.920 36.618 ■urn 0.470 89.916 , 0.116 22.239 ft** 166.619 42.566 81.189 20.741 ft** rror B 40 0.134 0.005 3.914 otal 99 5.932 0.059 23-993 CALCIUM MAGNESIUM POTASSIUM epllcatlons 4 0.015 0.308 n.s 0.065 4.366 ft* 2.047 0.396 n.s anples 9 1.608 33.190 ««» 0.038 2.529 ft 132.581 25.687 «*« rror A 36 0.048 0.015 5.161 reatments 1 52.201 1256.238 3.015 129.002 ftftft 2020.323 226.845 *«* nteraction 9 0.624 15.017 «** 0.111 4.952 ft* 38.344 4.305 «« rror B 40 0.041 otal 99 "0.765 ’ * .• ' 0.023 8.906 0.061 t 41.5041 ^ SODIUM COPPER ) ZINC % epllcatlons 4 92.502 1.284 nif.tfst O.O6I 0.711 n.s 0.004 0.229 n.siiples 9 1024.625 14.228 0.117 1.354 n.s 0.576 35.294 ««• rror A 36 72.014 . 0.086 0.016 reatments 1 74.822 1.186 n.s 0.128 1.572 n.s - 1-736 O.s 123.363 *«« r.teraction 9 684.867 10.860 A 0.121 1.479 0.019 1.384 n.s rr<3r B ,40 63.062 ■ 0.082 0.014 otal 99 211.568 0.089 0.083 ♦ = P<0.05 * = P<0.01 * . f * = P<0.001 ' ! 5 = not significant (P>0.05) '1 ' W " - - ' - P i k analysis of v o t e t a b u s f o r effot of aggie heat e a t o * of pigs _ PROTEIN SAChE ACTIVITYAChE ACTIVITY mean squares F. VALUE PEAN SQUARES F. VALUE P mean squares F. VALUE P PCE OF VARIATION DF 0.188 n.s j 0.413 1.913 n*.s 0,006 0.720 n.s 0.508 ** cftft 1639.431 607.482 ««« 56.006 9 108.861 504.987 "0.512 2.699 27 Q.216 0.009 ftft 99.944 *«* s 8.004 41.057 HI 0.037 4.037 328.207 *** / 2.684 13-769 •t* 0.040 4.408 ««« 127.413 38.799 4 5 3/284 150 0.195 0.009 4.924 94.964239 “ > - :• 0.034• - . » * * * I4>f CALCIUM MAGNESIUMIRON i 0.866 n.s 0.052 3-582 * j 0.002 5.755 * 0.006463.045 ««* I. 191 I54.38O »*» 0.618 42.173 *** 9 0.165 , 27 0.356 x 10“i 0.008 <*« 0.015 631.404 1.036 45.324 *** 5 0.147 689.253 »*« 4.411 «»» 0.156 6.838 *** 45 0.029 134.438 HI 0-415 45-123 0.023 150 0.213 x 10-3 0.007 0.091 239 0.015 0.202 COPPER . POTASSIUM • SODIUM n.s 3 2.030 0.581 n.s 4.546 .386 n.s 0.001 2.324 HI 7-955 HI 25.867 7.409 21.066 .425 i»* 0.005 _ 9 3.278 0.602 x 10“3 27 3.491 ««• 11.465 t*» 0.019 26.563 »*« 5 360.708 165.664 27.476 4.693 HI 0.003 4.809 *** 45 38.326 17.602 HI II. 253 150 2.177 2.396 0.715 x 10-3 0.002 239 17.523 5.418 ZINC n!s plications 3 0.869 x 10-3 1.171 ariples 9 6.499 673.671 ««* ■ipr A____ 27 0.742 X 10-3 Hi UNIVERSITY OF IBADAN LIBRARY 30k rv> i—1 IV) M ro t-1 rvi m 1 UO VJ1 t r IV) VO VC1 f IV) VvOo von vtnr vn —ro3 vovo VO vn t r IV) VO O U l 'J l 'J ^ O U ) V D O U lU l- v l^ lW vo o vn vn -4 vo vo h-1 VO OO o O < 3 0 0 0 O O O oo o o o o o INI OVVO t v o v o o vo T) - f O VO -VI iTH H O O M vo O CT\ O F cn O -4 -4 m - f vo •O O O H1 O J^O 0 —4 i—■ i—1 -c=" i—■ vo vo 0 ) 0 i—1 vn O I—1 o O vo vo vn cn o -Vi .cn > vIVo) cOr\ VfO VrOe Oro —Moi O O O O V O V O H t o o V J IH W S H1 VO VO VO OO O t - M t r VO 10 —1 3 cn on cr\ rv) vo m iv> co —4 pr Pr M CT\ OO U oo ro -ni -VI M VJ1 VJI oo ro ro m CT\ p )r VJI o vo o oo • • • • • • • • • • • • • • • • CF\ oo M o ro ro vo o o o o —4 VJI o\ -4 VJI pr U~l M -Cr 1—1 Pr vo CO —4 O VJI VJI 00—4 ro —4 OO CO VJI VJI oo OO -pr VJI oo -prVO ro vo OO Pr X X x 3 X X X 3 X X * 2 X X X X * X x • * X X • * X X • X X X X X X X If) * X X U) X XU) X X X X Tl t-1 vn t -v o oovo oo t r O O O M o o o o o o o o o o —si ro CO trVO CO M O O I—1 OVO CT\ o O O O O O o 'oi oo rvi vo vo vo ru VO O -VI -Cr o VO vo IV) OVOVO O H O VO CO CO O CO IV) M VO Cn-4 V0 VO oo o vo i—1 I—1 rv> i—1 o vn Vr) IV) ro cr\ —4 ro ro o ro Pr ro i—1 OO pr• • • • • • C• T\U•O —• 4 •oo •o ov•n ro ro ro o VJI -pr ro oo OO VJI ro ro F vo vo VJI -Pr 1—» OO O 1-* VJI o -o M —4 —4 CT\ Pr O —4 03 CT\ Pr —4 M \ X X x 3 X X * * * * * * X • X X X * X X X U) * X X * X X o o o o o o o O M O O W O V O o v n o t v o i—1 vo vo I—1 cn t - h 1 lO X X X O O O IV) o o o -vi vo vo envo vo • • • • • • • • • • • • <1 O VO Pr UO VO VO O Pr vn pr ro o > M O -o UO cn cn cr\ oo rv> co m vo f—* UO o vn oovn oo iv) t CO VO -4 F X X X 3 • X X X X X X x 3 X X X • X X X X X X X • •D X X X U) X X X X X X U) UNIVERSITY F IBADAN LIBRARY 305 % % n> (<§Drt> £ !Q 5 U )fO H LO IV) H CO fO »-* 1U fU I—1 U) W H OO HOJ CO CO CO o o o o o o t r ru IU H t r r\J VO —J VO ©coco —j c VOJl Ot r Ot r ro vji ÎV)OJ (ft cMo voji oov eon o cmo o o o M trV O o U1U1U1 ru cruo vrut r CO ON on o ru ru r t r t r rU r u s o c OoN rOuN oO\N VJl O uN O rv) -t- H-CU1 ̂ ru t o o o O rĈ-̂l -tr ru- oru t r t r u rH iu rOu OruN VvJol rtur o1+ VoJl V1+ o JI 14- riu+ riu+ riu+ vo ru oo in ruu)h-1 H 14- 1+ 1+ IruVD H OO ON oo H o ro iu fh r1u+ o14- • • • H H FV3 rou mo o Crm on ru O t r t r o irnu o lU Hru fNJ -<1 t r o ru o o o o o oru - t n VJl VJl VJl ru ru rtu S ON-~J M rO C ON ON ON no ru ru o o o ro oo o • • O •CO c• o •o •ru X■D O• N N•O • • •o o o VJl tr VJl r o o oo ru u tnr rHu ituH ru trur 4©-* o r tuC oOo VcJol OruN VJlvji o14- oit o1+ C14O- 1 ru co CO 5 CO ONV14O- 1 1 ru o vji t4r- 1r4u- 1t4r- J1~4-* ritut- r14u- r1u4- 1—’ ru ^ htr rtur CtO o -It CO tro rruu tto -ru—i tr U1 £h-* vji ru oo o ru ru iUJ tr VJl rtu oru tru VrJul ru rtur cogvo (DO O ru h ru jru) oru rtu -rto- frco> ro 0O~- o ru vji o VJl VJl tr coo vru 1 OO • ON ON o hru H- 14- 14- 1+ 14- 14- 1 1 1 14- 14- 14- ru iu h h-* co ru O O O CD O O crou trur oru iO—N1 1I—U1 CtOr ON-̂ 1 tr (ODHO HM ru m co ru ru o ru ru ru tr ru ru rtur- Vrul Mru rruu ru ru on vji vo vo vo J r-tH f l 1r+u Mru- 1r4u- 1O+ HO- H H I U 1O4- I £ £ S c s § CD CD 1P/ 5* er 8Tsr ts> Lt* N> t—* W M H C/4 N f > W N H . 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