"Land and Food Systems, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Robinson, Julie A."@en . "2011-02-16T18:46:22Z"@en . "1991"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Feeds often contain molybdenum (Mo) and sulfur (S) in excess of recommended allowances for adequate copper (Cu) absorption by ruminant livestock. Two randomized-block experiments were conducted using lambs given a cereal-based diet (90% of dry matter (DM) intake), containing 8 mg Cu, 0.7 mg Mo and 2.1g S per kg DM, that was either unsupplemented or supplemented with ammonium molybdate or sodium sulfate alone or in combination, to determine the effects of Mo, S and Mo+S on growth, hematology, serum Cu and Mo concentrations and reproductive function.\r\nShort term intake (4 weeks) by rams, aged 18 and 20 weeks, of 26 mg Mo alone or in combination with 2g S per kg DM, had no effect on growth, hematology or the concentration of total Cu in serum (TCu). However, supplemented groups had lower (P<0.05) concentrations of serum Cu soluble in trichloroacetic acid (TCA-Cu), but the group given Mo+S had the highest (P<0.05) concentration of serum residual Cu (RCu). Luteinizing hormone (LH) peak amplitude was affected by age*diet interaction (P<0.05), because of high amplitude LH peaks in the serum of lambs, aged 24 wks, given Mo alone. Testosterone serum secretory profiles did not differ among diet groups, but testosterone peak frequency was higher (P<0.05) for older ram lambs.\r\nLong term intake (32-39 wks) by ewe, ram and wether lambs (gonadal influence) of 12 mg Mo, or 2g S alone or combined per kg DM also had no effect on hematology or TCu. Food intake and liver weights were higher (P<0.05), but TCA-Cu was lower (P<0.05) for groups given S. Serum concentrations of total Mo (TMo) were higher (P<0.05) for Mo-supplemented groups, but RCu was highest only for the Mo+S group. Growth was affected by Mo*S*gonadal influence*time interaction (P<0.05); until autumn, the body weight of Mo-supplemented groups were higher than those of ram and wether lambs given Mo+S. Ovarian or testicular functions were delayed more severely for Mo- than Mo+S-supplemented groups. Mean concentrations of LH were affected by Mo*S*gonadal influence*time interaction (P<0.05); the mean LH values of Mo-supplemented ram and ewe lambs were higher than the Mo+S-supplemented\r\ngroup, whereas a reverse trend was observed for wether lambs. The amplitude of LH peaks was affected by Mo*S*gonadal influence*time interaction (P<0.05); for ram lambs, LH peak amplitude was higher for Mo than Mo+S-supplemented groups, whereas for wether and ewe lambs the reverse trend was noted. Mean concentrations of serum Cortisol and Cortisol peak amplitude were affected by Mo*S interaction (P<0.05); the mean Cortisol concentration and peak amplitude for the Mo-supplemented group were higher than those for the Mo+S-supplemented group. The frequency of LH and Cortisol peaks did not differ (P>0.10) among diet groups.\r\nIn conclusion, Mo supplementation of cereal-based diets containing a high concentration of Cu did not adversely affect lamb growth. However, the effect of high dietary Mo on reproductive function appears to depend on the dietary level of S and the induction of high serum RCu (thiomolybdate). Further investigations on the effects of Mo and thiomolybdate on endocrine function may provide a nutritional basis for improving reproductive efficiency in ruminants."@en . "https://circle.library.ubc.ca/rest/handle/2429/31319?expand=metadata"@en . "T H E EFFECTS O F DIETARY M O L Y B D E N U M AND SULFUR O N S E R U M COPPER CONCENTRATIONS, G R O W T H A N D REPRODUCTIVE FUNCTION IN LAMBS. by JULIE A N N ROBINSON B.Sc, The University of Guelph, 1980 M.Sc, The University of Manitoba, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR T H E D E G R E E O F DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Animal Science We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA March 1991 \u00C2\u00AEJulie Ann Robinson, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date DE-6 (2/88) ii ABSTRACT Feeds often contain molybdenum (Mo) and sulfur (S) in excess of recommended allowances for adequate copper (Cu) absorption by ruminant livestock. Two randomized-block experiments were conducted using lambs given a cereal-based diet (90% of dry matter (DM) intake), containing 8 mg Cu, 0.7 mg Mo and 2.1g S per kg D M , that was either unsupplemented or supplemented with ammonium molybdate or sodium sulfate alone or in combination, to determine the effects of Mo, S and Mo+S on growth, hematology, serum Cu and Mo concentrations and reproductive function. Short term intake (4 weeks) by rams, aged 18 and 20 weeks, of 26 mg Mo alone or in combination with 2g S per kg D M , had no effect on growth, hematology or the concentration of total Cu in serum (TCu). However, supplemented groups had lower (P<0.05) concentrations of serum Cu soluble in trichloroacetic acid (TCA-Cu), but the group given Mo+S had the highest (P<0.05) concentration of serum residual Cu (RCu). Luteinizing hormone (LH) peak amplitude was affected by age*diet interaction (P<0.05), because of high amplitude L H peaks in the serum of lambs, aged 24 wks, given Mo alone. Testosterone serum secretory profiles did not differ among diet groups, but testosterone peak frequency was higher (P<0.05) for older ram lambs. Long term intake (32-39 wks) by ewe, ram and wether lambs (gonadal influence) of 12 mg Mo, or 2g S alone or combined per kg D M also had no effect on hematology or TCu. Food intake and liver weights were higher (P<0.05), but TCA-Cu was lower (P<0.05) for groups given S. Serum concentrations of total Mo (TMo) were higher (P<0.05) for Mo-supplemented groups, but RCu was highest only for the Mo+S group. Growth was affected by Mo*S*gonadal influence*time interaction (P<0.05); until autumn, the body weight of Mo-supplemented groups were higher than those of ram and wether lambs given Mo+S. Ovarian or testicular functions were delayed more severely for Mo- than Mo+S-supplemented groups. Mean concentrations of L H were affected by Mo*S*gonadal influence*time interaction (P<0.05); the mean L H values of Mo-supplemented ram and ewe lambs were higher than the Mo+S-supplemented iii group, whereas a reverse trend was observed for wether lambs. The amplitude of L H peaks was affected by Mo*S*gonadal influence*time interaction (P<0.05); for ram lambs, L H peak amplitude was higher for Mo than Mo+S-supplemented groups, whereas for wether and ewe lambs the reverse trend was noted. Mean concentrations o f s e r u m C o r t i s o l and C o r t i s o l peak a m p l i t u d e were affected by Mo*S interaction (P<0.05); the mean Cortisol concentration and peak amplitude for the Mo-supplemented group were higher than those for the Mo+S-supplemented group. The frequency of L H and Cortisol peaks did not differ (P>0.10) among diet groups. In conclusion, Mo supplementation of cereal-based diets containing a high concentration of Cu did not adversely affect lamb growth. However, the effect of high dietary Mo on reproductive function appears to depend on the dietary level of S and the induction of high serum RCu (thiomolybdate). Further investigations on the effects of Mo and thiomolybdate on endocrine function may provide a nutritional basis f o r improving reproductive efficiency in ruminants. iv T A B L E O F CONTENTS TITLE P A G E A B S T R A C T ii T A B L E O F CONTENTS iv LIST O F TABLES vii LIST O F FIGURES viii LIST O F ABBREVIATIONS * A C K N O W L E D G E M E N T S ' x i Chapter 1 INTRODUCTION . 1 1.1 Subject and background 1 1.1.1 Overview of the pituitary-gonadal-adrenal axis 2 1.1.2 Overview of steroid synthesis and catabolism 4 1.1.3 Glossary 5 Chapter 2 L I T E R A T U R E REVIEW 8 2.1 Cu Mo S Nutrition 8 2.1.1 Cu Mo S Recommended dietary allowances 8 2.1.2 Causes of nutritional imbalance 9 2.1.3 Thiomolybdate Theory 10 2.2 Reproduction 12 2.2.1 Puberty in the ram and ewe 12 2.2.2 Pituitary gonadal development 14 2.2.2.1 Hypothalamo-pituitary development in utero 14 2.2.2.2 Pituitary-testes development 14 2.2.2.3 Pituitary-Ovarian development 17 2.2.2.4 Desensitization 18 2.2.3 Photoperiodism 18 2.2.4 Adrenal effects on the pituitary-gonadal axis 19 2.2.4.1 Stress and Cortisol secretion 19 2.2.4.2 Cortisol effects on pituitary-gonadal function 20 2.3 Nutrition-Reproduction 22 2.3.1 Nutrition Reproduction studies in sheep 22 2.3.2 Cu-Mo in reproductive function 23 2.3.2.1 In vivo 23 2.3.2.2 In vitro 24 Chapter 3 HYPOTHESIS 26 Chapter 4 MATERIALS A N D METHODS 28 4.1 Animals and housing 28 4.1.1 General 28 4.1.2 Experiment I (Ram Iambs) 28 4.1.3 Experiment II (Ram, wether and ewe lambs) 28 4.2 Dietary treatments 30 4.2.1 General 30 4.2.2 Experiment I (Groups I, II, III) 30 4.2.3 Experiment II (Groups I, II, III and IV) 30 4.3 Sampling procedures 31 V 4.3.1 Feed and feed intake (Experiments I and II) 31 4.3.2 Body weight (Experiments I and II) 32 4.3.3 Blood sampling 32 4.3.3.1 General 32 4.3.3.2 Experiment I (LH, testosterone, Cu) 32 4.3.3.3 Experiment II (LH, testosterone, P4, Cortisol, Cu, Mo) 33 4.3.3.4 Hematology (Experiments I and II) 34 4.3.4 Gonadal samples 34 4.3.4.1 Scrotal circumference (Experiment I and II) 34 4.3.4.2 Semen collection (Experiment II) 35 4.3.4.3 Vaginal smears (Experiment II) 36 4.3.5 Other tissues 36 4.4 Laboratory procedures 37 4.4.1 General 37 4.4.2 Nutrient composition of feed 37 4.4.2.1 Experiment I 37 4.4.2.2 Experiment II 38 4.4.3 Mineral analysis of serum and hematology 39 4.4.3.1 Experiment I (TCu, TCA-Cu and RCu) 39 4.4.3.2 Experiment II (TCu, TCA-Cu, RCu and TMo) 40 4.4.3.3 Hematology (Experiments I and II) 40 4.4.4 Hormone analysis of serum 41 4.4.4.1 Luteinizing hormone (LH) 41 4.4.4.1.1 Experiment I 41 4.4.4.1.2 Experiment II 42 4.4.4.2 Testosterone 42 4.4.4.2.1 Experiment I 42 4.4.4.2.2 Experiment II (Testosterone) 43 4.4.4.3 Progesterone (Experiment II) 43 4.4.4.4 Cortisol (Experiment II) 44 4.4.5 Evaluation of gonadal samples (Experiment II) 45 4.4.5.1 Semen evaluation 45 4.4.5.2 Estrus and Ovulation 45 4.5 Data Analysis 46 4.5.1 Hormone secretory profile characteristics 46 4.5.2 Statistical analysis 46 4.5.2.1 Experiment I 46 4.5.2.2 Experiment II 47 4.5.3 General 47 Chapter 5 RESULTS 49 5.1 Experiment I 49 5.1.1 Nutrient composition and intake of diets 49 5.1.2 Serum copper and hematology 49 5.1.3 Body weight and scrotal circumference 50 5.1.4 Serum secretory profiles of L H and testosterone 50 5.1.5 Other tissues 51 5.1.6 Partial correlations 51 5.2 Experiment II 57 5.2.1 Nutrient composition and intake of diets 57 5.2.2 Serum copper and molybdenum and hematology 58 vi 5.2.3 Body weight 58 5.2.4 Serum L H secretory profiles 59 5.2.5 Scrotal circumference and semen 59 5.2.6 Serum Testosterone twice weekly and secretory profiles 60 5.2.7 Estrous and serum P4 twice weekly profiles 61 5.2.8 Serum Cortisol twice weekly and secretory profiles 62 5.2.9 Other tissues 63 5.2.10 Partial correlations 63 Chapter 6 DISCUSSION 85 6.1 Experiment I 85 6.1.1 The effect of Mo and Mo+S on serum Cu 85 6.1.2 The effect of Mo on pituitary-testes function 86 6.2 Experiment II 87 6.2.1 The effect of S, Mo+S and gonadal influence on serum Cu and Mo 87 6.2.2 The effect of Mo on pituitary function 88 6.2.3 The effect of Mo on testicular function 88 6.2.4 The effect of Mo on ovarian function 89 6.2.5 The effect of Mo on adrenal function 90 6.3 General 91 6.3.1 The relation between Cu and Mo in diets and serum 91 6.3.2 Gonadal influence on serum Cu 92 6.3.3 The relation between Mo and S and endocrine function 93 Chapter 7 CONCLUSION 95 Chapter 8 LITERATURE CITED 99 APPENDICES 112 vii LIST OF TABLES Table 1. Nutrient composition of dry matter in experimental diets given to ram lambs for 30 days. . 52 Table 2. Copper (Cu) soluble in trichloroacetic acid (TCA), residual Cu and total Cu in serum of crossbred rams, aged 22 and 24 weeks, given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 30 days. 53 Table 3. Characteristics of luteinizing hormone secretory peaks in serum of crossbred rams, aged 22 and 24 weeks given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 30 days 55 Table 4. Characteristics of testosterone secretory peaks in serum of crossbred rams, aged 22 and 24 weeks, given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 30 days 56 Table 5. Nutrient composition of dry matter in experimental diets given to ewe, ram and wether lambs for 32 to 39 weeks 64 Table 6. Copper (Cu) soluble in trichloroacetic acid (TCA) and residual Cu in serum of ewe, ram and wether lambs given, cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 23-30 weeks (October) 66 Table 7. Total copper (Cu) and total molybdenum (Mo) in serum of ewe, ram and wether lambs given, cereal-based diets with and without supplemental Mo and sulfur (S), for 32-39 weeks (December) 67 Table 8. Luteinizing hormone (LH) mean concentration in serum of ram, wether and ewe lambs given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 32-39 weeks 72 Table 9. Cortisol mean concentration of secretory profiles in serum of ram, wether and ewe lambs given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S). . . 83 viii LIST OF FIGURES Figure 1. Schematic overview of the hypothalamo-pituitary-gonadal/adrenal axis and retino-hypothalamo transmission of light stimuli to the pineal gland. Refer to text for description (From Hafez 1985; Gorbman et al. 1983) 6 Figure 2. Schematic overview of principal pathways of steroid biosynthesis and catabolism. Unless specified, pathways are common to the adrenal and gonadal glands. Catabolism occurs mainly in the liver, but water soluble forms are excreted with urine (from Jefcoate 1986; Schwall et al. 1986; Dufau et al. 1984; Gorbman et al. 1983) 7 Figure 3. Luteinizing hormone (LH) and testosterone serum secretory profiles of individual ram lambs aged 22 and 24 weeks, given a cereal-based diet (Group I). Lambs were bled every 20 m. Secretory peak concentrations are circled 54 Figure 4. The effect of dietary sulfur level on feed intake by lambs (n=5) given cereal-based diets containing two levels of molybdenum (ANOVA sulfur*time P<0.05) 65 Figure 5. The effect of dietary molybdenum level on body weight of lambs (n=5) given cereal-based diets containing two levels of sulfur (ANOVA molybdenum*time P<0.05). The inset shows when molybdenum plus sulfur affected ewes (n=2) differently than rams or wethers (gonadal influence * molybdenum * sulfur * time P<0.05) 68 Figure 6a. Luteinizing hormone (LH) serum secretory profiles of individual ewe lambs given cereal- based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise 69 Figure 6b. Luteinizing hormone (LH) serum secretory profiles of individual ram lambs given cereal- based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise 70 Figure 6c. Luteinizing hormone (LH) serum secretory profiles of individual wether lambs given cereal- based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise 71 Figure 7. The effect of dietary molybdenum level on the amplitude of luteinizing hormone (LH) secretory peaks in serum of ram, ewe and wether lambs given cereal-based diets containing two levels of sulfur (ANOVA gonadal influence * molybdenum * sulfur * time P<0.05). The inset shows the gonadal influence*time (P<0.05) effect on L H peak frequency. (R=ram n=8, E=ewe n=8, W=wether n=4) 73 Figure 8. The effect of dietary molybdenum level on scrotal circumference (line graph) and sperm per ejaculate (histogram) of ram lambs (n=2) given cereal-based diets containing two levels of sulfur (ANOVA molybdenum*time P<0.05; *differs from other groups; dased line excludes a cryptorchid ram; refer to text) 74 Figure 9. The effect of dietary molybdenum level on testosterone concentrations in serum collected twice weekly from ram lambs (n=2) given cereal-based diets containing two levels of sulfur ( A N O V A molybdenum * sulfur * time P<0.05) 75 Figure 10. Testosterone serum secretory profiles of individual ram lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise 76 Figure 11. The effect of dietary molybdenum level on basal testosterone concentrations in serum of ram lambs (n=2) given cereal-based diets containing two levels of sulfur (ANOVA molybdenum*time, sulfur*time P<0.05). The inset shows the effect of time (P<0.05) on the frequency of serum testosterone secretory peaks 77 Figure 12. The effect of dietary molybdenum level on P4 concentrations in serum collected twice weekly from ewe, lambs (n=2) given cereal-based diets containing two levels of sulfur ( A N O V A molybdenum * sulfur * time P<0.05) 78 ix Figure 13. The effect of dietary molybdenum level on Cortisol concentrations in serum collected twice weekly from lambs (n=5) given cereal-based diets containing two levels of sulfur ( A N O V A molybdenum*time P<0.05) 79 Figure 14a. Cortisol serum secretory profiles of individual ewe lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise 80 Figure 14b. Cortisol serum secretory profiles of individual ram lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise 81 Figure 14c. Cortisol serum secretory profiles of individual wether lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise 82 Figure 15. The effect of dietary molybdenum level on the amplitude of Cortisol secretory peaks in serum of lambs (n=5) given cereal-based diets containing two levels of sulfur ( A N O V A molybdenum * sulfur * time P<0.05). The inset shows gonadal influence*time (P<0.05) effect on Cortisol peak frequency (R=ram n=8, E=ewe n=8, W=wether n=4) 84 LIST OF ABBREVIATIONS A D G average daily gain RBC red blood cell CO carboxy RCu residual copper Cu copper S sulfur C V coefficient of variation STDEV standard deviation D B H dopamine B hydroxylase SEM standard error of the mean D H T dihydrotestosterone T C A trichloroacetic acid D M dry matter TCu total copper in serum E 2 estradiol 17 B TCA-Cu T C A soluble copper in serum FSH follicle stimulating hormone a aplha (Figure 2 only) G n R H / L H R H gonadotrophin releasing hormone ~ approximately H B G hemoglobin wk week ICPMS inductively coupled plasma emission d day mass spectrophotometry h hour L H luteinizing hormone m minutes M C H mean corpuscular hemoglobin fl femtoliters M C V mean corpuscular volume mm millimeter M C H C mean corpuscular hemoglobin content g grams Mo molybdenum mg kg\"1 milligrams per kilogram M O A monoamine oxidase ng ml'1 nanograms per milliters O H hydroxy Mg L \ micrograms per liter PCV packed cell volume /imol L 1 micromoles per liter xi ACKNOWLEDGEMENTS I wish to express my thanks to the many people who were involved with the development and completion of this project. My special thanks and appreciation to my advisor, Dr Raja Rajamahendran, who guided my program to its completion. My thanks and appreciation to Dr W Kitts who introduced the PhD program to me, and Dr R Blair who secured the development of my program, and to Gay Huchelega and Andreanna Phillips for their administrative assistance. My thanks and appreciation for technical assistance from Francis Newsome, Sylvia Leung, Vicki Adams, Lisa Koehn and farm staff at UBC, Gay Wilson at the Agriculture Canada Research Station, Agassiz, BC, Heather McPherson at the University of Manitoba (UM), MB; Susan Cook at the University of Saskatoon, Sask, and Sherry Fillmore, Agriculture Canada, Nappan, NS. My thanks and appreciation to my colleagues Marg Crowley, Rob McCann, and Murray Drew and students Karen Forrest, and Jane Copeland and friend Beaven Gill for their help with sample collection. My thanks to Dr. B Howland (Oral Biology Dept, UM) and Dr M Palmer (Animal Science Dept, UM) for giving me the help and use of their laboratories for RIA. My thanks and appreciation for thought provoking questions and remarks given to me by my supervisory committee Dr W Buckley (Agriculture Canada, Agassiz, BC), Dr B March (Animal Science Dept, UBC), Dr C Krishnamurti (Animal Science Dept, UBC) Dr D Kitts (Food Science Dept, UBC) and Dr W Powrie (Food Science Dept, UBC) and my candidacy examining committee Dr B Owen (Animal Science Dept, UBC), Dr J Shelford (Animal Science Dept, UBC), Dr C Krishnamurti, Dr R Blair (Animal Science Dept Head, UBC), Dr W Buckley, Dr D Kitts and chair Dr D Shackleton (Animal Science Dept, UBC), and my final examining committee Dr W Buckley, Dr B March, Dr J Shelford, Dr P Leung (Obstetrics & Gynecology, UBC), Dr L Sanford (Animal Science and Obstetrics and Gynecology, McGill University, Quebec) and chair Dr J Vanderstoep (Food Science Dept, UBC). Last but not least, I wish to express my thanks and appreciation for morale support given to me by my friends BJ Dempsey, Brian Phillips, Bill Price, Jim Sceviour, Karin Wittenberg Alma Kennedy, Bev Keeling Trish Irvin, Rob Friars, Joan Flower-Ellis, Joan Styles, cousin Dian O'Brien and my many other friends that space prevents me from listing and especially to my two and four footed family. To David, Elaine, Dave, Jennifer and Ashley 1 Chapter 1 INTRODUCTION 1.1 Subject and background The subject of this thesis is the relationship between trace mineral nutrition and reproduction in ruminants. A dietary deficiency of copper (Cu) or excess of molybdenum (Mo) adversely affect reproduction in domestic ruminants and laboratory rodents, but in cattle and sheep, Cu and Mo requirements for reproduction are difficult to define because of complex antagonistic interactions with sulfur (S). The development of reproductive function in lambs is paramount to the efficiency of reproduction in adulthood. Puberty occurs with the establishment of mature endocrine function that is largely dependent upon nutrition, photoperiod and social environment. In ruminant animals S limits the availability of dietary Cu and Mo for metabolic functions by forming cupric and molybdate sulfides that are excreted in feces. However, the S rich environment created by microbial metabolism in the rumen leads to complex systemic interactions that, if prolonged over several months, will induce signs of Cu deficiency. The depleting effect of Mo and S on tissue concentrations of Cu have been studied extensively and may be due to the formation of a series of Mo-S compounds called thiomolybdates. However, long before symptoms of Cu deficiency can be diagnosed, an excess of Mo or thiomolybdates can be identified from a knowledge of the Cu, Mo, S content of feeds (Suttle 1983) and measures of Cu parameters in blood (Lamand et al. 1980). Metabolism of Cu and the antagonistic effects of Mo and S has shown differences between sexes and in sheep, among breeds and with age and stress (Underwood 1981). A better understanding of subclinical Mo and S interactions with reproductive function would improve our ability to formulate diets that meet the Cu, Mo, S requirements and prevent a cause of poor reproductive efficiency in ruminants. The lamb model, levels of Mo and S added to cereal based diets and parameters of Mo and S interaction with Cu metabolism used in this thesis were based on previous work (Robinson 1983; Robinson et al. 1987). In the previous work, various parameters of Cu metabolism, growth and sperm production 2 were studied in ram lambs of different sire breeds given cereal-based diets that contained different concentrations of supplemental Mo (6 and 12 mg kg1) and S (2 g kg1). Parameters of Cu metabolism which included concentrations of Cu in whole blood, plasma and liver and ceruloplasmin oxidase (Ferroxidase E.C. 1.16.3.1), showed differences among diet groups, typical of subclinical Mo and S interaction with Cu metabolism that persisted for 15 weeks. After 15 weeks, when lambs were aged 33 weeks, semen collected by electroejaculation showed that sperm production and maturation was impaired in lambs given Mo+S, but Line M and Suffolk sire breeds were affected more than the Dorset. Although the group given S alone had satisfactory semen quality, this was mostly due to exceptional semen quality for Line M lambs in this group, but the S alone diet had been contaminated with Mo (26 mg kg-1 DM) for a brief period early in the experiment and the effect of S was inconclusive. The results also showed better growth rate for supplemented groups and perhaps stress caused an increase in plasma ceruloplasmin oxidase activity. The study demonstrated that severe Mo and S treatments were not necessary to impair reproductive function in rams and that the condition was subclinical since supplemented lambs grew rapidly and appeared healthy. The effect of Mo and S on spermatogenesis may have been mediated by endocrine dysfunction. 1.1.1 Overview of the pituitary-gonadal-adrenal axis The ewe has estrous cycles of 16-17 days, each cycle normally begins with behavioural estrus (Day 0) and ovulation occurs ~ 24 h later (Karsch and Foster 1980). In the ram, the spermatogenic cycle is of 54 days duration and each wave of cell types at different stages of development along the seminiferous tubule reappears every 10 days (Ortavant et al. 1977). These gonadal cycles are regulated by dynamic long-loop feedback systems of hormone secretions among the hypothalamus, pituitary gland and gonads (testes or ovaries). Figure 1 presents an overview of the hypothalamo-pituitary-gonadal axis. The synchrony of hormone secretions originates from the intermittent (pulsed) release of small peptides synthesized in specialized 3 neurosecretory cells in the hypothalamus. In sheep, gonadotrophin releasing hormone (GnRH) cell bodies are diffusely distributed in forebrain regions that include the pre-optic area, diagonal band of Broca and septum (Lincoln 1988). GnRH is stored and released from axons that terminate in perivascular spaces surrounding capillary loops of the hypothalamo-pituitary, portal system in the median eminence (Polkawska 1981). In sheep, as in other mammals, GnRH is released in pulses and carried by the hypophyseal portal system to the anterior pituitary where GnRH is captured by specific receptors within the cell membrane of gonadotrophs. GnRH stimulation of gonadotrophs signals the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) into the peripheral circulation. Pulsatile release of GnRH induces corresponding GnRH dependent pulses in L H secretion (Clarke and Cummins 1982; Caraty and Locatelli 1988) whereas FSH is not pulsatile and its release can be autonomous once synthesis has been primed by G n R H (Lincoln and Fraser 1987 a,b). In the ewe, FSH stimulation of the ovary initiates follicular development; in the ram FSH stimulation of the testes initiates spermatogenesis (Courot 1988). Binding of L H to its gonadal receptor initiates a cascade of reactions governed by guanine nucleotide proteins within the cell membrane, which eventually activates a cascade of intracellular reactions involving cyclic adenosine monophosphate (cAMP) and phosphorylating enzymes to signal release of gonadal steroid hormones (Johnson and Dhanasekan 1989); principally luteal cell progesterone (P4) (Fitz et al. 1982; Rodgers, O'Shea and Findlay 1983; Leung 1985), or Leydig cell testosterone (Lincoln 1988). Peripherally circulating P4 and testosterone have an inhibitory effect (negative feedback) on L H secretion. In the ram and ewe, estradiol (E2) has an inhibitory effect on the tonic secretion of L H , however in the ewe, E 2 stimulation of specialized cells in the hypothalamus drives a surge release of L H (positive feedback) that induces ovulation. Similarly, hypothalamic pulsed release of corticotropin releasing hormone (CRF) stimulates release of adrenocorticotrophic hormone (ACTH) into the peripheral circulation (Plotsky et al. 1989). A C T H binds to receptors in the membranes of adreno-cortical fasciculata cells (ACF) signalling the release of Cortisol which exerts negative feedback on A C T H release. 4 The pulsed release of GnRH and CRF is regulated, in part, by seasonal variation in length of daylight (photoperiod). Photoperiod stimuli transmitted via the retino-hypothalmic tract eventually reaches the pineal gland through the superior cervical ganglia. The modifying effect of photoperiod on reproductive function (eg. seasonal breeders and delayed puberty in lambs born in autumn) is mediated, in part, by melatonin synthesized in pinealocytes (Malpaux 1988; Lincoln, Libre, Merriam 1989). 1.1.2 Overview of steroid synthesis and catabolism A schematic of steroid synthesis in the adrenal and gonadal organs is presented in Figure 2. Synthesis begins in the inner mitochondrial membrane with the action of cytochrome P 4 S 0 side chain cleavage enzyme catalyzing conversion of cholesterol to pregnenolone (Lambeth and Xu 1989; Jefcoate, McNamara and DiBartolomeis 1986). This hormonally controlled reaction is believed to be a rate limiting step governing the total amount of steroid produced in the adrenal gland and presumably, ovaries and testes. Pregnenolone leaves the mitochondria for the endoplasmic reticulum to serve as a precursor to P4 and androgens. In the adrenal cortex zona fasciculata, A C T H stimulation of this cascade of reactions leads to the production of Cortisol. In the gonads, testosterone, P4 and E 2 are major secretory products in proportions characteristic to the sex. A family of cytochrome P 4 5 0 enzymes catalyze steroidogenesis. For the purpose of this thesis, two Cu containing enzymes are noted; 17, 20 lyase (Cytochrome P 4 5 0 17a) and aromatase. Binding proteins pick up the majority of the steroids that enter the peripheral circulation. In domestic animals, plasma Cortisol is mostly bound to an a-globulin, transcortin for transport and a small amount may be bound to albumin, will the remainder is 'free' having been conjugated with glucuronide or sulfate in the liver (Wilkinson 1980). In human serum, 90% of Cortisol may be bound to globular protein, with a small amount bound to albumin (7%) (Mendel 1989). In male serum, about 50% of testosterone is bound to albumin (30% in females) and about 44% to globulin (66% in female). About 80% of P4 is bound to albumin and 17% to globular proteins. Similarly, albumin bound E 2 accounts for ~ 78% of E 2 in male serum (61% in females), with 20% (37% in female) bound to globular protein. The remaining (~ 2%) steroid is free (unconjugated), but may be conjugated in the liver with glucuronide, or 5 sulfate and excreted in bile. Steroid stimulation of target cells is mediated by specific cytosolic receptors that translocate to the nucleus and bind to specific sites on DNA, stimulating messenger R N A synthesis (gene expression). A plot of pituitary, gonadal-adrenal hormone concentrations in serum of blood samples collected at frequent (10-20 minutes) intervals over an extended period (4 to 24 h) of time will reveal the endogenous rhythm of high and low concentrations; the serum secretory profile. Peak concentrations shown by the serum secretory profile are indicative of endogenous stimulation of hormone release as opposed to peaks induced by an exogenous dose of releasing hormone or agonist. Peaks of L H are often called pulses, whereas Cortisol and testosterone peaks are called episodes. In this thesis, secretory peak refers to the endogenous rhythm of peak concentrations of LH, Cortisol and testosterone in serum. 1.1.3 Glossary Residual copper (RCu) originally used to describe a fraction of plasma containing Cu that does not react with diethyldithiocarbamate (Suttle and Field 1968), in this thesis, RCu refers to the difference, between the amount of Cu in untreated serum (TCu) and Cu in serum that is soluble in 5% trichloroacetic acid (TCA-Cu). The same fraction is also called T C A insoluble. Wether generally means gonadectomized male sheep. In this thesis, wether is used only for male lambs that have been castrated within six weeks of birth and in the experimental procedure refers to single born male lambs castrated at birth. L H R H and GnRH (luteinizing hormone releasing hormone and gonadotropin releasing hormone) in this thesis are used interchangeably for natural or synthetic analogues of the hypothalamic decapeptide that stimulate synthesis and release of LH. Clinical/subclinical - clinical refers to obvious signs of poor animal health that reduce performance such as feed refusal, scrotal hernia, achromatricia, scouring, reduced growth rate such that the animal appears abnormally small for its age. In contrast, subclinical is used to describe metabolic disorders that reduce the efficiency of livestock production without affecting the appearance of the animal, such as the early stages of Cu deficiency or poisoning and infertility (eg. failure to ovulate, poor semen quality, failure to maintain pregnancy). Hypothalamus Pineal gland Melatonin / s u p e r i o r \u00E2\u0080\u00A2 c e r v i c a l g a n g l i a Pituitary gland Adrenal glands Gonadal glands Legend PON preoptic nuclei + AHA anterior hypothalamic area + PVN paraventricular nuclei DHA dorsal hypothalamic area DMN dorsal medial nucleus VMN ventromedial nucleus \u00E2\u0080\u00A2 PHA posterior hypothalamic area ARC arcuate nucleus \u00E2\u0080\u00A2 SON supraoptic nuclei SCN suprachiasmatic nucleus + OC optic chiasm ME median eminence \u00E2\u0080\u00A2 GnRHgonadotropin releasing hormone CRF corticotropin releasing factor FSH follicle stimulating hormone LH luteinizing hormone ACTH adrenocorticotropic hormone T testosterone P4 progesterone E2 estradiol AM adrenal medulla ACF adrenal cortex + controls preovulatory LH and FSH \u00E2\u0080\u00A2 controls tonic secretion of LH and FSH axis Figure 1. Schematic overview of the h y p o t h a l a m o - p i t u i t a r y - gonadal/adrenal and re t ino-hypothalamo transmission of l ight s t i m u l i to the pineal gland. Refer to text for description (From Hafez 1985; Gorbman et al 1983). Cholesterol Pregnenolone (C21) 17aOHPregnenolone 17,20 Lyase (Cu) 20Dihydroprogesterone (ovarian luteal cells) \u00E2\u0080\u00A2^\u00E2\u0080\u00A2PROGESTERONE 17aOHlase -> 17aOHProgesterone 17,20 Lyase (Cu) Aldosterone Tetrahydroaldosterone Dehydrocorticosterone rticosterone -9> Deoxycorticosterone (adrenal zona glomerulosa) 11 -Deoxycortisol \u00E2\u0080\u0094 (adrenal zona fasciculata) \"C OH -J> CORTISOL \u00E2\u0080\u00A2P> Cortisone Tetrahydrocortisone Dehydroepiandrosterone (C19) Sulfatas DHA-SO. Androsterone Androstenedione Estriol 5aDihydrotestosterone (Testicular leydig cells) aromatase (Cu) H O ESTRADIOL-17b (C18) Catechol estrogen or E - S O , (liver/brain) Figure 2. Schematic overview of pr inc ipa l pathways of steroid biosynthesis and catabolism. Unless specified, pathways are common to the adrenal and gonadal glands. Catabolism occurs mainly in the l iver, but water soluble forms are excreted with ur ine ( from Jefcoate 1988; Schwall et a l . 1988; Dufau et al 1984; Gorbman et al . 1983). 8 Chapter 2 LITERATURE REVIEW 2.1 Cu Mo S Nutrition 2.1.1 Cu Mo S Recommended dietary allowances The Cu requirement for sheep ranges from 1 mg (16 /_mol) kg\"1 D M for the preruminant lamb, to 3 mg (47 /_mol) kg\"1 D M for growing lambs and 6 mg (95 jumol) kg'1 D M for all other classes of sheep (ARC 1980). For the preruminant calf and all other classes of cattle, respectively, 2 and 12 mg kg' 1 D M Cu are recommended (ARC 1980). The molybdenum (Mo) requirement for sheep and cattle is estimated to be less than 1.0 mg kg\"1 (10.4 /.mol kg\"1) D M (NAS-NRC 1975). However, this estimate is primarily a level found not to depress tissue Cu. The sulfur (S) requirement for sheep ranges from 1.8 to 2.6g kg\"1 D M for growing lambs, to 1.4 to 1.8g kg\"1 D M for other classes of sheep. A ratio of 1:10, S to nitrogen is also recommended in diets for sheep (NRC-NAS 1975). The availability of dietary Cu to cattle and sheep is largely influenced by the concentrations of Mo and S present in the diet. Ruminants, unlike non-ruminants, are sensitive to Cu-Mo-S interaction because of extensive S metabolism by rumen microbes. The effect of the rumen is clearly shown by the dramatic drop in Cu absorption associated with the onset of rumen function (Suttle 1975b). The coefficient of Cu absorption decreases from 0.90 for the pre-ruminant lamb, to 0.25 for growing lambs (ARC 1980). In sheep, there is also a further decline to 0.06 (ARC 1980). With cereal-based diets containing low S (~ lg kg\"1), low Mo (<1 mg kg\"1), the coefficient is high, 0.10, but a three fold increase in Mo and S concentrations reduces the coefficient to 0.03 (ARC, 1980). Only the magnitude of the effects of Mo and S on Cu absorption differs with forage based diets (Suttle 1983). Langlands et al (1981) recommended at least 6 mg kg\"1 D M Cu when pastures for sheep contained 2 mg Mo and 3g S per kg D M . 9 2.1.2 Causes of nutritional imbalance In ruminants, the trace mineral content of soil can initiate Cu-Mo-S nutritional imbalance by soil ingestion with feed (Suttle 1975a; Suttle, Abraham and Thornton 1984) and by soil-plant interactions that affect the initial trace mineral content of feeds (Reid and Horvath 1980; F A O 1976). A simple deficiency of Cu is usually associated with feeds grown on mineral based soils; whereas Cu-Mo-S interactions more commonly occur with alkaline, peat and calcareous soils (Underwood 1981). Soil and soil-plant interactions have been reviewed (Reid and Horvath 1980; F A O 1976); a synopsis is given here to show a fundamental cause of regional and seasonal effects on dietary trace mineral availability. The availability of minerals to soils begins with soil stability relative to natural weathering (Dudal 1976). The trace mineral content of mineral soils is about 300 kg ha\"1; but in peat soils it is only about 20 kg ha\"1 (FAO, 1976). Availability of trace minerals to plants is primarily a function of solubility as determined by soil properties such as soil organic matter, pH, texture, clay material, oxidation-reduction potential, trace mineral interactions and seasonal variation. Mineral deficiencies for plant growth are primarily due to availability, since an average normal crop yield will remove only about 50g Cu and lg Mo per ha (FAO 1976). Mineral availability is largely reduced by soil organic matter which binds minerals in an insoluble form. Reduced acidity lowers solubility and plant uptake of Cu, but increases that of Mo and S. The addition of lime or phosphate fertilizers, or increased soil pH dramatically increases plant uptake of Mo (Langlands 1981) which has been associated with induced Cu deficiency in sheep and diarrhea in cattle (Underwood 1981). Accumulation of Mo differs between legumes, grasses and grains. Legumes usually contain high concentrations of Mo, which accumulates in the seed and reaches peak concentrations midway through the growing season. In contrast, grasses tend to be low in Mo, which is concentrated in the leaf rather than the seed. Grains tend to accumulate Cu and concentrations are generally greater than those found in grasses or legumes (Winter 1987; Fletcher and Brink 1969). High Cu+low Mo concentrations characteristic of many cereals are potentially toxic to sheep (Todd 1972). 10 Range forage in south central British Columbia has been found to contain low concentrations of Cu, (less than 10 mg kg1) and variable concentrations of Mo ranging from less than one to 12 mg kg\"1 D M . Miltimore et al. (1964) used an injectable Cu supplement to improve average daily gain and hemoglobin in beef cattle on pastures grown on ground water soils in the interior. In 1970, a large scale survey of Cu concentrations showed that 95% of ruminant feeds in British Columbia contained an insufficient amount of Cu for cattle (<10 jig g\"1 D M ; Miltimore, Mason and Ashby 1970) and a subsequent study showed extreme variability in Cu and Mo concentrations in feeds (Miltimore and Mason, 1971). Concentrations of Cu in grass hays, legume hays and grains ranged from below one to 48 /_g g\"1 D M with means of 4.4 \u00C2\u00B1 4.7, 6.7 \u00C2\u00B1 5.5 and 7.4 \u00C2\u00B1 6.5 (n=74), respectively. Molybdenum concentrations in grass hays, legume hays and grains ranged from below one to ten ug g 1 D M with means of 2.0 \u00C2\u00B1 1.9, 1.8 \u00C2\u00B1 1.5 and 1.0 \u00C2\u00B1 0.7 (n=74). Stage of maturity, location and climate likely contributed much to the wide range of Cu and Mo concentrations. In 1977, Peterson and Waldern concluded from a large scale survey of dairy cattle in the Fraser Valley, that poor fertility, was associated with feeds that contained less than a 2:1 ratio of Cu to Mo. Although Cu deficiency was implied by this result, present knowledge of interaction among Cu, Mo and S in ruminant nutrition, does not fully support this hypothesis. 2.1.3 Thiomolybdate Theory There is evidence of complex interactions among Cu, Mo and S in the digestive tract, blood and other tissues, especially liver and kidney (Marcilese et al. 1970; Suttle 1974; Suttle 1975c; Suttle 1977; Bremner and Young 1978; Mason et al. 1978; Lamand et al. 1980). The thiomolybdate hypothesis is an explanation for many of the Cu deficiency symptoms associated with excess Mo in diets for ruminant animals (Mason 1986; Suttle and Field 1983). By definition, thiomolybdates are a series of compounds formed by the progressive substitution of O by S (H2S), in vitro, at neutral pH (Aymonino et al. 1969). The series of compounds produced are mono- (Mo0 3S =), di- (Mo0 2S 2 =), tri- (MoOS 3 =) and tetra (MoS 4 =) thiomolybdates. There has been considerable controversey over the chemical species that is responsible for systemic Cu, Mo and S interactions. Since the 1970's it has been recognized that the effect of Mo on Cu 11 metabolism is dependent upon the level of dietary S (Suttle 1974). Specific populations of bacteria in the rumen utilize S 0 4 = in either a dissimilatory pathway which yields free S = , or an assimilatory pathway which incorporates S in microbial protein (Bird and Hume 1971). Intra-ruminal infusion of M o 0 4 has been found to inhibit S0 4 reduction to S = resulting in an increase in S0 4 absorption from the rumen and a reduction in the amount of free S = (Gawthorne and Nader 1976). This cascade of events has been found to occur especially under more acidic conditions (Bird and Hume 1971), for example when ruminants consume a diet rich in soluble carbohydrate characteristic of cereals (Hungate 1966). In the intestine, Cu, M o 0 4 and S0 4 are absorbed and M o 0 4 and S0 4 are believed to compete for a non-specific transport system for Group IV oxyanions (Mason and Cardin 1977). However, when thiomolybdates are given in the diet or infused this mechanism does not seem to be involved (Mason 1986). It has been demonstrated, using the depletion-repletion technique and comparison between the pre-ruminant and mature Iambs given dietary supplemental di-, tri- and tetra- thiomolybdate, that interaction occurs when MoS 4 with an affinity for Cu is formed, in the rumen, or systemically, when sufficient amounts of each element have been absorbed (Suttle and Field 1983). Dose studies using isotopes of Mo (\"Mo) have shown that di- and tri- thiomolybdates may form systemically (Mason, Lamand and Kelleher 1980; Mason, Kelleher and Letters 1983), but most likely they are reduced to MoS 4 (Suttle and Field 1983). Indirect evidence of thiomolybdates in blood is residual Cu (RCu). Residual Cu is a non-homogeneous fraction containing Cu that does not appear in trichloroacetic acid (TCA) acidified blood serum or plasma collected from ruminant animals given Mo and S, as sulfide or sulfate, as a dietary supplement (Lamand et al. 1980), or an intra-rumen (Mason, Kelleher and Letters 1983;), or duodenal (Mason, Lamand and Kelleher 1980), or intravenous (Ishida, Yoshikawa and Kawashima 1982) dose. At one time it was believed that RCu represented firmly bound Cu-Mo-S complex assumed to be unavailable for metabolism. However, it is now realized that thiomolybdates increase the affinity of albumin for tissue Cu and, as a result, the concentration of albumin bound Cu (direct reacting Cu) in serum increases at the expense of tissue Cu (Mason 1986). 12 The thiomolybdate hypothesis explains the depleting effect of Mo and S on tissue Cu (Mason 1986). In the gut, interaction among Cu, Mo and S is probably influenced by turnover and rate of passage of rumen contents and pH extremes ranging from 5.5 to 7 in the rumen to ~ 2 in the abomasum. Beyond interactions in the digestive tract, Cu, Mo and S interactions occur in the circulation and affect Cu and Mo concentrations in nuclear, cytosolic and microsomal fractions of liver and kidney tissue (Mason 1986). The most serious difficulty with Cu-Mo-S interaction is that cattle and sheep endure a prolonged period of subclinical symptoms before clinical signs manifest. Accumulation or depletion of tissue Cu occurs over a period of several weeks to months, whereas RCu indicative of thiomolybdates can be measured in serum of ruminant animals within 10 to 35 days of Mo and S supplementation. It is possible that Mo or thiomolybdate interference with intracellular metabolic pathways precede significant effects on Cu metabolism. Thiomolybdate or M o 0 4 = interference with intracellular pathways of protein, or lipid synthesis, or hormone action could impair reproductive performance, or alter body composition without an obvious effect on growth, or the general health of the animal. 2.2 Reproduction 2.2.1 Puberty in the ram and ewe Puberty in spring-born ram lambs generally occurs in autumn, when they have reached 22-28 weeks of age and 40-70% of mature body weight, depending upon the breed. At this time, semen contains ~ 1 billion spermatozoa, whereas semen collected from adult rams may contain 6-7 billion sperm with approximately 70-80% motile and 10% abnormal sperm (Hulet and Ercanbrack 1962). Indices of testicular function of lambs include testicular size and tonicity, semen evaluation and serum testosterone (Ott and Memon 1980; Yarney and Sanford 1990a,b). Positive correlation between testicular size and sperm production have been higher for the immature (Notter, Lucas and McClaugherty 1981) and pubertal lamb (Yarney and Sanford 1990a) than the mature lamb (Ott and Memon 1980). However, testicular size measured over time, shows seasonal growth (or pubertal growth) and regression 13 of the testes associated with increasing and decreasing sperm production and motility, can be distinguished (Islam and Land 1977; Mickelson, Paisley and Dahmen 1981). Testicular size and spermatogenic function in yearling Suffolk rams are also positively correlated with mean serum testosterone concentrations at 50 and 150 days of age (Yarney and Sanford 1990b). Semen may be collected by either the artificial vagina or electroejaculation methods for evaluation of semen quality which includes sperm concentration, motility and morphology (Hulet, Foote and Blackwell 1964). First ovulation in spring-born ewes normally occurs in autumn when they have reached 31 weeks of age and a threshold weight ~41 kg, depending upon the breed. Ewes may then exhibit 5.5 ovulatory cycles if pregnancy is not established before the first breeding season ends (Fitzgerald and Butler 1982). Estrous without ovulation often precedes first ovulation and is associated with a transient luteal structure that is apparently uterine dependent (Keisler, Inskeep and Dailey 1983). First ovulation has been defined by P4 measured in blood serum samples taken twice (Fitzgerald and Butler 1982) or three times (Huffman, Inskeep and Goodman 1987) weekly, since concentrations of P4 greater than one ng ml\"1 in serum have occurred with the development of a functional corpus luteum. Small transient increases in serum P4 have occurred during the pre-pubertal/anestrous phase (Fitzgerald and Butler 1982), which subsequent study has indicated may be required for transition from anestrous to estrous phase of ovarian function (Legan et al. 1985). Pulsatile secretion of P4 during the late luteal phase, with no temporal relation to L H , has been reported recently in the ewe (Alecozay et al. 1988). Vaginal smears have been used to follow stages of differentiation of the vaginal epithelium associated with the estrous cycle (Sanger, Engle and Bell 1958; Ducker and Bowman 1970; Newsome and Kitts 1977). Using vaginal smears, extensive cornification of the vaginal epithelium has been characterized in response to high concentrations of phytoestrogens in legume feeds (Sanger, Engle and Bell 1958; Newsome and Kitts 1977). The rate of sexual maturation can be influenced by ram and ewe proximity (Illius et al. 1976; Fitzgerald and Butler 1988), nutrition (Fitzgerald, Michel and Butler 1982; Lindsay et al. 1984) and season of birth (Skinner and Rowson 1968; Fitzgerald and Butler 1982), photoperiod (Howies, Webster and 14 Haynes 1980; Ebling and Foster 1988) and stress (Downey, Gunn and Griffiths 1973). These effects can be explained, in part, by effects on pituitary secretion of L H . 2.2.2 Pituitary gonadal development 2.2.2.1 Hypothalamo-pituitary development in utero Pituitary secretion of L H is pulsatile as a result of intermittent release of L H releasing hormone (GnRH or LHRH) from hypothalamic neurons that terminate in the median eminence (Figure 1). A functional hypothalamo-pituitary portal system has been demonstrated in the fetal lamb as early as 45 days of gestation (Matwijiw, Thliveris and Faiman 1989) and GnRH dependent pulsatile release of L H , has been demonstrated as early as 81 days of gestation (Clarke et al 1982; Matwijiw and Faiman 1987). Pulsatile secretion of L H is evident in the neonate lamb by 1 week of age in the ram (Foster et al. 1978) and before 8 weeks of age in the ewe (Foster, Jaffe and Niswender 1975). 2.2.2.2 Pituitary-testes development At birth, androgen production by the testes is present, thereafter testosterone becomes the dominant androgen rather than androstenedione (Skinner et al. 1968). Pituitary gonadotrophin content and testicular weight begins to increase sharply after rams have reached 6 weeks of age (Skinner et al. 1968). Although spermatogenesis begins at around 8 weeks of age, spermatozoa do not appear in the ejaculate until about 16 weeks of age (Skinner and Rowson 1968). Pulsatile secretion of L H followed by increments of testosterone are evident in serum of neonate rams as early as one week of age (Foster et al. 1978). Carr and Land (1975) showed that mean serum L H at 12 weeks of age was positively correlated with testicular size and sperm output at puberty and concluded that ram lambs with higher mean L H at 12 weeks of age matured earlier as long as elevated levels disappear at a young age. Inhibition of L H secretion using P4 implants given to two week old ram lambs for 4, 8, or 12 weeks delayed puberty until L H peak frequency increased following implant removal (Echternkamp and Lunstra 1984). McNeilly et al. 15 1986 studied FSH, L H and testosterone secretory profiles in Finn-Dorset lambs from six to 76 wks of age. Lambs had been selected for high and low testicular diameter at six, ten and 14 wks of age. Testicular growth was delayed in lambs selected for low testicular diameter, but at 72 wks of age, body weight was about 10 kg heavier than lambs selected for high testicular diameter. Prior to 20 wks of age, lambs selected for low testicular diameter exhibited high amplitude low frequency L H peaks. In contrast, lambs selected for high testicular diameter experienced low amplitude high frequency L H peaks. Temporal patterns of L H and testosterone measured in serum collected from the developing ram have shown that L H peaks increase frequency, but decrease in magnitude while concentrations of testosterone gradually rise. Although testosterone and L H concentrations differ among breeds, the temporal patterns are conserved (D'Occhio, Schanbacher and Kinder 1984). Associated with temporal patterns are an increase in the number of L H receptors in the testes (Yarney & Sanford 1989; Monet-Kuntz 1984). A similar temporal pattern exists in the mature ram during testicular redevelopment in autumn; with the transition from long to short day photoperiod, L H peaks decrease in amplitude but increase frequency (Sanford, Howland and Palmer 1984a). The temporal patterns indicate that in the ram, the pituitary becomes less sensitive to the negative feedback of testosterone, while the testes become more sensitive to L H . The pituitary also becomes less sensitive to GnRH since L H peak amplitude in response to a 10 /_g single iv injection of GnRH was greater between spring and summer (May-Aug) than autumn and winter (September to April) (Sanford, Howland and Palmer 1984a). Pituitary sensitivity is also affected by social environment and mating. Mating in July or autumn increased L H frequency, whereas observing and mounting ewes caused depressed frequency and mounting depressed amplitude (Yarney and Sanford 1983). Variations in L H peak amplitude in the ram have been attributed to the influence of gonadal steroids on the pituitary (Schanbacher 1980a,b). The modulating effect of gonadal steroids on gonadotropin secretion in rams is revealled by the dramatic increases in serum L H concentration which follow gonadectomy (Schanbacher and Ford 1977). The post-castration rise in L H can be prevented with testosterone implants (Schanbacher and D'Occhio 1985), but not 5\u00C2\u00ABDHT, demonstrating the inhibitory effect of testosterone on L H secretion. However, testosterone given in combination with L H R H restores 16 the serum L H profile of castrate rams, indicating that the post castration rise in L H is due to increased L H R H discharge and that the hypothalamus, rather than the pituitary, is the principal site for androgen feedback, even though androgen receptors are present in pituitary cytosol in rams (D'Occhio 1984) and ewes (Clarke et al. 1982). Differential control of L H secretion by E 2 has been demonstrated in the intact and castrate ram. In the castrate ram implants of either E 2 , testosterone, E 2 plus testosterone, but not 5aDHT diminished L H and FSH, within six hours of treatment. Although testosterone maintained lower L H , there was a rebound increase with either E 2 treatments (Schanbacher and Ford 1977). Ram lambs immunized against E 2 had larger testes, slightly higher testosterone and normal L H . In contrast, testosterone immunization increased serum L H several fold, without affecting testicular size (Land, Baird and Carr 1981). Subsequently, it was demonstrated that Ej functions to regulate the frequency of L H peaks in the ram. Immunoneutralization of E 2 in mature rams effectively increased serum L H and induced short day L H profiles during long day photoperiod (Schanbacher 1984). Aromatization of testosterone to E 2 other than those specifically coupled to testicular steroidogenesis (eg. peripheral aromatization) may also affect pituitary gonadotropins (Schanbacher 1984). Differential regulation of L H secretion has also been demonstrated in rams in which one testes has failed to descend at birth. With cryptorchidism, which occurs naturally in ~0.5% of rams (Ott and Memon 1980), or with surgical cryptorchidism, testes weights, seminiferous tubule diameters and testicular sperm numbers are greatly reduced, but serum levels of testosterone remain normal while serum L H concentrations may be higher than normal (Hillard and Bindon 1975). Surgical cryptorchidism, at six weeks of age, has resulted in increased serum L H without affecting either pulsatile secretion of L H , or episodic secretion of testosterone, at nine months of age (Schanbacher and Ford 1977). In the cryptorchid ram, E 2 , but not testosterone, treatments effectively reduced L H , without a rebound increase that occured in rams (Schanbacher 1984).. Therefore, in the cryptorchid ram E 2 was the main inhibitor of L H , and testosterone provided a precursor that could be aromatized. It is generally accepted that E 2 formed peripherally and centrally mediates the action of testosterone on hypothalamic-pituitary function (Schanbacher 1984). 17 2.2.2.3 Pituitary-Ovarian development The hypothalamic pituitary ovarian axis of the lamb has the potential for mature function long before the normal age of puberty; however, follicular development to the preovulatory stage does not normally occur before 30 weeks. This delay in the onset of ovulation can be explained, in part, by pituitary secretion of L H . Pulsatile secretion of L H and E 2 during the follicular phase in ewes precedes ovulation (Baird 1978). For the ewe, pituitary L H secretion must acquire pulsatile (tonic release of LH) as well as cyclic (surge release of LH) patterns to establish and maintain normal estrous cycles. The initiation of the sequence of events that culminate in the timing of the first ovulation in the ewe is dependent upon increased L H peak frequency (Huffman, Inskeep and Goodman 1987). High frequency L H peaks have been detected in the postpubertal lamb (Foster et al. 1975) and mature ewe (Karsch et al. 1983) during the follicular phase when one or more large follicles develop and E 2 secretion becomes maximal. By contrast, low L H peak frequency (less than 1 per h) occur in prepubertal ewes (Foster et al. 1975). Physiological doses of L H given hourly, to the immature lamb (18-20 weeks) replicates the sequence of events that occur during the follicular phase, including the sustained increase in circulating E 2 (Foster, Ryan and Papkoff 1984). In the immature lamb (19 wks), an L H surge can be induced by endogenous E 2 in response to exogenous L H , indicating that the signal for the pituitary to release a surge of L H is as sensitive as that of the mature ewe to the stimulatory feedback of E 2 (Foster 1984). The hypothalamus-pituitary unit becomes less sensitive to E 2 feedback because doses that will suppress L H during the prepubertal period are ineffective in the mature ovariectomized ewe (Foster and Ryan 1979). Thus, as ewe lambs mature the pituitary becomes less sensitive to the inhibitory feedback action of E 2 , L H peak frequency increases with decreasing magnitude (Huffman, Inskeep and Goodman 1987) until induction of the L H surge prior to ovulation. The changes in L H secretion reflect change in GnRH pulse frequency which has also been shown to alter the amplitude of L H peaks by affecting the amount of releasable L H in the pituitary (Clarke and Cummins 1985). 18 2.2.2.4 Desensitization In the ram, as pubertal development procedes the pituitary becomes more responsive to the inhibitory effect of testosterone and less sensitive to the stimulating effect of GnRH. In the ewe, for ovulation to occur, the pituitary becomes less responsive to the inhibitory effect of E 2 and more sensitive to the stimulating effect of GnRH (Fitzgerald et al. 1985). Regulation of the frequency and amplitude of L H release by the hypothalamic release of GnRH is a dose and time dependent (biphasic) response, in which overstimulation can lead to desensitization. In the ram, prolonged exposure to high doses of GnRH, will suppress pituitary responsiveness to a single exogenous dose of GnRH (Lincoln 1979b). Rams were given either a 100 or 500 Lig dose of G n R H every two hours (pulsed infusion) for 23-57 weeks. Treatments effectively induced temporal patterns of L H and testosterone associated with increased testicular sizes. At the end of the trial, all rams were given a single i.v. dose of GnRH. The magnitude (amplitude) of the L H peak was diminished, without affecting testosterone, in rams treated with pulsed infusion of 500 jig GnRH. Pituitary desensitization was due to high titres of GnRH acting on the pituitary (Fraser and Lincoln 1980). In the ewe, GnRH agonists attenuated E 2 negative feedback on L H (Fitzgerald et al. 1985). The effect of E 2 on pituitary ovarian development is dose and time dependent since puberty can be delayed by 3 weeks (37 vs 34) in ewe lambs given chronic E 2 treatment (Foster et al. 1986). 2.2.3 Photoperiodism Photoperiod modulation of gonadal development is a cranial sympathetic mechanism, since removal of the superior cervical ganglia has prevented seasonal regression of the testes, or changes in L H secretion in the ram (Lincoln 1979a) and delayed puberty in the ewe (Yellon and Foster 1984). Ewe lambs require exposure to long followed by short photoperiod for puberty to occur at a normal age (Yellon and Foster 1985). Spring-born ewes maintained under constant long days during their first year of life, experienced either no ovulation, or short luteal phases. However, exposure of ewes to continuous short days also delayed ovulation (Yellon and Foster 1985). By the time the ewe reaches ~20 weeks of 19 age she is photoreceptive in that she recognizes that the combination of long days and short days for the first ovulation to occur at a normal age (Yellon and Foster 1985). In fall-born ram lambs raised in an environment of continuous long days (12D:12L), basal testosterone increased and L H decreased, but there was no association with change in peak amplitude or frequency (Klindt et al. 1985). Concentrations of L H and testosterone in serum also show differences among breeds, especially during natural (Sanford, Palmer and Howland 1982) or artificial short day photoperiods (D'Occhio, Schanbacher and Kinder 1984). Photoperiod influence on reproduction is mediated by the secretion of melatonin by the pineal gland. Melatonin concentrations are high at night and low during the daylight hours. Higher concentrations during the day have only occurred in the prepubertal lamb. Studies of puberty in ewes given melatonin at different ages have indicated that long day melatonin patterns enable the lamb to recognize the short patterns of autumn (Foster, Yellon and Olster 1985). In the ram, modulation of seasonal patterns of L H by melatonin may involve regulation of E 2 feedback on the pituitary since immunoneutralization of E 2 prevented a change in L H secretory profiles from temporal patterns of short days (high frequency, low amplitude L H peaks) to long days (low frequency, high amplitude L H peaks) (Schanbacher 1984). 2.2.4 Adrenal effects on the pituitary-gonadal axis 2.2.4.1 Stress and Cortisol secretion A variety of environmental factors and management practises impose stress which suppresses reproductive function in sheep and cattle (Moberg 1985a,b). Estrus, ovulation, implantation and spermatogenesis are affected adversely by stress such as heat and transportation. The effects on reproductive function may occur as a result of adrenal glucocorticoid or progestin actions on the pituitary or the gonad and may be independent of A C T H . Cortisol concentrations have generally been used to identify adrenal response to stress. The Cortisol response to stress can be induced with A C T H but not prolactin, which has also been reported to be elevated during periods of stress (De Silva, Kaltenbach and Dunn 1983). 20 Circadian rhythm and episodic secretion of Cortisol concentrations in blood have been shown in sheep (McNatty, Cashmore and Young 1972; Fulkerson and Tang 1979), the spotted leopard (Wildt et al. 1986) and other mammals and rodents (Calvano and Reynolds 1984). Newborn lambs have also had higher resting and stress response levels of serum corticosteroids than neonate or mature lambs (Moberg, Anderson and Underwood 1980). In sheep, serum Cortisol concentrations were found to be highest at night and lowest in midafternoon with rhythmic episodic peaks (greater than 10 /_g L 1 ) among and within (ultradian) hours (Fulkerson and Tang 1979). In cows, serum Cortisol concentrations were found to be highest at noon and lowest at midnight and also higher after nursing or machine milking (Wagner and Oxenreider 1972). Serial blood sampling by tail clip of male rats may disrupt corticosteroid rhythms (Calvano and Reynolds 1984), but serial blood sampling by jugular venipuncture of rams had no apparent effect on serum Cortisol concentrations (Yarney and Sanford 1983). Serum Cortisol rhythms may be regulated by the pineal gland since in mature ewes maintained in a natural outdoor environment, serotonin prevented a ~ 15 fold increase in serum Cortisol in response to restraint stress (Frey and Moberg 1981). Cortisol may also be involved with aldosterone secretion. Induced hypotension in ewes stimulated an increase (20 to 46 /_g L\"1) in serum Cortisol associated with depressed hematocrit (26% PCV) and inhibition of renin-angiotension response to low blood pressure (Wood and Silbiger 1987). 2.2.4.2 Cortisol effects on pituitary-gonadal function Correlations between high concentrations of circulating Cortisol and depressed L H and FSH secretion have occurred only during the early phases of stress and experiments have indicated that L H and FSH are not similarly affected and not all changes can be attributed to the adrenal axis (Moberg 1985b). However, in cattle and sheep an inhibitory effect of corticosteroids on L H secretion has been consistently demonstrated. Welsh and Johnson (1981) showed a temporal relationship between electroejaculation and increased circulating Cortisol and depressed plasma L H peak frequency and testosterone concentrations in bulls. However, in bulls pulsatile secretion of L H recommenced when circulating corticosteroids returned to basal levels after an exogenous A C T H induced a six hour peak in plasma corticosteroid concentrations 21 and suppressed L H secretion (Johnson, Welsh and Juniewicz 1982). In rams, restraint caused increased serum Cortisol, but only A C T H suppressed L H response to L H R H (Matteri, Watson and Moberg 1984). An increase in serum Cortisol immediately following ejaculation seems to be a normal part of sexual behaviour (Sanford, Palmer and Howland 1974; Yarney and Sanford 1983; Martin, Lapwood and Elgar 1984; Wildt et al. 1986). However, serum Cortisol concentrations are higher following electroejaculation than following collection of semen by artificial vagina (Martin, Lapwood and Elgar 1984), but it is not known if high serum Cortisol is the reason for poorer quality ejaculates after electroejaculation (Hulet, Foote and Blackwell 1964). The extremely high serum Cortisol concentrations that occur in the clouded leopard (up to 400 ug L 1 ) are thought to be related to the extremely high proportion of abnormal spermatozoa in their ejaculates (Wildt et al. 1986). In cows, restraint stress was found to depress L H only if the treatment caused a 20 fold increase in Cortisol; a two or four fold increase in Cortisol had no effect on L H (Echternkamp 1984). The preovulatory L H surge has been inhibited in dairy heifers given exogenous A C T H (Matteri and Moberg 1982). In the anestrous ewe, treatment with large doses of synthetic Cortisol (dexamethasone) or Cortisol did not prevent Ej stimulation of L H release (Moberg, Stoebel and Cook 1981). Incubation of ewe hypothalamic extracts with Cortisol increased activity of peptidases, indicating that Cortisol may enhance degredation of GnRH (Swift and Crighton 1979). Therefore, it seems that large increases in circulating Cortisol are required to induce adverse effects on the pituitary-gonadal axis in ruminant animals. The mechanism of stress induced suppression of reproductive function is poorly understood, but suppression of pituitary sensitivity to L H R H is at least partly responsible for short term or acute responses to stress. 22 2.3 Nutrition-Reproduction 2.3.1 Nutrition Reproduction studies in sheep Nutrition and puberty may be linked by gonadotropin secretion in sheep (Fitzgerald 1984), cattle (Day et al. 1986), humans (Ojeda 1980) and rats (Howland and Ibrahim 1973). Energy restricted diets, prepared by substitution of forage for concentrate (80:20% versus 30:70%), given to ewe lambs delayed the occurrence of first ovulation and reduced serum L H , but did not prevent ovulation in response to pregnant mare serum gonadotropin (Fitzgerald and Butler 1982). Undernutrition may delay puberty more severely in ewes than rams. For example, a low energy diet that suppressed growth rate of lambs, delayed the occurrence of first ovulation in ewes, but had no effect on testicular growth or the appearance of spermatozoa in the epididymis of rams (Fitzgerald 1984). However, in ewe and ram lambs, pituitary responsiveness to L H R H was suppressed, but there was no effect on the post-castration rise in L H concentrations or suppression of L H after replacement (Fitzgerald 1984). Nutritional treatments used by Foster and Olster (1985) involved restricted intake of a commercial cereal-based diet to a level that maintained body weight of ewes at 20 kg in contrast to 40+ kg body weight with unrestricted feed intake. At various ages, serum secretory profiles of L H were evaluated, before and after ovariectomy, refeeding and implants. Restricted feed intake delayed the onset of ovulation as determined by serum P4, reduced L H peak frequency, eliminated pulsatile L H secretion in response to, but did not prevent induction of an L H surge (positive feedback). Subsequent studies of growth-retarded ovariectomized lambs have shown that severe undernutrition causes low L H peak frequency, but maintains pituitary responsiveness to GnRH without affecting growth hormone or prolactin (Foster et al. 1989). Refeeding restores L H pulsatile secretion which is associated with increased synthesis storage and secretion of L H (Landefeld et al. 1989). The model used by Foster has also shown that FSH is also responsive to refeeding (Padmanabhan et al. 1989). Infusion of a free fatty acid mixture containing linoleic, oleic, 23 palmitic, linoleic, stearic and <1% myristic, arachidic and behenic had no effect on L H secretory profiles in the mature ewe (Estienne et al. 1989). Lindsay et al. (1984) examined the effect of restricted protein (66g versus 49 g/kg) and photoperiod on L H and testicular development of winter born He de France rams. Treatments were introduced at 10 months of age. Blood samples were collected at hourly intervals over a twelve hour period. Protein restriction reduced the total number of L H peaks, without affecting testicular diameter or volume, or L H response to photoperiod. The rate of decreasing daylength stimulated pulsatile release of L H and growth of the testes. Peak frequencies were lowest around the solstices and highest around the equinox. With energy or protein restricted diets, sexual maturation may be delayed due to impairment of hypothalamic regulation of L H peak frequency (Fitzgerald 1984; Lindsay et al. 1984). Although severe undernutrition of rats may amplify the negative feedback action of E 2 on L H secretion (Howland and Ibrahim 1973), this does not seem to be the case in sheep (Foster and Olster 1985). 2.3.2 Cu-Mo in reproductive function 2.3.2.1 In vivo Impaired reproductive function in ruminants and rodents has been associated with low Cu and added dietary Mo. Poor libido has been associated with low plasma Cu in rams grazed on low Cu pastures (Weiner, Hayter and Field 1976). In ewes given low Cu diets (~ 1 mg kg1) pregnancy has been maintained, but the lambs died during neonatal life because of incomplete development of the nervous system in utero. This disease, known as swayback or enzootic ataxia, also occurs in ewes given Mo to induce low concentrations of Cu in tissues (Suttle and Field 1968). In rats given supplemental Mo estrous cycles were prolonged and there was an increase incidence of fetal resorption and impaired embryogenesis, especially myelination in the spinal cord (Fungwe et al. 1987). In contrast to excess Mo, tungsten-induced Mo deficiency was associated with suppressed growth and a greater frequency of abortions and stillborn lambs (Anke et al. 1985). Poor semen quality has been reported for ram lambs given dietary supplemental 24 Mo+S (Robinson 1983) and sterility has been induced in bull calves given extremely high dietary concentrations of Mo (400 mg kg1) (Thomas and Moss 1958). Phillippo et al. (1982) found that pregnancy failure occurred in beef heifers given supplemental Mo (lOmg kg\"1), not in heifers with low tissue Cu induced with Fe. A subsequent study (Phillippo et al. 1987) showed that dietary Mo supplementation delayed puberty, decreased conception rate and caused anovulation in heifers. In rats, the effects of Mo on estrous and pregnancy were associated with increased serum E 2 , decreased FSH and increases in S and Mo enzymes; sulfate oxidase and xanthine oxidase (Liu et al. 1986). Several experiments have shown a positive relationship between steroid hormones and Cu levels in tissues of a variety of animals (Evans, Cornatzer and Cornatzer 1970; Schreiber, Pribyl and Jahodova 1980), which has been explained in part, by enhanced mobilization of hepatic Cu (Russanov, Banskalieva and Ljutakova 1981; Weiner and Cousins 1983). 2.3.2.2 In vitro Cuproproteins superoxide dismutase, ceruloplasmin, C17, 20-lyase and aromatase are all involved in aspects of gonadal function. Superoxide dismutase is present in ram semen and spermatozoa (Abu-Erriesh, Magnus and Li 1978) and ceruloplasmin ferroxidase activity has also been detected in ram seminal plasma (Robinson 1983), ceruloplasmin-like protein secretion by rat Sertoli cells has also been reported (Skinner and Griswold 1983). The Cu is believed to prevent oxidative damage to sperm. In steroid synthesizing cells, the enzyme 07,20-lyase competes with cytochrome P 4 5 0 21-hydroxylase for 17-OH-progesterone substrate, which serves as a precursor for androgens and glucocorticoids respectively; while aromatase is required for the conversion of testosterone to E 2 . Ceruloplasmin polyphenol oxidase (EC 1.10.3.2) and dopamine 8 hydroxylase (EC 1.14.17.1) and monoamine oxidase (EC 1.4.3.4) are involved with catecholamine metabolism and pituitary function. In the rat Cu attenuation of pituitary growth in response to has E 2 been reported (Schreiber, Pribyl and Jahodova 1980). Although dietary Cu deficiency has not been identified with deficiencies of these enzymes (Mills, Dalgarno and Wenham 1976), low 25 norepinephrin content of bovine adrenals coupled with depressed km of dopamine B hydroxylase for tyramine has been reported (Hesketh 1981). Copper and thiol regulation of GnRH stimulation of L H release has been demonstrated using pituitary membrane preparations from immature rats (Hazum 1983). The apparent affinity of the pituitary receptor for GnRH was depressed by Cu ions (30 nM), whereas higher doses (100 nM) induced an eight or 18 fold increase of basal L H secretion. In contrast, S (dithiothreithol) inhibited GnRH stimulated L H release without altering receptor binding affinity for GnRH (Hazum 1983). A physiological function of blood borne Cu may be rapid, energy dependent interaction with the L H R H granule to stimulate L H R H release since Cu-His has been a potent stimulant of L H R H release from isolated hypothalamic granules with an apparent km of 4 LtM Cu (Barnea and Cho 1984). Molybdenum as sodium or ammonium molybdate (1 to 10 mM), has been used to inhibit binding of the cytosolic steroid-receptor complex to the nuclear receptor where regulation of gene expression occurs. The activation of the cytosolic steroid-receptor is believed to be inactivated by non-specific ionic interactions with the receptor, or a cytosolic component required for activation (Noma et al. 1980; Meuller et al. 1982; Naray et al. 1983; Weisz, Baxter and Lan 1984). After duodenal infusion, Mo exists in blood as the M o 0 4 = ion, freely diffusable (Mason et al 1978) and perhaps the inhibiting effect of Mo on receptor activation occurs in vivo. Although M o O = was found to freely cross lymphocyte membranes, its action on the glucocorticoid receptor in vivo was questioned (Naray et al. 1983). By virtue of the nature of the interaction among Cu, Mo and S in diets for ruminants, the effects on reproduction may result from an excess or deficiency of Cu, or Mo, or thiomolybdates. From the literature reviewed, the interface between diet and subclinical impairment of reproductive function is possibly the pituitary. 26 Chapter 3 HYPOTHESIS Based on the literature reviewed the effects of Mo and S on reproduction may be mediated by pituitary dysfunction. There is evidence that a dietary deficiency of Cu (Underwood 1981), or dietary supplemental Mo (Phillippo et al. 1987) or Mo+S (Robinson 1983) impairs reproductive function in ruminant animals. Because of the limiting effect of Mo and S on Cu metabolism, there have been unsuccessful attempts to use supplemental Cu to treat fertility problems in cows (Whitaker 1982). The roles of Cu, Mo and S may not necessarily be interdependent. It will be necessary to identify the effects of Mo, S and Mo*S interaction on reproductive function before it is possible to formulate diets that promote reproductive efficiency. The experiments described herein, were designed to determine the effects of Mo, S and Mo+S on reproductive function. Cereal-based diets were used since they typically contain Cu in excess of requirements for sheep (Todd 1972) and therefore Cu intake was not expected to be severely limited. Cereal-based diets were also expected to supply adequate energy and with appropriate supplementation, adequate protein, macrominerals and trace elements so that these major nutrients would not be limiting. The level of supplemental S used (2g kg1) was based on previous work that demonstrated its limiting effect on Cu and Mo concentrations in tissues (Suttle 1974; 1975c; Robinson et al. 1987). Lambs were used because this stage of development is a critical determinant of adult reproductive performance and was therefore expected to be most sensitive to dietary treatments. Experiment I was designed to compare supplemental Mo (26 mg kg1) with supplemental Mo in combination with S on reproductive function. The level of supplemental Mo used was based on previous work that suggested that this level induced a shift in the distribution of Cu in plasma within two weeks (Robinson 1983). The objectives were to determine the effects of short term exposure to experimental diets on pituitary (LH) and testes (testosterone) function, serum Cu concentrations and growth. Experiment II was designed to identify the effects of supplemental Mo (12 mg kg1), supplemental S and supplemental Mo and S in combination on reproductive function. The level of Mo used was based 27 on previous work that demonstrated that this level of Mo impaired spermatogenesis in lambs (Robinson 1983). The objectives were to determine the effects of long term exposure to experimental diets on serum Cu and Mo concentrations, growth and pituitary (LH), gonadal (testosterone and P4) and adrenal (Cortisol) function. Since gonadal feedback on the pituitary may mediate the effects of undernutrition (Howland and Ibrahim 1973), ewe, ram and wether lambs were used to determine if gonadal feedback influenced the effects of the experimental diets on pituitary and(or) adrenal function. Wethers were ram lambs castrated at birth to ensure an absence of post-natal gonadal feedback on pituitary function. Wethers were also expected to provide a control for gonadal influence on photoperiod regulation of pituitary and adrenal function. Because it was only possible to use a limited number of animals, an attempt was made to minimize among animal variation by using purebred, genetically uniform (ie. same sire) animals, and to control variation due to photoperiod effects on hormone profiles, blood samples were collected at a fixed time relative to sunrise throughout the experimental period which spanned between the summer and winter solstices of 1985. 28 Chapter 4 MATERIALS AND METHODS 4.1 Animals and housing 4.1.1 General Both experiments utilized lambs selected from the flock maintained at the University of British Columbia, South Campus farm located 49\u00C2\u00B0 15' latitude and 123\u00C2\u00B0 14' longitude. 4.1.2 Experiment I (Ram lambs) Twelve ram lambs (11 Suffolk x Dorset and 1 Dorset x Dorset), were used to examine the effect of short term exposure to supplemental Mo alone or in combination with S, on pituitary-testes function. The lambs were born between March 21th and April 5th, 1984 and all were polled except one. At approximately three months of age, they were randomly assigned to one of three treatment groups. The lambs were allowed two weeks to adjust from mashed to pelleted diets and to increase feed intake from 1000 to 1300 g/lamb d\"1. This period also included one week for lambs to adjust from group to individual housing. During the experimental period, the lambs were kept in pens constructed of wood upon wooden slats in the south east corner of the main sheep barn at South Campus. To hold feed and water, two plastic pails were attached to each pen. Pens and feed pails were cleaned and hosed with water, daily. The sampling protocol of the experiment began on August 8th, 1984, when ram lambs were aged 18.4 \u00C2\u00B1 0.8 weeks and weighed 36.6 \u00C2\u00B1 2.0 kg. 4.1.3 Experiment II (Ram, wether and ewe lambs) Twenty polled Dorset x Dorset lambs, offspring of one ram, aged six years (#77K), were used to examine the effect of long term exposure to supplemental Mo and S alone or combined on 29 pituitary-gonadal and adrenal function. Twelve ram lambs, born between November 18th and December 8th, 1984, were selected at birth and either left intact (Rams n=8), or castrated (Wethers n=4). Eight ewe lambs, born between March 3rd and March 10th, 1985, were selected at weaning. Two rams, two ewes and one wether were randomly assigned to each treatment. Rams and wethers were introduced to experimental diets, on March 18th, 1985, when they were aged 13.7 \u00C2\u00B1 0.5 weeks and weighed 31.6 \u00C2\u00B1 3.9 kg. Ewes were introduced to experimental diets, on May 5th, 1985, when they were aged 8.6 \u00C2\u00B1 0.4 weeks and weighed 29.2 \u00C2\u00B1 3.6 kg. Lambs selected for the experiment, were moved from group housing with the main flock and individually penned on the south side of the Small Ruminant Research barn (formerly known as the Wildlife Unit) at South Campus. The barn had not been used for a long period of time and considerable effort was taken to accomodate the lambs. The large pens were partitioned into two pens by three foot fencing. Each pair of pens were partitioned by floor to ceiling fencing. Wooden feed troughs were constructed for the sheltered portion of each pen. Plastic piping was run from the small animal research unit to supply water to nipple-tipped taps located at the south east, or west corner of each pen. The pens were constructed on concrete which sloped south to allow drainage to a concrete gutter. Initially, it was planned not to use bedding and to maintain cleanliness by hosing the pens with water, however, the concrete floor chafed some lambs and it was decided to use wood chips for bedding. The pens were scraped and hosed as required, approximately once per week, but fresh wood chips were added daily to each pen. The lambs were shorn on July 16th and until September 3rd, ewes were kept in pens on the south east side of the barn. From September 4th to completion of the experiment, the lambs were arranged in repeated sequences of ram, ewe, wether. This arrangement was to allow male-female proximity, which has been known to enhance reproductive development of lambs. When the sampling protocol of the experiment began on June 20th, 1985, rams and wethers were aged 29.2 \u00C2\u00B1 0.8 weeks and weighed 52 \u00C2\u00B1 4 kg, while ewes were aged 14.9 \u00C2\u00B1 0.3 weeks and weighed 2 9 \u00C2\u00B1 4 kg. 30 4.2 Dietary treatments 4.2.1 General For both experiments, ammonium molybdate [(NH4)6Mo702 4-4H20] and sodium sulfate (Na2S04), were used to achieve different concentrations of Mo and S in cereal-based diets. The prescribed amount of supplement was substituted for wheat shorts to which a small amount of molasses was added to prepare a premix. The premix was then mixed with the cereal ingredients to provide 80 to 90% of the diet offered to lambs. All lambs were given 200 to 250g of hay per day and free continuous access to tap water. 4.2.2 Experiment I (Groups I, II, III) Three treatment groups consisted of lambs given diets that were either unsupplemented (Group I), to serve as a control, or supplemented with Mo alone (Group II; 26 mg kg\"1 Mo), or in combination with S (Group III; 26 mg Mo plus 2g S kg\"1). The diets were prepared from commercial sheep mash that was ground, mixed with one of the three premixes, then pelleted. The lambs were offered 650g of the pelleted cereal-based mixture with 250g of alfalfa cubes, plus fresh water (~4 L) twice daily, for a period of 30 days. 4.2.3 Experiment II (Groups I, II, III and IV) Four treatment groups consisted of lambs given diets that were either unsupplemented (Group I), to serve as a control, or supplemented with Mo (Group II; 12 mg kg\"1 Mo), or S (Group III; 2g kg\"1 S), or Mo+S in combination (Group IV). The diets were prepared from crushed barley (73.75%), wheat shorts (6.25%), 32% protein supplement (18.75%) and molasses (1.25%). After thorough mixing, the diets were stored in plastic bins. The cereal-based mixture and hay for each lamb, were weighed and transferred to a plastic pail that was labelled for each morning and afternoon feeding. The cereal-based ration was gradually increased, but the amount of hay, which was either alfalfa-brome or timothy hay, was held 31 constant at 200 g per day. Increasing increments (~ 50 g d1) of the concentrate ration were offered when lambs consumed all of the ration given the previous day. Rams and wethers were given 1100 to a maximum of 1450 g per lamb per day, but in September, the amount was gradually cut back to 1300 g per lamb per day. Ewes were offered 800 to a maximum of 1300 g per lamb per day. During the last 12 weeks of the experimental period (October 1st to December 20th, 1985), all lambs were offered 1300g of the cereal-based mixture per day. Ram, wether and ewe lambs were maintained on the experimental diets for 39, 39 and 32 weeks, respectively. 4.3 Sampling procedures 4.3.1 Feed and feed intake (Experiments I and II) Experimental diets were sampled at random throughout the experimental period. Approximately 250 ml samples were taken from bins where each cereal-based mixture was stored after each batch was prepared. For Experiment I, diets for each treatment group were prepared in one batch. Four samples for each treatment group were pooled, mixed, then duplicate subsamples were transferred to plastic bags and stored at room temperature. For Experiment II, seven batches of diets for each treatment group were prepared. Four samples per month, for each treatment group were pooled by month and prepared for storage as described for Experiment I. Samples of alfalfa cubes (Experiment I; 4 samples of cubes) and hay (Experiment II; 7 monthly composite samples of hay), were also taken at random from storage bins. The samples were pooled, subsampled and duplicate samples were stored as described for cereal-based mixtures. For both experiments, feed refused, which included the total ration of hay and grain, by each lamb was collected and weighed daily, before the morning ration of feed was offered. 32 4.3.2 Body weight (Experiments I and II) For both Experiments, body weight was measured before the morning ration of feed was offered. For Experiment I, lambs were weighed on the first and last day of the experiment. For Experiment II, lambs were weighed weekly from their introduction to experimental diets to the completion of the experiment. 4.3.3 Blood sampling 4.3.3.1 General For both Experiments, blood sampling was scheduled to facilitate hormonal (LH, testosterone, P4 and Cortisol) and mineral (Cu and Mo) analysis of serum and hematological evaluation. All blood samples were collected, before the morning ration of feed was offered from a jugular vein into 10 ml evacuated tubes (vacutainer), by means of venipuncture with a single draw 21-gauge needle. Samples for hormone and mineral analysis were collected into non-heparinized tubes (red-top vacutainer). For hematological evaluations, blood samples were collected into heparinized tubes (green-top vacutainer). In preparation for collection of blood samples for mineral analysis, glassware for handling and storage of serum, was acid-washed, as described in section 4.4. 4.3.3.2 Experiment I (LH, testosterone, Cu) For determination of L H and testosterone secretory profiles in serum, blood samples (~ 5 ml) were collected, every 20 minutes from midnight to eight am (0000-0800 h), on the first and last day of the trial. This time was chosen to avoid interference with the normal daily management of the sheep flock which were housed in the same barn. The lambs remained in their pens for the duration of the sampling regime. For the first series of samples, the barn lights were turned on one hour before blood sampling was started. For the second series, the barn lights had been left on since the previous afternoon. Serum was retreived 33 after the samples had clotted and were centrifuged at 1500xg. Duplicate aliquots of serum were transferred, using disposable pipets, to glass culture tubes (5 ml) which were capped with parafilm and then stored in the freezer until assay. For determination of total Cu (TCu) and trichloroacetic acid soluble Cu (TCA-Cu), duplicate blood samples (~ 10 ml) were collected before serial sampling was started. Serum was retrieved, transferred to glass tubes (10 ml) that were then capped with parafilm and stored frozen until assay. 4.3.3.3 Experiment II (LH, testosterone, P4, Cortisol, Cu, Mo) Twice weekly (Tuesday and Friday) blood samples (5 ml) were collected at two hours after sunrise, beginning on June 20th for ewes and August 13th for rams and wethers and continuing to December 20th, 1985. Aliquots of serum were transferred to four culture tubes (5 mm), of which two had been acid washed and then stored frozen. The samples that were stored in tubes that were not acid-washed were used to determine the time course of P4, testosterone and Cortisol associated with gonadal development and the transition from long to short day photoperiod. Samples stored in acid washed tubes were used for TCu, TCA-Cu and total Mo (TMo) determinations. Serial collections of blood samples for determination of L H , testosterone and Cortisol secretory profiles were performed on seven occasions, always for four hours between one and five hours after sunrise. More blood samples per animal per hour were taken in autumn because greater frequency of L H pulses was expected at this time (Lindsay et al. 1984; Fitzgerald, Michel and Butler 1982). On June 20th, the time of the summer solstice, rams and wethers were sampled every 20 minutes (n=12 per lamb), to determine the immature and long-day photoperiod secretory profile. Beginning on September 13th, the time of the autumn Equinox and every two weeks over a 12 week period (September 27th, October 11th, 25th, November 8th and 22nd), ewes, rams and wethers were bled every 12 minutes (n=20 per lamb per period), to determine secretory profiles associated with the first natural breeding season and the transition to short-day photoperiod. To facilitate the collection procedure, the lambs were moved to metabolism crates, adjacent to their pens and sampling began one hour after the lambs were moved. 34 Aliquots of serum were transferred to two culture tubes (5 ml) and then stored frozen. Excess serum from each serial sampling period was pooled for preparation of a reference sample. Pooled serum was later thawed and after thorough mixing, equal aliquots of serum were transferred using disposable pipets to 5 ml culture tubes that were then capped with parafilm and returned to the freezer where serial samples were stored. In total, one hundred pooled reference samples were prepared. One sample was included with each hormone assay described for Experiment II, for determination of inter- and intra- assay variation of samples. During the period of semen sampling of rams, blood was collected immediately before the electrical stimulus was applied (ram was in the squeeze), immediately after ejaculation (ram still in the squeeze) and one hour after ejaculation (the ram had been returned to his pen within 5 minutes of semen collection). Serum was stored as for serial samples, for subsequent determination of testosterone and Cortisol concentrations. At the end of the experiment, blood was collected before and after the lambs were transported to the abattoir. These samples were stored as for serial samples and used to determine the effect of stress on testosterone, P4 and Cortisol concentrations. 4.3.3.4 Hematology (Experiments I and II) A blood sample was collected from each lamb before and after serial blood sampling sessions for both experiments. The samples were used for hematological evaluations which are described in section 4.4.3.3. 4.3.4 Gonadal samples 4.3.4.1 Scrotal circumference (Experiment I and II) For both Experiments, testicular growth was estimated from scrotal circumference measured, as described by Robinson (1983). The scrotum was shaved, the testes were palpated until fully extended into 35 the scrotum, then the circumference of the widest area was measured with a vinyl tape. Scrotal circumference was measured after the lambs were weighed on the first and last day of Experiment I and on seven occasions (~ every two weeks) from August 13th to December 6th, of Experiment II. 4.3.4.2 Semen collection (Experiment II) Sperm output and motility score were determined from semen collected by electroejaculation of each ram on 12 occasions over a four week period between December 2nd and 19th inclusive. Samples were collected on three consecutive days, every five days and always at four hours after sunrise. Ejaculates were collected into graduated tubes (10 ml) immersed in warmed water (37\u00C2\u00B0 C). All solutions and glassware for handling the fresh ejaculate were kept in a water bath maintained at 37\u00C2\u00B0C. Immediately following collection, the volume and motility score of the ejaculate were recorded and semen was diluted with 30% glutaraldehyde for subsequent determination of sperm concentration. The procedure for collecting semen was as described previously (Robinson 1983), however the opportunity was taken to compare two types of electroejaculators, the Dairy Bull ejaculator (DE) and the Bailey ejaculator (BE), which were used on alternate days. The two ejaculators differed in voltage, rate of stimulus and the area of the posterior mesenteric plexus that was stimulated. The D E was designed for bulls, but adapted to rams by the use of a specially designed rectal probe, which possessed three longitudinal electrodes, embedded in insulating material, to deliver a 60 cycle alternating current (AC). Power to the probe was regulated by two settings; one for fixed maximum voltage (20 to 40 V) and one for controlling rate of increase to maximum voltage (0 to max). In contrast, the BE was designed specifically for rams. Two 1.5 V batteries delivered power to a probe which possessed two spherical electrodes at the tip of the probe. With the BE it was not possible to regulate the rate of increase to maximum voltage. Further information on the physiology of electrical stimulation of ejaculation by rams and the comparison between types of ejaculators have been given in Appendix IV. For collection, the ram was placed in a restraining device which was then shifted to a horizontal position to allow the ram free movement of its head and legs. Feces were removed from the rectum. A 36 sterile lubricant gel was applied to the probe, which was then inserted into the rectum. When the D E was used, power was first set to a minimum (1 volt) and was applied with increasing intensity (0 to max) over three seconds; held for a second, then removed. Depending upon the ram's response, the cycle would be repeated before beginning a stepwise increase to maximum voltage. Most rams would experience erection and ejaculation within two or three repetitions at the second maximum voltage setting (2 volts). However, the stimulus required was variable among rams and between collections within rams. When the BE was used, the probe was inserted and the stimulus was applied for three to five seconds and removed. Depending upon the ram's response, the probe was moved slowly along the rectal wall in an attempt to make better contact with lower lumbar or sacral nerves. Most rams would ejaculate, usually without erection, within 4 or 5 repetitions. 4.3.4.3 Vaginal smears (Experiment II) To determine the time of first estrus and number of estrous cycles, smears of vaginal epithelial tissue were taken from ewe lambs, at two hours after sunrise, every Tuesday and Friday, from June 20th to December 20th, 1985. Smears were fixed and stained with Papanicolaou solution (in Hafez, 1987), then stored. 4.3.5 Other tissues At the completion of each experiment lambs were taken for slaughter at the abattoir in Pitt Meadows, British Columbia. For Experiment I, the liver, kidneys and testes were removed and weighed. For Experiment II, the livers, testes and ovaries were removed and conditions of the organs were noted. The livers were weighed and subsamples of the testes and ovaries were taken for histological evaluation. 37 4.4 Laboratory procedures 4.4.1 General For both experiments chemical assay of mineral concentrations in serum and feed and radioimmunoassay (RIA) of hormone concentrations in serum were performed. All glassware used for chemical analyses were acid-washed prior to use. The acid-washing procedure involved ~ 10 hour submersion of glassware in 10% (v/v) nitric acid (70% HN0 3 ) , Glassware was then rinsed five times with distilled water and three times with distilled-deionized water and allowed to dry. This washing procedure had been tested previously (Robinson, 1983) and found to minimize Cu contamination. For steroid hormone RIA antibody specificity and quality control parameters are summarized for each hormone described in section 4.5, however, more detailed comparisons are shown in Appendix I. , 4.4.2 Nutrient composition of feed 4.4.2.1 Experiment I Air-dried feed samples were ground through a stainless steel screen (2 mm) attached to a Wiley mill and hay was reground through a 0.5 mm screen. Duplicate samples (~ 20g) were transferred to whirl top plastic bags and stored at room temperature. Copper, Mo and S concentrations in pellets and alfalfa cubes were determined by inductively coupled plasma emission mass spectrophotometry (ICPMS) after ashing of 0.5g duplicates of samples, station standards (pelleted barley) and international standards (orchard leaves) in concentrated nitric acid. This procedure was performed at the Agriculture Canada Research Station, Agassiz, B.C. Standard concentrations of Cu (0, 2, 4, 8, 10 (j,g ml'1), Mo (0, 2, 4, 10, 12, 14, 24, 26, 28 jig ml\"1) and S (0, 2, 4, 6, 8 mg ml\"1) were prepared in 2% nitric acid (70% nitric acid in distilled, deionized 38 water). In preparation for the ashing procedure, standards (1.0 ml) were added to the control diet samples (0.5g) and blank standard (1.0 ml) was added to the remaining samples (0.5g) in 50 ml Taylor tubes. Concentrated nitric acid (8 ml 70% HN0 3 ) was added and the samples were left at room temperature for 14 hours. After the predigestion period, hydrogen peroxide (1 ml) and two glass boiling chips were added to each tube. The tubes were then transferred to heating blocks which were gradually heated to 160\u00C2\u00B0C. After four hours most samples had completed digestion, but samples were removed as the volume of solution was reduced to ~one ml. Once cooled, the solution was brought to a volume of 25 ml using distilled deionized water and rhodium (50 ppb Rh) was added to provide an internal calibration standard during analysis by the mass spectrophotometer. The solutions were then filtered through ashless filter paper (#40) into scintillation vials (25 ml) and capped with polyethylene caps. Ion intensity data given by the mass spectrophotometer was corrected for drift (Rh) during the analysis and blanks and finally concentrations of Cu, Mo and S were calculated using the formula for standard additions. Other nutrients including dry matter, protein, calcium, phosphorus, magnesium, sodium, potassium, iron and manganese were determined by Association of Official Analytical Chemists (A.O.A.C.) methods used by the University of British Columbia, Department of Animal Science Nutrition Laboratory. Dry matter was determined by drying a lg sample to a constant weight at 100\u00C2\u00B0C. Samples were ashed with concentrated sulfuric acid prior to atomic absorption spectrophotometric measurement of specific mineral concentrations. The nitgrogen content was measured by the auto-analyzer after microkjeldal ashing of samples. 4.4.2.2 Experiment II Copper, Mo and S in cereal-based mixture and hay were determined by mass spectrophotometry after digestion of the ground sample (2 mm stainless steel screen) and aqueous standards in concentrated nitric acid as described for Experiment I. Other nutrients including dry matter, protein, energy and neutral 39 detergent fiber were determined by the A.O.A.C. procedures used by the University of British Columbia, Department of Animal Science Nutrition Laboratory. 4.4.3 Mineral analysis of serum and hematology 4.4.3.1 Experiment I (TCu, TCA-Cu and RCu) Total Cu (TCu) concentrations in serum were determined by atomic absorption spectrophotometry (Perkin-Elmer 1973). Standard solutions of aqueous Cu (0, 0.5, 1.0, 2.0, 5.0 j_g ml\"1) were prepared in glycerol (10% v/v). Serum (2 ml) and blanks (2 ml of 0 Cu standard) were diluted 1:1 in distilled deionized water. The absorbance read by the spectrophotometer was converted to blank-corrected Cu concentrations in /_mol L\"1 (/ig Cu ml\"1 serum * 1000 ml L\"1 * 63.546 /xmol Cu /ig\"1). Serum trichloroacetic acid soluble Cu (TCA-Cu) concentrations were determined by atomic absorption spectrophotometry after precipitation of serum protein using 10% and 5% solutions of TCA. The method was based on procedures described by Mason et al. (1978) and Lamand et al. (1980). The 10% and 5% T C A (w/v) solutions were prepared in distilled deionized water. Standard concentrations of aqueous Cu (0, 0.5, 1.0, 2.0, 5.0 j_g ml1) were prepared in 5% TCA. Serum (2 ml) and blanks (2 ml 0 Cu standard) were transferred to weighed flat-bottom vials (10 ml) and the total weight recorded; then 10% T C A (2 ml) was added. The mixture was vortexed for five seconds, then centrifuged (1500g for 20 minutes). The supernatant layer (TCA soluble fraction was transferred to tared test tubes. The precipitate was washed with 5% T C A (1.5 ml), recentrifuged and the supernatant was added to the T C A soluble fraction and the weight of the final volume was recorded. Absorbance readings were blank corrected and Cu concentrations were calculated and expressed in )_mol L'1. Residual Cu (RCu) was calculated as the difference between TCu and TCA-Cu concentrations. 40 4.4.3.2 Experiment II (TCu, TCA-Cu, RCu and TMo) Concentrations of TCu, TCA-Cu and RCu were determined by atomic absorption spectrophotometry as described for Experiment I (refer to 4.4.3.1.). In preparation for assay, equal aliquots of serum from each sample collected for each animal, over a two or four week period, was pooled to provide enough serum for assay (at least 2 by 2 ml). Serum collected during the last four weeks of the experiment was pooled to determine TMo and TCu by ICPMS after digestion of 0.5 ml serum and aqueous standards in concentrated nitric acid. The procedure was essentially the same as for determining the concentrations of Cu and Mo in feeds, however, duplicate samples contained standard additions of Cu (1.5 Ltg) and Mo (1.0 /ig). Standards were prepared in serum from control animals, but there was no need to filter the samples prior to analysis by ICPMS. 4.4.3.3 Hematology (Experiments I and II) For both Experiments, hematological evaluations were used for diagnosis of possible Cu deficiency, characterized by microcytic hypochromic anemia (Underwood 1981). The evaluations were performed as described by The Ministry of Agriculture Department of Fisheries and Food (1984). Hemoglobin (Hb) was determined by the cyanomethemoglobin method. Hematocrit (PCV) was measured using a microcapillary centrifuge and reader. To estimate the proportion of Hb in packed cells (PCV), mean corpuscular hemoglobin content (MCHC) was calculated from hemoglobin and hematocrit. For Experiment II, after the lambs had been given experimental diets for 22 weeks, a more thorough hematological evaluation was performed. Red blood cell number was determined using a standard hemocytometer. The mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV) were calculated. Red blood cell osmotic fragility was determined by counting the number of RBCs that did not hemolize in hypotonic saline solutions (0, 0.75, 0.65, 0.60, 0.55, 0.50, 0.45% saline). A fixed amount of blood (5 /xL ml1) was diluted with each saline solution (1:200), shaken for 15 minutes at 25\u00C2\u00B0C and 41 allowed to set for seven minutes before counting the number of intact RBC using the hemocytometer. From these measures it was possible to estimate the average amount of Hb per RBC (MCH), the average volume of a single RBC (MCV) and the integrity of the RBC membrane (red blood cell osmotic fragility). Total protein in serum was determined by the method of Lowry (1951). The colorimetric procedure utilizes Folin-Ciocalteau reagent to produce a blue color formed by the reaction of protein with the alkaline Cu in the reagent and the reduction of the phosphomolybdate-phosphotungstate salts in the reagent by the tyrosine and tryptophan residues in serum (Greenberg 1970). 4.4.4 Hormone analysis of serum 4.4.4.1 Luteinizing hormone (LH) 4.4.4.1.1 Experiment I Serum L H concentrations were determined by an established double-antibody radioactive iodine (125I) radioimmunoassay (Sanford et al. 1984a), which was performed at the University of Manitoba. All samples collected at the end of the trial (Day 30), were included in one assay which had 7.6% intra-assay variation and a minimum detectable level of 0.1 ng ml\"1 (defined as 95% B/BO). Standard concentrations of ovine L H (NIH-LH-S14; 0, 0.5, 1.0, 2.0, 4.0, 10.0, 30.0 ng ml1) were prepared in 1% egg-white phosphate azide buffer (pH 7.6). Serum stripped using dextran and charcoal were used for detection of non-specific binding (NSB). Duplicate samples (0.2 ml) of standards and serum were incubated (4\u00C2\u00B0C) with 125I-labelled ovine L H (~ 12,000 cpm) and anti-ovine L H serum (GDN #21; 1:100,000 initial dilution). After five days, anti-rabbit 8-globuin serum was added to separate bound from free hormone. The supernatant was discarded. The radioactivity in control tubes, totals (125I only) NSB and of bound hormone in samples and standards was measured on a gamma counter (1275 Mini Gamma LKB). The gamma counter was programmed for conversion to L H concentration, corrected for NSB. 42 4.4.4.1.2 Experiment II Serum concentrations of L H were determined by a specific double antibody radioimmunoassay (125I) (Rawlings et al. 1977; Rawlings, Jeffcoate and Rieger 1984) which was performed by the University of Sasakatchewan, Veterinary Services Laboratory. For six L H assays, the reference sample contained 3.25 \u00C2\u00B1 0.49 ng ml'1 L H (n=133) with intra- and inter- assay variation of 8.6% and 10.5%, respectively. The minimum detectable level was 0.01 /xg ml 4 . The first antibody was prepared in rabbits against NIH-LH-B6 which had some crossreactivity with ovine follicle stimulating hormone (NIAMDD-oFSH-13 1.1%), prolactin (NIH-B3 0.2%), thyroid stimulating hormone (NIH-TSH-S8 5%) and growth hormone (NIH-GH-B17 3.1%). Highly purified L H (LER 1017-2) was iodinated and standard concentrations of ovine L H (NIH-LH-S18) were prepared in a 5% BSA solution (5g BSA per 100 ml phosphate buffered saline) to yield 0, 0.06, 0.12, 0.25, 0.50, 1.0, 2, 4, 8, 16 ng m l 1 L H . The second antibody, had been raised against rabbit 5-globulins in sheep. 4.4.4.2 Testosterone 4.4.4.2.1 Experiment I Serum testosterone (17B-Hydroxy-4-androsten-3-one) concentrations were determined by competitive radioimmunoassay, extraction, radioactive hydrogen (3H) radioimmunoassay procedure (Sanford et al. 1984), which was performed at the University of Manitoba. Six assays for sera collected on day 30, had sensitivities of 0.6 \u00C2\u00B1 0.4 ng ml'1 (95% B0) and 12.9% intra- and 14.9% inter-assay variations. Standard concentrations of testosterone (0.0, 0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, 12.0, 16.0 , 20.0 ng ml\"1) were first prepared in phosphate buffered saline. However, for assay, standards (0.1 ml) were mixed with an equal volume (0.1 ml) of stripped serum (dextran-charcoal extracted serum). Lipids in samples (0.1 ml) and standards were extracted with absolute ether, then incubated (4\u00C2\u00B0C) for one hour with anti-testosterone (Sanford et al. 1978) and 3H-testosterone (~ 30,000 cpm). Charcoal-dextran was added to separate bound from free hormone, then the supernatant was diluted with scintillation fluid and the 3 H activity was read 43 by a scintillation counter. Percent 3H-hormone bound, less non-specific binding, was plotted against standard 3H-testosterone on semi-log paper for conversion to testosterone concentration (ng ml1). 4.4.4.2.2 Experiment II (Testosterone) Concentrations of testosterone in serum were determined by no extraction, solid-phase 125I radioimmunoassay (Coat-a-CountR). For nine testosterone assays, the reference sample contained 2.53 \u00C2\u00B1 0.13 ng ml'1 testosterone with an intra- and inter- assay variation of 4.7 and 5.1%, respectively. The procedure employed antiserum highly specific for testosterone with some cross-reactivity reported for P\u00E2\u0080\u009E (0.028%), Cortisol (0.002%), androsterone (0.03%), androstenedione (3.0%), 116-hydroxytestosterone (0.6%). The standard curve was derived from human serum containing 0, 0.3, 1, 3, 10 and 30 ng ml'1 testosterone. Average specific activity of the trace (total counts), maximum binding (MB), non-specific binding (NSB) and sensitivity (95% B/B0) were 28377 \u00C2\u00B1 6569 cpm, 57.9 \u00C2\u00B1 2.2%, 0.9 \u00C2\u00B1 0.3% and 0.17 \u00C2\u00B1 0.05 ng testosterone, respectively. The intra- and inter- assay coefficients of variation of the standard curve were 2.3 and 3.8%, respectively. Concentration of testosterone was determined by logit log regression of the standard curve as described for P4 (section 4.4.4.2) and expressed in /ig L\"1. 4.4.4.3 Progesterone (Experiment II) Progesterone concentrations in serum were determined by no extraction, solid-phase 125I radioimmunoassay (Coat-a-CountR). For seven assays, the reference sample contained 0.46 \u00C2\u00B1 0.04 ng ml\"1 P4, with intra- and inter- assay variation of 8.7 and 11.3%, respectively. The procedure employed antiserum highly specific for P4 which was reported to have nondetectable crossreactivity with pregnenolone and Cortisol, but some crossreactivity for 17a-hydroxyprogesterone (0.3%), 20a-dihydroprogesterone (2.0%), 11-deoxycorticosterone (1.7%), 11-deoxycortisol (2.4%) and corticosterone 44 (0.4%). Human sera containing 0, 0.1, 0.5, 2, 10, 20 and 40 ng ml\"1 P4 were used to derive the standard curve. Average specific activity of the trace (total counts), maximum binding (MB), non-specific binding (NSB) and sensitivity (95% B/B0) were 50585 \u00C2\u00B1 5058 cpm, 42.1 \u00C2\u00B1 4.6%, 1.9 \u00C2\u00B1 0.4% and 0.09 \u00C2\u00B1 0.13 /_g L' 1, respectively. The intra- and inter- assay coefficients of variation of the standard curve were 3.8 and 4.3%, respectively. Concentration of P4 was computed using a customized program written in the Statistical Analysis System (SAS Personal computer version 6.03) language. The SAS program included logit-log representation of the standard curve (equation i) and a correction factor (equation ii) for bias produced by back-transformation of logarithms (Baskerville 1972; Sprugel 1983) and a minimum detectable value equal to 95% of the maximum binding tube. (i) Y = [EXP(lnB:F - bO) + bl)]*CF, where Y = hormone concentration, EXP = antilogarithm, In = natural logarithm, B:F = ratio of bound to free trace, bO = regression line intercept, bl = slope of the regression line and CF = correction factor. (ii) CF = EXP(RMSE 2 -s- 2), where RMSE = standard error of the estimate 4.4.4.4 Cortisol (Experiment II) Cortisol concentrations in serum were determined by no extraction, solid-phase 125I radioimmunoassay (Coat-a-CountR). For 17 assays, the reference sample contained 13.4 \u00C2\u00B1 1.1 ng ml'1 Cortisol with intra- and inter- assay variation of 9.5 and 8.6%, respectively. The procedure employed antiserum highly specific for Cortisol with some crossreactivity reported for 11-deoxycorticosterone (1.4%), 11-deoxycortisol (0.25%), 21-deoxycortisone (0.04%), P4 (0.15%), 45 lla-hydroxyprogesterone (0%) and aldosterone (0.01%). The standard curve was derived from human serum containing 0, 10, 50, 100, 200 and 500 ng ml 1 Cortisol. Average specific activity of the trace (total counts), maximum binding (MB), non-specific binding (NSB) and sensitivity (95% B/B0) were 41083 \u00C2\u00B1 5233 cpm, 79.3 \u00C2\u00B1 4.5%, 1.1 \u00C2\u00B1 0.4% and 3.8 \u00C2\u00B1 0.8 ng Cortisol, respectively. The intra- and inter- assay coefficients of variation of the standard curve were 2.5 and 6.5%, respectively. Concentration of Cortisol was determined by logit log regression of the standard curve as described for P4 (4.4.4.2) and multiplied by a factor of 10 for conversion to /xg L 1 . 4.4.5 Evaluation of gonadal samples (Experiment II) 4.4.5.1 Semen evaluation An established procedure for scoring sperm motility had been used previously (Rhodes 1980; Robinson 1983). To estimate sperm motility, one drop of semen was placed on a prewarmed slide (37\u00C2\u00B0C), then examined by two persons, using low and high power light microscopy. The drop was observed from edge to edge to estimate the proportion of live active sperm and the center of the sample was observed for waves and eddies. Sperm motility was then scored on a scale of 0 (few cells; no motility) to 5 (>80% motile cells;excellent motility) Semen diluted with 30% glutaraldehyde was used to determine the concentration of spermatozoa in semen using a hemocytometer (Herman and Madden 1974). Total sperm per ejaculate was calculated as the product of semen volume and sperm concentration. 4.4.5.2 Estrus and Ovulation Stages of the estrous cycle were defined by cell types and stages of cellular differentiation observed on vaginal smears (Sanger, Engel and Bell 1958; Frandson 1980; Ross and Reith 1985). Smears were 46 examined in sequence (June to December) and estrus was said to have occurred when clumps of cornified epithelial cells were present on slides. Further detail is given in Appendix V. Ovulations were determined from serum P4 measured in samples collected twice weekly (section 4.3.3.3). A functional corpus luteum was said to have occurred when P4 concentration was greater than 1 j_g L' 1 (Fitzgerald and Butler 1982). 4.5 Data Analysis 4.5.1 Hormone secretory profile characteristics Secretory profiles were described by the basal, amplitude and frequency of peak concentrations of L H , testosterone and Cortisol in serum. For each profile, the mean and standard error were determined and an episode of hormone secretion was defined by concentrations greater than three times the standard error of the mean. The peak of the episode was the maximum value between nadirs of the rise. Basal value was the lowest value preceding each peak. The amplitude was the difference between a peak and its nadir. 4.5.2 Statistical analysis 4.5.2.1 Experiment I For experiment I, lambs were classified by age (<160 days; 22 weeks and >160 days; 24 weeks) and dietary treatment (Group I, II and III). Statistical analyses for a randomized block experiment were used to describe variation in L H , testosterone (mean, basal, peak and amplitude values), serum Cu and hematology parameters caused by the influence of age (df=l), diet (df=2) and age by diet group interaction (df=2). Data were first tested using lamb within group (df=7) as the error term (Snedecor and Cochran 1980). If age by diet group interaction was not significant (P>0.20), the main effects of age and diet were retested using the pooled error (df=9) (Snedecor and Cochran 1980). Body weight and scrotal circumference randomized block A N O V A included repeated measures (Day 0 and Day 30) and 47 lamb within diet within day (Error b) was used to test day effects and the interactions with age and diet effects. 4.5.2.2 Experiment II For Experiment II, lambs were classified by gonadal influence (ram (testicular)), wether (none), ewe (ovarian)) and dietary treatment (Groups I, II, III and IV) to form Statistical analyses for a randomized block experiment, were used to describe variation in feed intake, body and liver weight, serum Cu and hematology parameters, L H and Cortisol characteristics (mean, basal, peak and amplitude values), caused by gonadal influence (df=2), diet (df=3) and gonadal by diet group interaction (df=6). Data were first tested using lamb within group (df=8) as the error term (Snedecor and Cochran 1980). When gonadal influence by diet group interaction was not significant (P>0.10), the main effects of gonadal influence and diet were retested using the pooled error (df=14) (Snedecor and Cochran 1980). With repeated measures over time (sampling day or period), lamb within diet group within day (Error b) was used to test day effects and the interactions with gonadal and diet effects. For measures of gonadal function (testosterone, semen, P4, estrus and ovulation) lambs were classified by diet Group only and A N O V A for a complete random experiment was used (lamb within diet Error a, df=4) with repeated measures over time when applicable (lamb within diet within sampling period, Error b). Orthogonal contrasts were used to partition diet effects into variation caused by the addition of Mo (Groups I, III compared with II, IV; df=l), of S (Groups I, II compared with III, IV; df=l) and Mo by S effects (Groups I, IV compared with II, III; df=l). Gonadal effects were partitioned into two contrasts to compare absence with presence of gonads (wether compared with ram and ewe; df=l) and testicular with ovarian gonadal effects (ram compared with ewe). 4.5.3 General All data analysis were performed using the Statistical Analysis System (SAS-personal computer version 6.03). Homogeneity of variance among treatment groups were tested using Bartlett's Test (Snedecor 48 and Cochran 1980). For A N O V A , SAS general linear models (GLM), procedures with Type III sums of squares (SAS Institute Inc. 1986) was used. The G L M with Type III sums of squares analysis applied the method of weighted squares of means (complete least squares), which compared block (age or gonadal influences) and diet effects (Group) with an adjustment for block by group interaction. This allowed a comparison of block and group effects in the presence of interaction and was also unrelated to cell frequencies (number of lambs per age or gonadal grouping) (Freund and Littel 1981). Residual sums of squares were used to determine partial correlation coefficients (r) among feed intake, serum Cu and Mo, gonadal and hormonal variables. An effect was declared significant when the probability of a greater F value was <0.05. Probability of difference (PDIFF) was used to compare differences among least squares means. Standard errors of least squares means were determined from the appropriate error term used in the A N O V A . Unless specified, least squares means and the standard error of the least squares means are presented. 49 Chapter 5 RESULTS 5.1 Experiment I 5.1.1 Nutrient composition and intake of diets Table 1 shows the nutrient composition of dry matter in experimental diets given for 30 days to ram lambs in Groups I, II and III. The concentration of Cu in the pelleted cereal-based ration of the diet was 7.5 \u00C2\u00B1 0.4 mg kg\"1 D M (n=6). The concentration of Mo in the unsupplemented (Group I) and Mo- supplemented (Groups II and III) diets were 0.68 \u00C2\u00B1 0.09 (n=2) and 30.0 \u00C2\u00B1 0.3 (n=4) mg kg 1 D M , respectively. The concentration of S in the unsupplemented (Groups I and II) and S-supplemented (Group III) diets were 2.3 \u00C2\u00B1 0.1 g kg 1 D M (n=4) and 3.7 \u00C2\u00B1 0.2 (n=2) g kg 1 D M , respectively. All lambs consumed their daily allotment of grain, however most refused their ration of alfalfa cubes. Using the coefficients of Cu absorption (Table 1) and an average daily intake of 1300g pellets/lamb d'1, at 90% D M , the estimated available Cu intake per lamb per day was 6.75, 1.13 and 0.25 /.moles for Groups I, II and III, respectively. Estimates for total intake of Cu, Mo and S were 157, 149 and 157 jtimol, 9, 397 and 424 /_mol and 97, 93 and 150 mmol/lamb d'1, for Groups I, II and III, respectively. 5.1.2 Serum copper and hematology Serum TCA-Cu, RCu and TCu are shown in Table 2. TCA-Cu was lower for Mo-supplemented lambs (Groups II and III) and RCu was highest for lambs given Mo+S (Group III). In contrast, TCu did not differ among diet groups. Serum Cu concentrations did not differ between lambs aged 22 and 24 weeks. Hematological parameters did not differ among diet or age groups. For Groups I, II and III, respectively, HBG, PCV and M C H C were 99 \u00C2\u00B1 2, 107 \u00C2\u00B1 2 and 100 \u00C2\u00B1 3 mg L\"1, 29.8 \u00C2\u00B1 0.5, 30.9 \u00C2\u00B1 0.5 and 30.0 \u00C2\u00B1 0.6% and 33.4 \u00C2\u00B1 0.8, 34.8 \u00C2\u00B1 0.8 and 33.6 \u00C2\u00B1 0.9%, respectively. These parameters did not 50 differ between samples collected before and after serial blood sampling was performed on the first and last day of the trial. 5.1.3 Body weight and scrotal circumference Body weight and average daily gain did not differ among diet or age groups Body weight at the end of the trial and average daily gain over the 30 day period, were 36.3, 36.6 and 36.9 \u00C2\u00B1 1.01 kg and 0.36, 0.35, 0.36 \u00C2\u00B1 0.01 kg d 1 for Groups I, II and III respectively. Scrotal circumference did not differ among diet or age groups, but average daily increase in scrotal circumference tended (P=0.10) to be lower for the Mo-supplemented group. Scrotal circumference at the end of the 30 day period and average daily increase in scrotal circumference were 32.1 \u00C2\u00B1 1.1, 32.1 \u00C2\u00B1 1.1, 34.4 \u00C2\u00B1 1.2 cm and 0.16 \u00C2\u00B1 0.03, 0.07 \u00C2\u00B1 0.03, 0.21 \u00C2\u00B1 0.04 cm d 1 for Groups I, II and III, respectively. 5.1.4 Serum secretory profiles of L H and testosterone For all lambs, concentrations of L H and testosterone in serum revealed pulsatile secretory patterns as shown in Figure 3. Peaks of L H secretion in serum of lambs aged 22 weeks, occurred 40 \u00C2\u00B1 9 minutes prior to the first significant rise in testosterone. For lambs aged 24 weeks, in Groups I, II and III respectively, L H peaks occurred 30 \u00C2\u00B1 16, 60 \u00C2\u00B1 16 and 40 \u00C2\u00B1 23 minutes before testosterone peaks. However, the these differences were not significant (P>0.10). Characteristics of L H and testosterone secretory profiles are shown in Tables 3 and 4, respectively. Mean L H did not differ among diet groups, but tended to be lower for lambs aged 24 weeks (2.18 \u00C2\u00B1 0.59 and 1.75 \u00C2\u00B1 0.72 L4g L' 1, respectively; P=0.08). However, L H peak amplitude was affected by age*diet interaction, because of high peak amplitude for Mo-supplemented lambs (Group II), aged 24 weeks. L H peak frequency did not differ among diet or age groups. Basal testosterone and peak frequency did not differ among diet groups, but were lower for younger lambs. For lambs aged 22 and 24 weeks, basal testosterone, peak amplitude and peak frequency were 0.60 \u00C2\u00B1 0.34 and 1.71 \u00C2\u00B1 0.41 ng L' 1 (P=0.08), 51 4.44 \u00C2\u00B1 0.67 and 4.97 \u00C2\u00B1 0.82 Ltg L' 1 (P>0.20) and 0.20 \u00C2\u00B1 0.03 and 0.35 \u00C2\u00B1 0.04 peaks/h (P<0.05), respectively. 5.1.5 Other tissues For Groups I, II and III testes, liver and kidney weights were 0.33, 0.41, 0.45 \u00C2\u00B1 0.04 kg, 0.82, 0.80, 0.80 \u00C2\u00B1 0.03 kg and 0.12, 0.14, 0.12 \u00C2\u00B1 0.03 kg, respectively and did not differ among diet or age groups. 5.1.6 Partial correlations Partial correlation coefficients (r) among parameters are shown in Appendix VI. Significant correlation occurred between RCu and mean L H (r=0.78), TCA-Cu and mean L H (r=-0.72) and TCu and testosterone amplitude (r=0.64). 52 Table 1. Nutrient composition of dry matter in experimental diets given to ram lambs for 30 days. Treatment group Item I II III Number of Samples 2 2 2 Copper (mg kg1) 7.7 7.3 7.7 Molybdenum (mg kg1) 0.7 29.3 31.3 Coppenmolybdenum ratio 11.6 0.2 0.2 Sulfur (g kg1) 2.4 2.3 3.7 Copper availability (%)\u00CC\u0082 4.6 0.8 0.2 Based on laboratory analysis. Other nutrients measured in concentrate and alfalfa cubes, respectively, were, on a per kg dry matter basis, protein (18.3 and 17.3%), calcium (0.9 and 1.2%), phosphorus (0.6 and 0.2%), magnesium (0.2 and 0.3 g), sodium (0.8 and 0.6%), potassium (0.7 and 2.7%), iron (550 and 265 mg) and manganese (149 and 45 mg). Alfalfa cubes contributed 20% to D M intake and contained 6.0 mg kg'1 copper, 2.5 mg kg\"1 molybdenum and 2.7% sulfur. \u00C2\u00AE Coefficient of copper absorption was derived from the equation given by Suttle and McLaughlin (1976): Log T A = - 0.0019 Mo - 0.075 S - 0.0131 ((Mo)(S)) - 1.153 where T A Cu is the true availability of copper, Mo and S are molybdenum (mg kg1) and sulfur (g kg1) concentration in the diet dry matter. 53 Table 2. Copper (Cu) soluble in trichloroacetic acid (TCA), residual Cu and total Cu in serum of crossbred rams, aged 22 and 24 weeks, given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 30 days. Mineral Supplement (kg\"1) Age of ram lambs (wk) Mo S Group Group mg g 22 24 Mean (n=4) T C A copper (jizmol L\"1) I 0 0 12.3 \u00C2\u00B1 1.0 13.1 \u00C2\u00B1 1.0 127 \u00C2\u00B1 0.T II 26 0 10.3 \u00C2\u00B1 1.0 11.2 \u00C2\u00B1 1.0 10.8 \u00C2\u00B1 0.7\" III 26 2 8.7 \u00C2\u00B1 0.8 11.6 \u00C2\u00B1 1.4 10.2 \u00C2\u00B1 0.7\" Residual copper (/.mol L'1) I 0 0 0.0 \u00C2\u00B1 1.2 0.1 \u00C2\u00B1 1.2 0.0 \u00C2\u00B1 0.8a II 26 0 1.2 \u00C2\u00B1 1.2 2.0 \u00C2\u00B1 1.2 1.6 \u00C2\u00B1 0.8\" III 26 2 3.5 \u00C2\u00B1 1.0 9.0 \u00C2\u00B1 1.2 6.2 \u00C2\u00B1 1.0\u00C2\u00B0 Total copper (/xmol L 1 ) I 0 0 12.0 \u00C2\u00B1 2.4 13.1 \u00C2\u00B1 2.7 12.6 \u00C2\u00B1 1.7 II 26 0 11.5. \u00C2\u00B1 2.7 13.2 \u00C2\u00B1 2.7 12.3 \u00C2\u00B1 1.7 III 26 2 15.6 \u00C2\u00B1 2.2 20.6 \u00C2\u00B1 2.8 18.1 \u00C2\u00B1 1.8 , b Means in columns with different letters differ (P<0.05) 15 R282 aged 22 weeks R251 aged 24 weeks 0:00 1:20 2:40 4:00 5:20 6:40 8:0 0:00 1:20 2:40 4:00 5:20 6:40 8:0 Time of day (h:m) Figure 3. Luteinizing hormone (LH) and testosterone serum secretory profiles of individual ram lambs aged 22 and 24 weeks, given a cerea l - based diet (Group I). Lambs were bled every 20 m. Secretory peak concentrations are c ircled. 55 Table 3. Characteristics of luteinizing hormone secretory peaks in serum of crossbred rams, aged 22 and 24 weeks given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 30 days. Mineral Luteinizing hormone Supplement (kg1) Age of ram lambs (wk) Mo S Group Group mg g 22 24 Mean (n=4) Base value (jLtg L'1) I 0 0 0.2 \u00C2\u00B1 0.3 0.5 \u00C2\u00B1 0.3 0.3 \u00C2\u00B1 0.2 II 26 0 0.8 \u00C2\u00B1 0.3 0.7 \u00C2\u00B1 0.5 0.7 \u00C2\u00B1 0.2 III 26 2 0.8 \u00C2\u00B1 0.3 0.8 \u00C2\u00B1 0.5 0.8 \u00C2\u00B1 0.3 Peak amplitude (jig L'1) I 0 0 7.8 \u00C2\u00B1 1.5 3.2 \u00C2\u00B1 1.5a 5.5 \u00C2\u00B1 1.1 II 26 0 4.1 \u00C2\u00B1 1.5 8.6 \u00C2\u00B1 1.5\" 6.4 \u00C2\u00B1 1.1 III 26 2 6.4 \u00C2\u00B1 1.3 3.8 \u00C2\u00B1 2.2a 5.1 \u00C2\u00B1 1.3 Peak frequency (peaks/h) I 0 0 0.2 \u00C2\u00B1 0.0 0.2 \u00C2\u00B10 .0 0.2 \u00C2\u00B1 0.03 II 26 0 0.3 \u00C2\u00B1 0.0 0.2 \u00C2\u00B10 .0 0.2 \u00C2\u00B1 0.03 III 26 2 0.3 \u00C2\u00B1 0.0 0.2 \u00C2\u00B10 .0 0.3 \u00C2\u00B1 0.03 , b Means in columns with different letters differ (P<0.05) 56 Table 4. Characteristics of testosterone secretory peaks in serum of crossbred rams, aged 22 and 24 weeks, given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 30 days. Mineral Testosterone Supplement (kg1) Age of ram lambs (wk) Mo S Group Group mg g 22 24 Mean (n=4) Basal (/ig L 1) 1\" I 0 0 0.2 0.6 1.9 \u00C2\u00B1 0.6 1.1 \u00C2\u00B1 0.4 II 26 0 1.1 0.6 1.1 \u00C2\u00B1 0.6 1.1 0.4 III 26 . 2 0.5 0.5 2.1 \u00C2\u00B1 0.9 1.3 \u00C2\u00B1 0.5 Peak amplitude (/_g L 1 ) I 0 0 4.8 1.2 5.0 \u00C2\u00B1 1.2 4.9 + 0.8 II 26 0 4.3 1.2 4.6 \u00C2\u00B1 1.2 4.5 \u00C2\u00B1 0.8 III 26 2 4.1 1.0 5.3 \u00C2\u00B1 1.7 4.7 \u00C2\u00B1 0.8 Peak frequency (peaks/h)^ I 0 0 0.2 0.1 0.4 \u00C2\u00B1 0 . 1 0.3 0.04 II 26 0 0.2 \u00C2\u00B1 0.1 0.2 \u00C2\u00B1 0.1 0.2 0.04 III 26 2 0.2 0.0 0.4 \u00C2\u00B1 0.1 0.3 \u00C2\u00B1 0.05 T Testosterone base tends to be lower for lambs aged 22 weeks (P=0.08). ^ Testosterone peak frequency is lower for lambs aged 22 weeks (P<0.05). 57 5.2 Experiment II 5.2.1 Nutrient composition and intake of diets Table 5 shows the nutrient composition of dry matter in experimental diets given for 32-39 weeks to lambs in Groups I, II, III and IV. The concentration of Cu in the cereal-based ration was 7.7 \u00C2\u00B1 0.4 mg kg 1 (n=4). The concentration of Mo in the unsupplemented (Groups I and III) and Mo-supplemented (Groups II and IV) diets were 0.6 \u00C2\u00B1 0.1 mg kg 1 D M (n=2) and 16.2 \u00C2\u00B1 3.3 mg kg 1 D M (n=2). There was considerable variation in the Mo concentrations within Mo-supplemented groups caused by extremely high concentrations of Mo in diets prepared in May (40 to 50 mg kg1) and low concentrations in diets prepared in July (2 to 4 mg kg1). The concentration of S in the unsupplemented (Groups I and II) and supplemented (Groups III and IV) diets were 2.1 \u00C2\u00B1 0.1 (n=2) and 3.7 \u00C2\u00B1 0.1 (n=2) g kg-1 D M . Feed intake was affected by S*time interaction (Figure 4), regardless of higher feed intake by ram and wether lambs than ewe lambs until autumn, when the amount of food offered to ram and wether lambs was reduced to the same amount given to ewe lambs. The effect of S was due to better feed intake by lambs given S alone (Group III), or in combination with Mo (Group IV), especially during the first few weeks in July. In autumn, the effect was mostly due to depressed feed intake by lambs given the unsupplemented diet (Group I). Using the coefficients of Cu availability in the cereal based ration (Table 5) and an average daily feed intake of 1367, 1428, 1487 and 1493 g/lamb d 1 , at 90% D M , for Groups I, II, III and IV, respectively, . ' the estimates for intake of available Cu per lamb per day were 8.2, 3.1, 5.3 and 0.7 Ltmols, respectively. Estimates for total intake of Cu, Mo and S were 174, 173, 175 and 179 /zmol and 8, 215, 9 and 285 Ltmol and 89, 93, 181 and 172 mmol/lamb d\"1 for Groups I, II, III and IV, respectively. 58 5.2.2 Serum copper and molybdenum and hematology Concentrations of Cu and Mo in serum are shown in Tables 6 and 7. The distribution of Cu in serum was predominantly affected by S level, regardless of Mo level, or gonadal influence. In June, after ram and wether lambs had been given experimental diets for 13 weeks, TCA-Cu and TCu which were 14.2, 12.6, 10.6 and 10.1 \u00C2\u00B1 0.7 and 17.2, 16.9, 14.8, 17.1 \u00C2\u00B1 0.7 /.mol L 1 , for Groups I, II, III and IV, respectively, tended to be lower for S-supplemented lambs (Groups III and IV), but thereafter the effect of S level on TCA-Cu and RCu was significant. Serum TCu concentrations did not differ among diet groups, but serum TMo concentrations were higher for Mo-supplemented groups, regardless of S level. In autumn, the effect of gonadal influence on TCA-Cu and TCu was significant, but this did not show interaction with the effects of S. TCA-Cu and TCu were higher for wether, than ram or ewe lambs and were 15.9 \u00C2\u00B1 1.1, 11.4 \u00C2\u00B1 0.7 and 11.5 \u00C2\u00B1 0.7 /_mol L\"1 and 19.5 \u00C2\u00B1 1.3, 13.7 \u00C2\u00B1 0.9 and 13.6\u00C2\u00B1 0.9 /.mol L\"1, respectively. TCA-Cu was also analyzed in serial samples collected in June from ram and wether lambs and were pooled within lamb by hour. The results shown in Appendix II, tended (P=0.20) to show differences among hours. Hematological parameters and serum protein did not differ among diet or gonadal groups. However, red blood cell osmotic fragility tended (P=0.08) to be lowest for lambs given Mo alone (Group II). Means for Groups I, II, III and IV are shown in Appendix III. 5.2.3 Body weight Figure 5 shows Mo*time and gonadal influence*molybdenum*sulfur*time effects on body weight. Until October, higher body weight for lambs given Mo (Groups II and IV) was mostly due to low body weight for Group I. In autumn, Mo*time effect showed interaction with S level and gonadal influence, due to lower body weight for ram and wether lambs given Mo+S. Between 0 and 10 weeks after the equinox, average daily gain (110, 139, 116 and 139 g d\"1 for Groups I, II, III and IV, respectively; P<0.05) was higher for Mo-supplemented groups. At this time, the rate of growth was also more rapid for ewe than 59 ram or wether lambs (172, 119, 88 \u00C2\u00B1 8 g d'1, for ewe, ram and wether lambs, respectively P<0.05) and especially for ewes given Mo+S (inset Figure 5). 5.2.4 Serum L H secretory profiles Examples of serum L H secretory profiles for ewe, ram and wether lambs in each diet group are shown in Figure 6a-c. In autumn, extremely high peak L H values (50 to 145 ng ml1) occurred for Group III and IV wethers, especially on September 27th. One Group III ewe experienced an L H surge, during the last sampling period, which was characterized by a gradual decline in L H from 28.87 fig L' 1 to 1.96 /ig L' 1. These data were excluded from analysis of variance. Mean concentrations of L H (Table 8) were affected by gonadal influence*Mo*S interaction. For ram and ewe lambs, mean L H values were higher for Mo- (Group II) than Mo+S-supplemented (Group IV) groups, whereas the reverse trend occurred in wether lambs. The effects on mean L H were mostly due to the gonadal influence*Mo*S*time effect on peak amplitude and gonadal influence*time effect on L H peak frequency (Figure 7). For ram lambs, L H peak amplitude was higher for Mo- than Mo+S- supplemented groups, whereas the reverse trend occurred in wether and ewe lambs. The frequency of L H peaks was higher for wether than ram or ewe lambs and increased two to four weeks after the autumn equinox. The gonadal influence*time effect on L H peak frequency was mostly due to the gradual drop in frequency for ram lambs 4 to 8 weeks after the equinox. Basal L H did not change over time, but were affected by gonadal influence*Mo*S interaction as described for the mean L H . Residual sums of squares for L H mean and pooled basal value were correlated (r=0.77; df=7; P<0.05). 5.2.5 Scrotal circumference and semen Scrotal circumference and sperm per ejaculate are shown in Figure 8. For Group I, mean scrotal circumference, sperm per ejaculate and sperm motility (20 to 50%) were low because one ram had only one descended testicle (cryptorchism was confirmed at slaughter), but for Group II, sperm per ejaculate and motility score were low even though scrotal circumference indicated normal testes. Scrotal 60 circumference was affected by Mo*time interaction. Testicular growth was rapid from -2 to 4 weeks after the equinox (August to October), but testes regression between 4 and 12 weeks after the equinox (October to December) was less for rams given Mo (Groups II and IV). Sperm output and motility between 12 and 16 weeks after the equinox (December) was affected by S level. Sperm per ejaculate was lowest for Mo-supplemented group, but sperm motility score which was 1.4, 2.1, 2.0 and 3.4 \u00C2\u00B1 0.5 (P<0.05), for Groups I, II, III and IV, respectively, was highest for Group IV (50 to 75% sperm motility). 5.2.6 Serum Testosterone twice weekly and secretory profiles Twice weekly serum testosterone concentrations (Figure 9) were affected by Mo*S*time interaction. Testosterone was generally lower for S-supplemented groups, but without added S concentrations were lower for Mo-supplemented rams between 4 and 8 weeks after the equinox. Without added Mo (Groups I and III), testosterone was highest between October and November, when rams were aged 41-47 weeks and then returned to summer levels in December. The rise in testosterone occurred as scrotal circumference (Figure 8) increased and declined after scrotal circumference had significantly decreased. For rams given Mo (Groups III and IV) testosterone was highest in November, when lambs were aged 48 to 52 weeks and the sustained rise in testosterone occurred after scrotal circumference had begun to decrease. Examples of serum testosterone secretory profiles for ram lambs in each diet group are shown in Figure 10. For Mo-supplemented rams, there was no peak secretion of testosterone in one ram in June (Group IV) and three rams on October 25th (Groups II and IV). Mean concentrations did not differ among groups (9.7, 8.0, 7.8 and 4.6 \u00C2\u00B1 1.8 /_g L 1 ; Groups I, II, III and IV, respectively), but basal testosterone was affected by Mo*time and S*time interactions and peak frequency was affected by time (Figure 11). Basal values were lower for Mo-supplemented groups especially four to six weeks after the autumn equinox (October). Interaction between S level and time was due to the sharp decrease in basal value that occurred between eight and ten weeks after the equinox in lambs given S alone. For all groups, peak frequency was maximal two weeks after the equinox. Peak amplitude showed no differences among 61 treatment groups or time periods and was 4.25, 3.98, 3.68, 3.96 \u00C2\u00B1 0.96 jug L\"1; (P>0.20), for Groups I, II, III and IV, respectively. The lag time between peak concentrations of L H and testosterone tended (P=0.20) to be affected by Mo*S interaction. L H peak concentrations occurred 23, 34, 29 and 29 \u00C2\u00B1 5.5 minutes before the first significant rise in testosterone, for Groups I, II, III and IV, respectively. 5.2.7 Estrous and serum P4 twice weekly profiles First estrous, as determined by vaginal smear, occurred ~8 weeks earlier (P<0.05) in ewes given Mo (Groups II and IV). However, body weight at first estrous and total number of estrous cycles did not differ among groups (38, 32, 34 and 36 \u00C2\u00B1 4 kg and 4.5, 5.5, 6.0, 6.5 \u00C2\u00B1 0.8 cycles for Groups I, II, III and IV, respectively). The duration of estrous cycles for ewes in Groups I, II, III and IV, respectively, were 18, 22, 12, 23 \u00C2\u00B1 3 days and tended (P=0.10) to be longer for ewes given Mo (Groups II and IV). Serum P4 was measurable in ewes by 15 weeks of age (-12 weeks before the equinox). For each ewe, the profiles consisted of pre-ovulatory (when P4 <1 jug L'1) and ovulatory (P4 peaks >1 jug L'1) phases. The time of first ovulation for Groups I, II, III and IV, respectively, was 28, 34, 32 and 30 \u00C2\u00B1 2 weeks of age and did not differ among groups. However, this was mostly because of the extreme difference between ewes given the unsupplemented diet (Group I). For this Group, one ewe began ovulations on September 3rd, but the other had only one cycle which occurred at the end of November. Otherwise, ewes given Mo (Group II) tended to be older and heavier (41, 49, 46, 48 \u00C2\u00B1 5 kg for Groups I, II, III and IV, respectively), when ovulation occurred. The total number of ovulations and average duration of the luteal phase of estrous which were 3.0, 2.5, 3.5 and 4.5 \u00C2\u00B1 0.4 ovulations and 14, 17, 16, 16 \u00C2\u00B1 1.5 days, for Groups I, II, III and IV, respectively, also did not differ among Groups. Serum P4 concentrations were affected by Mo and Mo*S interaction (Figure 12). Pre-ovulatory (prepubertal) P4 concentrations were higher for ewes given Mo, regardless of S level. The Mo*S interaction was due to the steady rise in P4 concentrations between ovulations in Mo+S-supplemented ewes (Group IV). 62 Measurable concentrations of P4 in wether serum occurred in only 8, 3, 3 and 7 of 31 samples analyzed for each wether in Groups I, II, III and IV, respectively. The respective concentrations (mean \u00C2\u00B1 standard deviation n=4 assays) were 0.13 \u00C2\u00B1 0.02, 0.10 \u00C2\u00B1 0.01, 0.11 \u00C2\u00B1 0.01 and 0.10 \u00C2\u00B1 0.01 j_g L\"1 (Also refer to Appendix VHIb). 5.2.8 Serum Cortisol twice weekly and secretory profiles Twice weekly serum Cortisol profiles (Figure 13) were affected by gonadal influence and Mo*time interaction. Cortisol was higher for Mo-supplemented lambs before the autumn equinox and again eight weeks later, especially in the absence of added S. Generally, Cortisol concentrations were lower for ram, than ewe or wether lambs (11.2 \u00C2\u00B1 1.4, 16.7 \u00C2\u00B1 1.4 and 20.4 \u00C2\u00B1 2.0 /zg L 1 ; P<0.05). The highest concentrations of Cortisol were measured in serum collected immediately following transportation (~ lh) from U B C to the abbatoir in Pitt Meadows, British Columbia. Adjusted for concentration in samples collected before transport (covariate), after transport concentrations which were 61.4 \u00C2\u00B1 3.3, 55.8 \u00C2\u00B1 4.2, 62.7 \u00C2\u00B1 3.3 and 49.3 \u00C2\u00B1 4.2 jig L\"1 (P<0.05), for Groups I, II, III and IV, respectively, were lower for Mo- supplemented groups (Groups II and IV), depending upon gonadal influence and level of S. Unadjusted means and discussion of these observations are given in Appendix VII. Examples of serum Cortisol secretory profiles of ewe, ram and wether lambs in each diet group are shown in Figure 14a-c. Mean concentrations of Cortisol were affected by Mo*S interaction and gonadal influence (Table 9). The Mo*S interaction was due to S causing differences between Mo-supplemented groups. Mean Cortisol was lower for ram than wether or ewe lambs, but this did influence the effect of Mo*S interaction. Basal Cortisol was affected as described for the mean and the residual sums of squares were correlated (r=0.84; df=7; P<0.05). Cortisol peak amplitude was higher for Mo-supplemented groups, however, supplemental S eliminated this effect when concentrations were low two to four weeks after the equinox (Figure 15). Peak frequency showed gonadal influence*time interaction due to the gradual rise in frequency for ram lambs six to ten weeks after the equinox. 63 5.2.9 Other tissues Liver weight was greater for S-supplemented groups (0.76, 0.76, 0.78, 0.82 \u00C2\u00B1 0.02 kg for Groups I, II, III and IV, respectively P<0.05), regardless of gonadal influence. Details are given in Appendix VII. All ewes had several small and a few medium follicles on the right ovary. A single corpus luteum was present on the right ovary in 4/8 ewes, on the left ovary in 1/8 ewes and both ovaries in 3/8 ewes of which two were in Mo-supplemented groups. 5.2.10 Partial correlations Ontogeny of puberty and serum secretory profiles are shown in Appendices VIII and IX, respectively. Partial correlation coefficients among parameters are given in Appendix VI. Available Cu intake showed correlation with average daily gain (r=0.69), liver weight (r=0.69), pre-pubertal P 4 (r=0.89), L H peak frequency (r=-0.74) and Cortisol basal value (r=0.66). Total Mo intake showed correlation with testosterone peak amplitude (r=0.92), L H peak frequency (r=-0.74) and Cortisol basal value (r=0.72). Correlation also occurred between TCu and testosterone amplitude (r=0.88), L H peak amplitude (r=0.73) and L H peak frequency (r=-0.64), RCu and Cortisol peak amplitude (r=-0.75) and TMo and L H peak amplitude (r=0.64). 64 Table 5. Nutrient composition of dry matter in experimental diets given to ewe, ram and wether lambs for 32 to 39 weeks.11 Treatment group Item I II III IV Number of Samples 15 15 9 14 Copper (mg kg1) 8.1 7.7 7.5 7.6 Molybdenum (mg kg1) 0.6 14.5 0.6 18.3 Coppenmolybdenum ratio 13.5 0.5 12.5 0.4 Sulfur (g kg1) 2.1 2.1 3.9 3.7 Copper availability (%)\u00C2\u00A7 4.7 1.8 3.0 0.4 Based on laboratory analysis. Other nutrients measured in concentrate were, on a per kg dry matter basis, gross energy (19.2 MJ), protein (17.7%), lipid (2.0%) and acid detergent fibre (8.3%). Timothy hay contributed ~20% to dry matter intake and contained, on a dry matter basis, 17.9 MJ kg-1 gross energy, 11.5% protein, 2.5% lipid, 31.3% acid-detergent fibre, 58.3% neutral-detergent fibre. ^ Coefficient of copper absorption was derived from the equation given by Suttle and McLaughlin (1976): Log T A = -0.0019 Mo - 0.075 S - 0.0131((Mo)(S)) - 1.153 where T A Cu is the true availability of copper, Mo and S are molybdenum (mg kg\"1) and sulfur (g kg1) concentration in the diet dry matter. m b / d 7 H\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i\u00E2\u0080\u0094i i i i i i i i i i r~i i i i i i i i i i i i i i i i i i i i i i i i -12 -8 - 4 . 0 4 8 12 -12 -8 -4 0 4 8 12 Time f rom autumn equinox (weeks) Figure 4. The effect of dietary sul fur level on feed intake by lambs (n=5) given cereal-based diets containing two levels of molybdenum (ANOVA s u l f u r H i m e P<0.05). 66 Table 6. Copper (Cu) soluble in trichloroacetic acid (TCA) and residual Cu in serum of ewe, ram and wether lambs given, cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 23-30 weeks (October). Mineral Supplement (kg1) Gonadal influence Mo S Group Group mg g Ewe Ram Wether Mean (n=5) T C A Copper (/xmol L 1) 7\" I 0 0 11.9 13.6 19.7 14.6a II 12 0 11.2 13.6 18.3 13.5a III 0 2 11.9 11.5 15.3 11.9b IV 12 2 11.4 7.0 10.4 10.1\" pooled SEM \u00C2\u00B1 1.2 \u00C2\u00B1 1.2 \u00C2\u00B1 1.3 \u00C2\u00B1 0.8 Residual copper (/Ltmol L\"1) I 0 0 2.4 0.7 2.5 1.8a II 12 0 1.9 2.3 2.6 2.1a III 0 2 1.0 1.9 2.1 1.6a IV 12 2 4.1 5.4 8.4 5.9\" pooled SEM \u00C2\u00B1 1.6 \u00C2\u00B1 1.6 \u00C2\u00B1 2.2 \u00C2\u00B1 0.9 a b Means in columns with different letters differ (P<0.05) 7 T C A means are higher for wether, than ram or ewe lambs (P=0.05). 67 Table 7. Total copper (Cu) and total molybdenum (Mo) in serum of ewe, ram and wether lambs given, cereal-based diets with and without supplemental Mo and sulfur (S), for 32-39 weeks (December). Mineral Supplement (kg1) Gonadal influence Mo S Group Group mg g Ewe Ram Wether Mean (n=5) Total copper (Ltmol L\"1)1\" I 0 0 11.6 15.6 22.6 16.6 II 12 0 16.3 15.0 21.4 17.7 III 0 2 16.1 14.5 18.6 16.4 IV 12 2 17.1 13.6 25.6 18.8 pooled SEM \u00C2\u00B1 1.9 \u00C2\u00B1 1.9 \u00C2\u00B1 2.7 \u00C2\u00B1 1.3 Total molybdenum (Ltmol L\"1) I 0 0 0.2 0.2 0.3 0.2a II 12 0 3.0 2.6 2.7 2.8\" III 0 2 0.1 0.1 0.1 0.1a IV 12 2 2.5 3.5 2.8 2.9\" pooled SEM \u00C2\u00B1 0.5 \u00C2\u00B1 0.5 \u00C2\u00B1 0.7 \u00C2\u00B1 0.3 a b Means in columns with different letters differ (P<0.05) T Total Cu is higher for wether, than ram or ewe lambs (P<0.05) Figure 5. The effect of dietary molybdenum level on body weight of lambs (n=5) given cereal-based diets containing two levels of sulfur (ANOVA molybdenurn*time P<0.05). The inset shows when molybdenum plus sulfur affected ewes (n=2) differently than rams or wethers (gonadal influence*molybdenum*sulfur*tirne P<0.05). L H 3H Group I \u00E2\u0080\u0094 E410 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 Group III E406 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 Group IV \u00E2\u0080\u00A2 E365 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 Date (m/d) and time from autumn equinox (weeks) Figure 6a. Luteinizing hormone (LH) serum secretory profilesof individual ewe lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise. Group I \u00E2\u0080\u0094 R333 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 Group III R345 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 Group IV \u00E2\u0080\u0094 R329 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 Date (m/d) and time from autumn equinox (weeks) Figure 6b. Luteinizing hormone (LH) serum secretory profilesof individual ram lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise. 6/20-9/13 9/27 10/11 10/25 11/8 11/22 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 -12 0 2 4 6 8 10 Date (ra/d) and time f rom a u t u m n equinox (weeks) Figure 6c. Luteinizing hormone (LH) serum secretory profilesof individual wether lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise. 72 Table 8. Luteinizing hormone (LH) mean concentration in serum of ram, wether and ewe lambs given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 32-39 weeks. Mineral Luteinizing hormone Supplement (kg1) Gonadal influence Mo S Group Group mg g Ewe Ram Wether Mean (n=5) L H (fig Vl)\u00C2\u00A7 I 0 0 0.44 0.6V 6.89a 1.80a II 12 0 0.40 0.84a 3.47\" 1.19\" III 0 2 0.31 0.55a 7.15a 1.77a IV 12 2 0.32 0.21\" 8.16a 1.84a , b Means in columns with different letters differ (P<0.05) ^ Significant effects Mo*S and gonadal influence*group (P<0.05; SE=0.02; 7 df). 10-13 ug/L Time f rom autumn equinox (weeks) Figure 7. The effect of dietary molybdenum level on the amplitude of lute iniz ing hormone (LH) secretory peaks in serum of r a m , ewe and wether lambs given cereal-based diets containing two levels of sulfur (ANOVA gonadal influence* molybdenum*sul fur* t ime P<0.05) The inset shows the gonadal inf luence*t ime (P<0.05) effect on LH peak frequency. (R=ram n=8, E=ewe n=8, W=wether n=4). Figure 8. The effect of dietary molybdenum level on scrotal c ircumference (line graph) and sperm per ejaculate (histogram) of r a m lambs (n=2) given cereal-based diets containing two levels of sulfur (ANOVA molybdenum*time P<0.05; * differs f rom other dashed lines exclude the cryptorchid r a m , refer to text). 0 I i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i > i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i -4 0 4 8 12 -4 0 4 8 12 Time from a u t u m n equinox (weeks) Figure 9. The effect of dietary molybdenum level on testosterone concentrations in serum collected twice weekly from r a m lambs (n=2) given cereal-based dietsontaining two levels of sul fur (ANOVA molybdenum*sul fur* t ime P<0.05). 6/20 9/13 9/27 10/11 10/25 11/8 11/22 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 -12 0 2 4 6 8 10 Date (m/d) and time from autumn equinox (weeks) Figure 10. Testosterone serum secretory profiles of individual ram lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise. 24 n 0 Sulfur g/kg - 2 Sulfur g/kg B a Molybdenum mg/kg Molybdenum mg/kg s J H 1 i r 1 1 1 i 1 1 1 1 1 1 \u00E2\u0080\u009412 0 2 4 6 8 10 -12 0 2 4 6 8 10 Time from autumn equinox (weeks) Figure 11. The effect of dietary molybdenum level on basal testosterone concentrations in serum of ram lambs (n=2) given cereal-based diets containing two levels of sulfur (ANOVA molybdenumHime, sulfur*time P<0.05). The inset shows the effect of time (P<0.05) on the frequency of serum testosterone secretorypeaks. -a \u00E2\u0080\u0094 i 0 Sulfur g / k g \" 2 Sul fur g / k g - 1 2 - 8 - 4 0 4 8 12 . -12 - 8 - 4 0 4 8 12 Time from autumn equinox (weeks) Figure 12. The effect of dietary molybdenum level on progesterone concentrations i n serum collected twice weekly f rom ewe lambs (n=2) given cereal-based diets containing two levels of sulfur (ANOVA molybdenum*sulfur* t ime P<0.05). 0 Sulfur g / k g Molybdenum mg/kg 0 \u00E2\u0080\u0094 1 \u00E2\u0080\u0094 12 1 1 1 1 1 I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 -12 -8 -4 0 4 8 12 2 Sul fur g / k g Molybdenum mg/kg \u00E2\u0080\u0094 0 \u00E2\u0080\u0094 1 \u00E2\u0080\u0094 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 -12 -8 -4 0 4 8 12 Time from autumn equinox (-weeks) Figure 13. The effect of dietary molybdenum level oncortisol concentrations in serum collected twice weekly f rom lambs (n=5) given cereal-based diets containing two levels of sul fur (ANOVA molybdenum*time P<0.05). Group I \u00E2\u0080\u0094 E410 6/20 9/13 9/27 10/11 10/25 U / 8 11/22 -12 0 2 4 6 8 10 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 6/20 9/13 -12 0 9/27 10/11 10/25 11/8 11/22 2 4 6 8 10 Group III - E408 6/20 9/13 9/27 -12 0 2 4 6 Date (m/d) and time from autumn equinox (weeks) 10/11 10/25 8 11/8 11/22 10 Figure 14a. Cortisol serum secretory profiles of individual ewe lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken from 1 to 5 h after sunrise. 80 n 6/20 9/13 9/27 10/11 10/25 11/8 11/22 -12 0 2 4 6 8 10 Group III - \u00E2\u0080\u0094 R345 6/20 9/13 9/27 10/11 10/25 U / 8 11/22 -12 0 2 4 6 8 10 Figure 14b. Cortisol serum secretory profiles of individual r a m lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken f rom 1 to 5 h after sunrise. oo \u00C2\u00B0 6/20 9/13 9/27 10/11 10/25 11/8 11/22 6/20 9/13 9/27 10/U 10/25 11/8 11/22 -12 0 2 4 6 8 10 -12 0 2 4 6 8 10 Date (m/d) and time f rom autumn equinox (weeks) Figure 14c. Cortisol serum secretory profiles of indiv idual wether lambs given cereal-based diets with and without (I) added molybdenum (II) or sulfur (III) alone or combined (IV). Serial samples were taken f rom 1 to 5 h after sunrise. 83 Table 9. Cortisol mean concentration of secretory profiles in serum of ram, wether and ewe lambs given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S). Mineral Supplement (kg1) Gonadal influence Mo S Group Group mg g Ewe Ram Wether Mean (n=5) Cortisol (/ig L\"1) \u00C2\u00A7 I 0 0 16.4 9.5 22.2 14.8a II 12 0 23.9 17.1 29.8 22.3a III 0 2 19.9 13.1 25.8 18.4a IV 12 2 17.2 10.4 23.1 15.6b * Significant effects Mo*S and gonadal influence (SE=0.31; df=7; P<0.05) '\"b Means in columns with different letters differ (P<0.05) 28 0 Sulfur g / k g Molybdenum mg/kg 0 \u00E2\u0080\u0094\u00E2\u0080\u00A2\u00E2\u0080\u0094 12 2 Sul fur g /kg Molybdenum mg/kg \u00E2\u0080\u0094\u00E2\u0080\u0094 0 \u00E2\u0080\u0094I\u00E2\u0080\u0094 12 2.5- -12 0 2 4 6 8 10 2 4 6 8 10 -12 0 2 4 Time f r o m autumn equinox (weeks) \u00E2\u0080\u0094i 10 Figure 15. The effect of dietary molybdenum level on the amplitude of Cortisol secretory peaks i n serum of lambs (n=5) given cereal-based dietssontaining two levels of sul fur (ANOVA molybdenum*sulfur*t ime P<0.05). The inset shows gonadal inf luence*t ime (P<0.05) effect oh Cortisol peak frequency (R=ram n=8, E=ewe n=8, W=wether n=4). 85 Chapter 6 DISCUSSION 6.1 Experiment I 6.1.1 The effect of Mo and Mo+S on serum Cu The basal levels of Cu, Mo and S in the experimental diets were higher than recommended minimum requirements for sheep (ARC 1980). The expected level of supplemental Mo (26 mg kg1) in experimental diets was achieved using ammonium molybdate and similar to levels tested by Bremner and Young (1978), Smith and Wright (1975) and the period of feed contamination in previous work (Robinson 1983). The level of S in the basal diet and the expected level of supplemental S (2 g kg1) in the diet for Group III achieved using sodium sulfate, was the same as previous work (Robinson et al. 1987), similar to Bremner and Young (1978) and Mason et al. (1978), but considerably higher than ~0.73% used by Smith and Wright (1975). Since only one batch was prepared for each treatment group, Mo intake was consistent throughout the 30 day period. The Cu:Mo ratio did not differ between supplemented groups, but the estimated amount of available Cu was 4 times lower for the latter group given Mo+S (Table 1). The effect of Mo and Mo+S on serum Cu concentrations further illustrate the importance of known levels of dietary S when addressing Cu and Mo requirements for ruminant animals (Peterson and Waldern 1977). In agreement with the long term studies cited above, the current work has shown that high dietary Mo+S resulted in the appearance of RCu in serum within a short period of time. Although Cu:Mo ratio and Cu availability of less than 1 were associated with low but adequate concentrations TCA-Cu in serum, neither parameter was associated with the highest level of serum RCu in the Mo+S-supplemented group (Table 2). There was some RCu present in serum of lambs given Mo-alone (Group II) but this was likely due to high dietary Cu and S (Suttle and Field 1983). 86 6.1.2 The effect of Mo on pituitary-testes function The high level of serum RCu in the Mo+S-supplemented group (III) is indicative of thiomolybdates in serum (Suttle and Field 1983), however this did not affect growth or the pulsatile secretion of L H and testosterone within the time frame of the experiment. In contrast, the age*diet effect of supplemental Mo alone on L H peak amplitude is indicative of an effect of Mo on pituitary function. The pulsatile patterns of serum L H and testosterone for the control group (Figure 3) agreed with the developmental patterns for spring-born rams reported by Yarney and Sanford (1989). Higher testosterone for older rams was indicative of increased responsiveness of the testes to L H stimulation. The amplitude of L H serum secretory peaks was expected to be lower for older ram lambs (Yarney and Sanford 1989), but this did not occur because of the abnormally high L H peak amplitude for rams given Mo (Table 3). Pituitary content of L H is maximal by 21 weeks of age, but by 28 weeks of age, drops by 50% (Yarney and Sanford 1989). During this same period, pituitary FSH remains unchanged. Thus, the experimental diets were introduced during a critical stage of hypothalamic-pituitary development. Between 17 and 21 weeks of age, characteristics of L H and testosterone serum secretory profiles do not differ, but between 21 and 28 weeks of age show striking change; the amplitude of L H peaks drops by ~33% while basal L H and L H peak frequency increases by ~50%; basal testosterone increases 1200 fold, frequency and amplitude doubles (Yarney and Sanford 1989). Because basal testosterone tended to show differences between age but not diet groups, it would seem testicular responsiveness to L H was not impaired severely, but abnormally high L H peak amplitude in older ram lambs given Mo indicated that high dietary Mo enhanced pituitary release of L H , perhaps due to impaired pituitary responsiveness to GnRH. 87 6.2 Experiment II 6.2.1 The effect of S, Mo+S and gonadal influence on serum Cu and Mo As in Experiment I, the basal levels of Cu, Mo and S in experimental diets were above minimum allowances recommended for sheep (ARC 1980). The variation in Mo concentrations in diets (refer to 5.2.1) occurred within Mo-supplemented groups only and may have contributed to some of the Mo*time interaction but without affecting comparisons with groups not given Mo. As in Experiment I, the Cu:Mo ratio did not differ between Mo-supplemented groups, but the estimate of available Cu was 4.5 times lower when S was added with Mo. The effect of Mo+S on the distribution of Cu in serum agreed with other work (Lamand et al. 1980), but in the current work, the highest level of RCu occurred in animals given Mo+S, even though S and gonadal function had a depleting effect on TCA-Cu (Table 7). Serum Mo concentrations increased ~ 12 fold regardless of S level, similar to the effect of Mo added in diets given to cattle (Wittenberg and Devlin, 1988). Serum TMo was lower for the S-supplemented than the control group in a two group, but not four group comparison because TMo was so high for Mo-supplemented groups. However, whether or not S could induce Mo deficiency can not be concluded from this work. Unlike the serum Cu, TMo was not influenced by gonadal function. Body weight, serum L H , testosterone, P4 and Cortisol concentrations were affected by Mo*S interaction indicating that the effect of Mo on these parameters are dependent upon the presence of thiomolybdate. The experiment was conducted over a long period of time during a dynamic period of reproductive development and thus Mo*S interaction with time effects on growth and endocrine function were expected. The enhancing effect of Mo and the gonadal influence*Mo*S*time effect on lamb growth in autumn (Figure 5) is important from a physiological and production point of view. In practise this subtle effect of Mo on growth would probably go unnoticed as normal variation among animals, but physiologically is associated with an effect on endocrine function, primarily pituitary secretion of L H . 88 6.2.2 The effect of Mo on pituitary function Pulsatile patterns of L H concentrations in serum were in general aggreement with previous studies in sheep (Sandford, Howland and Palmer 1984ab; Fitzgerald, Michel and Butler 1982). The higher L H concentrations in serum of wethers was expected and reflects pituitary function in absence of gonadal feedback (Sandford, Howland and Palmer 1984b). Increased L H pulse frequency occurred in the presence or absence of gonadal feedback indicating that this event is regulated primarily by photoperiod and occurs beginning two weeks after the equinox when the rate of decreasing daylength is most rapid (see also Appendix VHIa-c). Sandford, Howland and Palmer (1984b) also showed that hypothalamic-pituitary function in long term castrate rams is photoperiod sensitive. Within the time frame of the present experiment there was an inverse effect of Mo and Mo+S on L H peak amplitude in ram, wether and ewe lambs. The inverse affects of Mo and Mo+S were also not dependend on low serum Cu, because low serum Cu induced with supplemented S had no effect on L H . Therefore, high serum Mo and high serum thiomolybdate (RCu) have unique effects on the pitutary not associated with depressed TCA-Cu in serum. This does not preclude the metabolic function of Cu was affect by Mo. The effect of Mo and Mo+S on L H secretion was different in rams than ewe or wether lambs which likely reflected sexual differences in hypothalamic-pituitary function. 6.2.3 The effect of Mo on testicular function Molybdenum delayed maturation of the pituitary-testes axis as indicated by the delayed increase in serum testosterone that has normally occurred with maturation of the hypothalamic-pituitary-testicular axis, in autumn, (Yarney and Sanford 1989; Sanford et al. 1984). Delayed rise in serum testosterone has also been induced in rams given P4 implants (Echternkamp and Lunstra 1984). In agreement with Sanford and Yarney (1989), the rise in testosterone in autumn was due to increased base and frequency (Figure 9) rather than amplitude. In ram lambs Mo depressed sperm output but S alleviated this effect. With reference to Figure 13, temporal association between rising testosterone and peak scrotal circumference agrees with other work showing the seasonal pattern of scrotal circumference, testosterone and semen 89 quality in rams (Mickelsen 1981). For lambs given the unsupplemented diet or added S (Groups I and III) twice weekly testosterone was highest between October and November and then returned to summer levels in December. The rise in testosterone occurred as scrotal circumference increased and declined after scrotal circumference had significantly decreased. For rams given Mo (Groups III and IV) a sustained rise in testosterone did not occurr until November after scrotal circumference had begun to decrease. In December, when semen collections were performed Groups I and III had already passed their optimum time for sperm production and better motility for Mo+S-supplemented lambs (Group IV) may have resulted from higher testosterone at this time, since sperm motility is affected by seasonal regression sooner than sperm production (Mickelsen 1981) (see also Appendix Villa). 6.2.4 The effect of Mo on ovarian function Dietary supplemental Mo affected ovarian function less severely than testicular function. The effect of Mo on ovarian function was predominant during the pre-ovulatory phase, which may explain, in part, why estrous occurred earlier in ewes given Mo (Groups II and IV). Ewes given the basal diet (Group I) experience a short first luteal cycle which frequently in lambs but there has been some debate over the importance of this event for subsequent cycles (Lehmen 1986; Keisler, Inskeep and Dailey 1983). A short first luteal cycle did not occur in ewes given Mo-supplemented diets, this may or may not have been due to the effect of Mo on preovulatory P4. In cycling ewes, prolonged estrous cycles may be due to high serum P4 concentrations and this may partially explain prolonged estrous in rats (Fungwe et al. 1987) and heifers (Phillippo et al. 1987) given Mo. High serum P4 was not likely of adrenal origin since P 4 was essentially non-detectable in wether serum (refer Appendix VHIb). In ewes given Mo+S, high P4 was associated with low Cortisol. A deficiency of the cuproprotein C,720-lyase would theoretically prevent conversion of P4 to androgens and/or 11-deoxycortisol (Figure 2). Thus Mo+S or thiomolybdates have an undefined effect on P 4 and Cortisol metabolism. 90 6.2.5 The effect of Mo on adrenal function Cortisol concentrations in serum were within ranges reported for sheep (Wilkinson 1980). Twice weekly serum Cortisol profiles were affected by Mo*time because of chronically higher concentrations for Mo-supplemented lambs from -8 to 0 and 4 to 12 weeks after the autumn equinox, when concentrations were low for the other groups (Figure 13). However, serum secretory profiles were affected by Mo*time because basal Cortisol and peak amplitude were higher for Mo- than Mo+S-supplemented groups (Table 9 and Figure 15). No publications describing this effect of Mo on serum Cortisol were found. However, circannual and diurnal rhythms in Cortisol secretion are known (Wilkinson 1980; Fulkerson and Tang 1979). Higher concentrations during estrus and in gonadectomized sheep have been reported (Wilkinson 1980) and was shown in the present study by gonadal influence*time variation. Cortisol concentrations in serum were lowest two to four weeks after the equinox but at the same time frequency of Cortisol pulses were highest. This pattern occurred in presence or absence of gonadal influence and thus indicates that the frequency of pulsed Cortisol secretion is regulated by photoperiod as for L H . From two to eight weeks after the equinox Cortisol peak was low, especially for Mo-supplemented groups, but basal Cortisol was higher for Mo- than Mo+S-supplemented groups. Thus Mo and thiomolybdates (RCu) must affect adrenal function by different mechanisms. The effect of Mo and Mo+S on adrenal secretion of Cortisol did not differ significantly among ram, wether and ewe lambs and thus represents a common mechanism. The adrenal-pituitary-gonadal relationship has not been well defined and there are positve and negative effects of Cortisol on reproductive function (Moberg 1985ab). Acute or chronic increases in serum Cortisol has had a depressing effect on L H , testosterone, and ovarian function (Welsh, McCraw and Johnson 1979; DeSilva, Kaltenbach and Dunn 1983; Welsh and Johnson 1981; Welsh, Bambino and Hsueh 1982; Matteri, Watson and Moberg 1984; Moberg 1985ab). However in most cases, severe restraint stress or injection of A C T H (>40 I.U. ACTH) were used. Further investigation is required to determine if higher serum Cortisol due to Mo mediated the effect of Mo on pituitary-gonadal function or if high serum Cortisol was due to the effect of Mo on hypothalamic-pituitary release of CRF and A C T H . Further investigation is also required 91 to more clearly define the dampening effect of Mo+S on serum Cortisol as this finding suggests that it may be possible to use thiomolybdates to lower adrenal secretion of Cortisol in response to stress or suckling (Wagner and Oxenreider 1972) without reducing P4 (Figure 12). 6.3 General 6.3.1 The relation between Cu and Mo in diets and serum Concentrations of Mo and S in diets for both experiments were similar to naturally occurring levels in various feeds for ruminant animals (Fletcher and Brink 1969; Miltimore and Mason 1970). The grain ration of the diets represented ~90% of dry matter intake and therefore hay did not contribute substantially to the amount of Cu, Mo and S consumed each day. On a cereal-based diet, rumen pH is normally slightly acidic, ~ pH 6.5 and favours the production of glucose precursor, propionate, by microbes (Hungate 1966). Protein, nitrogen to sulfur ratio, iron and other elements were probably not limiting nutrients in the diets since they met or exceeded recommended minimum allowances for lambs (NAS-NRC 1975; A R C 1980). The estimates of Cu availability (Tables 1 and 5) ranging from 0.2 to 4.7% are applicable to cereal, but not forage concentrations of Cu, Mo and S (Suttle 1983). However, when compared with total intake of Cu they illustrate the limiting effect of Mo and S on Cu absorption. The absorption of Mo is also normally very low (Mason et al. 1978) and the estimates of total intake of Cu, Mo and S are more indicative of the abundant supply to rumen microflora. The stimulating effects of Mo and S on microbial metabolism (Gawthorne and Nader 1976) may have caused the enhancing effect of S level on feed intake (Figure 4). The pH of the rumen may (Clark and Laurie 1980) or may not (Aymonino et al. 1969) have favored the formation thiomolybdates, however, the high levels of RCu (Tables 2 and 6) for Mo+S-supplemented groups indicate that sufficient amounts of each element were absorbed and thiomolybdates were present and perhaps formed in blood (pH ~7.4) (Suttle and Field 1983). 92 The dietary levels of Mo and S achieved using ammonium molybdate and sodium sulfate, induced changes in the distribution of Cu in serum (Tables 2, 6 and 7) that were consistent with previous studies in rams (Mason et al 1978), castrate rams (Marcilese et al. 1969) and ewes (Lamand et al 1980). For all lambs, TCA-Cu concentrations were indicative of adequate Cu status, even though concentrations were lower for S-supplemented groups. Some RCu was detected for all groups, but was ~6 fold higher for Groups given Mo+S. The RCu for groups given the basal diet or S alone was probably caused by high concentrations of Cu and S despite low Mo and may have formed during T C A acidification of serum (Mason 1986). However, high concentrations of Mo coupled with high RCu were indicative of thiomolybdates in blood (Mason 1986). Although Mo in the T C A component of serum was not measured in the present work, it is likely that there was more freely diffusable M o 0 4 = in serum of lambs given Mo alone, whereas most of the Mo in serum of lambs given Mo+S was probably thiomolybdate as indicated by high levels of RCu. The absence of an effect of Mo and S on TCu concentrations agreed with others (Marcilese et al. 1969; Mason et al. 1978; Lamand et al. 1980; Ishidia et al. 1982) although an increase has been reported by some authors (Mason 1986). These discrepancies may be due to methodology since RCu is a nonhomogeneous fraction subject to some degree of artifact or perhaps real differences in metabolism caused by diet ingredients (forage or cereal based) and gonadal factors. Serum Cu concentrations were higher in absence of gonadal factors (Table 7) and studies using wethers are probably more likely to show increased TCu when given Mo and S. 6.3.2 Gonadal influence on serum Cu Significant effects of gonadal influence, regardless of the effects of Mo and S on serum Cu, occurred in autumn, but not in June. This evidence of a relationship between Cu metabolism and gonadal function supports several other reports of steroid hormone effects on Cu in tissues and blood. An implant of E 2 , but not P4, has been shown to maintain serum Cu at the expense of liver Cu in the ovariectomized ewe (Russanov, Banskalieva and Ljutakoba 1981). Glucocorticoids also mobilize hepatic Cu (Weiner and 93 Cousins, 1982) and perhaps higher serum Cu for wether lambs, was due to higher concentrations of circulating Cortisol (Table 9). Testosterone inhibits the action of E 2 on serum Cu (Schreiber and Pribyl 1980), but then mobilization of hepatic Cu in the ram may be dependent upon aromatization of testosterone to E2. These relationships between Cu and steroid hormones may explain, in part, why the effects of Mo, S and Mo+S on growth, pituitary, gonadal and adrenal function were time dependent and showed interaction with gonadal influence especially in autumn when the rate of reproductive development was accelerated by rapidly decreasing photoperiod. 6.3.3 The relation between Mo and S and endocrine function Both experiments showed significant effects of Mo on the amplitude of L H peaks in serum (Table 3 & Figure 7). This effect of Mo differs from depressed L H peak frequency, indicative of impaired hypothalamic function, in lambs given energy or protein restricted diets (Fitzgerald 1982; Foster et al. 1984ab, Lindsay et al 1984). In agreement with Phillippo et al. (1987), the effect of Mo on L H was not dependent upon low concentrations of Cu in serum. The dampening effect of Mo on L H peak amplitude in wether and ewe lambs agrees with the effect of Mo on L H in heifers (Phillippo et al. 1987). If Mo+S had caused depressed activity of Cu enzymes such as aromatase then one would expect higher concentrations of testosterone (Figure 2), but this did not occur. However, if Mo did impair E 2 feedback on L H secretion this may explain why scrotal circumference was largest for Mo-supplemented lambs twelve weeks after the autumn equinox (Figure 8; Land, Baird and Carr 1981) and higher L H in rams given Mo alone (Schanbacher 1984). In the ewe, suppression of Ej during the prepubertal period may have promoted the onset of estrus since at this stage the pituitary is very sensitive to the low levels of E 2 secreted by the developing follicle. However, with the advent of ovulation a higher level of serum E 2 is required for the induction of the L H surge. An effect of Mo on E 2 metabolism may also explain why Mo affected L H secretion differently in wethers and ewes than rams. In ewes, the pituitary becomes less sensitive to E 2 where as, in rams it becomes more sensitive. In contrast, to ewe and wether lambs, Mo had an enhancing 94 effect on L H peak amplitude in rams (Table 6, Figure 7) especially when concentrations of testosterone were high (Table 8). The inverse effects of Mo and Mo+S on L H may be useful for manipulating pituitary secretion of L H . To depress L H amplitude Mo+S given to rams and Mo given to wether and ewe lambs should be effective. In converse, to enhance L H amplitude Mo given to rams and Mo+S given to wether and ewe lambs should be effective. The dampening effect of Mo on L H amplitude in wethers may explain some of the discrepancy between regions concerning photoperiod effects on L H secretion (Sanford et al. 1984b). In Manitoba, feeds typically contain one to two mg kg 1 D M which may be sufficient to eliminate the seasonal effect on L H amplitude in wethers maintained on low Mo feeds (<1.0 mg kg 1 D M ; Lincoln and Short 1980). It is evident that Mo and Mo+S have unique effects on pituitary, gonadal and adrenal function in lambs. The effects of Mo may be mediated by a non-specific effect on steroid action on target cells. This may explain why the effects of Mo were anabolic-like i.e. altered amplitude of L H peaks, increased Cortisol, increased P4, increased growth, suppressed testicular regression and reduced osmotic fragility. In contrast, the effects of Mo+S may be mediated by the effects of thiomolybdates on the affinity of albumin for circulating steroids and enzyme activity (Mason 1986). Thiomolybdate is known to alter the affinity of albumin for Cu. However, the proportion of steroid bound to albumin is generally low when compared with globular conjugates. It is possible that the non-specific thiomolybdate inhibition of enzyme activity observed in vitro, occurs in vivo altering the activity of biosynthetic (Figure 2) or signal transmission enzymes (Figure 1). The effects of Mo+S may involve Cu metabolism but at a level undetected by serum Cu concentrations. 95 Chapter 7 CONCLUSION A total of 32 lambs were used in two randomized-block experiments to investigate the effects of supplementation of a cereal-based diet containing 8 mg Cu, 0.7 mg Mo and 2.1 g S kg 1 D M , with either Mo or S alone or in combination, on growth, serum Cu and Mo concentrations and reproductive function. Experiment I examined the effects of short-term intake (4 weeks) by ram lambs, aged 16 and 18 weeks, of the basal diet either unsupplemented or supplemented with Mo (26 mg kg-1 DM) alone or in combination with S (2 g kg-1 DM), on growth, serum Cu, hematology and pituitary-testes function. Conclusions drawn from Experiment I were: 1) Serum TCu, feed intake, body weight, scrotal circumference and hematology (PCV and MCHC) were not affected by Mo or Mo+S or age of lamb (P>0.10). 2) Serum TCA-Cu was lower for Mo and Mo+S groups (P<0.05), but serum RCu was highest for the Mo+S-supplemented group (P<0.05) and age of lamb did not influence this result (P>0.10). 3) Serum L H secretory profiles were affected by age*diet interaction (P<0.05); L H peak amplitude, was highest for Mo-supplemented lambs aged 24 weeks (P<0.05), but basal L H and peak frequency were not affected (P>0.10). 4) Serum testosterone secretory profiles were not affected by diet (P>0.10), but age influence was significant (P<0.05); testosterone peak frequency was higher in lambs aged 24 weeks. Basal testosterone and peak amplitude did not differ between lambs aged 22 and 24 weeks (P>0.10). Experiment I showed that short term intake of high dietary Mo enhanced pituitary secretion of L H , without affecting testosterone secretion in ram lambs at a more advanced stage of puberty. This effect of 96 Mo on pituitary function was not due to high serum thiomolybdates (RCu) or low TCA-Cu because, for the Mo+S-supplemented group, RCu was highest and TCA-Cu was low, but L H did not differ from the control group. Experiment II examined the effects of long-term intake (32-39 weeks) by wether, ram and ewe lambs (gonadal influence), of the basal diet with and without added Mo (12 mg kg\"1 DM) or S (2 g kg\"1 DM) alone or in combination on growth, serum Cu and Mo, hematology, pituitary, gonadal and adrenal function. Conclusions drawn from Experiment II were: 1) Feed intake was higher for S-supplemented groups regardless of the level of Mo (P<0.05) and gonadal influence (P<0.05). 2) Serum TCu was not affected by Mo or S (P>0.20), but was affected by gonadal influence; TCu was higher for wether than ram or ewe lambs beginning in autumn (P<0.05). 3) Serum TMo was highest for Mo-supplemented groups regardless of the level of S (P<0.05) and there was no significant effect of gonadal influence (P>0.20). 4) Serum TCA-Cu was lower for S-supplemented groups regardless of the level of Mo and gonadal influence (P<0.05); TCA-Cu was higher for wether than ram or ewe lambs (P<0.05). 5) Serum RCu was highest for the Mo+S-supplemented group (P<0.05) and there was no significant effect of gonadal influence (P>0.20). 6) Growth was affected by Mo and Mo*S interaction depending upon gonadal influence; throughout the study growth was generally better for the Mo-supplemented group, but in autumn, growth rate decreased in wether and ram lambs given Mo+S. 7) Liver weight was higher for S-supplemented groups regardless of Mo level and gonadal influence (P<0.05). 8) Hematology (PCV, MCHC, RBC and MCV) was not affected by Mo, S or Mo+S or gonadal influence (P>0.20), but red blood cell osmotic fragility tended to be lowest for the Mo-supplemented group (P=0.10). 9) Serum L H secretory profiles were affected by Mo depending upon the level of S and gonadal influence (P<0.05); the amplitude of L H peaks, but not basal L H or peak frequency were affected by Mo*S interaction and the nature of this effect was dependent upon gonadal influence. L H peak amplitude was lower for wether and ewe lambs but higher for ram lambs given Mo. Mo+S reversed this response. There was no significant effect of Mo,S and Mo+S on basal L H or peak frequency (P>0.10), but peak frequency was highest 4 to 6 weeks after the autumn equinox (P<0.05). 10) Mo affected testicular function more severely than ovarian function. Testicular regression 12 weeks after the autumn equinox, was less for Mo-supplemented groups regardless of S level. Serum testosterone secretory profiles were affected by Mo, regardless of S level (P<0.05); a sustained rise in basal testosterone was delayed from 0 to 4 weeks to 8 to 10 weeks after the autumn equinox and sperm output in December was lowest for lambs given Mo (P<0.05). The age of ewes when first estrus occurred was lower and prepubertal P4 was higher for Mo-supplemented ewes, regardless of S level (P<0.05), but the occurrence of first ovulation tended to be delayed and the duration of subsequent estrous cycles longer for Mo-supplemented ewes (P=0.10). 11) Twice weekly serum Cortisol was higher for Mo-supplemented groups, regardless of S level and gonadal influence (P<0.05). Cortisol was lower for ram than wether or ewe lambs and also lowest between 4 and 6 weeks after the autumn equinox (P<0.05). Serum Cortisol secretory profiles, were affected by Mo*S interaction; specifically basal Cortisol and peak amplitude were higher for Mo- than Mo+S-supplemented groups, regardless of gonadal influence. Cortisol peak frequency was not affected by Mo,S or Mo+S (P<0.10), but peak frequency was highest 2 to 6 weeks after the autumn equinox (P<0.05). 98 Experiment II has shown that the effects of Mo on growth and endocrine function are dependent upon the level of dietary S. This is probably because high dietary Mo will increase serum Mo, but only high dietary Mo+S will cause high concentrations of thiomolybdates (RCu). As in Experiment I, the effect of Mo was not dependent upon low serum TCA-Cu. Pituitary secretion of L H was affected by Mo with or without gonadal feedback, but the nature of the effect ie. either increased or decreased peak amplitude, is dependent upon the dietary level of S and its interaction with gonadal feedback. Maturation of gonadal function is delayed more by Mo than Mo+S and is more evident in rams than ewes. Adrenal secretion of Cortisol is enhanced more by Mo than Mo+S, regardless of gonadal influence. Experiment II has shown that the level of dietary Mo and S required by replacement lambs is dependent upon their sex and stage of development and that Mo and Mo+S are two possible agents to manipulate the secretion of L H and Cortisol. Short-term investigations such as dose-response studies in rams, synchronized ewes and gonadectomized lambs should be performed to verify the use of Mo and Mo+S to manipulate endocrine function. In conclusion, systemic interaction among Cu, Mo, S, have unique subclinical effects on pituitary, gonadal and adrenal function in lambs and the effect of Mo preempts obvious interaction with Cu as defined for this thesis. In both experiments, Mo affected the amplitude, but not frequency of L H peaks indicating that Mo affected pituitary secretion of L H , but not the hypothalamic pulse signal (pulse generator). Similarly Cortisol peak amplitude but not frequency was affected by Mo. 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Endocrinology 116:2090-2097. 112 APPENDICES T A B L E OF CONTENTS APPENDIX I 114 Steroid hormone radioimmunoassay (RIA) characteristics 114 Experiment I 114 Experiment II 114 APPENDIX II 118 Hourly concentrations of copper in serum 118 APPENDIX III 120 Hematological status, in October, of lambs given cereal-based diets with or without i supplemental molybdenum (Mo) and sulfur (S), for 25-32 weeks (Experiment II) . . 120 APPENDIX IV 121 The influence of electroejaculation method on serum testosterone and Cortisol, libido and semen quality in ram lambs given cereal-based diets with or without added molybdenum and sulfur (Experiment II) 121 Introduction 121 Materials and Methods 123 Results and Discussion 123 Cortisol and testosterone 124 Semen parameters 124 Conclusions 125 APPENDIX V 131 Vaginal Smear evaluation of estrous cycles 131 APPENDIX VI 133 Tables of partial correlation coefficients (r) among copper and molybdenum, growth and pituitary (LH), gonadal and adrenal function examined in this thesis 133 APPENDIX VII 138 The effect of molybdenum (Mo) and sulfur (S) and the influence of the gonads, on transportation induced increased serum Cortisol and liver weight of lambs (Experiment II) 138 Introduction 138 Materials and Methods 138 Results 139 Discussion and conclusions 139 APPENDIX VIII 143 Ontogeny of Puberty 143 APPENDIX IX 146 Ontogeny of serum secretory profiles 146 113 LIST OF TABLES A N D FIGURES IN T H E APPENDICES i Table la. RIA antisera crossreactivity (%) as given by the manufacturer 116 Table lb. Quality of RIA of progesterone (PRGN), testosterone (TESTO) and Cortisol (CORT) in the present study and as given by the manufacturer (in brackets) 117 Table II. Serum copper (Cu) soluble in trichloroacetic acid (TCA), at one, two and three hours after sunrise on June 20th, in ram and wether lambs given cereal-based diets with or without supplemental molybdenum (Mo) and sulfur (S), for 13 weeks 119 Table III. Hematological status, in October, of lambs given cereal-based diets with or without supplemental molybdenum (Mo) and sulfur (S), for 25-32 weeks (Experiment II) 120 Table IVa. Cortisol concentrations in serum before and after, two types of electroejaculation of ram lambs given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 39 weeks 127 Table IVb. Testosterone concentrations in serum before and after, two types of electroejaculation of ram lambs given cereal-based diets with or without supplemental molybdenum (Mo) and sulfur (S), for 39 weeks 128 Table IVc. Frequency and proportion of ejaculations with penile erection, induced, using two types of electroejaculators, in ram lambs given cereal-based diets with or without supplemental molybdenum (Mo) and sulfur (S), for 39 weeks 129 Figure IV. Mean concentrations of Cortisol (cort) and testosterone (testo) in serum immediately before, after and one hour after ejaculation, total sperm per ejaculate (SPJ) and motility score (MTY) for each collection from rams given diets with or without (I) added Mo (II) or S (III), alone or combined (IV) 130 Table Via. Partial correlations (r) between serum copper and luteinizing hormone (LH), testosterone (TESTO) and growth parameters, in crossbred ram lambs (Experiment I) 134 Table VIb. Partial correlations (r) between copper, molybdenum and sulfur intake and growth, gonadal, pituitary and adrenal parameters in Dorset lambs. (Experiment II) 135 Table Vic. Partial correlations (r) between copper, molybdenum in serum and gonadal, pituitary and adrenal parameters in Dorset lambs 136 Table VId. Partial correlations (r) between pituitary and growth, gonadal and adrenal parameters in Dorset lambs 137 Table Vila. Cortisol in serum before and after transportation of ewe, ram and wether lambs given cereal based diets with and without Mo and S for 32-39 weeks 141 Table VHb. Body weight and liver weight of ewe, ram and wether lambs given cereal based diets with and without Mo and S, for 32-39 weeks 142 Figure VHIa-c. Ontogeny of Puberty 143 Figure IXa-c. Ontogeny of serum secretory profiles 146 114 APPENDIX I Steroid hormone radioimmunoassay (RIA) characteristics Experiment I An established ether-extraction 3H-RIA procedure was used to determine serum testosterone (TESTO) concentrations (Sanford et al. 1984). The procedure employed antiserum that had been raised in sheep immunized with testosterone-3-carboxy-methyloxime conjugated to bovine serum albumin (Sanford et al. 1976). Antiserum crossreacted with 1,4 - androstediene-17G-ol-3-one (14%), 4-androstene-3, 1713-diol (10%), androstan-178-ol-3-one (9%), 5\u00E2\u0080\u009E-androstan-3a, 176-diol (5%), but all occurr in such small amounts in ram serum that they do not interfer with TESTO estimations (Sanford et al 1974). Purified TESTO (sigma T1500), reconstituted in stripped (dextran-charcoal extraction) ram serum and containing 0, 0.4, 0.6, 1.0, 2.0, 4.0, 6.0, 8.0, 12.0, 16.0 and 20.0 ng ml\"1 was used to derive the standard curve. Final concentrations were expressed as /_g L\"1 serum. Experiment II Diagnostic Products Corporation - Intermedia) Coat-a-Count kits were used for RIA of progesterone (catalogue number TKPG), total testosterone (TKTT) and Cortisol (hydrocorticosterone, Compound F; TKCO). The assays employed highly specific antisera (Table la) and good repeatability with constant conditions (Table lb). The incubation period affected the 20% binding intercepts of the standard curve. When the incubation period was extended, there was a shift to the right (inflated values) for progesterone (PRGN) and testosterone (TESTO), but to the left (depressed values) for Cortisol (CORT). The 80% intercept was the least affected, which was where most of the samples fell. Manufacturer coefficients of variation (CV) were based on human serum containing 1.5 \u00C2\u00B1 0.13 ng ml\"1 (n=20) PRGN, 2.07 \u00C2\u00B1 0.09 ng ml\"1 (n=20) TESTO and 48 \u00C2\u00B1 3 ng ml\"1 (n=720) CORT for within assay (intra- CV) and 1.6 \u00C2\u00B1 0.16 115 ng ml\"1 PRGN (n=20), 3.97 \u00C2\u00B1 0.20 ng ml'1 TESTO (n=20) and <50 ng ml\"1 C O R T (from graph) for between assay CV (inter- CV) and showed greater CV at lower concentrations. The reference sheep serum used in the present study contained 0.46 \u00C2\u00B1 0.04 ng ml -1 (1.46 nM) PRGN (n=6), 2.53 \u00C2\u00B1 0.13 ng ml\"1 (8.77 nM) TESTO (n=18) and 13 \u00C2\u00B1 1 ng ml 1 (36.2 nM) CORT (n=57). Note PRGN ng L 1 * 3.18 = nmol L\"1 TESTO ng ml 1 * 3.467 = nmol L 1 C O R T jug d L 1 * 27.86 = nmol L\"1 Table la. RIA antisera crossreactivity (%) as given by the manufacturer. Antisera Compound PRGN TESTO C O R T Crossreactivity (%) Progesterone (PRGN) 100.0 0.028 0.150 Testosterone (TESTO) n.d^ 100.000 % Cortisol (CORT) n.d 0.002 100.000 Pregnenolone n.d 20a-dihydroprogesterone 2.0 17\u00C2\u00AB-Hydroxyprogesterone 0.3 n.d 11-Keto testosterone 3.200 Androstenedione 3.000 19-Hydroxyandrostenedione 1.500 Androstenediol n.d 0.710 Androsterone 0.030 Corticosterone 0.4 0.015 1.400 11-Deoxycorticosterone 1.7 1.500 11-Deoxycortisol 2.4 0.250 Estradiol n.d n.d Estrone 0.025 Aldosterone 0.07 0.010 n.d not detectable; . not given 117 Table lb. Quality of RIA of progesterone (PRGN), testosterone (TESTO) and Cortisol (CORT) in the present study and as given by the manufacturer (in brackets). Antigen Parameter PRGN TESTO C O R T Total cpm 50859 (62214) 28377 (44751) 41083 (57587) NSB (%) 1.9 (0.6) 0.9 (0.8) 1.1 (0.5) BO (100%) 42.0 (41.0) 57.9 (43.3) 79.3 (62.0) Sensitivity^ (ng ml1) 0.08 (0.07) 0.17 (0.11) 3.8 (2.0) 20%B/B0 (ng ml1) 11.1 (10.6) 36.6 (38.9) 196.0 (269.0) 50%B/B0 (ng ml1) 1.8 (2.1) 5.3 (5.9) 49.0 (61.0) 80%B/B0 (ng ml1) 0.3 (0.4) 0.8 (0.9) 12.0 (14.0) Intra-assay CV (%) 3.8 (8.4) 4.7 (4.6) 9.5 (~5.0) Inter-assay C V (%) 4.3 (10.0) 5.1 (6.7) 8.6 (6.3) 11 sensitivity based on 95% of the maximum binbing tube (B0) for incubation periods of 3 hours for PRGN and TESTO and 45 minutes for CORT. 118 APPENDIX II Hourly concentrations of copper in serum 1 Equal aliquots of serum, from blood samples collected, every 20 minutes, from one to four hours after sunrise, on June 20th, from ram and wether lambs, in diet Groups I, II, III and IV (Experiment II), were pooled by hour, by animal and the concentration of copper in serum soluble in trichloroacetic acid was determined. The means for rams and wethers in Groups I, II, III and IV are shown in Table II. The effect of S tended to be significant (P=0.06) and differences among hours tended to differ among groups (P=0.20). More frequent sampling and a more specific measure of cuproprotein activity may identify relevant fluctuations in circulating Cu. 119 Table II. Serum copper (Cu) soluble in trichloroacetic acid (TCA), at one, two and three hours after sunrise on June 20th, in ram and wether lambs given cereal-based diets with or without supplemental molybdenum (Mo) and sulfur (S), for 13 weeks. Mineral T C A Copper (/xmol L _ 1 ) T Supplement Hour after sunrise (kg1) Mo S Group mg g One Two Three Ram (n=2) I 0 0 12.6 12.3 13.0 II 12 0 12.4 12.4 12.1 III 0 2 11.8 11.5 11.2 IV 12 2 10.7 10.2 10.8 pooled SEM \u00C2\u00B11 .6 \u00C2\u00B11 .8 \u00C2\u00B12 .0 Wether (n=l) I 0 0 17.6 18.2 18.1 II 12 0 14.2 14.1 14.5 III 0 2 10.1 9.3 8.5 IV 12 2 10.0 9.0 9.7 pooled SEM \u00C2\u00B12.3 \u00C2\u00B12 .5 \u00C2\u00B1 2 . 9 T Least squares means \u00C2\u00B1 pooled standard error of the mean (SEM df=4). TCA-Cu tends to be lower for lambs given S (Groups III and IV) (P=0.06). Differences among hours tends to differ among groups (Hour by treatment group; P=0.20). 120 APPENDIX III Table III. Hematological status, in October, of lambs given cereal-based diets with or without supplemental molybdenum (Mo) and sulfur (S), for 25-32 weeks (Experiment II). Group (n=5)\u00C2\u00A7 pooled Criterion I II III IV SEM Serum protein (g L 1 )^ 9.8 9.4 8.7 10.8 \u00C2\u00B16 .8 Red blood cells (RBC* 109) 10.9 10.4 10.5 11.9 \u00C2\u00B1 0 . 9 RBC fragility (%)T 78.1 70.6 78.3 82.9 \u00C2\u00B12 .8 Packed cell volume (PCV %) 29.8 30.3 30.0 27.3 \u00C2\u00B11 .3 Hemoglobin (HBG g L\"1) 1.2 1.2 1.2 1.0 \u00C2\u00B10 .4 Mean corpuscular H B G (MCHC%) 39.4 38.3 39.4 38.8 \u00C2\u00B10 .7 H B G per RBC (MCH pg) 11.4 11.3 11.9 9.3 \u00C2\u00B11 .3 Red cell volume (MCV fL) 29.5 29.6 30.4 24.6 \u00C2\u00B1 3 . 9 \u00C2\u00A7 Group I was unsupplemented, Group II was given 12 mg Mo, Group III was given 2 g S and Group IV was given 12 mg Mo plus 2 g S, per kg DM. ^ Protein determined by the method of Lowry (1958). T % hemolysis in hypotonic saline (0.7%) 121 APPENDIX IV The influence of electroejaculation method on serum testosterone and Cortisol, libido and semen quality in ram lambs given cereal-based diets with or without added molybdenum and sulfur (Experiment II). Introduction Ejaculation is a reflex evacuation of the epididymi, urethra and accessory sex glands of the male, that occurs in two stages; emission ie. delivery of sperm and seminal plasma into the urethra and ejaculation or discharge of semen (Hafez 1984). The reflex mechanisms are initiated by psychic or physical stimuli that are integrated in the lumbar (L2-L5) and sacral (S1-S3) spinal cord (Gunn 1936). Penile erection is caused by parasympathetic impulses that relax the retractor penis muscle. Sympathetic impulses from L2-L5 initiate emission, which subsequently sends rhythmic parasympathetic impulses through the pudendal nerve (nervus erigens) in the sacral region of the spinal cord which culminate to ejaculation. However, the autonomic and central nervous system pathways involved in ejaculation are extremely complex and very diverse responses occurr depending upon the pathway stimulated. Electrical stimulation of ejaculation facilitates semen collection when animals are not trained or refuse the artificial vagina method or injury prevents the animal from being able to mount. The technology for rams was first described by Gunn (1936). The technique was based on the estimate that spinal nerves transmit 40-60 impulses per second for a total stimulation and refractory period of 0.012 seconds. Thus, with a 60 cycle alternating current (AC) current the nerve would be stimulated every 0.016 seconds. In the ram, erection and ejaculation could be induced by direct stimulation of both lumbar and sacral nerves by passing an AC, 30 volt current between a needle inserted into the longissimus dorsi muscle and a blunt electrode inserted into the rectum. At low voltage (8 V 60 milliamps) only slight muscle contraction would occurr, whereas 30 V (160 milliamps) applied to the mid-lumbar (L2-L5), without the rectal stimulation, induced ejaculation, but without penile erection. Gunn also found that four inch insertion of 122 a rectal probe stimulated sacral nerves, whereas eight inch insertion stimulated the last lumbar nerves. Low voltage and amperage (8 V; 60 milliamps) stimulation of sacral nerves would induce erection of the penis, secretion of accessory gland fluid and in some cases ejaculation, but not necessarily with spermatozoa. However, direct stimulation across the spinal cord caused extreme motor reactions. Since this time, the method of stimulation has been improved by the use of a bipolar rectal probe which stimulates the mesenteric nerves by the spread of current round the poles. Lower voltage (2-5 Volts) and amperage (0.9 amps) provide a sufficient stimulus when delivered in low frequency (25 c/s) A C sine wave pulses (Dziuk et al. 1954). Although, semen collected from the reproductive tract of the ewe after copulation or after the artificial vagina method have been of better quality than electroejaculates (Hulet, Foote and Blackwell 1964), for this thesis, electroejaculation was an efficient method to determine the effect of dietary supplemental Mo and S on sperm output by inexperienced ram lambs. An inexpensive Bailey ejaculator (BE) for rams was purchased from Western Instrument Company, Denver Colarado, but subsequently, the Langley Animal Clinic, Langley, B. C , kindly provided a Dairy Bull ejaculator (DE). The D E was adapted to rams by the use of a specially designed rectal probe which was provided by the Agriculture Canada Research Station, Leithbridge, Alberta. Apart from size and cost, the main differences between the two ejaculators were the type and distribution of electrical stimulus delivered to and by the rectal probe. The two ejaculators are described in the Materials and Methods section (4.3.2.5). Libido and copulation, in adult rams, have been associated with spontaneous change in the secretion of luteinizing hormone (LH) and testosterone (Sanford, Palmer and Howland 1974). Also first time exposure of bulls to electroejaculation has been associated with increased blood C O R T that was negatively correlated with TESTO secretion (Welsh and Johnson 1981). Unfortunately, these studies did not include examination of semen. Weiner, Hayter and Field (1976) reported poor libido for rams with low concentrations copper (Cu) in plasma (~ 8 /_mol L 1 ) . The objectives of this study were to determine the effect of Mo and S and the influence of ejaculation method on serum Cortisol (CORT) and testosterone (TESTO), libido and sperm output and 123 to compare the BE and D E methods, with the expectation of determining which method would be best suited for future research involving semen evaluation of rams. Materials and Methods Details of the sampling procedure have been given in the Materials and Method section (4.3.2.5) and only key points are given here. In December 1985, when rams were aged 51 weeks and had been given experimental diets since 12 weeks of age, semen was collected by electro-ejaculation of each ram on three consecutive days followed by a two day rest for a total of 12 collections over 18 days. The D E and BE were used on alternate days. The first two samples were discarded and comparisons were based on ten samples collected from each lamb (5 collections per method per lamb). Blood samples were taken by jugular venepuncture immediately before ejaculation, when the ram was secured in the squeeze and immediately after ejaculation (the ram was still in the squeeze) and one hour after ejaculation (the ram had been returned to his pen within five minutes of collection). For analysis of variance (ANOVA), lamb within group (Error a; df=4) was used to test the effect of Mo, S and Mo*S interaction, lamb within group by method (Error b; df=4) was used to test the effects of method and method by group interaction and lamb within group by method by collection (Error c; df=8) was used to test the effects of consecutive collections (ie. first, second or third consecutive day) and interaction with group and method effects. Repeated measures A N O V A was used to test differences in TESTO and C O R T within collection. Standard errors of the least squares means were determined from the error term used in the A N O V A . Results and Discussion Figure IV shows mean ( \u00C2\u00B1 standard error of the mean) concentrations of Cortisol and testosterone in serum immediately before, after and one hour after ejaculation, total sperm per ejaculate and motility score for each collection from rams given dietary treatments I, II, III and IV. 124 Cortisol and testosterone Serum C O R T (Figure IV; top panel), regardless of method or group or day of collection, was highest immediately following ejaculation. The means, by type of ejaculator, before and after ejaculation are shown in Table IVb. Because there was no difference between sampling days or methods, it can be concluded that either method caused a transient, acute stress response. One hour post ejaculation CORT tended (P=0.08) to be lowest for lambs given S (Group III), which may be indicative of more rapid clearance of C O R T from the circulation. Serum TESTO (Figure IV; panel 2) before and after ejaculation tended to be highest for Group IV (P=0.08) and lowest one hour after ejaculation (P=0.11). This latter effect may have eluded to the transient negative correlation between CORT and TESTO after electroejaculation which has been demonstrated in bulls (Welsh and Johnson 1981). Lower TESTO for Group III was consistent with the twice weekly and secretory serum TESTO profiles for this Group. Semen parameters Both ejaculators were used successfully to collect semen from all lambs and only one ram, a cryptorchid in Group I, failed to ejaculate spermatozoa with repeated collections. Hulet and Erchanbrack (1962) reported a 61% (n=148) success rate with electroejaculation of rams. With each collection, the occurrence of ejaculation subsequent to penile erection was noted as an indirect measure of libido and/or the ability of each type of ejaculator to induce a more natural ejaculation. Frequency of penile erection (Table IVa) did not differ between types of ejaculators, but was affected by Mo*S interaction. The role of Cu in the metabolism of catecholamines in nerve transmission may have resulted in a lower frequency for Group IV (P=0.05). Weiner, Hayter and Field (1976) reported poor libido for rams with low plasma copper. Total sperm per ejaculate (Figure IV; panel 3) was higher when the D E was used regardless of group or collection. Higher sperm per ejaculate when the D E was used was due to higher volume (0.65 and 1.28 \u00C2\u00B1 0.16 ml; P<0.05) and, to a lesser extent, higher sperm concentration (236.4 and 308.3 \u00C2\u00B1 52 125 million; P=0.11). These effects indicate that stimulation of emission of sperm and seminal plasma was greater when the D E was used. Sperm motility was not affected by method (2.1 and 2.4 \u00C2\u00B1 0.3 motility score) or collection. Various collection frequencies for semen evaluation have been recommended, eg. evaluation of the second of three consecutive samples taken in one day (Hulet and Erchanbrack 1962), evaluation of samples collected two and three days apart over a period of five weeks (Mattner and Voglmayer 1962), evaluation of samples collected seven to ten days following an introduction to the experimental collection frequency (Berndsten, 1977). There was no significant day within method effect, the data was reclassified by type of ejaculator, to first, second and third consecutive day of collection and subjected to A N O V A . Table IVb shows mean sperm per ejaculate for the first, second and third consecutive sample collected, using the B E and DE, from lambs in Groups I, II, III and IV. Sperm per ejaculate showed differences among Groups that were dependent upon method and collection (Table IVc). However, this effect was mostly due to method by collection interaction, since both methods showed higher sperm output for S- supplemented lambs (Groups III and IV). Method by collection effects were primarily due to increasing sperm output with subsequent collections using the BE (Collection 1 vs 2) and decreasing sperm output with subsequent collections using the D E (Collection 1 vs 3). This effect indicated that the D E depleted extragonadal sperm reserves more rapidly than the BE. Conclusions The Bailey (BE) and Dairy (DE) electroejaculators may be used successfully to collect semen from ram lambs. Electroejaculation, regardless of method or collection period, caused an immediate transient increase in serum Cortisol (CORT). Within one hour of ejaculation, CORT returned to precollection levels, especially for lambs given supplemental S alone (Group III). More frequent blood sampling was required to determine conclusive effects on TESTO, but the trend for lower TESTO one hour after ejaculation was consistent with a transient inhibitory effect of CORT on TESTO secretion in bulls (Welsh and Johnson 1981). However, further research is required to determine if this transient rise in CORT is 126 actually detrimental to sperm output. Since before collection CORT concentrations did not differ among collection periods, it is concluded that either electroejaculation procedure caused only a transient acute stress response. The frequency of penile erection did not differ between types of ejaculators, but was lowest for lambs given Mo+S (Group IV). The possibility of Mo+S induced low cuproprotein activity in nervous pathways requires further investigation. For subsequent research, the Dairy ejaculator is recommended, because of higher sperm output and lower between animal variation. Differences among groups were dependent upon method and collection. Therefore, the maximum output for each lamb per week was used to evaluate the effect of diet group on sperm output reported in the main body of this thesis. 127 Table IVa. Cortisol concentrations in serum before and after, two types of electroejaculation of ram lambs given cereal-based diets with and without supplemental molybdenum (Mo) and sulfur (S), for 39 weeks. Supplement (kg4) Type of ejaculator^ Mo S Group Group mg g Bailey Dairy Mean (n=2) Before ejaculation I 0 0 11.4 15.7 13.5 II 12 0 16.5 18.0 17.2 III 0 2 26.6 33.1 29.8 IV 12 2 29.8 23.2 26.5 pooled SEM \u00C2\u00B13.9 \u00C2\u00B1 3 . 9 \u00C2\u00B110.3 Immediately after I 0 0 32.4 33.3 32.8 II 12 0 38.3 41.5 39.9 III 0 2 47.6 51.0 49.3 IV 12 2 38.1 39.6 38.8 pooled S E M \u00C2\u00B14 .0 \u00C2\u00B14 .0 \u00C2\u00B18 .1 One hour after I 0 0 22.6 19.5 21.1 II 12 0 28.3 20.1 24.2 III 0 2 19.1 16.6 17.8 IV 12 2 31.4 22.3 26.9 pooled SEM \u00C2\u00B14.3 \u00C2\u00B14 .3 \u00C2\u00B16 .2 Cortisol, regardless of type of ejaculator, is highest immediately after ejaculation (P<0.05) and tends to be lowest for Group III at one hour after ejaculation (P=0.08). 128 Table IVb. Testosterone concentrations in serum before and after, two types of electroejaculation of ram lambs given cereal-based diets with or without supplemental molybdenum (Mo) and sulfur (S), for 39 weeks. Supplement Group Mo mg (kg1) S g Type of ejaculator^ Bailey Dairy Group Mean (n=2) I 0 0 3.6 Before ejaculation 3.4 3.5 II 12 0 3.7 5.1 4.4 III 0 2 1-7 1.3 1.5 IV 12 2 5.7 6.8 6.3 pooled SEM \u00C2\u00B1 1.1 \u00C2\u00B1 1.1 \u00C2\u00B1 1.8 I 0 0 3.2 Immediately after 3.7 3.4 II 12 0 3.5 5.5 4.5 III 0 2 2.0 1.6 1.8 IV 12 2 7.0 7.6 7.3 pooled SEM \u00C2\u00B1 1.2 \u00C2\u00B1 1.2 \u00C2\u00B1 2.2 One hour after I 0 0 2.5 2.8 2.7 II 12 0 2.5 4.0 3.2 III 0 2 2.3 2.3 2.3 IV 12 2 4.3 5.3 4.8 pooled SEM \u00C2\u00B1 0.6 \u00C2\u00B1 0.6 \u00C2\u00B1 0.8 ^ Testosterone, regardless of type of ejaculator, tends to be lowest one hour after ejaculation and highest for Group IV (P=0.09) 129 Table IVc. Frequency and proportion of ejaculations with penile erection, induced, using two types of electroejaculators, in ram lambs given cereal-based diets with or without supplemental molybdenum (Mo) and sulfur (S), for 39 weeks.\u00CC\u0082 1 Mineral Supplement (kg1) Type of ejaculator Mo S Group Group mg g Bailey Dairy Mean (n=2) (no/10) (no/10) (%) I 0 0 2 5 35.0ab II 12 0 7 5 60.0a III 0 2 7 6 65.0a IV 12 2 1 2 15.0b pooled SEM \u00C2\u00B1 13.0 Type Mean (n= =8) (%) 42.5 \u00C2\u00B1 5.0 52.5 \u00C2\u00B1 5.0 a b means with different letters differ (P<0.05). ^ total number of penile erections, out of 10 collections (5 per ram per method). Group I ^ ^ ^ - t i EB 35 _ 55 P 1 J _ s ; l | J P\u00E2\u0080\u0094S ? J Group III 48 24 AAA^Af 8 4 - ^ 1.6 0.8 ' p i r g| 1 3 0 J I . I L i i 1 | ; 4 7 S 9 12 13 14 17 IS 19 Date (Dec 1985) Group II V A A A _ = 55 H 1 71 Cort (ug/L) Testo (ug/L) Spj (bil) Mty (Score) Group IV i 1 i 1 i i i H i ; i _ l 9 12 13 14 17 18 19 Date (Dec 1985) Cort (ug/L) Testo (ug/L) Spj (bil) Mty (Score) Time Day 131 APPENDIX V Vaginal Smear evaluation of estrous cycles This procedure was based on the cellular differentiation of the vaginal epithelium that occurs during the estrous cycle. Smears were stained with Papinocoleau stain and stages of the estrous cycle were defined as described by Sanger, Engle and Bell (1958). During proestous, rising follicular estrogens induce growth of the vaginal squamous epithelium with cornification of the superficial layers. The thickened epithelium limits the movement (diapedidis) of neutrophils or leucocytes or red blood cells into the vagina and as a result the smear contains basophilic (purple granules) and a few with magenta-colored cytoplasm around the nucleus indicating early cornification, scattered groups of small cells with pycknotic nuclei and perhaps vacuoles (Sanger, Engle and Bell 1958) and erythrocytes (Frandson 1986). At estrous, cornification of the vaginal epithelium has been maximal and the smear contains very few cells, but some basophilic and acidophilic (pink) superficial squamous cells, few neutrophils, some cornified cells with irregular shaped pyknotic nuclei (Sanger, Engle and Bell 1958). At metestrous, the drop in estrogen causes flaking of the cornified endometrial cells and leucocytes are able to enter by diapedidis, into the vagina. The metestrous smear contains clumps of large cornified cells and a few scattered basophilic, parabasal and squamous cells. During diestrous, rising progesterone stabilizes the vaginal epithelium and therefore, non-cornified cells and neutrophils have appeared on the slides. Smears taken during anestrous contain bacteria and a few basophilic parabasal and squamous cells with some cornification (Sanger, Engle and Bell 1958). In the ewe folliculogenesis occurrs over 4 to 5 days and includes proestrous, estrous and metestrous, whereas the luteal phase or diestrous lasts approximately 10 to 11 days. In rats, cats and dogs all stages of the estrous cycle have been identified clearly by vaginal smears taken daily. In the present study, the use of the smears was limited to the identification of metestrous/diestrous by the presence of clumps of cornified cells and the number of days between the occurrence of 132 metestrous/diestrous smears was designated one vaginal epithelial cycle. During anestrous, the smears were heavily infested with bacteria. The vaginal smears were used to identify an estrous cycle which may or may not have been occompanied by ovulation, which has been known to occur frequently, prior to puberty in ewes (Robinson 1954). Progesterone in serum was used to determine the number of estrous cycles that were accompanied by ovulation and subsequent development of a corpus luteum. 133 APPENDIX VI Tables of partial correlation coefficients (r) among copper and molybdenum, growth and pituitary (LH), gonadal and adrenal function examined in this thesis.\u00CC\u0082 The sampling error lamb within group was used. 134 Table Via. Partial correlations (r) between serum copper and luteinizing hormone (LH), testosterone (TESTO) and growth parameters, in crossbred ram lambs (Experiment I). Serum copper (Limol L'1) Item Total T C A Residual Partial correlation coefficient (r) L H mean (ng ml\"1) 0.54 -0.72* 0.78* L H baseline (ng ml1) 0.32 -0.58 0.52 L H peak (ng ml1) 0.07 -0.43 0.23 L H amplitude (ng ml1) 0.02 -0.35 0.16 L H peak frequency (no/8h) 0.18 -0.51 0.36 TESTO mean (ng ml\"1) 0.55 0.49 0.31 TESTO base(ng ml1) -0.00 0.31 -0.12 TESTO peak (ng ml\"1) 0.49 0.28 0.33 TESTO amplitude (ng ml1) 0.64* 0.15 0.53 TESTO frequency (no/8h) -0.22 0.47 -0.38 Body weight (kg) 0.60 0.34 0.42 Average daily gain (g d\"1) 0.26 -0;35 0.38 Scrotal circumference (cm) -0.01 0.53 . -0.22 Testes growth (mm d\"1) -0.11 0.28 -0.21 Testes weight (g) -0.17 0.36 -0.30 * df=8 r=0.632 P<0.05, r=0.549 P<0.10 135 Table VIb. Partial correlations (r) between copper, molybdenum and sulfur intake and growth, gonadal, pituitary and adrenal parameters in Dorset lambs. (Experiment II) Nutrient (intake/lamb/day) Cu Mo S Available Cu Item (/Ltmol) (/_mol) (mmol) (/_mol) Growth (df=7) A D G (Jun-Sep)(g d1) 0.63 0.24 0.57 0.69 A D G (Sep-Nov) 0.14 -0.12 0.01 0.24 Liver weight (kg) 0.73* 0.56 0.75* 0.69* Gonadal (df=3) Progesterone (PRGN) Transient (days) 0.86 0.79 0.89* 0.82 Transient peak 0.82 0.40 0.79 0.89* Testosterone (TESTO) Base (j_g L 1 ) -0.38 0.51 0.01 0.28 Amplitude (/_g L 1 ) 0.24 0.92* 0.61 -0.23 Frequency (peaks/h) -0.64 -0.66 -0.73 -0.39 Pituitary L H (df=7) Base (/ig L l ) -0.24 -0.04 -0.22 -0.28 Amplitude (jug L\"1) 0.04 -0.15 0.03 0.10 Frequency (pulse/h) -0.83* -0.74* -0.91* -0.74 Adrenal Cortisol (df=7) Base (jLtg L 1 ) 0.72* 0.72* 0.67* 0.66* Amplitude(/ig L 1 ) 0.56 0.58 0.55 0.50 Frequency (episodes/h) -0.12 -0.39 -0.14 -0.03 * * * P<0.05 136 Table Vic. Partial correlations (r) between copper, molybdenum in serum and gonadal, pituitary and adrenal parameters in Dorset lambs. Total Cu T C A Cu Residual Total Mo Item (/xmol L 1 ) Gonadal (df=3) Transient C L (days) -0.76 0.65 -0.79 0.86* Weight 1st estrous -0.98* 0.64 -0.92* 0.79 Number of C L cycles -0.33 0.91* -0.69 0.10 PRGN peak (Ltg L 1 ) -0.73 0.93* -0.93 * 0.75 PRGN base (Ltg L 1 ) -0.88 0.76 -0.93* 0.46 TESTO base (/tig L 1 ) 0.28 0.33 0.07 0.42 TESTO amplitude (Ltg L 1 ) 0.88* 0.67 0.17 0.64 TESTO frequency (peaks/h) -0.68 -0.76 -0.15 -0.57 Pituitary L H (df=7) Base (Ltg L'1) 0.53 0.30 0.34 0.60 Amplitude (Ltg L 1 ) 0.73* 0.57 0.32 0.63 Frequency (pulse/h) -0.64* -0.28 0.15 -0.14 Adrenal Cortisol (df=7) Base (Ltg L 1 ) -0.19 -0.19 -0.43 0.16 Amplitude(/ig L'1) -0.33 0.34 -0.75* 0.17 Frequency (peaks/h) 0.25 0.01 0.28 -0.02 * P<0.05 I 137 Table VId. Partial correlations (r) between pituitary and growth, gonadal and adrenal parameters in Dorset lambs. L H parameter Item Base Amplitude Frequency (/Umol L\"1) (/xmol L\"1) (pulse/h) Gonadal (df=3) Transient C L (days) 1st CL (days) Luteal PRGN TESTO base (j_g L'1) TESTO amplitude (/xg L\"1) TESTO frequency (peaks/h) 0.00 0.75 -0.39 0.30 0.09 0.02 -0.26 -0.97* 0.26 -0.14 -0.01 -0.35 -0.87* -0.12 -0.84 0.12 -0.47 0.68 Adrenal Cortisol (df=7) Base (/xg L\"1) Amplitude(/ig L\"1) Frequency (peaks/h) 0.04 0.03 -0.12 -0.06 -0.01 -0.13 -0.40 -0.36 -0.52 * P<0.05 138 APPENDIX VII The effect of molybdenum (Mo) and sulfur (S) and the influence of the gonads, on transportation induced increased serum Cortisol and liver weight of lambs (Experiment II). Introduction Venipuncture, restraint and transportation have been shown to induce stress in cattle, sheep and pigs (Moberg 1985ab). Stress defined by increased concentration of serum Cortisol (CORT) can be mimicked by acute or chronic injection of adrenocorticotrophin (ACTH). In the present study, lambs had been accustomed to twice weekly and serial venipuncture and rams had been exposed to repeated electroejaculation. The lambs also contained different concentrations of copper (Cu) in serum as a result of prolonged exposure to diets that contained different concentrations of Mo and S. Transportation to the abattoir was expected to stress the lambs and the primary objective of this sub-study was to determine the effect of Mo and S treatments and gonadal influence on post-transportation Cortisol (CORT). Fresh liver weight was recorded for subsequent Cu and Mo determinations, but unfortunately liver samples were lost prior to analysis. Materials and Methods Lambs were fasted over night, weighed and bled before loading onto the truck (~0800 h). The drive to the abattoir was about 1 h. A blood sample was taken as the lambs were unloaded. Determination of C O R T and statistical analysis (except for the inclusion of covariate analysis in this study) were as described in the Materials and Methods section of this thesis (Chapter 4). 139 Results Serum C O R T before and after transportation of ewe, ram and wether lambs given dietary treatments I, II, III and IV is shown in Table Vila. Post-transportation CORT was lower for lambs given Mo (Groups II and IV), depending upon gonadal influence and level of sulfur (S). However, these interactions were due to Group I ram and wether and Group IV ewe and wether lambs. Body weight, liver weight and liver weight adjusted for body weight (covariate) of ewe, ram and wether lambs given dietary treatments I, II, III and IV are shown in Table Vllb. Liver weight was higher S-supplemented, Groups III and IV, regardless of gonadal influence or level of Mo. Discussion and conclusions Post-transportation CORT was was higher than pre-transportation levels in an order of magnitude similar to sheep given acute injections of A C T H (Wood and Silbiger 1987). Therefore, in the present study, the adrenal response to a pituitary signal of stress was not inhibited by dietary treatments, but the magnitude of the response was depressed by Mo. Serum CORT, regardless of gonadal influence, was lower for lambs given Mo alone (Group II) than S alone (Group III), indicating a general suppressing effect of Mo on C O R T response to stress. With reference to Cu parameters (Tables 6 and 7), serum Cu was higher for Group II than III, indicating that the depressing effect of Mo on C O R T was not due to low serum Cu per se. However, ewes responded differently than ram or wether lambs to the combined effect of Mo and S (Group IV). Additional S eliminated the depressing effect of Mo on serum CORT, in ewes, but not in ram and wether lambs. Groups IV represented the extreme low serum Cu concentrations indicating that serum Cu may also affect CORT response to stress. Low activity of Cu containing enzymes in steroid synthesis could be expected to cause increased output of CORT, which may be an explanation for high C O R T in Group IV ewes, but in ram and wether lambs the depressing effect of Mo on C O R T was obviously greater than potential enhancing effects of Cu deficiency per se. 140 Certainly, these results indicate the necessity for further investigation of independent effects of Cu and Mo on the pituitary-gonadal-adrenal axis. In the short term, Mo treatment may be a simple way to reduce or prevent deleterious effects of transportation stress on reproductive function (eg. stress induced abortion or temporary infertility in bulls and rams) and/or carcass quality (for example dark cutters). If the enhancing effect of S on liver weight is taken in consideration of generally lower circulating C O R T and TESTO for lambs given S alone, perhaps the effect of S on liver weight reflected a consequence of high levels of sulfated steroid metabolites. However, differences between S groups was correlated with differences between high and low serum Cu groups and therefore the enhancing effect of S on liver weight may have alternatively or in conjunction, been due to low circulating Cu. Investigation of Cu and S effects on hepatic metabolism may be a useful model to further our understanding of Cu and S influence on metabolism. 141 Table Vila. Cortisol in serum before and after transportation of ewe, ram and wether lambs given cereal based diets with and without Mo and S for 32-39 weeks. Mineral Cortisol (jug L\"1) Supplement (kg1) Gonadal influence Mo S Group Group mg g Ewe Ram Wether Mean (n=5) Before transportation I 0 0 14.3 17.7 18.2 16.7 II 12 0 13.4 8.1 4.0 8.5 III 0 2 17.3 11.8 17.9 15.7 IV 12 2 29.9 12.5 26.0 22.8 pooled SEM \u00C2\u00B1 4.9 \u00C2\u00B1 4.9 \u00C2\u00B1 6.9 \u00C2\u00B1 3.2 After transport I 0 0 49.6a 37.4C 98.1a 61.7a II 12 0 44.3ab 49.3b 67.1\" 53.8\" III 0 2 52.3ab 58.9a 76.8ab 62.6a IV 12 2 71.6C 49.5b 33.4C 51.5\" pooled SEM \u00C2\u00B1 4.9 \u00C2\u00B1 4.9 \u00C2\u00B1 6.9 \u00C2\u00B1 3.2 a c Means with different letters within period differ (P<0.05). ^ A N O V A Wether vs Ram and Ewe lambs, Mo, Mo*S (P<0.05). 142 Table Vllb. Body weight and liver weight of ewe, ram and wether lambs given cereal based diets with and without Mo and S, for 32-39 weeks. Supplement (kg1) Gonadal influence Mo S Group Group mg g Ewe Ram Wether Mean (n=5) Body weight (kg) I 0 0 55.0 75.5 61.5 65.2 II 12 0 56.7 76.7 75.0 69.1 III 0 2 57.7 74.5 80.0 69.6 IV 12 2 61.2 71.7 70.5 68.0 pooled SEM \u00C2\u00B1 4.0 \u00C2\u00B1 4.0 \u00C2\u00B1 5.7 \u00C2\u00B1 2.6 Liver weight (kg) I 0 0 0.71 0.73 0.71 0.74 II 12 0 0.73 0.84 0.77 0.78 III 0 2 0.71 0.86 0.87 0.80 IV 12 2 0.80 0.88 0.81 0.83 pooled SEM \u00C2\u00B1 0.03 \u00C2\u00B1 0.03 \u00C2\u00B1 0.05 \u00C2\u00B1 0.02 Adjusted liver weight (kg)11 I 0 0 0.81 0.76 0.70 0.76a II 12 0 0.82 0.75 0.70 0.76a III 0 2 0.79 0.80 0.76 0.78ab IV 12 2 0.85 0.84 0.78 0.82b pooled SEM \u00C2\u00B1 0.01 \u00C2\u00B1 0.01 \u00C2\u00B1 0.01 \u00C2\u00B1 0.02 b Means with different letters within parameter differ (P<0.05). ^ A N O V A body weight covariate, wether vs ram and ewe lambs and S (P<0.05). 143 APPENDIX VIII Figure VHIa-c. Ontogeny of Puberty. Figures VHIa-c. The time course of feed intake7 (Fi; panel 1), body weight (Bwt; panel 2) and serum Cortisol^ (CORT; panel 5) for ram (Figure Villa), wether (Figure VHIb) and ewe (Figure VIIIc) lambs given dietary treatments I, II, III and IV\u00C2\u00A7. Gonadal development of ram lambs (Figure Villa) is represented by scrotal circumference and sperm per ejaculate^ ( \u00C2\u00A7 c a n ( j spj; panel 3) and serum testosterone^ (TESTO; panel 4). Gonadal development of ewe lambs (Figure VIIIc) is represented by age of first estrus and ovulationTT (Es and Ovn; panel 3) and serum progesterone^ (PRGN; panel 4). Figure VHIb also shows the natural rate of change of decreasing light of day (sine wave) and sunrise times (panel 3) and PRGN in wether serum (panel 4). Values are means \u00C2\u00B1 standard error of the mean (n=2), although some are two small to show. Tripods on the X-axis indicate blood sampling days for hormone secretory profiles shown in Figures IXa-c. T Weekly average daily intake per lamb per day is based on daily weigh back of feed refused. \u00C2\u00A7 Cereal-based diests that contained 8 mg kg-1 copper and were either unsupplemented (I) or supplemented with molybdenum alone (II; 12 mg kg'1, sulfur alone (III; 2 g kg'1) or in combination (IV). ^ Semen was collected, by electroejaculation, on three consecutive days per week and the best of three samples was used for comparisons. & Blood samples were collected every Tuesday and Friday at two hours after sunrise. T T Estrous based on vaginal smears taken every Tuesday and Friday. Ovulation based on serum progesterone greater than 1 jig L\"1. 147 APPENDIX IX Figure IXa-c. Ontogeny of serum secretory profiles. Figures IXa-c. Secretory profiles\u00C2\u00A7, of luteinizing hormone (LH: panel 1) and Cortisol (CORT; panel 3) in serum of Dorset ram (Figure IXa), wether (Figure IXb) and ewe (Figure IXc) lambs, given dietary treatments I, II, III and IV, for 32-39 weeks .\u00CC\u0082 Secretory profiles of testosterone for ram lambs are also shown (TESTO; Figure IXa; panel 2) (Experiment II). \u00C2\u00A7 Values are mean ( \u00C2\u00B1 standard error of the mean; n=2 ram or ewe, n=l wether) concentrations in samples collected, from one to four hours after sunrise, every 20 minutes, in June (rams and wethers) and every 12 minutes, in autumn (ram, wether and ewe lambs). Some SEM are too small t o show. ^ Treatments as explained for Appendix VIII. June 20th September October November 13th 27th 11th 25th 8th - 22nd Group I 20 10 30 Group 1 1 1 1 r 1 1 1 1 1 1 1 1 1\u00E2\u0080\u0094 1 3 1 3 1 3 1 3 1 3 1 3 1 3 4 Time after sunrise (h) / 9 u r-e. June September October November 20th 13th 27th 11th 25th 8th 22nd Group II LH (ug/L) Testo (ug/L) Cort (ug/L) -I 1 1\u00E2\u0080\u0094 i i M Group IV a B a ' , \" \" i r [ [ g LH (ug/L) Testo (ug/L) Cort (ug/L) 1 1 1 1 1 1 1 1 1 1 ! 1 1 1\u00E2\u0080\u0094 1 3 1 3 1 3 1 3 1 3 1 3 1 3 4 Time after sunrise (h) 7E*- June September October November 20th 13th 27th 11th 25th 8th 22nd 25 i 1 1 1 1 1 25 10 H h 69 105 Group 60 -i 1 1 1 1 1 1 1 i 1 1 1 1 1\u00E2\u0080\u0094 1 3 1 3 1 3 1 3 1 3 1 3 1 3 4 Time after sunrise (h) June September October November 20th 13th 27th 11th 25th Sth 22nd Group II Wuv-iW\u00C2\u00BB /^v*ta* ^Lfc PA- n 1 1 1 1 1 1 1 1 1 1 r- LH (ug/L) Cort (ug/L) H : \ VvJWiy 71 Group IV LH (ug/L) Cort (ug/L) T 1 1 1 1 1 1 1 1 1 1 r 1 3 1 3 1 3 1 3 1 3 1 3 1 3 4 Time after sunrise (h) 6 ^ June September October November 20th 13th 27th 11th 25th 8th 22nd I I I I \u00E2\u0080\u0094\u00E2\u0080\u00941 1 Group I _ i i u Group 1 1 1 1 1 1 1 1 1 1 1 1 1 1\u00E2\u0080\u0094 3 1 3 1 3 1 3 1 3 1 3 1 3 4 Time after sunrise (h) June September October November 20th 13th 27th 11th 25th 8th 22nd i i i i i i Group II 52 i . jWM, L i 1 1 1 1 1 1 1 1 r LH (ug/L) Cort (ug/L) -1 u Group IV \u00E2\u0080\u0094L: LH (ug/L) Cort (ug/L) i 1 1 1 1 1 1 1 1 1 1 1 1 1\u00E2\u0080\u0094 1 3 1 3 1 3 1 3 1 3 1 3 1 3 4 Time after sunrise (h) o"@en . "Thesis/Dissertation"@en . "10.14288/1.0100628"@en . "eng"@en . "Animal Science"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "The effects of dietary molybdenum and sulfur on serum copper concentrations, growth and reproductive function in lambs"@en . "Text"@en . "http://hdl.handle.net/2429/31319"@en .