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The effect of feeding hay containing high levels of molybdenum on reproduction in beef heifers Stephens, Lisa Anne 1999

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The Effect of Feeding Hay Containing High Levels of Molybdenum on Reproduction in Beef Heifers by Lisa Anne Stephens B.Sc, The University of Victoria, 1992  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE DOCTOR OF PHILOSOPHY in  THE FACULTY OF GRADUATE STUDIES (Animal Science, Faculty of Agricultural Sciences)  We accept this thesis as conforming to the requjrejd-standarcl  THE UNIVERSITY OF BRITISH COLUMBIA January 1999 © Lisa Anne Stephens, 1999  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. I 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 The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  Ap/  7°l  11  ABSTRACT The effects of feeding hay containing high levels of molybdenum on reproduction in beef heifers were investigated.  Sixty Hereford and Hereford-cross beef heifers,  approximately 6 months of age, were randomly assigned to one of five treatment groups: Treatment 1 (TR1): 1 mg/kg Mo, 16 mg/kg Cu; TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; and TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu. Three main areas of reproduction were investigated: onset of puberty, estrous cycle characteristics and fertility (including pregnancy and post-partum reproduction).  Onset of puberty was assessed through  twice monthly ultrasound examinations to determine the presence of the first corpus luteum (CL) and blood sampling to measure the associated increase in progesterone (P ) levels (>1 ng/ml). There were no statistically significant differences between the 4  treatment groups. Once the heifers were known to be cycling, estrus was synchronized to allow for comparisons between the length of the estrous cycle, diameters of the dominant follicle and CL and maximum P levels. There were no statistically significant 4  differences except for cycle length in which mean cycle length in TR4 was significantly shorter than TR1 (p<0.05), although still within the normal range.  Fertility was  assessed by comparing conception rates after artificial insemination. There were no significant differences in conception rates, number of inseminations required for pregnancy, gestation length, calving rate or calf birth weight between the treatment groups.  Four weeks after calving, weekly ultrasound examinations were done and  blood samples were taken to determine the presence of the first post-partum CL and associated P levels to determine the length of the post-partum anestrus period. There 4  were no statistically significant differences between the treatment groups. After the resumption of estrous cycles, superovulation and embryo recovery were used to assess  post-partum fertility. There were no significant differences in superovulatory response, ova recovery or embryo quality between the groups.  On three occasions during the study, liver biopsies and blood samples were taken to monitor Mo and Cu concentrations.  Although there were significant differences  between the treatment groups, the levels of Mo and Cu appeared to reflect the intake level of each group. There did not appear to be any interactions between Mo and Cu. Only when an ancillary experiment was done in which heifers were fed inorganic Mo and additional S were any symptoms of molybdenosis encountered. Therefore, it is concluded that high Mo hay can safely be fed to beef heifers without any adverse effects on reproduction.  TABLE OF CONTENTS Abstract  ii  List of Tables  vii  List of Figures  ix  List of Abbreviations  xii  Acknowledgments  xiii  Chapter 1: GENERAL INTRODUCTION  1  Chapter 2: LITERATURE REVIEW  4  2.1 Molybdenum Biochemistry 2.1.1 General 2.1.2 Interactions between Molybdenum, Copper and Sulfur 2.1.3 Molybdenosis  4 4 4 6  2.2 Reproductive Function 2.2.1 Puberty 2.2.2 Estrous Cycles 2.2.3 Conception, Pregnancy and the Post-Partum Period 2.2.4 Hormonal Manipulation of Reproductive Function 2.2.4.1 Estrus Synchronization 2.2.4.2 Superovulation  8 8 11 15 20 20 22  2.3 Nutrition - Reproduction Interactions 2.3.1 General Interactions  25 25  2.3.2 Molybdenum and Reproduction 2.4 Hypothesis Chapter 3: GENERAL MATERIALS AND METHODS 3.1 Animal Management 3.1.1 General 3.1.2 Dietary Treatments  34 38 40 40 40 41  3.2 Heifer Weights  46  3.3 Ultrasonography 3.3.1 Puberty and Estrous Cycles 3.3.2 Fertility, Post-Partum and Superovulation 3.4 Sampling Procedures 3.4.1 Blood Sampling  48 48 49 50 50  3.4.1.1 General 3.4.1.2 Serial Blood Sampling 3.4.2 Liver Biopsies and Blood Samples for Mineral Analysis  50 50 51  3.5 Laboratory Analysis 3.5.1 Hormonal Analysis of Plasma 3.5.1.1 Progesterone 3.5.1.2 Gonadotropins 3.5.2 Mineral Analysis  52 52 52 52 53  3.6 Statistical Analysis  54  Chapter 4: THE EFFECT OF FEEDING HAY CONTAINING HIGH LEVELS OF MOLYBDENUM ON PUBERTY AND ESTROUS CYCLE DYNAMICS 55 4.1 Abstract 4.2 Introduction  55 56  4.3 Materials and Methods 4.3.1 Puberty Assessment 4.3.2 Serial Blood Sampling 4.3.3 Estrous Cycle Dynamics 4.3.4 Samples for Mineral Analysis 4.3.5 Statistical Analysis  57 57 57 58 59 59  4.4 Results 4.4.1 4.4.2 4.4.3 4.4.4  59 59 64 68 72  Puberty Assessment Gonadotropin Levels Estrous Cycle Dynamics Mineral Concentrations  4.5 Discussion  75  Chapter 5: THE EFFECT OF FEEDING HAY CONTAINING HIGH LEVELS OF MOLYBDENUM ON FERTILITY AND POST-PARTUM REPRODUCTION 80 5.1 Abstract  80  5.2 Introduction  81  5.3 Materials and Methods 5.3.1 Fertility Assessment 5.3.2 Gestation Period and Calving 5.3.3 Post-Partum Assessment 5.3.4 Superovulation 5.3.4.4 Trial la 5.3.4.2 Trial Ib 5.3.4.3 Trial II 5.3.4.4 Trial III  82 82 83 84 84 84 84 85 85  vi  5.3.4.5 Ova / Embryo Recovery 5.3.5 Liver and Plasma Samples for Mineral Analysis 5.3.6 Statistical Analysis 5.4 Results 5.4.1 5.4.2 5.4.3 5.4.4  Fertility Gestation Period and Calving Post-Partum Reproduction Superovulation 5.4.4.1 Trial la 5.4.4.2 Trial Ib 5.4.4.3 Trial II 5.4.4.4 Trial III  5.4.5 Mineral Concentrations 5.5 Discussion Chapter 6: ANCILLARY EXPERIMENTS  86 87 87 87 87 93 92 96 96 96 96 97 100 103 108  6.1 Non-Pregnant Heifers and Molybdenosis 6.1.1 Trial I 6.1.2 Trial II 6.1.3 Trial III  108 108 108 109  6.2 Spares  110  6.3 Greenhouse Experiment  111  Chapter 7: GENERAL DISCUSSION  112  Chapter 8: CONCLUSIONS AND RECOMMENDATIONS  118  8.1 Conclusions  118  8.2 Recommendations  119  References  121  Appendices  131  LIST OF TABLES Table 1 44 Nutrient Content (dry matter basis) of the Basal Hays and High Mo hays (Brenda mines) fed to the heifers during the duration of the study (one composite sample for each). Table II Composition of the UBC custom mineral mix.  45  Table III 63 Mean age (±SD) and mean (+SD) weight at first observed corpus luteum (CL) and corresponding mean (+SD) plasma progesterone (P ) concentrations of the first CL in heifers fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5). 4  Table IV 65 Mean (±SD) prepubertal plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations in 6 heifers from each group fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5). Table V 67 Mean (±SD) luteinizing hormone (LH) pulse frequency and pulse amplitude in serial plasma samples (one sample every 20 min for 6h) from 6 heifers in each group fed varying levels of hay containing high levels of molybdenum and supplemental copper (treatment (TR) groups 1-5). Table VI 70 Mean (±SD) cycle length and plasma progesterone (P ) levels on day 14 of the cycle in heifers fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5). 4  Table VII 89 Cumulative conception rates (CR) after 1, 2, and 3 inseminations and the total number of inseminations (mean+SD) required for pregnancy in heifers fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 -5). Table VIII 91 Mean (±SD) gestation lengths, calf birth weights and calving rates in heifers fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5). Table IX 95 Mean (+SD) length of the post-partum anestrus period and maximum plasma progesterone (P ) levels corresponding to the first corpus luteum in heifers fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5). 4  Table X 98 Superovulatory response and embryo recovery from cows superovulated with 8 decreasing doses of follicle stimulating hormone given over 4 days in 3 dietary treatment (TR) groups fed varying levels of high molybdenum hay and supplemental copper and a group of non-suckled control heifers. Table XI 99 Quality of embryos recovered from cows superovulated with 8 decreasing doses of follicle stimulating hormone given over 4 days in 3 dietary treatment (TR) groups fed varying levels of high molybdenum hay and supplemental copper and from a group of non-suckled control heifers.  LIST OF FIGURES Figure 1 47 Mean weights of the heifers in each of the treatment (TR) groups fed varying levels of high molybdenum hay and supplemental copper. Figure II 61 The mean number of class I, II and III follicles 1 (A), 3 (B) and 4(C) months after the start of the dietary treatments (TR) in heifers fed varying levels of high molybdenum hay and supplemental copper (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure III 62 Percentage of heifers with a corpus luteum (A) and mean progesterone concentrations (B) 1,3 and 4 months after the start of the dietary treatments (TR) in heifers fed varying levels of high molybdenum hay and supplemental copper (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure IV . 66 Characteristic pattern of prepubertal luteinizing hormone (LH) release in plasma of a representative heifer from each treatment (TR) group (TRs 1 - 5 fed varying levels of high molybdenum hay and supplemental copper) after serial blood sampling for 6 h (one sample every 20 min). Figure V 66 Characteristic pattern of prepubertal follicle stimulating hormone (FSH) release in plasma of a representative heifer from each treatment (TR) group (TRs 1 - 5 fed varying levels of high molybdenum hay and supplemental copper) after serial blood sampling for 6 h (one sample every 20 min). Figure VI 69 Mean number of follicles > 5mm (A), diameter of the dominant follicle (B) and diameter of the corpus luteum (C) during the estrous cycle in 6 heifers from each group fed varying amounts of hay containing high levels of molybdenum and supplemental copper (Treatment 1(TR1): 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 3040 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure Vll 71 Mean progesterone concentrations during a cycle after synchronized estrus in 6 heifers from each treatment (TR) group (TRs 1 - 5 fed varying levels of high molybdenum hay and supplemental copper).  Figure VIII 73 Mean concentration of molybdenum (Mo) in liver samples taken from heifers fed varying levels of high Mo hay prior to and 4 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure IX 73 Mean concentration of molybdenum (Mo) in plasma samples taken from heifers fed varying levels of high Mo hay prior to and 4 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure X 74 Mean concentration of copper (Cu) in liver samples taken from heifers fed varying levels of high molybdenum (Mo) hay prior to and 4 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure XI 74 Mean concentration of copper (Cu) in plasma samples taken from heifers fed varying levels of high molybdenum (Mo) hay prior to and 4 months after the start of the treatment (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure Xll 93 The number of class I (A), class II (B) and class III (C) follicles starting 4 weeks after calving in heifers fed varying levels of high molybdenum hay and supplemental copper (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure XIII 94 Mean progesterone concentrations starting 4 weeks after calving in heifers fed varying levels of high molybdenum hay and supplemental copper (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure XIV..... 101 Mean concentration of molybdenum (Mo) in liver samples taken from heifers fed varying levels of high Mo hay prior to, 4 months and 15 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  Figure XV 101 Mean concentration of molybdenum (Mo) in plasma samples taken from heifers fed varying levels of high Mo hay prior to, 4 months and 15 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure XVI 102 Mean concentration of copper (Cu) in liver samples taken from heifers fed varying levels of high molybdenum (Mo) hay prior to, 4 months and 15 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 3040 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu). Figure XVII 102 Mean concentration of copper (Cu) in plasma samples taken from heifers fed varying levels of high molybdenum hay (Mo) hay prior to, 4 months and 15 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  LIST O F A B B R E V I A T I O N S  ADF  acid detergent fibre  min  minute  Al  artificial insemination  ml  rnillilitre  ANOVA  analysis of variance  mm  millimeter  BSA  bovine serum albumin  Mn  manganese  Ca  calcium  Mo  molybdenum  CL  corpus luteum  n  number of observations  cm  centimeter  Na  sodium  Cu  copper  ng/ml  nanograms per rnillilitre  °C  degrees Celsius  P  phosphorus  DE  digestible energy  P  DM  dry matter  PGF  E  estrogen  PGs  prostaglandins  eCG  equine chorionic gonadotropin  PROT  protein  Fe  iron  PVP  polyvinylpyrrolidone  FSH  follicle stimulating hormone  rpm  revolutions per minute  GH  growth hormone  S  sulfur  GnRH  gonadotropin releasing hormone  TCA  tricloroacetic acid  h  hour  TCM  tissue culture media  hCG  human chorionic gonadotropin  TDN  total digestible nutrients  IGF-1  insulin-like growth factor 1  TR  treatment  K  potassium  Zn  zinc  kg  kilogram  LH  luteininzing hormone  Mg  magnesium  mg/kg  milligrams per kilogram  mg/L  milligrams per litre  2  progesterone  4  2a  prostaglandin F  2a  Xlll  ACKNOWLEDGMENTS / wish to express my thanks and appreciation to the many people who were involved in the development and completion of this project. First to my supervisor, Dr. Rajamahendran, for his guidance throughout the project and his faith in my transfer to the Ph.D. program. I will also be eternally grateful for his countless hours spent at the farm scanning and blood sampling and flushing (and many other things too numerous to list here). Also, my thanks go out to Dr. Tait for his patience with my lack of "nutrition knowledge" and for being the best editor I know. I also appreciate the neverending batches of mineral mix that so miraculously appeared whenever necessary. My thanks and appreciation to Dr. Owen for bringing the project to UBC and ultimately to me in the first place. Thanks for being there to answer my endless questions, for keeping my thoughts on the bigger picture and your support along the way. My thanks to Dr. Christensen for the use of his laboratory for mineral analysis. Also thanks for reviewing this manuscript and for your thought-provoking comments. To the farm staff, in particular Ted Cathcart, Paul Willing and Jamal Kurtu: thanks for taking such good care of my "girls" and for being patient with regards to my many schedule changes and demands. For technical assistance my thanks go out to Mohan Mahesh for his help blood sampling and scanning, to Divakar Ambrose for teaching me the non-surgical, closed method of flushing (and his willingness to help with the flushing even though the animals did not co-operate), to Siva for teaching me the proper way to do an RIA, to Giri Giritharan for his help assessing and grading embryos and to the many undergraduate students who helped with the weekly weighing, and data collection when necessary, in particular, Linda Pawloff, Sharon Bruce, Corrine Searle and Melanie Kerr. On a personal note, I thank my parents, Brian and Margaret Johnson, for their moral support and for the enthusiasm they showed throughout my time at UBC. And lastly to my husband, Mike Stephens, who probably feels like he's been through this whole endeavor with me. Thank-you for your unfailing faith in me and your patience. Your encouragement along the way meant more than you can ever imagine... P.S. and thanks to my son, Matthew, forgiving me the incentive to complete this thesis and for staying put until after my defense (even if he did arrive before the corrections were done).  1  Chapter 1: GENERAL INTRODUCTION  Mining is the second largest industry in British Columbia and therefore makes a substantial contribution to the province's economic development and prosperity. With approximately 30, 000 hectares of the province's land mass occupied by major coal and metal mines (as of 1990), 58% linked specifically to mineral mines, reclamation of exhausted mines is one way to achieve sustainable development.  Since 1969,  legislation in British Columbia has required that any land or water resources disturbed by mining practices be reclaimed to a level of productivity not less than that which existed prior to the mining, and that water released from a mine site meet long term water quality objectives. The British Columbia Mines Act (1996), requires that all new mining operations provide a detailed outline of the procedures for mine reclamation including ongoing research and monitoring programs.  The mining companies are  required to maintain continual and progressive reclamation from the time the mining operations begin until all reclamation objectives are met.  There are several mining companies in British Columbia involved in the mining of molybdenum (Mo) or the production of Mo as a by-product when another mineral such as copper (Cu) is being mined. The main mines of concern with respect to Mo are Brenda, Endako, Gibraltar and Highland Valley Copper mines. Several of these mines are in the process of examining their options for reclamation at this point in time. One option is to use the land for agricultural purposes such as grazing and/or forage production. The advantage of this is the relative ease of growing forage on these highly disturbed and deficient soils as compared to growing other agricultural crops or rebuilding forests. Forage crops can also act as an important developmental step in  2  which the soil organic material can be built up until it is possible to switch from forage to other agricultural crops or forestry. There is a concern, however, with the potential impact of high concentrations of minerals (e.g. Mo) on land and water resources. Vegetation from the mine sites is known to contain high concentrations of Mo which have the potential to adversely affect the health of any animals consuming vegetation from these areas, especially any wild or domestic ruminants. Beef cattle would be most at risk from feeding in these areas since they are more susceptible to problems associated with elevated Mo levels than other ruminants (Ward, 1991) and beef cattle are more likely to be feeding on vegetation from these areas.  Molybdenum is an essential mineral element, meaning that it has been proven to have a role in metabolism and must be present in the diet of all animals. Molybdenum is present in all tissues and fluids of the body in concentrations of approximately 1.0 to 4.0 mg Mo/kg of body weight (McDonald et al., 1988). Molybdenum becomes available to animals after its uptake from soil into plants. The uptake is influenced by soil properties such as soil organic matter, pH, texture, clay material, oxidation reduction potential, trace mineral interactions and seasonal variation. The accumulation of Mo in plants differs between legumes, grasses and grains. Grasses usually have lower levels of Mo than legumes (Winter and Gupton, 1987).  The toxic effects of Mo in ruminants (molybdenosis, which is primarily an induced Cu deficiency) have been well documented, but few studies have been done to investigate the effects of high Mo on reproductive function. Those studies which have been done found that Mo adversely affects reproduction by causing a delay in the onset of puberty, altering estrous cycle dynamics leading to anestrus, impairing fertility and affecting the  3  development of the early embryo (O'Gorman et al., 1987; Phillippo et al., 1987). The adverse effects of Mo on reproduction are important since the economic viability of the beef industry is sensitive to even small changes in reproductive efficiency. If the mine sites are found to be unsuitable for forage production, then a new approach to mine reclamation or a new end use designation is required. A study at the Highland Valley Copper mine site in British Columbia found that cattle grazing high Mo forage (40mg/kg) for up to 7 months showed no adverse effects on animal health and no signs of induced Cu deficiency were observed. Although the Mo levels in serum and liver samples from the cows were elevated, they were within safe limits and decreased to normal levels when the cows were returned to control hay. There was no long term accumulation of Mo in the tissues. It was hypothesized that other factors such as the form of Mo in the forage, the high concentration of Cu in the diet and the interaction of those minerals with sulfur (S) may have prevented a Mo induced Cu deficiency from occurring (Gardner, 1997).  4  Chapter 2 : LITERATURE REVIEW  2.1 Molybdenum Biochemistry 2.1.1 General Molybdenum is widely distributed in nature with a concentration of approximately 1 mg/kg in the earth's crust. It is one of the most highly concentrated trace minerals in sea water with a concentration of 10 ug/L (Ward, 1991). Molybdenum is an essential mineral element, which means that it has been proven to have a metabolic role in both plants and animals. Therefore, it must be present in the diet of all animals. In 1953, Mo was found to be a component of the enzyme xanthine oxidase which is important for purine metabolism. Since then, it has also been found to be a component of other metalloenzymes such as aldehyde oxidase and sulphite oxidase. Another role is in the reaction of enzymes with cytochrome C which is involved in oxidative phosphorylation (McDonald etal., 1988).  2.1.2. Interactions between Molybdenum, Copper and Sulfur. Besides its independent role in metabolism, Mo can also have certain effects through its interactions with Cu and S.  Molybdenum toxicity (molybdenosis) was first  discovered in 1938 in Somerset County, England when cattle feeding on high Mo pastures ("teart" pastures) were found to be suffering from symptoms of copper deficiency (anemia, diarrhea, stiff gaits and weight loss) (Ferguson et al., 1938). Soon after, it was reported that the problem could be corrected by feeding copper sulfate to the affected animals (Ferguson et al., 1943).  It was found that molybdenosis was  mediated through the formation of thiomolybdates (molybdate, Mo0 " interacting with 2  4  sulfide, S ") in the rumen (Clarke and Laurie, 1980). The formation of thiomolybdates is 2  5  progressive; that is, monothiomolybdates (Mo0 S ) are formed first, followed by di2  3  (Mo0 S -), tri2  2  2  (M0OS3 -) 2  and tetra (MoS -) thiomolybdates (Mason, 1986). 2  4  The  proposed sequence of thiomolybdate formation is as follows: the reduction of sulfate to sulfide in the rumen and the reaction of sulfide with molybdate to form thiomolybdates. Thiomolybdates form a highly insoluble and hon-utilizable Cu-thiomolybdate complex (CuMoS ) with Cu and the solid phase of the digesta (Allen and Gawthorne, 1987). As 4  thiomolybdates progress from mono- to tetrathiomolybdates, the complexes become progressively more stable and resist acid hydrolysis in the abomasum (Suttle, 1991). Thiomolybdates are absorbed through the intestinal mucosa, as evidenced by the increase in the concentration of plasma Cu. The formation of the Cu thiomolybdate complex prevents Cu from being available for absorption into the tissues (McDowell, 1992). Thiomolybdates interfere with Cu metabolism by modifying normal transport through the cell membrane, competing for functional sites within the cell and affecting the normal distribution of Cu between cell organelles. This affects the entry of Cu into the cells, intracellular metabolism and also increases the excretion of Cu via the bile canaliculi and renal tubular membranes (Gooneratne et al., 1994). Trithiomolybdates, after being released from association with the digesta, are absorbed then react with plasma albumin, changing the site at.which Cu is bound and increasing the strength of binding (Woods and Mason, 1987). The formation of thiomolybdates occurs mainly in the rumen because of the presence of sulfur reducing bacteria. The rates of Mo absorption, retention and excretion are inversely related to dietary S levels. A current working hypothesis is that S inhibits membrane transport of Mo, thereby decreasing absorption of Mo in the intestine (McDowell, 1992). As dietary S concentrations increase, the stable tetrathiomolybdate becomes the dominant rumen metabolite (Suttle, 1991).  6  2.1.3 Molybdenosis The clinical signs of molybdenosis are primarily due to a secondary copper deficiency because of the interactions between Mo, S and Cu as outlined above.  The first  symptom of molybdenosis in cattle is a debilitating diarrhea leading to emaciation, weight loss and even death. After prolonged exposure, the hair loses its colour and luster (achromotrichia) and lameness with a characteristic stiff gait may develop (Ward, 1991). Sheep develop steely wool and exhibit decreased growth but do not develop the severe diarrhea seen in cattle. In a case of Mo poisoning in feedlot cattle, a diet to which sodium molybdate (1.9% of the total ration) was accidentally added, resulted in a loss of appetite within 3 d and deaths commencing 6 d after its introduction. The cause of death was determined to be hepatic necrosis and acute renal tubular necrosis (Swan et al., 1998).  Molybdenum may also affect the immune system (Gengelbach et al.,  1997; Ward et al., 1997).  It is hypothesized that this is through a decrease in Cu  concentration within the neutrophils, impaired respiratory burst activity, an affect on hydrogen peroxide generation and increased lipid peroxidation (Gengelbach et al., 1997; Ward et al., 1997; Cerone et al., 1998). Molybdenum and Cu also alter body temperature and feed intake responses to disease by affecting tumor necrosis factor and perhaps other cytokines (Gengelbach et al., 1997) The clinical signs of molybdenosis may not be solely due to the sequestering of Cu. The severe diarrhea associated with molybdenosis, which is specific to cattle, is more likely a direct effect of Mo or thiomolybdates since it is not a characteristic of Cu deficiency (Ward, 1978). Dietary factors, in addition to Mo intake, that are clearly related to molybdenosis or Cu deficiency are: 1. level of Cu intake, 2. Cu availability, 3. S intake, 4. iron (Fe) intake and 5. the physical form of the feed (Ward, 1991). It has been recognized that grazed  7  forage that causes molybdenosis loses its potency when dried and fed as hay (Ferguson et al., 1943; Britton and Goss, 1946; Ward, 1991). Whether this is also true of the effects of Mo, independent of a secondary Cu deficiency, is unknown at this time.  Evidence suggests that cattle are the most susceptible to Mo toxicity of any species followed by sheep. Rabbits were shown to be very tolerant to Mo (Arrington and Davis, 1953) as were guinea pigs, rats, pigs and chickens (Underwood, 1977). This suggests that the functions of the rumen create an environment which enhances the toxicity of Mo (i.e. the formation of thiomolybdates). More recent information suggests that it may not be rumen function alone which accounts for species variation since mule deer (Ward and Nagy, 1976) and goats (Anke et al., 1985) can tolerate up to 1000 ppm Mo in the diet, or the same as rabbits, rats and chickens (Ward, 1991).  Diagnosis of molybdenosis can be difficult since the expression of symptoms such as reduced growth rate are difficult to distinguish from other disorders of cattle (Mills and Davis, 1987). The concentration of Mo in plasma, milk or urine is a fairly good indicator of intake levels when the intake is high. Plasma and liver concentrations of Cu, on the other hand, may not always be reliable indicators of Mo toxicity or the Cu status of the animal.  Suttle (1986a) concluded that there is no experimental basis for selecting  threshold levels for liver Cu since there is a range of acceptable levels that coincide with a marginally deficient state. Although there is some question as to the accuracy of determining Cu status through plasma and liver Cu concentrations, this is still the method most widely practiced by researchers. Ceruloplasmin is the principle carrier protein for Cu in the blood and can be used as an indicator of a Cu deficient state but is not closely correlated to Cu status or Mo toxicity. Superoxide dismutase is an enzyme  8  in erythrocytes and its activity is a better indicator of Cu status (Suttle, 1986b). However, determination of this enzyme requires specialized equipment. Clawson et al., (1972) and Suttle (1996) suggest that the best diagnosis of molybdenosis or Cu deficiency is through the response of the animal to Cu supplementation.  2.2 Reproductive Function 2.2.1 Puberty Beef heifers typically attain puberty at 11 to 15 months of age and at weights of 45 50% of their mature body weight.  Hereford heifers, specifically, attain puberty on  average at approximately 375 days of age or a body weight of 272 kg (Alberta Agriculture, 1992). The main determinant dictating the timing of the onset of puberty is weight as there is a strong correlation between heifer weight and puberty attainment (Sorenson et al., 1959; Hafez, 1993).  Puberty has been defined many ways. According to Robinson (1977), puberty is the process whereby an animal becomes capable of reproducing itself. However, this does not always correspond to the heifer's first ovulation as it is often followed by a shortened luteal phase which is incapable of supporting a pregnancy (Moran et al., 1989; Del Vecchio et al., 1992). Moran et al., (1989), defined puberty as being attained at the first estrus that is followed by a luteal phase of normal length. For the purposes of this report, puberty is defined as the time of the first ovulation as determined by the appearance of the first corpus luteum (CL) and the corresponding rise in progesterone (P ) regardless of the length of the following luteal phase (Hafez, 1993). 4  9  The first ovulation in heifers is accompanied by sudden changes in the endocrine patterns seen during the prepubertal period. The "gonadostat" theory is the classical theory used to explain the mechanism of hormonal control of the onset of puberty. In prepubertal heifers, the hypothalamic centre controlling the secretion of gonadotropins is highly sensitive to the negative feedback of estradiol from the ovaries. Because of this sensitivity, the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) is inhibited. This can be demonstrated by the resulting increase in LH secretion after the removal of the ovaries from pre-pubertal animals (Kinder et al., 1987). The gonadostat theory states that the sensitivity of the hypothalamic-pituitary axis decreases as puberty approaches, allowing the release of gonadotropin releasing hormone (GnRH) from the hypothalamus, which in turn causes an increase in the release of LH and FSH. The circulating concentrations of LH and FSH in the blood increase sufficiently to stimulate follicular growth and development which ultimately leads to the first ovulation (Dodson et al., 1988; Moran et al., 1989).  Prior to puberty in heifers, the mean level of LH in the blood increases from the lowest point at approximately 5 to 6 months of age to a peak at about 9 months of age. At the same time, the frequency of LH pulses also increases while the amplitude decreases (Schams et al., 1981; Dodson et al., 1988; Moran et al., 1989). Overall, the incidence of LH pulses remains low during the prepubertal period, most likely explained by the gonadostat theory as previously discussed (Kinder et al., 1987). Although there is no change in mean LH concentrations just prior to the first ovulation, LH pulse frequency increases, most likely due to the decreased sensitivity of the hypothalamus to estrogen (E ), which is thought to enhance follicular growth and cause further E secretion from 2  2  the ovaries. This in turn appears to stimulate the preovulatory surge of LH from the  10  pituitary, ultimately resulting in the first ovulation (Kinder et al., 1987). The increase in LH pulse frequency, and not an increase in the amplitude of LH episodes, is thought to be the initiator of the first ovulation in heifers (Dodson et al., 1988; Moran et al., 1989; Jones et al., 1991).  Gonzales-Padilla et al., (1975), found, that there are two distinct peaks in the prepubertal secretion of LH, one approximately 8 to 10 days before the other. The first peak is considered a "priming" LH peak, while the second peak is considered the preovulatory surge of LH, occurring on day 0 of the first estrous cycle. Both peaks were found to be closely followed by peaks in P concentration. Progesterone is not thought 4  to induce puberty, rather it enhances the secretion of gonadotropins from the pituitary, especially prior to puberty. Both the adrenal glands and the ovaries are thought to be sources of P in the prepubertal animal. The first priming peak of P is thought to 4  4  originate, at least partly, from the adrenal glands, while the second is thought to originate from the ovaries (Gonzales-Padilla et al., 1975). Besides these two peaks, P  4  levels remain quite low until the formation of a functional CL after the first ovulation (Dodson et al., 1988). It has also been suggested that, although transient increases in P concentration sometimes occur before puberty, the are not necessary for the correct 4  functioning of the CL after the first estrus (de Pinho et al., 1997).  Follicle stimulating hormone appears to follow the same fluctuating pattern of mean levels from birth to puberty as LH, rising from the lowest levels at approximately 5 months of age to a peak at about 9 months of age (Schams et al., 1981). Contrary to the change in LH levels prior to first ovulation, there does not appear to be much change in FSH just prior to the onset of puberty. Therefore plasma concentrations of  11  FSH appears to play a permissive role in the puberty attainment (Gonzales-Padilla et al., 1975; Dodson et al., 1988).  Follicular development occurs in prepubertal heifers (Adams et al., 1994) as it does in post-pubertal heifers (Pierson and Ginther, 1984; Savio et al., 1988; Sirois and Fortune, 1988; Taylor and Rajamahendran, 1991). There is the simultaneous emergence of a group of small follicles, followed by the selection of one which will become dominant while the others regress. This occurs in a wave-like pattern as shown by the 8 day interval between the maximum and minimum number of follicles and serum FSH concentrations and between the emergence of successive dominant follicles (Adams et al., 1994).  Despite these similarities, there are differences between pre- and post-  pubertal follicular dynamics which include: i) all waves are anovulatory in pre-pubertal heifers; ii) the growing phase of the dominant follicle is shorter; iii) the mean diameter of the dominant follicle is less; iv) the static phase of the dominant follicle is shorter; and v) the interwave interval is shorter. Wave emergence and dominant follicle selection are associated with a pre-wave surge of plasma FSH (Adams et al., 1994). Large antral follicles in prepubertal animals appears to be deficient in steroidogenic capabilities (Evans and Rawlings, 1994) which is a contributing factor for the lack of ovulation.  2.2.2 Estrous Cycle The estrous cycle has been defined as the interval from the beginning of one estrus period to the beginning of the next. Mature cows have estrous cycles lasting from 1724 d with an average of 21 d. Heifers have slightly shorter cycles lasting an average of approximately 20 d (Frandson and Spurgeon, 1992). There are several phases of the estrous cycle, including proestrus, estrus, metestrus and diestrus. The physiological  12  and endocrinological changes that occur during the estrous cycle prepare the female's reproductive tract for estrus, ovulation and pregnancy.  The following sequence of  events outlines a normal 21 day cycle in which pregnancy did not occur.  Day 0: This is the day of estrus when the cow is in standing heat and is sexually receptive.  It lasts approximately 18 h in the bovine.  High E levels act on the 2  behavioural centre of the brain to initiate estrus behaviour which includes restlessness, sniffing and licking of the vulva, resting her chin on the rump of other cows, mounting other cows and standing to be mounted (Hafez, 1993). The high levels of E also act 2  on the hypothalamus to stimulate the release of GnRH, leading to the ovulatory surge of LH. This surge of LH causes final maturation of the oocyte, ovulation and initiates luteinization of the follicle (Roche and Boland, 1991). At the same time, there is a sudden increase in FSH levels due to the drop in E and inhibin which occurs after 2  ovulation. Another effect of the high levels of E is that the cervix dilates slightly and 2  the cervical mucus becomes less viscous, allowing the passage of spermatozoa. Uterine and oviductal contractions, stimulated by E , increase to aid transport of sperm 2  towards the site of fertilization and oocyte transport down the oviduct. Ovulation occurs approximately 24 - 30 h after the onset of standing estrus (Rajamahendran et al., 1989).  The remaining cells of the follicle (granulosa and theca cells) begin rapid  division, accompanied by the infiltration of blood vessels and connective tissue forming a corpus hemorrhagicum (Rutter, 1995).  Day 1 - 2 : This is the post-ovulatory phase. During this time, E levels are decreasing 2  and P levels are increasing due to the luteinization of the follicular cells which leads to 4  the formation of the CL on the ovary at the site of ovulation (Rutter, 1995). Due to the  13  sudden drop in E levels following ovulation, the profusion of uterine vessels collapse 2  and rupture, resulting in a small amount of bloody mucus being released from the vulva which is known as metestrus bleeding (Beardon and Fuquay, 1992).  Day 3 - 5 : The CL is growing in size and increasing its P -producing capabilities. This 4  elevating level of P thickens the uterine lining, increases the number of uterine glands 4  and their secretions and hastens the development of mammary tissue, all in preparation for pregnancy.  It also quiets oviductal and uterine contractions and increases the  viscosity of the cervical mucus (Rutter, 1995).  Day 6 - 1 6 : The CL continues to grow and reaches its maximum size and function by day 10 -12. The high level of P inhibits the development of a preovulatory surge of 4  gonadotropins. There is continued follicular growth during this time, but the follicles become atretic because of the high levels of P and the high amplitude, low frequency 4  pattern of LH secretion (Rutter, 1995).  Day 16 - 18: If there is no pregnancy, the CL regresses due to the release of prostaglandin F  2a  (PGF ) from the uterus (Hafez, 1993). If pregnancy is achieved, the 2a  cow will enter a period of anestrus and the cycle is temporarily halted due to the release of interferon tau which acts as the signal for maternal recognition of pregnancy and prevents regression of the CL thereby maintaining P levels to support the pregnancy 4  (Bazer etal., 1994)  Day 18 - 1 9 : Gonadotropins (LH and FSH) are released because of the removal of the negative feedback effects of P on the hypothalamus (Hafez, 1993). 4  Gonadotropin  14  secretion follows a high frequency, low amplitude pattern which stimulates follicular growth and allows for the selection and dominance of one follicle. This follicle will eventually go on to ovulate while the others become atretic (Rutter, 1995).  Day 1 9 - 2 0 : Rising E levels produced by the ovulatory follicle stimulate preparation of 2  the reproductive tract for standing estrus (i.e changing the viscosity of the cervical mucus and allowing the opening of the cervix to permit the passage of spermatozoa). The rise in E concentrations causes an increase in LH pulse frequency which will give 2  rise to the LH surge . As E levels peak, standing estrus is once again initiated and 2  another cycle begins at Day 0 (Rutter, 1995).  The advent of ultrasonography has allowed monitoring of the ovaries and reproductive tract on a frequent basis. Pierson and Ginther (1984) were the first to monitor follicular growth throughout the estrous cycle and concluded that the number and size of follicles present on the ovaries changes depending on the day of the cycle.  In 1987, they  reported that there were 2 waves of follicular growth occurring in heifers. It is now known that most cows have two waves of follicular growth (Taylor and Rajamahendran, 1991), whereas heifers often have three waves (Savio et al., 1988; Sirois and Fortune, 1988; Taylor and Rajamahendran, 1991). A follicular wave is characterized by the appearance of a cohort of small follicles (10-20) followed by the selection and dominance of one of these follicles while the rest of the cohort regresses and becomes atretic. The first dominant follicle can be detected as early as day 4, reaches maximum size on day 6, remains stable between days 6 - 1 0 and can no longer be seen on day 15. The second dominant follicle is detectable on day 10, reaches maximum size on day 16 and will go on to ovulate in a two wave cycle (Knopf et al., 1989). In a three  15  wave cycle, the second dominant follicle regresses, the third dominant follicle is detectable on day 16, grows to a maximum on day 21 and ovulates. (Savio et al., 1988; Taylor and Rajamahendran, 1991). The ovulatory dominant follicle in heifers with 3 wave cycles emerges later and ovulates sooner after emergence than in heifers with 2 wave cycles (Ginther et al., 1989). The second dominant follicle will ovulate in cycles of shorter duration suggesting it is the time of regression of the CL which plays a major role in whether there will be a 2 or 3 wave cycle (Roche and Boland, 1991).  2.2.3 Conception, Pregnancy and the Post-Partum Period Reproduction is centered around fertilization, which is essentially the fusion of two gametes, a spermatozoa and an oocyte, to form a single cell called a zygote. In the cow, this fusion takes place in the ampullar-isthmic junction of the oviduct. The oocyte arrives at this location approximately 1 5 - 3 0 min after ovulation. The fertile life of the bovine oocyte in the oviduct is very short, only 8 - 12 h. After being deposited in the female reproductive tract, either naturally or through artificial insemination (Al), spermatozoa must undergo capacitation and acrosome reaction before penetration of the oocyte can occur. Capacitation is essentially a modification of the sperm's surface membrane in preparation for penetration of the oocyte. In cattle, this process can take 4-5 h (Ambrose, 1995). The acrosome reaction only occurs after capacitation in the female reproductive tract and attachment of the sperm to the zona pellucida of the oocyte. The acrosome reaction involves fusion of the outer acrosomal membrane with the plasma membrane of the spermatozoa to allow the release of enzymes necessary for penetration of the zona pellucida of the oocyte (Beardon and Fuquay, 1992). Once a spermatozoa has entered the oocyte, it forms the male pronucleus which fuses with the female pronucleus, in a process called syngamy, to form the zygote. Syngamy  16  marks the end of fertilization. The duration from the time of penetration of sperm to the first cleavage is estimated to be 20 - 24 h in the bovine (Rutter, 1995).  Cleavage continues through the 8 cell stage to the 16-32 cell stage at which point the embryo is known as a morula. It moves into the uterus and becomes free-floating, usually 4-5 d after ovulation. Fluid begins to accumulate between the cells and an inner cavity,  the blastocoele, begins to form.  When this cavity expands, the embryo is  known as a blastocyst. Some embryonic loss can occur at this point and is usually due to chromosomal defects. In cattle, the blastocyst stage occurs approximately 7 - 8 d after ovulation and this is the time a donor cow would be flushed for embryo collection. A single layer of trophoblast cells (eventually forming the placenta and embryonic membranes) surrounds the inner cell mass (which will give rise to the fetus). In the cow, the blastocyst hatches from the zona pellucida on about day 8 or 9 after ovulation and the elongation of the embryo begins a few days later.  Maternal recognition of  pregnancy takes place on about day 1 6 - 1 7 . It involves the production and secretion of interferon tau which blocks the activity of the enzymes necessary for the production of PGF  2a  by the uterine tissue (Thatcher et al., 1989; Bazer et al., 1994). The CL must  continue to release P in order to maintain the pregnancy. Significant embryo losses 4  can occur at this point due to failure of the embryo to produce the signal or failure of the mother to recognize it (Rutter, 1995)  While the embryo is undergoing cleavage and blastocyst formation, the uterus is also undergoing changes in order to provide a suitable environment for the developing embryo and to prepare for implantation. Implantation is said to occur when the embryo is in a fixed position and physical contact with the uterus is established. By 33 d after  17  insemination, the fetal chorionic membrane has formed a fragile attachment with 2 to 4 cotyledons surrounding the fetus; within a few days, the maternal caruncles and fetal cotyledons will become so interdigitated that the embryo is completely nourished through the cotyledons. Pregnancy diagnosis through ultrasonography is done on day 35 since the embryo is in a fixed position and can be readily observed. The other period of significant embryo loss occurs at the implantation stage. If an embryo is lost at this time, the cow will return to estrus 40 - 42 days after mating (Rutter, 1995).  There are clearly defined periods of follicular growth and turnover in early pregnancy with an interval of 8-10 d between the emergence of dominant follicles. There is no difference between cyclic and pregnant heifers in the emergence, persistence or regression of the first dominant follicle, therefore the presence of the CL of pregnancy and the embryo in the uterus does not appear to effect dominant follicle turnover (Gintheret al., 1989; Savio et al., 1990).  The gestation length in cows extends from the time of fertilization to calving and usually lasts an average of 283 d (ranges from 279 - 285 d). In well managed cattle, fetal death after implantation is rare, occurring less than 2.5% of the time. The placenta is the major hormone-producing unit during pregnancy.  Hormone production by the  placenta regulates the growth of the fetus and maternal function to ensure adequate environmental conditions. As pregnancy progresses, the uterus undergoes gradual enlargement to permit the expansion of the fetus. Three phases can be identified in the adaptation of the uterus to the growing fetus: proliferation, growing and stretching. The structural changes taking place in the pregnant uterus are reversible and are restored at different rates after parturition (Rutter, 1995).  18  During pregnancy, the CL persists at a maximal size and continues to produce P . After 4  approximately 150 - 250 d gestation, the fetal-placental unit is capable of producing enough progesterone to support the remainder of the pregnancy should the CL regress (Rutter, 1995). During pregnancy, in spite of the high P concentration, suspension of 4  the release of LH and FSH is not complete and there continues to be follicular wave development but without estrus or ovulation (Taylor and Rajamahendran, 1991). In late pregnancy, there is a reduction in the number of FSH surges with each heifer having 12 ineffective surges and LH concentrations are decreased (Ginter et al., 1986; Crowe et al., 1998).  The first stage of labour is the preparation for birth, which can take from 3 hours in a cow to 72 h in a first-calf heifer.  During this stage, the ligaments of the pelvis and  associated structures relax due to elevated levels of the hormone, relaxin (Hafez, 1993). The cervix, and vulva dilate and the mucous plug dissolves. These changes facilitate the passage of the calf. The cow becomes restless and often will want to separate from the herd. She may stand with her back arched and her tail raised and may attempt straining. The second stage of labour is signaled by the presence of the fetal membranes and the head and forelimbs of the calf at the cervical canal. The expulsion of the calf through the birth canal will last from !4 h in the cow to 2 - 3 h in a first calf heifer.  Expulsion of the placenta comprises the third stage of labour. The  placenta is usually expelled within a few hours of birth (Rutter, 1995).  The post-partum period is characterized by involution of the uterus and a period of anestrus which lasts approximately 35 - 70 d in well managed beef cows and up to 80-  19  90 d in first-calf beef heifers (Short et al., 1994). The length of this interval can be affected by season, breed, parity, and presence of a bull, but the greatest effects are from suckling and nutrition. Pregnancy has an inhibitory effect on the sensitivity of the pituitary to GnRH (Schallenberger et al., 1978). The sensitivity gradually decreases after calving, evident by increasing plasma LH concentrations. These changes are principally emphasized during the days preceding the first increase in progesterone and the first ovulation after calving (Hanzen, 1986). Suckling can induce a difference in the time of appearance and intensity of this response. The inhibitory effects of suckling after calving acts more on the release of GnRH and LH rather than their synthesis. Concentrations of progesterone in the plasma of cows is high throughout gestation, diminishing slowly during the last 3 - 4 weeks and abruptly 2 - 3 days before calving. Studies of post-partum endocrinology have shown that there are two types of luteal activity seen during the post-partum period. The first is a short luteal phase lasting approximately 6 - 12 d and the second is a phase of more normal duration accompanied by lower than normal progesterone levels (inadequate luteal phase) (Hanzen, 1986). The resumption of estrous cycles post-partum is analogous to the events leading to the initiation of cyclicity at puberty.  In suckled beef cows, a cohort of follicles (5-8 mm) develops for 2-8 d with one follicle becoming dominant approximately 10 d post-partum. In a small percentage of cows, this first dominant follicle will ovulate but usually there are 3 dominant follicles prior to ovulation. The prolonged interval to first ovulation in suckled beef cows is due to failure of ovulation rather than the failure of the dominant follicle. It is likely that infrequent LH pulses, due to the suppressive effect of suckling on LH secretion, are a major cause of prolonged anestrus in beef cows (Murphy et al., 1990)  20  2.2.4 Hormonal Manipulation of Reproductive Function 2.2.4.1 Estrus Synchronization Estrus synchronization is a management tool that can be used to help improve production efficiency and profitability. The purpose is to control estrus and ovulation so that breeding can be accomplished in a short period of time.  For any estrus  synchronization program to be effective, high management quality and strict adherence to the program is essential. There are numerous benefits to estrus synchronization, including: 1. decrease time and labour required for heat detection; 2. greater use of superior sires through Al; 3. concentration of breeding and calving periods; 4. more uniform calf crop; 5. uniform feeding of groups based on gestation requirements; and 6. shortened post-partum interval (Odde, 1990). The three main ways to synchronize estrus include inhibition of ovulation, induction of ovulation and induction of CL regression.  Synchronization is mainly accomplished through the administration of  appropriate natural or synthetic hormones.  There are several different hormones that  can be used to synchronize estrus. Progesterone / progestegins act by suspending the secretion of GnRH which prevents the onset of estrus and ovulation. Oral and implant progesterones have received the greatest attention, but it can also be administered through vaginal pessaries or injection. For progesterones to have an effect they must be administered for the duration of the cycle. When treatment is stopped, all animals will be in the follicular phase. The decrease in P concentration triggers the release of 4  GnRH and estrus can be expected 2 - 5 d later. Synchrony using this method is good (70 - 90%), but a marked decrease in fertility is seen (Hansel, 1967; Hafez, 1993).  21  Prostaglandins (PGs) such as P G F , cause the CL to regress within 24-72 h of 2a  administration, but the CL will respond to PGs only at certain time of the cycle (Cooper, 1974). For the first 5 d after ovulation (early luteal phase) the CL is unresponsive to PGs.  If PGs are administered 5 to 17 d after estrus, prompt luteolysis will occur.  Spontaneous luteal regression occurs from days 1 7 - 2 1 and PGs will have no effect if given at that time. Because of this, an injection of P G F  2a  is often followed by a second  injection within 10 - 1 2 d. If cattle are equally distributed across the days of the estrous cycle, then 70% should show estrus after the first treatment.  This 70% and the  remaining 30% of the cows should have a CL and be at a stage of the cycle to respond to the second treatment (Odde and Holland, 1994).  Although the double injection  system reduces the amount of time required for estrus detection, more of the drug is required. In addition, there are after effects of prostaglandin treatment. There could be an increase in cycle length and a decrease in fertility after synchronization due to the presence of persistent follicles (Larson and Ball, 1992).  Often, these compounds are used in combination in order to try to improve efficiency of the synchronization program. For example, progtaglandins and estrogens have been used together to force CL regression. When used in combination, they work faster and more completely than when used separately (Dailey, 1986). Recently, the use of GnRH and prostaglandins has been investigated. GnRH followed by P G F  2a  This involves treating the animals with  after 7 days. This methodology was developed to apply  knowledge regarding the control of follicular dynamics and the CL to the problem of estrus synchronization.  GnRH is given to ovulate any large functional follicles and  induce the emergence of a new follicular wave in order to increase the likelihood of there being a new large growing follicle at the time of luteolysis. This has been shown  22  to reduce the incidence of persistent follicles and to increase the number of animals that will respond to the PGF  2ot  treatment, resulting in an increased precision of estrus  (Pursley et al., 1995). While there has been success with this method, there has been the occurrence of short estrus cycles as well as poor conception and pregnancy rates possibly due to the failure of CL regression or incomplete maturation of the follicle and/or oocyte at the time of ovulation especially in heifers (Schmitt et al., 1996). This response in heifers may be due to differences in the timing of spontaneous ovulation between cows and heifers after estrus synchronization.  Greater success can be  expected if a second injection of GnRH is given 24 - 48 h after PGF  2ct  to ovulate the  preovulatory follicle at a precise time (Pursley et al., 1995). This allows for more control over the ovulation of the dominant follicle and results in a greater degree of synchrony and increased precision of estrus.  2.2.4.2 Superovulation Cows are monotocous animals, meaning that they usually release a singe oocyte during each estrous cycle (Hafez, 1993). Superovulation is a technique that has been developed to stimulate the release of multiple oocytes. It can be used to increase the incidence of twins in cattle but is primarily used in embryo transfer programs to increase the number of offspring from valuable animals. The main limitation is the variation in superovulatory response to the treatment which results in variations in the number and viability of ova recovered (Rajamahendran et al., 1987; Bo et al., 1994; Kelly et al., 1996). On average, superovulation results in 6 transferable embryos, but the typical range is 0-25 (Rajamahendran and Calder, 1993).  There are many factors, both  intrinsic and extrinsic, that can affect the response to superovulation.  One intrinsic  factor affecting superovulatory response is the number of small follicles on the ovaries  23  before treatment. Romero et al., (1991), found that the number of small follicles at the time superovulation was initiated affected the number of ovulatory follicles generated by the treatment.  Several researchers have attempted to induce superovulation at  different stages of the estrous cycle to determine the optimal time to start a superovulatory protocol. The best response was found to occur when superovulation was initiated on day 8 - 1 2 (Phillippo and Rowsan, 1975; Sreenan and Gosling, 1977; Lindsell et al., 1985; Rajamahendran et al., 1987).  Extrinsic factors affecting  superovulation include age, nutrition, body composition, type and amount of hormone administered and purity of the hormonal preparation (Bellows et al., 1969; Monniaux et al., 1983; Murphy et al., 1984; Rajamahendran and Calder, 1993).  The main hormones used for superovulation include FSH, LH, and GnRH which are all naturally occurring during the estrous cycle. Equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) are reproductive hormones from other species that are used for superovulation in cattle.  One of the first superovulatory methods  developed involved the use of a single injection of eCG on day 15 of the estrous cycle with Al done 12 h after the onset of estrus. The advantages of this method were that it was simple, inexpensive and involved limited handling of the animals. However it did not synchronize estrus so more labour was required for estrus detection. Overall, this method results in a lower response as compared to other methods that have been developed more recently. If eCG is administered on day 10 after estrus, followed by an injection of P G F  2o  on day 12 and a second injection of P G F  2a  12 h after the first, then  the superovulatory response can be increased although this still is not as effective as other methods (Hafez, 1993; Rajamahendran and Calder, 1993)  24  The most commonly used method of superovulating cattle is a series of FSH injections given over 3 - 5 d starting on approximately day 10 of the estrous cycle. The FSH can either be given in equal or decreasing doses, although the latter is more effective (Bellows et al., 1969, Chupin and Procureur, 1982; Rajamahendran et al., 1987). An injection of P G F  2a  is usually given on day 12.  This method stimulates follicular  development and induces CL regression leading to ovulation.  The reason for the  multiple injections is that the half-life of FSH has been reported to be 5 h or less (Demoustier et al., 1988) therefore multiple injections are needed to maintain circulating levels. Although this method is successful, the intensive labour and increased handling of the animals combine to make the search for alternate methods desirable.  One such alternate method is to use a single injection of FSH (same total dose as the multiple injections).  The FSH is first dissolved in a slow release agent such as  polyvinylpyrrolidone (PVP). If a single injection of FSH + PVP is given on day 7 of the estrous cycle followed by an injection of P G F  2a  on day 10, FSH is slowly released into  the animal's system and follicular growth and ovulation is stimulated. It was found that the superovulatory response and embryo recovery was the same in cows given a single injection of FSH in PVP as compared to control cows given 8 decreasing doses of FSH over 4 days (Yamamoto et al., 1993). More recently, the use of a single injection of FSH without prior dissolving in PVP has been investigated.  It was found that the  superovulatory response and embryo recovery was the same in cows given a single injection of FSH as compared to control cows given 8 decreasing doses of FSH over 4 days (Bo et al., 1994) although other studies have not shown as good a response (Monniaux et al., 1983; Staigmiller et al., 1995; Kelly et al., 1996).  25  2.3 Nutrition - Reproduction Interactions 2.3.1 General Interactions The nutritional quality of forages and other feedstuffs can have a tremendous influence on the reproductive performance of cattle. Although reproductive failure can happen for many reasons, management and the environment are often important contributing factors. Part of the management and environment of any animal is nutrition.  Energy is probably one of the most important considerations in cattle production, second only to water. Cows need energy to maintain milk production and as well as to meet the need for initiating and maintaining pregnancy. Carbohydrates are the primary source of energy in the diet. Besides being a source of energy, carbohydrates are also building blocks for other nutrients. Excess energy is stored as fat which provides insulation and protection for the body and which also can be catabolized as an energy source (Kreplin and Yaremcio, 1995).  The clinical signs of an energy deficiency as related to  reproduction are delayed onset of estrus in small thin heifers with inactive ovaries, and inactive ovaries, repeat breedings and a decrease in progesterone production by the corpus luteum in adults (Morrow, 1980). There can also be lower birth weights, higher death rates of newborns, decreased milk production, lower weaning rates, longer postpartum anestrus periods and reduced conception rates (Kreplin and Yaremcio, 1995). Lactation is a stressful period for cows in which the energy levels are crucial. For heifers, nutrients are required for maintenance and growth as well as milk production. Because of this, it is almost impossible to prevent postpartum weight loss due to a negative energy balance (Morrow, 1980). However, energy restriction which is a common practice to reduce production costs can delay the first ovulation (Grimard et al., 1996). During this period cows should be fed the highest quality feed and recent evidence suggests that  26  supplementing the diet with fat to be used as an alternate energy source can be beneficial. The fat may improve reproductive performance by stimulating P G F synthesis 2a  and secretion and by the enhanced utilization of blood cholesterol for progesterone synthesis (Grummer and Carroll, 1991). Supplementing fat in post-partum cows has also been shown to enhance ovarian follicular growth before the resumption of estrous cycles as well as increasing body condition scores and pregnancy rates without altering the length of the post-partum interval or P concentrations (de Fries et al., 1998) 4  Protein is the next critical nutrient in most rations. It is the building block of most tissues. If the level of energy provided by the diet is not sufficient, it can be supplied by the breakdown of fat and muscle in the body or, alternately, proteins will be broken down and the resulting amino acids will be used as a energy source. But if the level of protein is not sufficient, there is no way for the body to compensate (Kreplin and Yaremcio, 1995). The clinical signs of infertility associated with a protein deficiency are: a delay in the onset of puberty, and an increase in the number of days open (Morrow, 1980). It is difficult to separate a protein deficiency from a concurrent energy deficiency although it is generally believed that an energy deficiency has a much greater effect on reproduction. Protein is essential for proper fetal development and function of the reproductive organs. Delayed sexual maturity is observed in heifers that are fed protein deficient diets. In lactating cows, a protein deficiency results in emaciation and low milk production. A fine line exisists between too little and too much protein on which the reproductive performance of cows is optimal (Kreplin and Yaremcio, 1995).  Besides energy and protein, the other components of the diet can also have serious consequences on reproductive efficiency. Vitamin needs are frequently met by rumen  27  and tissue synthesis and natural feeds. Also, commercial feed preparations usually contain supplemental vitamins and, therefore, the probability of infertility due to a vitamin deficiency is minimal. Nonetheless, they should still be mentioned in a discussion of reproduction/nutrition interactions. Vitamin A is involved in the maintenance of body tissues, so requirements in the pregnant cow are higher in the last third of pregnancy and immediately after calving. The clinical signs of a vitamin A deficiency are: delayed onset of puberty, abortion or birth of weak, blind, uncoordinated calves, degeneration of the placenta and metritis (Morrow, 1980; Hurley and Doane, 1989). Cows with a vitamin A deficiency can conceive normally but will return to heat due to the early death of the embryo (Ganguly et al., 1980).  If supplemental levels of vitamin A, in excess of  requirements are given, there is no effect on circulating progesterone levels or on other measures of reproductive performance, therefore, providing high levels of vitamin A are not warranted (Tharnish and Larson, 1992).  p-carotene is a precursor of vitamin A but also has an independent role in reproduction. Cattle receiving supplemental p-carotene have an increased intensity of estrus, increased conception rates, and reduced frequency of follicular cysts. The CL of the cow has a higher p-carotene concentration than any other organ (Tharnish and Larson, 1992). There has also been observed a positive relationship between p-carotene and luteal cell progesterone during times when plasma p-carotene and vitamin A are decreased. Other studies have shown that there are no effects or even adverse effects of p-carotene supplementation on the fertility of dairy cattle. Because of these conflicting results, further research into the effects of p-carotene is required.  28  Vitamin D is essential for proper calcium and phosphorus absorption, normal bone growth, and prevention of rickets. Vitamin D deficiencies are rare since cattle usually form adequate amounts through exposure to the sun and from sun-cured forages (Kreplin and Yaremcio, 1995). If a cow is fed a diet that is deficient in vitamin D there is a reduction in fertility possibly by suppressing the signs of estrus and delaying the onset of estrous. If vitamin D supplementation is provided in the diet, the first postpartum estrus and conception occur earlier, but there is a delay in uterine involution (Morrow, 1980).  Minerals are the nutrients most commonly implicated in infertility in cattle. The large number of interrelationships that exist between the absorption and utilization of minerals make it difficult to delineate relationships between a specific mineral and fertility.  A  calcium deficiency is believed to have an indirect effect on reproduction in cattle. Calcium intake is important in preventing milk fever (hypocalcemia).  Milk fever leads to an  increased incidence of dystocia and retained placentas, therefore calcium is indirectly involved in the prevention of these two problems (Morrow, 1980).  Phosphorus is the mineral most often associated with infertility in cattle. Diets that are low in energy and protein are usually low in phosphorus as well. The effects of a phosphorus deficiency are variable depending on the severity of the deficiency: a severe deficiency results in delayed onset of puberty and postpartum estrus because of inactive ovaries, and a moderate deficiency may be associated with repeat breeding. The clinical impression is that there is a relationship between phosphorus intake and the development of cystic follicles, although this has yet to be proven (Morrow, 1980). A phosphorus deficiency can be expressed as a delay in the onset of puberty and an increased number  29  of services for conception. Insufficient amounts can also result in reduced milk production and consequently lower calf weaning rates (Kreplin and Yaremcio, 1995).  Iodine exerts its influence on fertility through its action on the thyroid gland. The basic interaction between low levels of thyroid function and reproduction appears to be an impairment of ovarian activity.  Toxic levels of iodine can cause abortion. An iodine  deficiency in the diet of pregnant cows can cause the premature birth of dead or weak calves and cows affected with goiter (Morrow, 1980).  Connective tissue, erythrocytes and many enzymes in the body require copper for proper functioning. Copper deficiency can manifest itself as reproductive disorders in females. Early embryonic death is quite common. Suboptimal ovarian activity, delayed estrus, reduced conception rates, increased incidence of retained placenta, and calving difficulties have all been associated with Cu deficiency. Administration of Cu sulfate has induced normal breeding patterns in heifers with low fertility associated with Cu deficiency and supplemental Cu has increased conception rates in cows with marginally low blood Cu.  Copper-related reproductive disorders may be due to actual Cu deficiency or  interference with Cu utilization (Hurley and Doane, 1989; Morrow, 1980).  Manganese is essential for the formation of condroitin sulfate, which is a component of bone organic matrix. Thus, it is essential for normal bone formation (Church and Pond, 1988). The clinical signs of the effect of a manganese deficiency on reproduction are: silent estrus, abortions, and the birth of calves with twisted or deformed limbs (Morrow, 1980).  30  The only known animals requirement for cobalt is as a constituent of vitamin B (Church 12  and Pond, 1988). A cobalt deficiency results in a delay in the onset of puberty, a delay in the onset of postpartum estrus, and anemia (Morrow, 1980).  Zinc is a constituent of numerous metalloenzymes, including carbonic anhydrase, carboxypeptidases  A  and  B,  several  dehydrogenases,  alkaline  phosphatase,  ribonuclease, and DNA polymerase. It activates some enzymes and plays a role in configuring DNA and RNA (Church and Pond, 1988). It is also an important mineral in reproduction. A deficiency can lead to a delay in testicular development in young bulls, testicular atrophy in adult bulls, reduced fertility in the female, and parakeratosis which is the hyperkeratinization of the epithelial cells (Morrow, 1980).  Selenium is a major component of the glutathione peroxidase enzyme system. Deficiency signs include a decrease in fertilization rate, and an increase in the incidence of retained placenta and muscular dystrophy (Morrow, 1980).  Three hypotheses have been developed to explain the effects of nutrition on the reproductive system. The first hypothesis relates to body weight and composition. It is known that the onset of puberty is correlated with the attainment of a certain body weight in most species. This can be modified to state that the onset of puberty corresponds to the attainment of a minimum lean to fat ratio and to the achievement of a minimum percentage of body fat (Aherne and Kirkwood, 1985). Body weight or relative fatness may influence reproduction through steroid metabolism.  Fat contains a significant  concentration of progesterone and is capable of converting androgens to estrogens.  31  Because of this, any changes in body weight are associated with a corresponding change in estrogen metabolism (I'Anson et al., 1991).  An alternate hypothesis for the effects of nutrition on reproduction states that food intake and its associated effects on metabolic rate may be the triggering stimulus for the change in the reproductive state of the animal. Nutrition has a profound effect upon circulating substrates and metabolic hormones which in turn have the potential to affect the hypothalamic-pituitary-gonadal axis (I'Anson et al., 1991). After an animal has finished a meal and after the absorptive period there are fluctuations in the levels of plasma hormones and substrates. These may provide signals to the brain that link metabolic status to the activation of the reproductive system.  Support for this comes from  numerous experiments in which the administration of metabolites such as glucose and amino acids elicited significant increases in LH and FSH secretion in castrated juvenile monkeys without a corresponding change in body weight (Steiner et al., 1983).  Metabolites related to energy balance, body weight, fatness or protein reserves may provide short and/or long term signals that influence reproduction.  In particular  physiological situations, such as growth either in a general sense or in relation to the development of certain tissues, metabolites may be the limiting factor for reproductive development and maturation (I'Anson et al., 1991). In the adult animal it has been shown that major changes in body weight can affect reproduction, but it has also been shown that the short-term metabolic state can have immediate and profound effects on reproductive function in the absence of any change in body weight or tissue composition. It is not known at this time whether the short term and long term effects are mediated by the same physiological mechanisms (I'Anson et al., 1991).  32  The third major hypothesis is that nutrition affects the reproductive system through the central nervous system and the release of peptides from the gastrointestinal system. The release of these peptides is stimulated by specific nutritional constituents.  These  peptides are active within the central nervous system, therefore it is possible that a gutneural connection may influence  neural and neuroendocrine function  directly.  Cholecystokinin, gastrin, neurotensin, and gastric inhibitory peptide have all been shown to suppress plasma gonadotropin levels in rats probably by inhibiting GnRH release (I'Anson etal., 1991).  The previous hypotheses were related to nutrition affecting the reproductive system through the hypothalamus. During periods of altered nutrition, the ability of the pituitary to secrete gonadotropins may or may not be affected. Whether the pituitary can respond to GnRH appears dependent on the type of metabolic disturbance.  It is possible that  changes in pituitary function may result from altered exposure to endogenous hypophysiotrophic hormones rather than any direct effect on the pituitary  itself.  Decreased GnRH neuronal activity, rather than pituitary responsiveness, may be compromised by changes in nutritional state to produce reproductive dysfunction or delayed sexual maturity. This conclusion is made from the results of numerous studies in which pituitary gonadotropin secretion can be activated (as during development) or restored (during adulthood) by exogenous GnRH, suggesting a deficit in GnRH secretion (I'Anson et al., 1991). There is no current information regarding GnRH synthesis during periods of altered nutritional states. Recent evidence does however suggest that it is unlikely that the primary effect of chronic poor nutrition is to inhibit synthesis. This suggests that GnRH release is limited during periods of restricted nutrition. The effect of  33  low nutritional intake on GnRH release can be mediated independently of gonadal steroids or by causing an increase in sensitivity to the negative feedback effect of steroid hormones (I'Anson et al., 1991).  There is extensive evidence that metabolic hormones can act at the ovarian level as well. They can either desensitize the ovary to gonadotrophic stimulation or amplify the response to other stimuli. Insulin is one such metabolic hormone. It has been shown to regulate general cellular morphology and development, and can either facilitate cell maintenance or stimulate the proliferation of porcine granulosa cells in vitro (Channing et al., 1976; Grimard et al., 1996). This granulosa cell proliferation could be due to the maintenance of basal and gonadotropin-induced progesterone production and by the enhancement of FSH-stimulated LH receptor induction (Channing et al., 1976).  Somatostatin or growth hormone inhibiting hormone is another metabolic hormone that has been shown to influence the activity of the ovary. It has been reported to inhibit GnRH release from the rat hypothalamus, and has been identified in porcine ovaries where it is thought to modulate the action of the other growth hormones. Conversely, growth hormone (GH) acting at the ovarian level augments FSH-stimulated LH receptor induction and progestin biosynthesis by cultured porcine granulosa cells. These action may be mediated by GH-stimulated insulin-growth factor I (IGF-I) synthesis in these cells, whereby IGF-I acts in an autocrine and a paracrine manner (Hammond et al., 1985). In other words, GH acts on granulosa cells to stimulate the production of IGF-I which then acts on the granulosa and thecal cells.  34  2.3.2 Molybdenum and Reproduction As discussed previously, Mo is an essential micromineral that can have adverse effects on metabolism through its interactions with Cu and S.  In addition to this, several  studies have been done to determine the effects that elevated levels of Mo can have on reproduction. The effect seems to be species specific, with increased severity being seen in ruminants as opposed to monogastrics (McDowell, 1992).  There are also  differences within ruminants, with cattle being more severely affected by high levels of Mo than sheep. The most comprehensive study looking at the effects of elevated levels of Mo on cattle was done by Phillippo et al., (1987), in which they were examining the effects of supplementing the diet with 5 mg Mo/kg DM to determine the effect this would have on puberty, fertility and estrous cycle regularity. The major results of this study were that heifers in the Mo-supplemented group reached puberty up to 12 weeks later than the control animals. This was independent of the live weight gain since all animals were similar in weight at puberty regardless of their group assignment. Phillippo et al., (1987), also suggest that this effect was not due to low Cu status, rather that it may be due to an alteration of LH secretion since a decrease in the concentration of plasma LH were evident as early as 11 weeks after the start of the experimental diets. After estrus synchronization and Al, conception rates were determined and found to be significantly lower in the Mo-supplemented animals as compared to the control group (12-33% Vs. 58-80%).  Mo supplementation also increased the number of animals that failed to  ovulate and eventually became anestrus. This had previously been seen in cattle with low Cu status but had not been attributed to elevated Mo.  Another study by O'Gorman et al., (1987), examined the effect of Mo induced Cu deficiency in cycling beef heifers with low Cu status after grazing high Mo pastures.  35 The animals were divided into two groups: one receiving supplemental Cu (200mg Cu, subcutaneous) and the other receiving 15-20 mg dietary Mo/kg.  After estrus  synchronization, Al and non-surgical embryo recovery it was found that there was no difference in fertilization between the two groups. There was, however, a difference in the normality of the recovered embryos (16% Vs. 63% in the Mo-supplemented and control animals, respectively).  Although this was only a preliminary study, it does  suggest that high levels of Mo may have a detrimental effect on early embryonic development.  Also in 1987, Wittenberg and Devlin examined the effects of high levels of dietary Mo on lactation and performance of suckling calves. In this experiment, 12 cow/calf pairs were assigned to one of three treatment groups where they received 0, 20 or 40 mg Mo/kg dry matter (DM). The animals on the high levels of Mo showed a significant decline in total plasma copper, TCA soluble copper and ceruloplasmin activity. Cows supplemented with Mo showed reduce milk yield and increased Mo levels in milk (severity depending on amount of Mo supplementation). This led to lower calf daily gains but did not affect the calf plasma Cu, ceruloplasmin, or liver Cu concentrations.  A study similar to that of Phillippo et al., (1987), was done by Fungwe et al., (1990), in rats. In this experiment the effects of supplemental Mo on estrous activity, fertility and reproduction were investigated.  The rats were assigned to five dietary treatment  groups, (control, 5, 10, 50 and 100 mg Mo/L deionized water). Supplementation of Mo at 10 mg/L or higher significantly prolonged the estrous cycle, resulted in significant decreases in the gestational weight gain and had detrimental effects on fetal development. The number of intrauterine deaths was few, but the number of resorbed  36  fetuses increased with supplemental Mo. Since there was a conception rate of over 80% in all groups, Mo did not seem to have an effect on fertility in the rat although it did result in a persistent estrus.  Another study investigated the effects of molybdenosis of LH, FSH and estradiol concentrations in rats (Igarza et al., 1996). There were 3 treatment groups in this investigation:  control,  copper deficiency  and  molybdenum  (500  ppm  sodium  molybdate). Vaginal smears showed that high Mo prolonged the length of the estrous cycle and altered the cytological characteristics of the different phases of the cycle. Peak values of FSH were significantly lower in the high Mo group. Serum estradiol concentrations were lower in the Cu deficient and high Mo groups.  The authors  concluded that high dietary Mo decreased FSH levels and the combination of high Mo and Cu deficiency decreased serum estradiol levels although different mechanism may be involved. They also speculated that any changes in the estrous cycle in animals receiving high Mo may be subsequent to the alterations in FSH and estradiol secretion.  Another study investigated the effects of molybdenosis on LH, FSH and estradiol concentrations in rats (Igzara et al., 1996). There were 3 treatment groups: control, copper deficiency and molybdenum (500 ppm sodium molybdate).  Vaginal smears  showed that high mo prolonged the length of the estrous cycle and altered the cytological characteristics seen during the different phases of the cycle. Peak values of FSH were significantly lower in the high Mo group.  Serum estradiol concentrations  were lower in the Cu deficient and high Mo groups. The authors concluded that high dietary Mo decreased FSH levels and the combination of high Mo and Cu deficiency decreased serum estradiol levels although different mechanisms may be involved.  37 They also speculated that any changes in the estrous cycle in animals receiving high Mo may be subsequent to the alterations in FSH and estradiol secretion.  The mechanism whereby Mo exerts its effect on reproduction is unknown at this time. In their study, Phillippo et al., (1987), suggested that Mo may act through a decrease in the release of LH which might be associated with an alteration in ovarian steroid secretion.  A decrease in LH peak concentration was also evident in a preliminary  experiment in which 5 heifers were fed a diet supplemented with 30 ppm Mo and 225 ppm sulfate ions. Blood samples taken every 2 h at estrus showed a decrease in peak LH concentrations in treated heifers (Igarza et al., 1995).  The effect of Mo (as  molybdate) on estradiol has been investigated previously by Noma et al., (1980) and Muller et al., (1982). Both of these groups examined the effect of molybdate on the activation of the receptors for estradiol.  Noma et al., (1980), found that the activation of steroid receptors by heating or dialysis was inhibited by molybdate, but that molybdate had no effect on the nuclear binding of previously activated steroid receptor complexes. They determined that the inhibition of receptor activation was concentration dependent and reversible when the molybdate was removed. They proposed three possible mechanisms through which molybdate may exert its effect: 1. it may inhibit phosphatase, 2. it may act directly with the receptor or, 3. it may affect the interaction between the receptor and a small molecular weight inhibitor.  Through further study of the small molecular weight inhibitor, the authors  proposed that molybdate directly interferes with the dissociation of this inhibitor from the receptor, thereby preventing its activation.  38  At the same time this work was being done, other studies concluded that inhibition of the estradiol receptor activation was not reversible when the molybdate was removed (Shyamala and Leonhard, 1980). Muller et al., (1982), set out to clarify this issue. They isolated estrogen receptors from the uteri of 8 calves, 3 rats and 1 mouse. Their results were similar to Noma et al., (1980). They found that the receptor activation was inhibited by the presence of molybdate and that it was completely reversible. They also concluded that molybdate had no effect if added after activation of the receptor had already occurred. They suggest that the mechanism of action is through nonspecific ionic interactions with the receptor or a cytosolic component required for activation. If this is the case and molybdate does affect the activation of the estrogen receptor, then it stands to reason that the increase of E that occurs during the approach to estrus 2  would not be seen and the subsequent increase in LH pulse frequency leading to the LH surge would be affected resulting in ovulation failure.  2.4 Hypotheses The current study was designed to determine if hay containing elevated concentrations of Mo could be safely fed to beef heifers without adversely affecting reproduction. Based on the literature review, the following is a summary of the expected results with the Null hypothesis being that there would be no significant differences between control heifers and heifers consuming elevated levels of Mo: 1. heifers being fed higher concentrations of Mo would reach puberty at a later age than control heifers; 2. heifers being fed higher concentrations of Mo would exhibit abnormal estrous cycles or become anestrus;  39  3. heifers being fed higher concentrations of Mo would have lower conception rates than control heifers, and 4. heifers being fed higher concentrations of Mo would have a longer interval from calving to the resumption of estrus post-partum and poor post-partum fertility.  Previous studies showing an effect of Mo on reproduction (O'Gorman et al., 1987; Phillippo et al., 1987; Fungwe et al., 1990) have utilized inorganic form of Mo to achieve the desired concentrations. This study is unique in that an organic form of Mo, in the hay from reclaimed mine sites, was used to incorporate the desired levels of Mo into the diets.  40  Chapter 3: GENERAL MATERIALS AND METHODS  3.1 Animal Management 3.1.1 General In October, 1995, sixty-five newly weaned Hereford and Hereford-cross heifer calves were delivered to the Teaching and Research Farm at the University of British Columbia (UBC) which is located at 49° 15' latitude and 123° 14' longitude and where the ambient temperature during the summer months ranges from 16 - 25 °C and from -2 - 9 °C during the winter months. The calves originated from a single farm near Merritt, BC. After their arrival, the heifers were housed in the Beef Barn in groups of 5 to 7 and were offered a good quality basal hay. They were initially observed for a short period of time and those with coughs, nasal discharge or a temperature greater than 40°C were treated with an antibiotic (single i.m. injection; 7.5 mg Micotil).  All heifers were  vaccinated to provide protection against the following: Infectious Bovine Rhinotracheitis (IBR), Parainfluenza Virus (PI3), and Bovine Respiratory Syncytial Virus (BRSV). Additionally, all heifers were vaccinated against Clostridial diseases, dewormed and ear-tagged to facilitate identification. Four weeks after the initial vaccination, all animals were given a BRSV booster shot and dehorned where necessary. Before the initiation of the trial, the heifers were moved to the Dairy Barn which is a closed freestall barn. The barn was modified to allow for 5 separate groups of animals. The heifers used during this study were handled and cared for according to the guidelines established by the Canadian Council on Animal Care (1993).  41  3.1.2 Dietary Treatments Two weeks after their arrival at UBC, the heifers were weighed.  Based on these  weights, they were assigned to one of five different treatment groups. The five heaviest were placed into a "spare" group since it is known the attainment of puberty is closely correlated to weight and it was desired that none of the heifers be cycling prior to the initiation of the dietary treatments. The remaining 60 heifers were randomly assigned to the five dietary treatment groups (12 heifers in each group) with equal weight distribution between the groups.  The heifers began their dietary treatments on December 18, 1995. The groups were as follows (all concentrations are for the total diet on a dry matter (DM) basis with supplemental Cu added as necessary): Treatment 1 (TR1):  1 mg/kg Mo, 16 mg/kg Cu (Control)  Treatment 2 (TR2):  6-8 mg/kg Mo, 16 mg/kg Cu  Treatment 3 (TR3):  6-8 mg/kg Mo, 6-8 mg/kg Cu  Treatment 4 (TR4):  30-40 mg/kg Mo, 60-80 mg/kg Cu  Treatment 5 (TR5):  50-67 mg/kg Mo, 115-134 mg/kg Cu  Desired Mo concentrations in the diet were achieved by feeding high Mo hay in combination with a necessary amount of basal hay containing normal concentrations of Mo and Cu. The treatment groups were designed to fully investigate the range of Mo concentrations found at the mine site. Treatments 2 and 3 were established in an attempt to duplicate the research done by Phillippo et al., (1987), and to determine if low Cu status was a pre-requisite for Mo to affect reproduction. The range of Mo shown for each treatment group arose from the necessity of feeding various blends of basal  42  and 1994, 1995, and 1996 harvests of the high Mo hay in order to achieve the desired levels of Mo in the diets. Copper supplementation, as copper sulfate, provided for a ratio of 2:1 Cu:Mo to prevent a secondary copper deficiency. The experimental rations were formulated using a computer program (Nutrition Module of Cowchips) developed by Alberta Agriculture Food and Rural Development based on NRC nutrient requirements for beef cattle (Alberta Agriculture, Food and Rural Development, 1993) (see appendix for an example of the ration formulation). The high Mo hay was obtained from Brenda mines (located approximately 18 km northwest of Peachland, BC) which was a Cu and Mo mine that closed in 1990. The area over which the mine tailings was spread was seeded to a grass-alfalfa mixture with the intention of preventing erosion and to provide grazing for cattle and wild ungulates. The basal hay used during the course of this experiment were also grass-alfalfa mixtures. The first batch of basal hay was obtained from the Rock Creek, BC area and subsequent batches were obtained from Merritt, BC. Analysis of the hay is summarized in Table I. All forage analysis was done by Northwest Laboratories, Lethbridge, AB. Mineral and vitamin supplements (not including Mo and Cu) were included in the diets to meet the requirements established by the National Research Council (1984) through the addition of a custom mineral mix in the ration (see Table II for the composition of the custom mineral mix). The actual rations as of February 15, 1996 (amount per head per day) were as follows:  TR1: Basal Hay - 5.5kg Barley - 0.4kg (as a carrier for the mineral supplements) Mineral Mix - 28g (incorporated into the barley to ensure consumption) C u S 0 - 0.2g (incorporated into the barley to ensure consumption) 4  43 TR2:  Basal Hay - 5.0kg Brenda '94 - 0.25kg Brenda '95 - 0.25kg Barley 0.4kg Mineral Mix - 28g C u S 0 - 0.2g 4  TR3: Basal Hay - 5.0kg Brenda '94 - 0.25kg Brenda '95 - 0.25kg Barley - 0.4kg Mineral Mix - 28g TR4: Basal Hay - 2.5kg Brenda '94 - 1.25kg Brenda '95 - 1.25kg Barley -1.4kg Mineral Mix - 30g C u S 0 -1.5g 4  TR5:  Brenda'95 - 4.5kg Barley - 2.0kg Mineral Mix - 38g C u S 0 - 2.75g 4  The heifers were first fed the custom mineral mix followed by any high Mo hay using individually locking head gates to insure individual consumption. They were fed twice a day at 08:00 and 15:00 h. They had access to water at all times.  Rations were  adjusted monthly and any time a new batch of hay was introduced. Adjustments were also made to meet requirements at three critical phases: growth, the last trimester of pregnancy and lactation.  44  Table I. Nutrient Content (dry matter basis) of the Basal Hays and High Mo hays (Brenda) fed to the heifers during the duration of the study (one composite sample for each). ELEMENT  BASAL '95  BASAL '96  BRENDA '94  BRENDA '95  BRENDA '96  A D F (%)  37.7  40.2  49.4  41.5  40.6  T D N (%)  60  57.6  50  56  57.3  DE (Mcal/kg)  2.6  2.53  2.2  2.5  2.52  P R O T (%)  12.5  12.6  10.4  11.7  11.2  C a (%)  0.8  1.17  0.96  1.19  0.88  P (%)  0.24  0.27  .0.1  0.15  0.17  K (%)  1.65  1.93  1.01  2.05  1.6  Mg (%)  0.22  0.21  0.13  0.12  0.13  Na (%)  0.08  0.03  0.03  0.02  0.05  Zn (mg/kg)  18  18  20  20  34  Mn (mg/kg)  31  25  57  54  65  Cu (mg/kg)  6  8  7  7  12  Mo (mg/kg)  1  1  89  90  74.5  S (%)  0.24  0.15  0.14  0.15  0.14  Table II. Composition of the UBC custom mineral mix.  NUTRIENT  AMOUNT  % Calcium  2  % Phosphorus  18  % Magnesium  5  % Potassium  0.3  % Sulfur  0.2  Iron (mg/kg)  3000  Zinc (mg/kg)  8000  Manganese (mg/kg)  6000  Copper (mg/kg)  0  Iodine (mg/kg)  200  Cobalt (mg/kg)  50  Selenium (mg/kg)  60  Vitamin A (lU/kg)  750000  Vitamin D (lU/kg)  120000  Vitamin E (lU/kg)  1500  % Salt  10  46  3.2 Heifer Weights The heifers were weighed weekly after the initiation of the treatments to ensure adequate growth.  At the start of the trial the average weight of the heifers was  approximately 230 kg. Approximately 3 weeks after the initiation of the treatment diets, heifers in TR5 began losing weight.  It was suspected that the relatively low energy  values and poor palatability of the Brenda '94 high Mo hay was responsible. The heifers were switched to only the Brenda '95 hay, although this did not completely correct the problem. It was only after the addition of rolled barley to make the diets isocaloric that they begin to gain weight again and caught up to the other groups. Starting at the time of breeding, the heifers were weighed every two weeks until later in their pregnancies when weighing was stopped. reinitiated.  After calving, bi-weekly weighing was  The weights were statistically analyzed at random times throughout the  study period and no statistical differences were observed between the treatment groups (Figure I).  CM  CO  TJ-  if)  cc cc cc cc  Ht  T -  cc  o  JO > O)  c  "E" ra > at  c  1 3 <A C  o u  (A Q. 3 O &D) i -  ^ fl> Di ^  fc&  O  c — o> re  E E E a>  0)  *-  Q. Q. 3  ro  ^  S2 |  ^51s o  •S E re O) O)  "5  S o >  re 3  E 3 O  c re o  5  3  o m if)  (6)() mB!3M  48  3.3 Ultrasonography Ultrasonography is a popular diagnostic and research tool in veterinary and animal science.  Its applications in the field of reproductive physiology include: monitoring  follicular and CL dynamics, pregnancy diagnosis, fetal  monitoring, fetal sex  determination, and monitoring abnormalities of the reproductive tract.  It is relatively  non-invasive, simple and effective, safe to both the subject and the operator, portable and  ultrarapid  since it  allows  for  immediate  interpretation  of  the  images.  Ultrasonographic examinations of the reproductive tracts were done on several occasions during the course of this study. All examinations were done using a real-time ultrasound scanner fitted with a 5MHz rectal probe. The examination itself was carried out as per the method of Taylor and Rajamahendran (1992). After the evacuation of the feces, the probe was placed intrarectally, and the reproductive tract was examined. The ovaries were scanned in several planes to identify all ovarian structures.  The  uterine horns and body were also scanned over their entire length and in several planes in order to examine the uterine contents and, if warranted, to identify the embryonic vesicles and the embryo proper. Desired images were frozen on the screen to allow measurements to be made.  3.3.1 Puberty and Estrous Cycles Rectal palpation began before the start of the experimental diets to ensure that there were no anatomical abnormalities of the reproductive tract in any of the heifers. Each month after the start of the diets, for two consecutive weeks, the heifers underwent ultrasound examinations of their reproductive tracts (except the second month due to serial blood sampling). During this examination, the number of class I (<5mm), class II  49  (5-1 Omm) and class III (>10mm) follicles was recorded as was the presence or absence of a CL. This continued until all heifers in each group were cycling.  Once the heifers were cycling normally, estrus was synchronized, and weekly ultrasound examinations were done to determine the diameter of the dominant follicle as well as to time the appearance of the resultant CL (to help determine cycle length) and determine its diameter.  3.3.2 Fertility, Post-partum and Superovulation After the estrous cycle investigation, the heifers were bred by Al. Twenty-eight days after breeding, ultrasonography was used to diagnose pregnancy. The presence of the embryo and/or embryonic vesicles was used as a positive indicator of pregnancy. The pregnancy diagnosis was confirmed by another ultrasound scan on d 35. The heifers were scanned monthly for the first 100 days of pregnancy to monitor the development of the embryos and to diagnose any cases of early embryonic mortality.  Beginning approximately 4 weeks after calving, weekly ultrasound examinations were initiated to monitor the appearance of the first CL which would indicate the animals return to cyclicity.  The number of days between calving and the first CL was  considered the length of the post-partum anestrus period.  After estrous cycles were reinitiated, the heifers were superovulated to determine their response to superovulation and whether the dietary treatments had any effect on the quality of the resulting embryo.  Ultrasonography was done after the superovulation  50  treatments, just prior to embryo retrieval to determine the number of CL on the ovaries. This was used as an indicator of superovulatory response.  3.4 Sampling Procedures 3.4.1 Blood Sampling 3.4.1.1 General During the study, blood samples were taken to analyze for P concentrations. Blood 4  samples were taken at the same time as each ultrasound scan. They were taken from a coccygeal blood vessel using 20 gauge, 1" Vacutainer needles. The samples were collected into 7ml heparinized Vacutainer tubes.  The base of the heifers' tail was  cleaned with paper towel then wiped with an alcohol swab before the sample was taken.  Blood samples were centrifuged at 3500 rpm for 10 min. The plasma was  separated into plastic vials, labeled with the animal ID # and sample date.  Plasma  samples were then frozen for future analysis.  3.4.1.2 Serial Blood Sampling Before the initiation of estrous cycles, serial blood samples were taken as per the method of Manikkam et al., (1995), to analyze for the prepubertal levels of gonadotropins (LH and FSH). Half the animals in each group were selected for sampling.  Briefly, the heifers were fitted with a jugular catheter by threading  approximately 30 cm of silastic tubing through a 13 gauge trocar into the jugular vein. The other end of the catheter was fitted with an 18 gauge needle to which a two-waystopcock was attached. It was through this stopcock that the blood sample was taken. The external length of the catheter (about 75cm) was lightly coiled and placed in a cloth pouch taped to the neck of the heifer. Sampling lasted for 6 h with a sample taken  51  every 20 min (18 samples per heifer). heparinized Vacutainer tubes.  The samples were collected into 7 ml  Between samples, the catheters were filled with a  saline+heparin (60 units heparin/ml) solution to prevent blood clots from forming in the tubing. Before each sample was taken, this solution was removed.  Blood samples  were centrifuged at 3500 rpm for 10 min. The plasma was pipetted into small plastic vials which were labeled with the heifer's ID # and the sample number. Samples were frozen for future analysis.  3.4.2 Liver Biopsies and Blood Samples for Mineral Analysis Blood and liver samples were collected three times during the study period in order to monitor the concentrations of Mo and Cu in the heifers. The first samples were taken just prior to the initiation of the dietary treatments to give a mean baseline value for each group. The second liver biopsy was 4 months after the start of the diets and the final sample was taken 15 months after the start of the diets. Liver biopsies were done by a veterinarian using a modification of the method of Smart and Northcote (1985). Briefly, the area surrounding the 11 intercostal space on the right side was shaved and th  a local anesthetic was applied 20 cm below the tip of the transverse process. A 1 cm stab incision was made through the thickness of the intercostal wall. A trocar and canula were introduced through the incision and advanced through the liver with a twisting motion. The liver samples were removed and transferred first to absorbent material (gauze pads), then to a pre-weighed test-tube. The liver samples were frozen for future analysis.  At the time of the biopsy, a blood sample was taken to monitor plasma concentrations of Mo and Cu. Blood samples were taken from a coccegeal vessel and collected into  52  7ml heparinized Vacutainer tubes. Samples were centrifuged at 3500 rpm for 10 min. The plasma was decanted and frozen for future analysis.  3.5 Laboratory Analysis 3.5.1 Hormonal Analysis of Plasma 3.5.1.1 Progesterone Progesterone concentrations in plasma were detected by a no-extraction, solid-phase 125  l radioimmunoassay (Coat-A-Count Kit, Diagnostic Products Corp. Los Angeles, CA)  as previously verified in our laboratory by Taylor and Rajamahendran (1992).  This  procedure employs antiserum highly specific for P and was designed for the direct, 4  quantitative measurement of P in serum or plasma. Plasma or reference standard 4  (100u,l) was added to antibody coated tubes. Standards ranged from 0 - 4 0 ng/ml P . 4  Buffered  125  l-labeled P (1.0ml) was added to all tubes which were incubated at room 4  temperature for 3 h. Tubes were then decanted to remove all excess  125  l-labeled P . 4  Tubes were counted for 1 min in a gamma counter (Packard Auto-Gamma 500, Packard Instruments, Donner's Grove, IL) which was located in the Department of Biology at the University of British Columbia.  Intra- and interassay coefficients of  variation were 6.2% and 9.8%, respectively. The detection limit of the assay was 0.05 ng/ml P . 4  3.5.1.2 Gonadotropins Plasma samples from the serial blood sampling were sent to the laboratory of Dr. N. Rawlings at the Western College of Veterinary Medicine at The University of Saskatchewan for determination of LH and FSH concentrations.  LH concentrations  were determined using a double-antibody radioactive iodine ( l) radioimmunoassay 125  53  (Sanford, 1987). Briefly, samples of standards and serum (200p.l) were incubated at 4°C with l-labelled LH and anti-LH serum. After 5 days, anti-rabbit 6-globulin serum 125  was added to separate bound from free hormone. The supernatant was discarded. Radioactivity was measured in a gamma counter which was programmed for conversion to LH concentration. Intra- and interassay coefficients of variation were 8% and 15%, respectively. The sensitivity of the assay was 0.1 ng/ml. FSH concentrations were measured in 200u.l plasma samples in a similar double-antibody technique (Rawlings et al., 1984). The first anti-body was raised in rabbits against the p-subunit of bovine FSH and used at a dilution of 1:100,000.  Highly purified bovine FSH was  enzymatically radioiodinated using a commercial preparation of lactoperoxidase and glucose oxidase bound to microspheres (Enzymobeads, Biorad, Richmond, CA) and standard concentrations were prepared. rabbit 8-globulins.  The second antibody was raised against  Intra- and interassay coefficients of variation were 5% and 11%,  respectively. The detection limit was 0.2ng/ml.  3.5.2 Mineral Analysis Liver and plasma samples for mineral analysis were taken to the Department of Animal and Poultry Science at the University of Saskatchewan for mineral analysis. Before analysis, liver samples were freeze-dried in a vacuum desiccator (Labonco Freeze Dry System, Kansas City, Ml) for 24h. The dried samples were digested using a rapid nitric-perchloric acid digestion method for multi-element tissue analysis as per Zasoski and Burau (1977). The liver samples (approximately 0.5mg) were transferred to Taylor tubes (graduated 25mm x 200mm) and 3.5 ml concentrated nitric acid was added. To this, 1.5 ml perchloric acid was added. This mixture was left standing for 6 h, after  54  which the tubes were placed in a block heater with two timers to allow for the setting of different temperatures. Timer #1 was set for 2.5 h at 80 - 90 °C and timer #2 was set for 4 h at 180 °C. Afterwards, deionized water was added to the samples up to 50 ml, and the tubes were mixed well.  Plasma samples (1ml) were first digested in 1 ml 1.4N HCI, then mixed well and left to stand for 30 min. This was followed by the addition of 1 ml 20% TCA solution; the tubes were then vortexed and centrifuged at 1000 rpm for 10 - 1 5 min as per the Methodology of Analytical Toxicology (1975).  The supernatant was removed and kept for  determination of mineral concentrations.  For both liver and plasma samples, Cu concentrations were determined with the use of an atomic absorption spectrophotometer (Model 4000, Perkin-Elmer, Norwalk, CT). Mo concentrations were analyzed by inductively coupled plasma atomic emission spectroscopy at the Saskatchewan Research Council.  3.6 Statistical Analysis The study was laid out as a completely randomized design. All data analysis was done using Microsoft EXCEL (Microsoft Excel for Windows 95, Version 7.0a, 1996). Mean comparisons were done through the use of a one-way analysis of variance (ANOVA). Whenever a significant difference between the means was indicated, Tukey's studentized range test was done (Zar, 1984). A 5% level of significance was accepted throughout.  Any other statistics performed or modifications to the study design are  noted in the relevant sections.  55  Chapter 4: THE EFFECT OF FEEDING HAY CONTAINING HIGH LEVELS OF MOLYBDENUM ON PUBERTY AND ESTROUS CYCLE DYNAMICS 4.1 Abstract The effect of feeding hay containing high levels of molybdenum on puberty and estrous cycle dynamics in beef heifers was investigated. Sixty Hereford and Hereford-X beef heifers were randomly assigned to 5 dietary treatment (TR) groups: TR1 - 1 mg/kg Mo, 16 mg/kg Cu; TR2 - 6-8 mg/kg Mo, 16 mg/kg Cu; TR3 - 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4 - 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5 - 50-67 mg/kg Mo, 115-134 mg/kg Cu. Puberty attainment was assessed through twice monthly ultrasound examinations and blood sampling. The number of class I, II and III follicles as well as the presence or absence of a CL on the ovaries was recorded. Puberty attainment was considered to be the time of the first CL and a corresponding plasma P level of >1 ng/ml. There were 4  significant differences between the groups in the number class I follicles 4 months after the start of the treatment diets, but this could be attributed to an increase in the number of cycling animals at different stages of the cycle rather than the dietary treatments. There were no significant differences in age or weight of the heifers at the time of puberty attainment. Once all the heifers were cycling, Kamar heat detectors were used to determine the length of the estrous cycle. Ultrasound examinations determined the total number of follicles >5mm and the diameters of the dominant follicle and CL. Blood samples were taken to analyze for P levels. There were no significant differences in 4  any parameter except cycle length. The mean cycle length for heifers in TR4 was significantly shorter than in TR1 (18.3±1.3 Vs. 20.8+0.43, respectively) (p<0.05). However, it was still within normal limits and did not appear to have been affected by Mo.  Liver biopsies and blood samples taken to monitor the levels of copper and  molybdenum within the heifers showed significant differences (p<0.05) in mineral  56  concentrations reflecting the dietary intake levels. These results indicated that feeding hay containing high concentrations of molybdenum did not affect the onset of puberty or estrous cycle dynamics in beef heifers.  4.2 Introduction Elevated levels of dietary Mo have been shown to have detrimental effects on puberty and normal estrous cycles (Phillippo et al., 1987; Fungwe et al., 1990; Igarza et al., 1996).  A comprehensive study investigating the effects of inorganic Mo on  reproduction in cattle was done by Phillippo et al., (1987), in which a diet supplemented with 5mg Mo/kg DM delayed the onset of puberty for up to 12 weeks as compared to control animals.  Also during this study, it was found that significantly more Mo  supplemented animals failed to ovulate following prostaglandin induced estrus synchronization as compared to the control animals (7% vs. 67%). They hypothesized that this delay was caused by a decrease in the release of LH from the pituitary due to an alteration in ovarian steroid secretion. They also suggested that these effects of Mo on reproduction were due to the presence of elevated levels of Mo rather than a secondary Cu deficiency (Phillippo et al., 1987).  The objective of this study was to investigate the effects of feeding hay containing elevated levels of Mo on attainment of puberty and estrous cycle dynamics in beef heifers and to determine if these effects were independent of the Cu status of the animal.  57  4.3 Materials and Methods 4.3.1 Puberty Assessment: At the initiation of the puberty assessment, the heifers were approximately 7 to 7.5 months of age. Twice a month (for 4 months) after the initiation of the trial, for two consecutive weeks, the heifers underwent transrectal ultrasound examination of their reproductive tracts.  There were no ultrasound examinations two months after the  initiation of the diets because of the serial blood sampling. During each examination, the number of class I (<5 mm), class II (5-10 mm) and class III (>10 mm) follicles were recorded as well as the presence or absence of a CL. At the same time as the ultrasound scan, a blood sample was taken from each heifer for determination of P  4  levels. Attainment of puberty was considered to be the time at which the presence of the first CL was observed along with a corresponding P level > 1 ng/ml. In the case of 4  a discrepancy between the ultrasound data and the P levels, the presence of a CL on 4  ultrasound was the deciding factor. The ultrasound examinations and blood sampling continued until all the heifers in each group had reached puberty and were cycling. Approximate age at onset of puberty was determined by assuming the heifers were all approximately 6 months of age on arrival at the University of British Columbia. The mean weight at puberty of all the heifers in each treatment group was also calculated.  4.3.2 Serial Blood Sampling In addition to the ultrasound examinations and the blood sampling for P levels, serial 4  blood samples were taken with the main objective of determining the mean levels of LH and FSH during the prepubertal period. These samples were taken 2 months after the initiation of the treatments when the heifers were approximately 9-10 months of age. Sampling occurred over a one week period. One treatment group was sampled each  58  day and 6 heifers from that group were randomly selected. Also, the mean LH pulse frequency and amplitude within the 6 h sampling period was calculated to determine if comparisons could be made between the treatment groups.  The mean LH pulse  frequency and amplitude were calculated using criteria defined by Cook et al., (1991). An LH pulse peak was one having an increase equal to or greater than one standard deviation from the mean or previous reading, a peak reading one standard deviation greater than the mean or subsequent reading, or two consecutive decreasing readings after the peak. The frequency of LH pulses was calculated as the mean number of pulses in each group occurring during the 6 h sampling period. The amplitude of the LH pulse was defined as the average height above basal levels.  4.3.3 Estrous Cycle Dynamics Once estrous cycles had been established, estrus was synchronized to allow for comparisons between the treatments in terms of estrous cycle dynamics. Estrus was synchronized with an injection of 125 ng GnRH i.m. (Factrel; Ayerst Laboratories, Montreal, QC) followed by 25 mg P G F after 7 days. At the time of the P G F  2a  2a  i.m. (Lutalyse; The Upjohn Co. Kalamazoo, Ml) injection, a Kamar heatmount detector (Kamar,  INC, Steamboat Springs, CO) was placed on the tailhead of the heifers to aid in heat detection. Heat detection was done from this day on by observing the heifers for 1h at both 08:00 and 20:00 h. On the day of the P G F  2a  injection and for the following four  days, a blood sample was taken to analyze for plasma P concentrations. Every 4 days 4  after this, another blood sample was taken to monitor P levels until the start of the next 4  cycle. Following the P G F  2a  injection, weekly ultrasound examinations were performed  to determine the diameter of the dominant follicle and the resultant CL. Approximately  59  16 days after heat had been detected, Kamar heat detectors were again placed on the tailhead of the animals and heat detection was done as before.  Cycle length was  determined by the number of days elapsing between the two red Kamar heat detectors and was corroborated by the CL data from the ultrasound examinations.  4.3.4 Samples for Mineral Analysis Liver samples were taken before and 4 months after the start of the treatments. Blood samples were taken at the same time. Liver and plasma samples were frozen for future analysis to determine Mo and Cu concentrations.  4.3.5 Statistical Analysis: This experiment was analyzed as a completely randomized design. Mean comparisons were done through the use of a one-way analysis of variance (ANOVA). A 5% level of significance was accepted throughout.  In case of a significant difference, Tukey's  studentized range test was used to compare the means (Zar, 1984).  4.4 Results 4.4.1 Puberty Assessment: The number of class I, class II, class III follicles and CL were compared at 1, 3 and 4 months after the start of the dietary treatments. There were no significant differences between the treatment groups 1 and 3 months after the initiation of the treatment diets. At 4 months, however, there was a difference in the number of class I follicles with TR4 having significantly more than TR1 and TR 5 (p<0.05) (Figure II).  There was no  significant difference in the percentage of heifers with a CL at any time during the puberty assessment. There was also no significant difference in the concentrations of  60  P between the treatment groups at 1, 3 or 4 months after the start of the experimental 4  diets or in the maximum P levels corresponding to the appearance of the first CL 4  between the dietary treatment groups (Figure III and Table III).  Age at puberty was  determined by calculating the age of the heifers (in days) at the time of the appearance of the first CL, assuming the heifers were all approximately 6 months (180 d) of age on arrival at UBC (Table III). There were no significant differences between the treatments. There was also no significant difference in the mean weight at the time of puberty attainment between the treatment groups. Any missing values are due to difficulties performing an ultrasound scan or collecting blood samples from an individual heifer.  61  0TR1  I  • TR2  5  • TR3  •5 4  1-  1 §  E1TR4  3  E3TR5  2  Class I  Class II  Class III  Class II  Class III  B  0)  6  •5 4 13  „  •9  3  Class I  Class III  Figure II. The mean number of class I, II and III follicles 1 (A), 3 (B) and 4(C) months after the start of the dietary treatments (TR) in heifers fed varying levels of high molybdenum hay and supplemental copper (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  62  HTR1 • TR2 • TR3 • TR4 HTR5  1 month  3 months  4 months  B  •  TR1  •  TR2  • TR3 0TR4 FJTR5  One month  three months  Four months  Figure ill. Percentage of heifers with a corpus luteum (A) and mean progesterone concentrations (B) 1,3 and 4 months after the start of the dietary treatments (TR) in heifers fed varying levels of high molybdenum hay and supplemental copper (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  63  Table III. Mean (±SD) age and mean (±SD) weight at first observed corpus luteum (CL) and corresponding mean (±SD) plasma progesterone (P ) concentrations of the first CL in heifers fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5*). 4  TREATMENT GROUPS  MEAN AGE (d) AT FIRST CL  MEAN WEIGHT (kg) AT FIRST CL  MEAN P (ng/ml) OF FIRST CL  TR1  349.5±31.46 (12)  306.4±28.06 (11)  6.8+3.82 (10)  TR2  356.7±26.71 (12)  304.6±28.36 (11)  6.2±1.92 (11)  TR3  348.0±44.08 (12)  301.9±27.08 (12)  4.9±2.77 (11)  TR4  328.7+42.40 (11)  297.1+37.67 (10)  5.4+2.37 (8)  TR5  363.7+29.62 (12)  308.7±32.78(12)  6.1±3.44 (7)  4  number in brackets represents the number of observations used in the calculation * TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115134 mg/kg Cu  64  4.4.2 Gonadotropin Levels Mean prepubertal concentrations of LH and FSH were compared 2 months after the start of the dietary treatments. There were no significant differences between any of the groups (Table IV).  The pattern of prepubertal LH and FSH release in a  representative heifer from each of the dietary treatment groups is shown in Figures IV and V. The mean LH pulse frequency and amplitude were not significantly different between the TR groups (Table V).  65  Table IV. Mean (±SD) prepubertal plasma luteinizing hormone (LH) and follicle stimulating hormone (FSH) concentrations in 6 heifers from each group fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5*). TR GROUPS  LH (ng/ml)  FSH (ng/ml)  TR1  0.24 + 0.16  0.43 ±0.15  TR2  0.14 ±0.04  0.36 ±0.17  TR3  0.13 ±0.07  0.45 ±0.15  TR4  0.23 ±0.14  0.32 ±0.14  TR5  0.22 ±0.10  0.46 ±0.13  * T R 1 : 1 mg/kg Mo, 16 mg/kg C u (Control); T R 2 : 6-8 mg/kg Mo, 16 mg/kg C u ; T R 3 : 6-8 mg/kg Mo, 6-8 mg/kg C u ; T R 4 : 30-40 mg/kg Mo, 60-80 mg/kg C u ; T R 5 : 50-67 mg/kg Mo, 115134 mg/kg C u  66  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  Sample number Figure IV. Characteristic pattern of prepubertal luteinizing hormone (LH) release in plasma of a representative heifer from each treatment (TR) group (TRs 1 - 5 fed varying levels of high molybdenum hay and supplemental copper) after serial blood sampling for 6 h (one sample every 20 min).  0.7  o - l — , — , — , — , — , — , — , — S — , — , — , — , — , — r — , — , — , 1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  Sample Number Figure V. Characteristic pattern of prepubertal follicle stimulating hormone (FSH) release in plasma of a representative heifer from each treatment (TR) group (TRs 1 - 5 fed varying levels of high molybdenum hay and supplemental copper) after serial blood sampling for 6 h (one sample every 20 min).  67  Table V. Mean (iSD) luteinizing hormone (LH) pulse frequency and pulse amplitude in serial plasma samples (one sample every 20 min for 6h) from 6 heifers in each group fed varying levels of hay containing high levels of molybdenum and supplemental copper (treatment (TR) groups* 1-5).  TR GROUPS  LH PULSE FREQUENCY (6 h")  LH PULSE AMPLITUDE (ng/ml)  TR1 TR2 TR3 TR4 TR5  1.0±0.89 0.510.84 0.810.41 1.511.05 1.010.63  1.110.55 0.710.41 0.610.36 0.710.48 1.010.57  1  *TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu  68  4.4.3 Estrous Cycle Dynamics: The following parameters were compared between the treatment groups on the various sampling days (i.e. days 0, 6, 14 and 21 of the cycle): the total number of follicles <5mm, the diameter of the dominant follicle and the diameter of the CL (Figure VI). Mean progesterone concentration on day 13 of the cycle (i.e. at the time the CL reaches its maximum diameter) and the duration of the cycle were also compared (Table VI). There were no significant differences between the treatments in any of these parameters except the length of the cycle. The mean length of the cycle for the heifers in TR4 was significant shorter (p<0.05) than in TR1 (18.8+1.3 Vs. 20.8±0.4, respectively) (Table Vll). The mean concentrations of P on the various sampling days 4  during the estrous cycle for each treatment group are shown in Figure Vll. Any missing values are due to difficulties performing and ultrasound scan or collecting blood samples from an individual heifer.  69  A  B  C  Day 6  Day 14  Figure VI. Mean number of follicles > 5mm (A), diameter of the dominant follicle (B) and diameter of the corpus luteum (C) during the estrous cycle in 6 heifers from each group fed varying amounts of hay containing high levels of molybdenum and supplemental copper (Treatment 1(TR1): 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 68 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  70  Table VI. Mean (±SD) cycle length, and plasma progesterone (P ) levels on day 14 of the cycle in heifers fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5*). 4  TR GROUPS TR1  CYCLE LENGTH (d) 20.8 ± 0.43 (4)  P LEVELS (ng/ml) 7.8 ± 1.04 (6)  TR2  20.2 ± 0.75  AB  (5)  4.3 ±2.05 (6)  TR3  19.7±0.75  AB  (6)  4.7 ± 2.05 (6)  TR4  18.3±1.30 (4)  5.1 ±2.77 (6)  TR5  A  B  20.5 ± 1.12  AB  (4)  4  7.5 ± 6.52 (6)  number in brackets represents the number of observations used in the calculation -> values with different superscripts are significant different (p<0.05) * TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115134 mg/kg Cu  71  Day-2  Day-1  DayO  Day 1  Day 6  Day 10  Day 14  Day 19  Day 21  Day of the Cycle Figure Vll. Mean progesterone concentration during a cycle after synchronized estrus in six heifers from each treatment (TR) group (TRs 1 -5 fed varying levels of high molybdenum hay and supplemental copper) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  72  4.4.4 Mineral Concentrations. There were significant differences between the levels of liver Mo and Cu between the treatments as well as in the levels of plasma Mo and Cu which reflected the dietary intake levels. There were small but significant differences in the basal levels of liver Mo and plasma Cu in the samples taken before the heifers began their treatment diets. For the liver Mo, the mean concentration was higher in TR5 than in TRs 1, 2, and 3. The mean concentration of Cu in plasma was significantly higher in TR1 than in TRs 3, 4, and 5 and the mean concentrations was higher in TR2 than TR4. Four months after the start of the experimental diets, there were a significant differences in both the liver and plasma Mo concentrations as well as the liver and plasma Cu concentrations. For the liver Cu levels, TRs 2 and 3 had significantly lower levels than TR 1, 4 and 5, and TR5 was significantly higher than TR1 and TR4. For the liver Mo concentrations, TRs 4 and 5 were significantly higher than TRs 1, 2 and 3. As well, TR4 was significantly lower than TR5. These trends were repeated in the results for the plasma Cu and Mo. For the plasma Cu, TR3 was significantly lower than all others, and TRs 4 and 5 were significantly higher than TR2. The plasma Mo results show that levels in TR5 were significantly higher than TRs 1, 2, 3 and 4. See Figures VIII, IX, X, and XI for graphical representation of these results. All values for liver mineral concentrations are given on a dry matter basis. Any missing values are due to difficulties obtaining a liver or blood sample from an individual heifer.  73  18 16  TR1  TR2  TR3  TR4  TR5  Figure VIII. Mean concentration of molybdenum (Mo) in liver samples taken from heifers fed varying levels of high Mo hay prior to and 4 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  • Before T R s • A t 4 months  TR1  TR2  TR3  TR4  TR5  Figure IX. Mean concentration of molybdenum (Mo) in plasma samples taken from heifers fed varying levels of high Mo hay prior to and 4 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  74  DM  300  250  O)  "5> 200 E c o  (0  l_ "E a> u c o u3  O  • Before TRs 150  a At 4 months  100  50  TR1  TR2  TR3  TR4  Lit TR5  Figure X. Mean concentration of copper (Cu) in liver samples taken from heifers fed varying levels of high molybdenum (Mo) hay prior to and 4 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  TR1  TR2  TR3  TR4  TR5  Figure XI. Mean concentration of copper (Cu) in plasma samples taken from heifers fed varying levels of high molybdenum (Mo) hay prior to and 4 months after the start of the treatment (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  75  4.5 Discussion The results of this experiment show that feeding hay containing high levels of molybdenum had no effect on the attainment of puberty in beef heifers. The normal age for puberty attainment is approximately 9 - 1 0 months of age for heifers (Moran et al., 1989), although it can be as early as 6 months and as late as 24 months (Robinson, 1977; Glencross, 1984). Hereford heifers, specifically, reach puberty at approximately 375 d. The mean age at puberty for heifers in the different treatment groups of the current study ranged from approximately 328 - 364 d.  There was no significant  difference between any of the treatment groups in age at puberty based on the presence of the first CL or P levels >1 ng/ml. This is contrary to results of Phillippo et 4  al., (1987), where up to a 12 week delay was seen in animals supplemented with 5mg/kg inorganic Mo. The was also no significant difference between the treatment groups in the weight of the heifers at puberty attainment. The mean weights ranged from 297-309 kg which is slightly higher than the average weight at puberty for a Hereford heifer which is 272 kg. This did not appear to be due to the dietary treatments since the trend was evident within all the TR groups including the control. This also is contrary to the results of Phillippo et al., (1987), in which heifers receiving supplemental Mo tended to be lighter at puberty than the controls. The results of the current study also indicated that the copper status of the heifers did not affect the attainment of puberty. There was no difference in age at first CL in heifers receiving supplemental Cu and those which were not. This agrees with Phillippo et al., (1987), in which low copper status did not affect onset of puberty, although they argued that a low Cu status might be a pre-requisite for Mo to act. The liver Cu levels for heifers in TR3, which did not receive any Cu supplementation, were significantly lower than those in the other TR groups, although there was no significant difference in age at first CL. The function of  76  the first CL was also investigated (i.e. P levels) and again there was no significant 4  difference between the TR groups. The P levels corresponding to the first CL ranged 4  from 4.9±2.77 to 6.8±3.82 ng/ml.  This is in accordance with levels seen in the  literature. Dodson et al., (1988), reported that the maximum P concentration from the 4  CL of the first cycle in crossbred beef heifers was approximately 5.2±1.6 ng/ml.  During the prepubertal period, the number of class I, II and III follicles were compared between the TR groups.  This was to determine whether follicular dynamics was  occurring is a wave-like pattern which is known to occur during the prepubertal period (Adams et al., 1994). Waves of follicular growth in the prepubertal period occur as during the post-pubertal period (i.e recruitment of a cohort of follicles followed by selection and dominance of one follicle), the only differences being that all waves are anovulatory in prepubertal animals, the duration of the waves is shorter and the dominant follicle does not reach the same diameter. For follicles to progress from class I to class II and class III, there must be specific gonadotropin support available at the correct time. Any impairment in the synthesis and/or secretion of LH and FSH would result in a lack of any follicles passing the class I stage of development. Therefore, by monitoring the number of follicles of different classes, any problems with gonadotropin production can be identified and in the current study, any effects due to the dietary treatments can also be identified. There was a difference in the number of class I follicles 4 months after the start of the dietary treatments (TR 4 had significantly more than TRs 1 and 5), however, it is not likely that these differences were due to the elevated levels of Mo being consumed. It is more likely that as more animals began cycling within a group then there was more variation between the groups because not all animals were at the same stage of the cycle at the same time.  77  In prepubertal heifers,  LH secretion is inhibited by estrogen secretion (Foster and  Ryan, 1981). Attainment of puberty involves a reduction of this inhibition resulting in the release of pulses of LH which eventually leads to normal estrous cycles. Phillippo et al., (1987), found that elevated levels of dietary Mo altered the secretion of LH (lower basal secretion of LH compared to controls). In the current study, serial blood samples were taken and analyzed for the mean concentrations of LH and FSH. There were no significant differences between the TR groups in the mean levels of LH and FSH. Normal LH concentrations can rise as high as 1.2 ng/ml in prepubertal heifers and FSH concentrations can rise as high as 45 ng/ml (Dodson et al., 1988). Mean gonadotropin concentrations in the current study were well within these ranges. In order to derive further information regarding LH and FSH, the release patterns of these hormones was determined (i.e. pulse frequency and amplitude). Dodson et al., (1988), reported that the frequency of LH pulses in heifers at 35 weeks of age was approximately 5/24h which agrees with the current findings where the mean pulse frequency ranged from 0.5 to 1.5 pulses during the 6 h sampling period. Dodson et al., (1988), also state that the levels of FSH fluctuate rapidly during a 24h period in contrast to the clearly episodic release of LH which was also seen in the current study. There were no measured differences between the treatment groups in the LH pulse frequencies or amplitude or in the release of FSH. A limitation to this comparison was the length of the sampling period. Although the results compared favourably to previously published observations, a longer sampling period would have allowed more conclusive results.  Once all the heifers were cycling, estrus was synchronized using GnRH followed by PGF  2a  to allow for comparisons between different characteristics of the estrous cycle,  78  namely, the mean number of follicles >5mm, the length of the cycle, the diameter of the dominant follicle, the diameter of the CL and the P levels corresponding to the Cl of 4  maximum diameter (day 14 of the cycle). These comparisons were made on the various sampling days. No significant difference was found in the mean number of follicles >5mm, the diameter of the dominant follicle, the diameter of the CL or the P  4  levels on day 14 of the cycle. The values were all comparable to previously published values. One study by Taylor and Rajamahendran (1991) examined these same criteria in non-pregnant cows and found that the mean diameter of the dominant follicle was 16.6±0.6mm, the diameter of the CL was 29.2±1.0mm and the P levels peaked at 4  5.9±0.9 ng/ml. In the current study, there was a significant difference in cycle length between the TR groups (p<0.05). The mean length of the cycle for heifers in TR4 was significantly shorter than for those in TR1 although the length of the cycle for TR4 heifers was still within the normal range. The difference is more likely due to individual variation rather than an effect of Mo since the same effect was not seen in TR5 which was receiving the highest concentration of Mo. This is in contrast to the results of Phillippo et al., (1987), in which supplemental Mo actually led to some of the study animals becoming anestrus. They believe that this was because Mo altered estrogen secretion which altered the release of LH, ultimately leading to ovulation failure.  The levels of Mo and Cu in both the liver and plasma samples before the start of the experimental diets represented the basal levels of these minerals. There were small but significant differences (p<0.05) in liver Mo concentrations and plasma Cu concentrations between the TR groups at that time, but the reasons for this are unknown. They were most likely due to individual variation as the heifers had not begun their experimental diets at that time.  Four months after the start of the  79  treatments, there were also significant differences between the TR groups (p<0.05) which reflected the dietary intake levels of these minerals by the heifers. These results demonstrate that the Mo was being absorbed and stored in the livers of the heifers.  80  Chapter 5: THE EFFECT OF FEEDING HAY CONTAINING HIGH LEVELS OF MOLYBDENUM ON FERTILITY AND POST-PARTUM REPRODUCTION 5.1 Abstract: The effects of feeding hay containing high levels of molybdenum on fertility, pregnancy and post-partum reproduction were investigated. Sixty Hereford and Hereford-X beef heifers were randomly assigned to 5 dietary treatment groups: TR1 - 1 mg/kg Mo, 16 mg/kg Cu; TR2 - 6-8 mg/kg Mo, 16 mg/kg Cu; TR3 - 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4 - 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5 - 50-67 mg/kg Mo, 115-134 mg/kg Cu. Estrus was synchronized in the heifers using either GnRH followed by P G F double P G F  2a  2a  after 7 days or  11 days apart. Artificial insemination was performed 72 and 96 h after  the second injection regardless of synchronization method used. Any heifers returning to heat were inseminated at that time to a maximum of 3 breedings. Pregnancy was diagnosed at 28 days using ultrasonography and was confirmed at 35 days. There was no significant difference due to dietary treatment in the number of heifers pregnant after 1, 2 or 3 inseminations. There was also no significant difference in the number of inseminations required for pregnancy to occur. There was no significant difference in the length of the gestation period between the treatment groups. The calves were weighed within 24h of birth and there were no significant differences in weight between the groups. Starting 4 weeks after calving, weekly ultrasound examinations were done to determine the appearance of the first CL which indicated the resumption of estrous cycles. At the same time blood samples were taken and analyzed for P . There were 4  no significant differences in the time between calving and the appearance of the first CL or in the P levels corresponding to the first CL. 4  81  The effects of elevated levels of dietary molybdenum on superovulatory response, ova recovery and embryo quality were also investigated.  These criteria were used as  indicators of post-partum fertility. The heifers were separated into either control, low Mo (10 mg/kg Mo) or high Mo (40-80 mg/kg Mo) groups. Superovulation was to be done using a single injection of FSH (400mg) (preliminary testing on 2 heifers). Unfortunately, there was no superovulatory response in most cows. Since dissolving FSH in PVP prior to injection was also not successful, it was decided to superovulate the 2nd group of cows with 8 decreasing doses of FSH (total 400mg) over 4 days. Molybdenum had no significant effects on superovulatory response or embryo recovery. However the presence of a suckling calf did have a significant effect (p<0.05). Cows with suckling calves had significantly fewer embryos recovered (3.0±2.8 vs. 10.8±3.2) and the quality of these embryos was also lower. These results indicated that feeding hay containing high concentrations of molybdenum did not have an effect on fertility and post-partum reproduction.  5.2 Introduction: Elevated levels of dietary Mo have been shown to have detrimental effects on fertility and the early development of the embryo in ruminants (Phillippo et al., 1987; O'Gorman et al., 1987). A study by Phillippo et al., (1987), linked the consumption of elevated levels of inorganic Mo (5 mg/kg) with a decrease in fertility of beef cattle. Conception rates decreased from 57 - 80% in the control animals to 12 - 33% in the Mo-treated animals. It was postulated that this effect was due to Mo decreasing the release of LH from the pituitary gland through an alteration in ovarian steroid secretion.  82  In a study looking at the effects of Mo on early embryo development, embryos from cows receiving supplemental Mo (15-20 mg/kg) were collected in order to compare with embryos from control animals (O'Gorman et al., 1987). It was found that the number of embryos recovered and fertilization rates were not different between the treatment groups. However, there was a significant difference in the quality of the early embryos. Unfortunately, this was only a preliminary study and no further work in this area has been done.  The first objective of the current study was to investigate the effects of elevated levels of dietary Mo on conception rates, pregnancy, calving, gestation length, calf birth weights, length of the post-partum period and P levels from the first CL post-partum 4  and to determine whether any observed effects were independent of the copper status of the heifers. The second objective of this study was to look at the effect of elevated levels of dietary Mo on superovulatory response, ova recovery and embryo quality in post-partum first-calf beef heifers.  5.3 Materials and Methods 5.3.1 Fertility Assessment: The heifers used in the following experiments were previously part of the investigations into puberty attainment and estrous cycle dynamics. Estrus was synchronized in all heifers by one of two methods: either injection of 125 u.g GnRH i.m. (Factrel; Ayerst Laboratories, Montreal, QC) followed by 25mg P G F  2a  i.m. (Lutalyse; The Upjohn Co.,  Kalamazoo, Ml) after 7 days or two injections of P G F  2a  (25mg i.m.) 11 days apart.  These two synchronization methods were equally represented in the treatment groups. Regardless of method used, the heifers were monitored for signs of estrus at 08:00 and  83  20:00 h daily following the second injection. Artificial insemination (Al) was performed by a trained technician using frozen-thawed semen from two Hereford bulls at 72 and 96 h following the second injection. Sires were equally represented in each treatment to remove error due to bull differences. If a heifer showed standing estrus before 48 h, Al was performed 12 h after it was observed. Any heifer returning to heat during the next cycle was inseminated at that time. Pregnancy was diagnosed 28 d after Al by rectal palpation and ultrasound examination of the uterus and was confirmed at 35 d. For heifers that were not pregnant at that time, estrus was re-synchronized using 2 injections of P G F  2a  (25 mg i.m.) 11 days apart and Al was again performed by a trained  technician using frozen thawed semen at 72 and 96 h after the second injection. Each heifer was allowed a maximum of three inseminations to become pregnant.  The  conception rates for each group after 1, 2, and 3 inseminations and the number of inseminations required for pregnancy were determined.  5.3.2 Gestation Period and Calving The heifers were monitored for the duration of their pregnancy. Monthly ultrasound examinations occurred during the first 100 d of pregnancy to observe the growth and development of the embryo. At the time of calving, the heifers were observed and assistance was given when necessary.  Calves were delivered onto straw and  colostrum was given within 24 h if the calf did not initiate suckling on its own. The calves were weighed within 24 h of birth and the calving rate and gestation lengths were calculated. Calculation of the gestation lengths included all calves that were born both alive and dead.  84  5.3.3 Post-partum Assessment Beginning approximately 4 weeks after calving, weekly ultrasound examinations were performed to determine the number of class I (<5mm), class II (5-1 Omm) and class III (>10mm) follicles and the presence of the first corpus luteum (CL). At the same time as the ultrasound examination, a blood sample was taken from a coccygeal vessel of each heifer for P determination. The time elapsing between calving and the presence of the 4  first CL based both on ultrasonography and a P level >1 ng/ml was considered the 4  duration of the post-partum anestrus period. Once cyclicity was established, the heifer was no longer included in the weekly examinations.  5.3.4 Superovulation 5.3.4.1 Trial la Superovulation was to be achieved using a single injection of FSH (400 mg Folltropin-V, Vetrepharm Inc., London, ON). Prior to using this method on the first experimental group, this method was tested on two heifers that did not conceive and were therefore taken out of the Mo study. The single injection of FSH (400 mg) was given on d 9 of the estrous cycle after estrus had been synchronized by double P G F after the second P G F treated with P G F  2a  2a  2a  injections (i.e. 12 d  injection). Three days after the FSH injection, the heifers were  (25mg). Artificial insemination was carried out using frozen-thawed  semen at 60 and 72h following P G F . Ova / embryos were recovered 7 d after Al. 2a  5.3.4.2 Trial Ib Due to the success of the preliminary trial, it was decided to use the single injection method on the first group of experimental animals (n=23). These cows all had suckling calves at the time of superovulation. Estrus was synchronized as in Trial 1a and the  85  single injection of FSH (400 mg) was given 12 days after the second P G F  2a  injection.  The cows were superovulated in staggered groups to allow for organization of the ova / embryo recovery. Three days after the FSH injection, P G F  2a  (25 mg) was administered.  Artificial insemination was performed at 60 and 72 h after P G F  2a  and ova / embryo  recovery occurred one week later.  5.3.4.3 Trial II Due to the very poor response to the single injection of FSH in Trial lb, Trial II was designed to determine if FSH (400 mg) dissolved in a 30% polyvinylpyrrolidone (PVP) solution, which acts as a slow release agent, would be more successful. Two of the cows used in Trial Ib were used for this test. The protocol was the same as in Trial Ib except with the use of PVP.  5.3.4.4 Trial III Faced with the poor response in Trial II, it was decided to use the conventional method of superovulating the final group, since the goal was to retrieve ova / embryos for comparison between the dietary treatment groups. This final group consisted of 13 cows from the molybdenum treatment groups as well as an additional 4 non-suckled heifers to form a non-suckling control (NSC). Because of the small number of cows in each TR group, TR1 remained as the control (n=3), TRs 2 and 3 were combined to form a low Mo group (n=4) and TRs 4 and 5 were combined to form a high Mo group (n=6). Superovulation was done with 8 decreasing doses of FSH over 4 days (total dose 400mg). Prostaglandin F  (25 mg) was given with the 6 injection of FSH and Al th  2a  was performed 60 and 72h after that. Ova / embryo recovery occurred 7 days later.  86  5.3.4.5 Ova / Embryo Recovery One week after Al, each heifer was examined ultrasonographically to determine the number of CL present on the ovaries (superovulatory response). Ova / embryos were collected using a non-surgical method. Briefly, the heifers were given an injection of a local anesthetic (Lidocaine 2%, Austin Laboratories, Joliette, QC) between the second and third coccygeal vertebrae to anesthetize the area. A foley catheter was inserted through the vagina into each uterine horn in turn. The cuff of the catheter was inflated to prevent the flushing media from flowing back into the uterine body. The flushing media used was Dulbecco's phosphate buffered saline (D-PBS) with 0.4% bovine serum albumin (BSA). The media was flushed through the catheter into the uterine horn, which was gently massaged through the rectal wall. The media was collected back through the catheter into the collection filter.  After collection, the filter was taken to the laboratory and the fluid was collected in a petri dish to search for ova / embryos. Ova were assessed for developmental stage and quality as described by Rajamahendran et al., (1987), (i.e. poor - embryos developed to the 8 cell stage; good - embryos from the 16-32 cell stage up to morula; excellent - embryos developed to blastocysts with normal morphology) and placed into maturation media (tissue culture medium (TCM) 199 + 5% superovulated cow serum, phosphate buffer, gentimycin and insulin).  Embryos of transferable quality were  maintained at 37°C then frozen in liquid nitrogen at -196°C.  87  5.3.5 Liver and Plasma Samples for Mineral Analysis Liver samples were taken 15 months after the start of the treatments (1-4 months before calving).  A blood sample was taken at the same time.  Liver and plasma  samples were frozen for future analysis of Mo and Cu concentrations.  5.3.6 Statistical Analysis: This experiment was analyzed as a completely randomized design. Mean comparisons were done through the use of a one-way analysis of variance (ANOVA). In the case of a significant difference between the means, Tukey's studentized range test was used (Zar, 1984). Data for heifers pregnant after 1, 2 and 3 inseminations underwent an arcsine transformation prior to being statistically analyzed by ANOVA. Due to the lack of results for superovulation Trials la, Ib and II, in the superovulation experiment, they were not analyzed. Only the results of Trial III were statistically analyzed. The response to superovulation (number of CL) and the number of embryos recovered were statistically analyzed using ANOVA. Embryo quality was compared between the low and high Mo groups using a t-test. A 5% level of significance was accepted throughout.  5.4 Results 5.4.1 Fertility There were no significant differences in terms of synchronization rate between heifers synchronized by GnRH followed by P G F  2a  or those synchronized by two P G F  2a  treatments, therefore no distinction was made between the heifers based on synchronization method.  There was no significant difference in conception rate  between the treatment groups after either 1, 2 or 3 inseminations. There were also no significant differences in the number of inseminations required for pregnancy to occur  88  (Table Vll). There were 2 cases of embryonic mortality between 28 and 35 d following the first breeding after which the heifers failed to conceive: 1 in TR3 and 1 in TR4. Any missing values are due to the fact that some heifers failed to conceive and were removed from the study.  89  Table VII. Cumulative conception rates (CR) after 1, 2, and 3 inseminations and the total number of inseminations (mean±SD) required for pregnancy in heifers fed varying levels of high molybdenum hay and supplemental copper (treatment (TR) groups 1 -5*). TR GROUPS  CR 1 %  CR 2 %  CR 3 %  # INSEMINATIONS  TR1  50(12)  100 (12)  100 (12)  1.5 + 0.52 (12)  TR2  75(12)  92 (12)  100 (12)  1.3 ±0.65 (12)  TR3  42 (12)  83 (12)  92 (12)  1.6 ±0.67 (11)  TR4  36 (11)  64(11)  82 (11)  1.8 ±0.83 (9)  TR5  50 (12)  75(12)  92 (12)  1.6 ±0.81 (11)  number in brackets represents the number of observations used in the calculation * TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115134 mg/kg Cu  90  5.4.2 Gestation Period and Calving Within 24 h of calving, the calves were weighed to determine birth weights. There were no significant differences in calf birth weights between the treatment groups. There were also no significant differences in terms of calving rate (number of calves as a percentage of the number of heifers inseminated) or in gestation lengths between the groups (Table IX). There were no significant problems associated with calving that were attributable to the dietary treatments. In some instances (27%), assistance had to be given (i.e. the calves had to be pulled with chains), but this was most likely due to the inexperience of the heifers and the large calves. There were some calves that were either born dead or died shortly after birth: 2 in TR1 (one born prematurely and one stillborn), 1 in TR3 (stillborn), 2 in TR4 (one crushed and euthanized and one born with congenital malformations of the digestive tract), and 1 in TR5 (stillborn) but these were not considered reflective of the dietary treatments.  91  Table VIII. Mean (±SD) gestation lengths, calf birth weights and calving rates in heifers being fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 - 5*). TR GROUPS TR1  GESTATION LENGTH (d) 279.3 + 10.23 (12)  CALF BIRTH WEIGHT (kg) 38.8 ±7.76 (11)  CALVING RATE (%) 92  TR2  283.3 ±4.68 (10)  39.4 ±4.27 (10)  100  TR3  282.1 ±4.83 (10)  38.7 ±6.22 (10)  75  TR4  280.8 ± 6.02 (8)  36.2 ±9.18 (8)  73  TR5  284.0 ±2.62 (10)  41.6 ± 5.19 (10)  83  number in brackets represents the number of observations used in the calculation * TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115134 mg/kg Cu  92  5.4.3 Post-Partum Reproduction: There were no significant difference between the treatments in the number of class I follicles 4, 5, 6, 7, and 8 weeks after calving. However, 9 weeks after calving there was a significant difference with the number of class I follicles in TR4 being significantly higher than TR3. This appeared to be due to the small number of observations in TR4 (n=3) and was not reflective of the treatment diets since there was no difference between TR1 (control) and TR5 (high Mo). There was no significant difference in the number of class II follicles at any time during the post-partum assessment. As with the class I follicles, there was no difference between the treatments in the number of class III follicles until 9 weeks after calving when the number of follicles in TR4 was significantly lower than the other treatments.  Again, this was most likely due to the  small number of observations in TR4 as was not affected by the treatment diets. See Figure XII for a summary of these results.  There were no significant differences between the treatment groups in the number of days required before the appearance of the first CL after calving. There were also no significant differences in the P concentrations from the weekly samples post-partum 4  (Figure Xlll) or in the maximum P concentrations corresponding to the first CL after 4  calving (Table IX). Missing values are due to the fact that the heifers were not included in any further assessments once cyclicity had been resumed.  93  A  4 weeks  5 weeks  6 weeks  7 weeks  8 weeks  9 weeks  4 weeks  5 weeks  6 weeks  7 weeks  8 weeks  9 weeks  4 weeks  5 weeks  6 weeks  7 weeks  8 weeks  9 weeks  B  Figure Xll. The number of class I (A), class II (B) and class III (C) follicles starting 4 weeks after calving in heifers fed varying levels of high molybdenum hay and supplemental copper (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  94  Figure XIII. Mean progesterone concentrations starting 4 weeks after calving in heifers fed varying levels of high molybdenum hay and supplemental copper (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  95  Table IX. Mean (±SD) length of the post-partum anestrus period and maximum plasma progesterone (P ) levels corresponding to the first corpus luteum in heifers being fed varying levels of high molybdenum hay and supplemental copper (treatments (TR) 1 5*). 4  TR GROUPS TR1  LENGTH OF POST-PARTUM INTERVAL (d) 51.1 ±7.40 (7)  MAX P LEVELS (ng/ml) 5.9 ±2.55 (8)  TR2  53.2 ±8.74 (12)  5.2 ± 1.17 (11)  TR3  49.7 ± 10.89 (10)  4.7 ± 1.59 (8)  TR4  50.2 ± 12.22 (6)  4.4 ±2.32 (5)  TR5  54.1 ±3.89 (8)  3.3 ±1.81 (7)  4  number in brackets represents the number of observations used in the calculation * TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TRS: 50-67 mg/kg Mo, 115134 mg/kg Cu  96  5.4.4 Superovulation 5.4.4.1 Trial la: This trial involved the use of 2 heifers, each receiving a single injection of FSH. Ultrasound examination prior to flushing showed that each heifer had 12-15 CL present on their ovaries.  Only one of the heifers was flushed, resulting in 11 embryos  recovered, 7 of which were of good quality. Because of the success of this method, it was decided to use it on the first group of experimental animals.  5.4.4.2 Trial Ib: Twenty-three cows received the single injection of FSH in this trial. This time there was no response to the single injection of FSH. Through ultrasound examination, it was determined that only 6/23 cows (26%) had more than one ovulation (one of these exhibited excessive overstimulation of the ovaries). The other 17/23 heifers (74%) had only one ovulation. For those heifers that responded to the FSH, the mean number of ovulations was 6.4±5.18 and the mean # of ova recovered was 3.6±2.7, all of poor quality. There appeared to be no effect on the response or the quality of recovered embryos due to Mo.  5.4.4.3 Trial II: It was decided to try dissolving the FSH in PVP before injection prior to continuing on with the second group of animals on the dietary treatments. This was tried on 2 cows that had been used in Trial Ib. There was no superovulatory response and neither of the cows were flushed.  97  5.4.4.4 Trial III: The second group of experimental animals was superovulated using 8 decreasing doses of FSH over 4 days. The response to superovulation was much better. All cows responded with more than 1 ovulation. Table XI shows the response to superovulation based on the number of CL as well as the number of embryos recovered. Results are shown for the three dietary treatment groups as well as for the non-suckled controls. There was no significant difference in either of the two parameters between the dietary treatment groups, suggesting that molybdenum does not have an effect.  Table Xll  shows the quality of the embryos from the different groups. Again there was no effect of molybdenum on the quality of the recovered embryos.  The number of ova recovered from the suckled cows was significantly lower (p<0.05) when compared to the non-suckled controls, 3.0±2.8 and 10.8±3.2, respectively. This is despite the fact that the superovulatory response was not significantly different between the non-suckled controls and suckled animals. In 3 of the 13 suckled cows (23%), there were no embryos recovered despite the good response.  98  Table X. Superovulatory response and embryo recovery from cows superovulated with 8 decreasing doses of follicle stimulating hormone given over 4 days in 3 dietary treatment (TR) groups receiving varying levels of high molybdenum hay and supplemental copper* and a group of non-suckled control heifers. TR GROUPS TR1 (n=3)  NO. OF CORPORA LUTEA (MEANtSD) 13.3 + 0.58  NO. OF EMBRYOS (MEAN+SD) 0.33+0.58  TR2+TR3 (low Mo) (n=4)  11.8 ± 1.71  4.8 ±2.99  TR4+TR5 (high Mo) (n=7)  9.2 ± 3.54  3.2 ±2.56  Non-suckled control (n=4)  13.0 ± 1:83  10.8 ±3.20  * TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115134 mg/kg Cu  99  Table XI. Quality of embryos recovered from cows superovulated with 8 decreasing doses of FSH given over 4 days in 3 dietary treatment (TR) groups receiving varying levels of high molybdenum hay and supplemental copper* and from a group of nonsuckled control heifers. TR GROUPS  TOTAL EMBRYOS  POOR  GOOD  EXCELLENT  TR1 (n=3)  1  0  1 (100%)  0  TR2+TR3 (n=4)  19  1 (5%)  6 (32%)  12(36%)  TR4+TR5 (n=7)  19  2(11%)  9(47%)  8 (42%)  Non-suckled control (n=4)  43  6 (14%)  25 (58%)  12 (28%)  * TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115134 mg/kg Cu  100  5.4.5 Mineral Concentrations There were significant differences between the concentrations of liver and plasma Mo and Cu between the treatments 15 months after the start of the treatment diets. The level of Cu in the livers of the heifers in TR3 was significantly lower than those in TR 1, 2, 4, and 5. Liver Mo levels were significantly lower in TR1 than in any of the other TR groups. TRs 2 and 3 were significantly lower than TRs 4 and 5. TR4 was significantly higher than TR5. These trends were repeated for the plasma mineral levels. There was no significant difference in the plasma Cu concentrations. Plasma Mo levels were significantly lower in TR1 than in any of the other TRs and TRs 2 and 3 were significantly lower than TRs 4 and 5. See Figures XIV, XV, XVI and XVII for graphical representation of these results. All values for liver mineral concentrations are given on a dry matter basis. Note that plasma and liver mineral concentrations are given for all three sampling periods to allow for comparison.  Any missing values are due to  difficulties obtaining a liver or blood sample from an individual heifer.  101  Figure XIV. Mean concentration of molybdenum (Mo) in liver samples taken from heifers fed varying levels of high Mo hay prior to, 4 months and 15 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  • Before TRs HAt 4 months • A t 15 months  TR1  TR2  TR3  TR4  TR5  Figure XV. Mean concentration of molybdenum (Mo) in plasma samples taken from heifers fed varying levels of high Mo hay prior to, 4 months and 15 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  102  DM  300 -]  250 -  "3> 200 E c o  ra k_  co o c o u 3 O  • Before T R s • A t 4 months  150 -  • A t 15 months 100 •  50 -  TR1  bUh TR2  •I 1 TR3  TR4  TR5  Figure XVI. Mean concentrations of copper (Cu) in liver samples taken from heifers fed varying levels of high molybdenum (Mo) hay prior to, 4 months and 15 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  • Before TRs • A t 4 months • A t 15 months  TR1  TR2  TR3  TR4  TR5  Figure XVII. Mean concentration of copper (Cu) in plasma samples taken from heifers fed varying levels of high molybdenum hay (Mo) hay prior to, 4 months and 15 months after the start of the treatments (TR) (TR1: 1 mg/kg Mo, 16 mg/kg Cu (Control); TR2: 6-8 mg/kg Mo, 16 mg/kg Cu; TR3: 6-8 mg/kg Mo, 6-8 mg/kg Cu; TR4: 30-40 mg/kg Mo, 60-80 mg/kg Cu; TR5: 50-67 mg/kg Mo, 115-134 mg/kg Cu).  103  5.5 Discussion There are several possible causes of reproductive failure in heifers. These include ovulation failure, fertilization failure, maternal recognition of pregnancy not signaled or not interpreted properly or failure of implantation. The results of this experiment show that feeding hay containing elevated levels of molybdenum had no effect on conception rates, pregnancy, gestation lengths, calf birth weights, length of the post-partum period or corresponding progesterone levels.  There was no significant difference between any of the TR groups in the number of inseminations required for pregnancy or in the conception rate after 1, 2 or 3 inseminations.  After 2 inseminations, however, there was a trend towards lower  conception rates in the animals receiving the highest levels of Mo (TRs 4 and 5). A previous study (Phillippo et al., 1987) showed a significant decrease in conception rates after supplementation with 5 mg/kg DM of inorganic Mo. However, another study done the same year (O'Gorman et al., 1987) found that supplementation of inorganic Mo (1520 mg/kg) had no effect on conception rates but did affect the development of the early embryo. Vaughan et al., (1994), also found that short-term feeding (21 days) of high Mo (15mg Mo/kg DM) or a low-Cu status did not significantly affect the pregnancy rate.  The normal gestation length for Hereford cows is 283 d (ranges from 279 - 285 d) (Rutter, 1995). There were no significant differences between the TR groups in term of gestation length and the mean values for each group were well within the normal range. The mean gestation lengths of the experimental animals ranged from 279-284 days. Therefore, gestations lengths were not affected by the diets.  104  Calving rate was defined for the purposes of this paper as the number of calves per heifers inseminated.  The calving rates ranged from 73 - 9 2 % and there were no  statistical differences between the treatment groups. Again, there was a trend toward a lower calving percentage in T R s 3, 4, and 5 although the reasons for this are unknown.  The calves were weighed within 24h of birth. There was no significant difference in the birthweights of the calves, although, on average, the calves were heavier than the average weights of Hereford calves which is 32kg.  The mean birthweights in the  current study ranged from 36-42kg. This increased size of the calves was seen in all T R s (including the control) and is therefore not a reflection of the dietary Mo levels.  There are several factors affecting the return to estrus post-partum.  Suckling can  extend the post-partum interval 2-3 times over a cow without a suckling calf.  Other  factors include poor nutrition, calving difficulties, retained placenta and time for involution of the uterus. In the current study, the number of class I, II and III follicles were compared between the treatment groups starting 4 weeks after calving. There were no significant differences that could be attributed to the experimental diets. The length of the post-partum period and the P levels of the first C L were also compared 4  between the T R groups and there were no significant differences. The mean number of days between calving and the appearance of the first C L was 50-54 d which is shorter than the average return to estrus for first-calf heifers which is normally 35 to 70 days, although it can be as long as 80 or 90 d (Short et al, 1994).  The maximum P  4  concentrations of the first post-partum C L in the current study ranged from 3.3±1.81 to 5.9±2.55 ng/ml, which is comparable to literature values in which the overall P  4  105  concentration in the first and second post-partum estrous cycles was 3.1±1.2 ng/ml and 4.4±1.0 ng/ml, respectively (Rajamahendran and Taylor, 1990).  There was no effect on superovulatory response, embryo recovery and embryo quality that could be attributed to Mo.  This is contrary to findings in which there was a  significant decrease in the normality of recovered embryos due to the addition of 15-20 mg/kg DM of inorganic molybdenum to the diet (O'Gorman et al., 1987). The normality of embryos in that study was 16% in the Mo-supplemented group as compared to 63% in the control group. The reasons for the lack of a similar response in the current study are not known.  However, the results were not unexpected since prior investigations  into puberty attainment, estrous cycles and fertility revealed no significant effects due to the consumption of high Mo hay. It is difficult to draw definitive conclusions from this experiment due to the small number of animals in the treatment groups and the high variability.  Further research using either cycling heifers or cows which have weaned  their calves is warranted.  A significant observation from this study was the effect that suckling had on the recovery of embryos.  This was seen in all 3 of the superovulatory trials. In Trial Ib,  suckling resulted in either a poor or non-existent response to the single injection of FSH as compared to the good response in the 2 non-suckled heifers (Trial la). This result also occurred when FSH was first dissolved in PVP then given as a single injection as it was done in Trial II. These results may be attributable to an increase in the clearance of exogenous FSH due to changes in the metabolism of post-partum, lactating cows. In Trial III (8 injections of FSH over 4 d) suckling did not affect the response to superovulation but did affect the recovery of ova / embryos.  106  Suckling creates a multitude of metabolic, neural (sensory and olfactory) and psychological messages (Short et al., 1994). It is known to cause a reduction in the pulsatile secretion of LH and consequently a lowering of ovarian activity (Lamming et al., 1981).  In cows, the suckling stimulus itself appears to responsible for the  suppression of GnRH and LH action by acting at the level of the hypothalamus (Williams, 1990).  When 8 injections of FSH were given in decreasing doses over 4 days (Trial III), there was a significantly better response as indicated by the  number of CL on  ultrasonography. However, there was a significant decrease in the number of embryos recovered from animals with suckling calves as compared to the non-suckled controls and the quality of the those embryos was also lower. A previous study investigating the effects of suckling on embryo quality and recovery (Brown et al., 1991) found that the response to superovulation was the same in dry and suckled cows (based on the number of CL).  Also, the number of recovered embryos was similar.  However,  suckling appeared to affect the viability of the recovered embryos. In the present study, there was a difference in the quality of the recovered embryos between the non-suckled controls and the suckled cows. Studies in humans have shown that prolactin acts at the ovarian level to inhibit estrogen production and decrease granulosa cells numbers, which results in the formation of a hypofunctional CL (Franchimont et al., 1988). Previously, it was found that weaned cows had a greater number of LH receptors/total follicular protein than did suckled cows (Walters et al., 1972). Whether prolactin affects the formation of LH receptors or there is a decrease in the number of follicular cells is unknown, most likely due to a combination of both. Regardless, this, in combination  107  with the fact that suckled cows have lower LH levels (Short et al., 1972), could result in an altered follicular milieu possibly affecting the developmental competency  of the  oocyte and/or the formation of a hypofunctional CL, which could be responsible for the lower embryo viability or lower embryo quality in suckled cows.  It is also interesting to note that in 23% of the suckled cows there were no embryos recovered, although there was a good response to superovulation. This difference can not be attributed to technique since all animals were flushed in exactly the same fashion and all materials and media were prepared the same way.  Possibly, the  hormones associated with suckling (prolactin and/or oxytocin) affected the transport of gametes to the site of fertilization or the embryos from the oviduct to the uterus.  In order to monitor the mineral levels of the heifers, liver biopsies were performed and blood samples were taken for analysis of Mo and Cu concentrations. These samples were taken after the heifers had been on their experimental diets for 15 months. They were compared to values of the previous samples taken before the start of the experimental diets (basal levels) and 4 months later. There were significant differences between the TRs, but these differences reflected the intake levels of the mineral and did not indicate that there were any interactions occurring between Mo and Cu. However, these mineral levels do indicate that elevated levels of Mo are being consumed, absorbed and stored by the heifers.  108  Chapter 6: ANCILLARY EXPERIMENTS 6.1 Non-Pregnant Heifers and Molybdenosis Throughout the study, no clinical symptoms of molybdenosis appeared in any of the animals. This suggested that either Mo in the hay was in some way rendered non-toxic as described by Ferguson et al., (1943), and by Britton and Goss (1946), or that the S content of the hay (approximately 0.15%) was too low to permit thiomolybdate formation in the rumen. A combination of both complexed Mo and low S was also a possibility. In order to test the underlying mechanism behind the lack of response, a series of ancillary experiments were conducted.  6.1.1 Trial I Six non-pregnant heifers were fed a ration of high molybdenum hay (80 mg/kg) with appropriate protein, mineral and vitamin supplements (no supplemental copper) and an additional 80 mg/kg of inorganic Mo in the form of ammonium molybdate for a total of 95 days. During this time no visible signs of molybdenosis were observed. These heifers had been fed varying levels of Cu and Mo prior to the start of this trial. They had been part of the main experimental group of animals but failed to conceive and were therefore removed.  6.1.2 Trial II The above lack of response prompted the addition of S, based on the premise that the S level was too low for the formation of thiomolybdates. The addition of sodium sulfate raised the sulfur level to 0.34% and the animals continued to receive ammonium molybdate as in Trial I.  Four of the heifers began to shown signs of molybdenosis  (stiffness, then diarrhea) within 9 - 1 5 days. The animals were returned to control hay  109  (1 mg/kg Mo) with supplemental copper and recovered. The recovery was very rapid and dramatic: the diarrhea was gone within 12 hours and the stiffness disappeared within 24 - 48 hours.  6.1.3 Trial III Two non-pregnant heifers not used in either Trial I or II were fed high molybdenum hay (80 mg/kg), but without added ammonium molybdate. Sodium sulfate was added to increase the total sulfur content of the ration to 0.44%. This ration was fed for 19 days. No symptoms of molybdenosis were observed by this time. Because these animals were only on the ration for 19 days, four other non-pregnant heifers were placed on the same ration.  They remained on the treatment for 35 days after which they were  returned to the control hay with copper supplementation. This level of sulfur in the absence of ammonium molybdate did not induce molybdenosis.  These trials were not properly controlled experiments and the results cannot be used to draw definitive conclusions. However, the results strongly suggest that since there was no response to added ammonium molybdate alone, low sulfur levels may have been a factor in preventing the development of molybdenosis.  Also, since there was no  response to added sulfur alone, the molybdenum in the high-Mo Brenda hay may have been complexed in some way making it unavailable for thiomolybdate synthesis in the rumen. It should be noted that the molybdenum was found not to be associated with fibre fractions (NDF or ADF) (Christensen, 1997), therefore a complex with protein seems likely. The rapid onset of molybdenosis after the addition of both ammonium molybdate and sodium sulfate suggests involvement of both low sulfur levels and  110  unavailable (complexed) molybdenum protecting against molybdenosis in heifers consuming the high molybdenum hay.  6.2 Spares As previously mentioned, 5 "spare" heifers were originally purchased in case there were any anatomical abnormalities of the reproductive tract in other heifers or in case another need for a replacement heifer arose. Since these heifers were never required as replacements, a sixth dietary treatment was created. This treatment was initiated at the same time as the main experiment (i.e. since before puberty). They were placed on a ration of high Mo hay without any supplemental Cu (50-60 mg/kg Mo, 8-10 mg/kg Cu). Only limited data were collected on the spares because of logistical problems. The following observations are of interest:  1. The conception rate after 2 inseminations was 100% and all five of these heifers delivered healthy calves (calving rate 100%).  2. Calf birth weights (50.0±5.66 kg), gestations lengths (286.0±1.22 d), length of the post-partum period (52.0+17.99 d) and post-partum progesterone levels (1.9+.0.86 ng/ml) did not appear to differ from those in other groups.  3. Liver biopsies were done on the spares only once (15 months after the start of the dietary treatments).  Liver copper levels were very low (6.4±1.32 mg/kg DM) and  indicated a possible deficiency state although there was no apparent effect on reproductive performance. Liver Mo concentrations (17.2±5.30 mg/kg DM) and the  Ill  concentrations of Mo (8.8+3.94 mg/L) and Cu (0.4±0.09 mg/L) in plasma appear to reflect the dietary intake levels of the minerals.  6.3 Greenhouse Experiment An additional experiment was initiated to obtain more information regarding the form of Mo in the hay in an attempt to explain the reasons for the lack of effects of the high-Mo hay on reproduction seen in this study. The overall objective of this experiment was to determine what proportion of the Mo was soluble, what proportion was associated with the protein fractions and whether this differed between fresh and dried material. To do this, both alfalfa and orchard grass were grown on Brenda mine tailings in 12cm pots. The tailing were fertilized with a non-soluble fertilizer (11:43:3) which did not contain any micronutients. The pots remained under greenhouse conditions during the course of the experiment.  The protocol called for the harvesting of these plants and the  analysis of both fresh and air-dried material. Although the seeds did germinate, the plants failed to thrive and therefore the analysis was not completed.  112  Chapter 7: GENERAL DISCUSSION  The results of the current study indicate that hay containing elevated levels of Mo can be fed to beef heifers without adversely affecting the reproductive parameters studied. Specifically, three key aspects of reproduction were investigated during the course of this study: onset of puberty, estrous cycle characteristics and fertility (including postpartum reproduction). There were no significant differences (p<0.05) in age at puberty, although there were some differences in the number of different sized follicles as more and more heifers began cycling. This was due to variation in the stage of cycle within the groups rather than an effect of the dietary treatments.  There were also no  significant differences between the groups in any of the estrous cycle parameters studied (i.e. diameter of the dominant follicle, diameter of the CL and corresponding P  4  concentration), except cycle length which was significantly shorter in TR4 than in the control heifers (p<0.05) although it was still within the normal range. Conception rates, gestation lengths and calf birth weights were also not affected by the dietary treatments.  Neither was the interval from calving to the resumption of estrus post-  partum or the maximum P concentrations from the first CL. When these animals were 4  superovulated to allow embryo recovery, there were no statistical differences between the treatment groups in terms of superovulatory response, ova recovery or embryo quality. These results are in contrast to other studies in which Mo at much lower levels than the current study has been shown to have an effect on reproduction in cattle, namely a delay in puberty, anestrus, decreased conception rates and impaired early embryonic development (O'Gorman et al., 1987; Phillippo et al., 1987). Most of these studies have used inorganic forms of molybdenum to ensure that adequate levels are being consumed. The current study was different in that an organic form of Mo, in hay grown on mine tailings, was fed to the experimental animals.  The addition of an  113  inorganic form of Mo to the rations would have compromised the study, as it was specifically designed to test the impact of the high levels of Mo contained in the hay.  In order to monitor the mineral concentrations in the heifers, periodic liver samples (prior to, 4 months and 15 months after the start of the dietary treatments) were taken and analyzed along with corresponding plasma samples for the concentrations of both Cu and Mo. Prior to the introduction of the experimental diets, there were small but significant differences in the liver concentration of Mo between the TR groups. The reasons for this are unclear, however it is possible that it was due to individual variation. Regardless, all values were within the normal range of 0.56-5.6 mg/kg DM (Puis, 1988). Four months after the start of the diets, there were significant differences between the groups, reflecting their dietary intake of Mo.  Puis, (1988), suggests that a dietary  concentration of Mo above 5 mg /kg DM in the presence of adequate copper reflects an elevated state; 10-203 mg Mo /kg DM is toxic where Cu levels are deficient (2.0 - 40.0 mg Cu/kg DM in liver). This was the situation for heifers in TR 3 and the "spares", although they conceived without difficulty at this time. These trends were also evident 15 months after the start of the diets.  There appeared to be a plateau effect on  accumulation of Mo in the liver, as the concentration in TR 5 did not increase at the same rate as in TR 4. The concentration of Mo in the liver of heifers in TR 4 was actually higher than those in TR 5, even though TR 5 received almost twice as much Mo in the diet.  At the start of the trial, plasma Mo levels were baseline and there were no significant differences between the groups. The plasma Mo levels in TR1 remained at these baseline levels for the duration of the study. Four months after the start of the trial, the  114  levels of Mo in the plasma of heifers in TRs 2 and 3 were still at baseline. It appeared that time is required for the appearance of Mo in plasma at a concentration of 10 mg/kg. This is in spite of the fact that an increase in liver Mo concentrations in these two groups was already apparent at that time. A greater increase was seen in these two groups in the samples taken 15 months after the start of the treatments.  The  concentration of Mo in the plasma of TRs 4 and 5 showed an increase in the samples taken four months after the start of the treatments. Fifteen months after the start of the diets, the levels in TR5 plateaued again and were not significantly different from TR4 at that point. This suggests that there must be a limit as to the amount of Mo that can be transported in the plasma.  Liver Cu concentrations indicated that all treatment groups were at adequate baseline levels at the start of the trial. Four months after the start of the diets, there was an increase in the liver Cu concentration in TR1 (control) which was probably due, in part, to the switch to the Basal '96 hay that had a higher Cu concentration than the previous basal hay. The liver Cu concentration in TR2 was significantly lower than in TR1 even though these two groups were receiving the same amount of copper supplementation. The liver Cu concentration in TR3 was even lower, which was expected as TR3 was not receiving any supplemental Cu. Liver Cu concentration in TR4 and 5 was significantly higher, reflecting the levels of supplemental Cu these groups were receiving. The concentrations in the samples taken 15 months after the start of the diets showed a similar pattern to the previous samples. TR 3 still had the lowest liver Cu concentration, although there was some increase due to the new basal hay containing a greater Cu concentration.  Other groups receiving a portion of this basal hay also showed an  increase. TR4 showed only a slight increase over the samples taken 4 months after the  115  start of the treatments and the concentration in TR5 decreased from the 4 months sample, indicating a possible plateau effect. This decrease could also be due to the increasing Cu demands of the developing fetus (Gooneratne et al., 1994).  As with the liver Mo concentrations, there was a significant difference in plasma Cu between the treatment groups before the initiation of the trial. The reasons for this are unknown. Four months after the start of the treatments, a decrease was seen in the concentrations in TRs 2 and 3 similar to that in the liver. The concentration of plasma Cu in TR 3 was suggestive of a deficiency state. Fifteen months after the start of the diets, the concentrations in TR 2 and TR3 increased as it did in the liver and a plateau effect was seen in the higher Mo groups.  These results indicate that Mo was being absorbed into the bloodstream and stored in the liver of the heifers.  Therefore, it was not inadequate Mo levels that were  responsible for the lack of significant effects on reproduction. Suttle, (1996), suggested that inconsistent responses may be due to differences in Cu status at important sites of endocrine activity and because of this, measurement of plasma and liver Cu may not be adequate indicators of Cu status. Suttle, (1996), continues by stating that it is possible that in the study of Phillippo et al., (1987), Cu concentrations in the pituitary may have been lower in the Mo-supplemented animals which affected pituitary function. However, this does not take into account the fact that the effects of elevated levels of Mo on reproduction may be due to Mo itself or to thiomolybdates and may be independent of the Cu status of the animal. Other studies have determined that Mo (as molybdate) acts to prevent steroid receptor activation (Noma et al., 1980; Muller et al.,  116  1982), which would account for the effect on reproduction (namely LH secretion) seen in the study of Phillippo et al., (1987).  Information in the literature indicates that the form of molybdenum can be a critical factor in the onset of molybdenosis. Huber et al., (1971), stated that ruminants can tolerate greater levels of Mo from Mo salts than Mo from fresh forage. There is also evidence in the literature that suggests that grazed forage, which causes Mo toxicity, loses this potential when dried and fed as hay (Ferguson et al., 1943; Britton and Goss, 1946; Ward, 1991). It is important to note that these previous studies were all using levels of Mo that were significantly lower than those in the current study.  The ratio of Cu to Mo is also important in determining whether molybdenosis will develop. Miltmore and Mason (1971) and Puis (1988) stated that if the ratio of Cu:Mo drops below 2:1 then molybdenosis might result.  It is for this reason that copper  supplementation was maintained at a 2:1 ratio during the current study (even though the appropriate ratio of Cu:Mo is unknown for Mo levels exceeding 10mg/kg). The aim was to determine whether the effects of Mo on reproduction were independent of the copper status of the animal and not due to a secondary Cu deficiency. Phillippo et al., (1987), did suggest the possibility that a low copper status was a prerequiste for elevated levels of Mo to affect reproduction.  There are numerous other factors that can affect the response to Mo. The availability of Cu in the ration can have an effect since Cu has a lower availability from pasture and a higher availability in hay and cereals (Lesperance and Bohman, 1969). Also, a higher protein content, as seen in alfalfa and concentrates, is associated with Mo toxicity  117  because of the increase in sulfide production that occurs (Lesperance et al., 1985). Interactions with other minerals can influence Mo toxicity. For example, iron can also bind to Cu, rendering it unavailable for use by the body (Phillippo and Humphries, 1987), and manganese and zinc can affect the degree of Mo toxicity (Lesperance and Bohman, 1969).  118  Chapter 8 : CONCLUSIONS AND RECOMMENDATIONS  8.1 Conclusions Sixty, Hereford and Hereford-cross beef heifers were used to investigate the effects of feeding hay containing elevated levels of Mo on reproduction in beef heifers. There were no significant effects on reproductive function. The reasons for this are unclear at this point since other studies investigating the effects of Mo on reproduction have shown that elevated dietary Mo levels can cause a delay in the onset of puberty, altered estrous cycles eventually leading to the animals becoming anestrus, decreased conception rates and impaired early embryonic development (O'Gorman et al., 1987; Phillippo et al., 1987) However, several definitive conclusions can be drawn from this research:  1. Mo from the hay was ingested by the heifers, absorbed into the bloodstream and accumulated in their livers. Liver and plasma concentrations of Mo reflected the dietary intake levels.  2. This accumulation of Mo did not have any statistically significant effects on the aspects of reproduction investigated during the course of this study (i.e. onset of puberty,  estrous  cycle characteristics, fertility,  pregnancy  and  post-partum  reproduction).  3. Even heifers with a low Cu status (based on liver Cu concentrations) did not have their reproductive function impaired (i.e. heifers in TR3 (6-8mg/kg Mo, 6-8 mg/kg Cu) and the "spares" (50-60 mg/kg Mo, 8-10mg/kg Cu)).  119  4. Inorganic Mo (ammonium molybdate) and sodium sulfate acted together to induce molybdenosis in a small number of heifers used in an ancillary experiment (although more heifers would have been needed to draw definitive conclusions). Therefore, it is likely that the form of Mo in the hay or inadequate S levels (or a combination of both) were responsible for the lack of any clinical signs of molybdenosis.  5. High levels of Cu (i.e. >100 mg/kg) can be fed to heifers for prolonged periods without the risk of Cu toxicity.  8.2 Recommendations The results of the current study suggest that feeding hay containing elevated levels of Mo to beef cattle is not likely to cause any adverse effects on the reproductive parameters studied. However, the following recommendations should be followed to ensure this:  1. for optimum reproductive efficiency, Mo levels should not exceed 10 mg/kg during the 3 months prior to puberty and during the breeding season since the reasons for the discrepancy between the results of this study and previous studies on the effects of elevated levels of Mo on reproduction in cattle are not known, and  2. levels of Mo in the hay should be monitored and copper supplementation given when necessary to maintain a 2:1 Cu to Mo ratio.  Although these results are promising, they can not be extrapolated to include cattle grazing on fresh forage since there is evidence in the literature suggesting that Mo in  120  fresh forage is more likely to induce molybdenosis. An intensive study looking at reproductive function in cattle grazing high Mo forage is warranted.  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Soil Science and Plant Analysis. 78:425-436.  131  APPENDICES TABLE OF CONTENTS Appendix I: RATION FORMULATION An example of ration formulation  132 133  Appendix II: WEIGHTS Mean weights for each group throughout the study  134 135  Appendix III: PUBERTY Statistical Analysis of age of puberty attainment Statistical Analysis of weight at puberty attainment Statistical Analysis of progesterone concentration of first corpus luteum Statistical Analysis of prepubertal LH concentrations Statistical Analysis of prepubertal FSH concentrations  136 137 138 139 140 141  Appendix IV: ESTROUS CYCLES Statistical Analysis of the diameter of the dominant follicle Statistical Analysis of the diameter of the corpus luteum Statistical Analysis of the maximum progesterone concentration Statistical Analysis of the length of the estrous cycle  142 . 143 144 145 146  Appendix V: FERTILITY, POST-PARTUM AND SUPEROVULATION Breeding Summary Statistical Analysis of the number of pregnant heifers after 1 insemination Statistical Analysis of the number of pregnant heifers after 2 inseminations Statistical Analysis of the number of pregnant heifers after 3 inseminations Statistical Analysis of the number of inseminations required for pregnancy Statistical Analysis of the gestation lengths Statistical Analysis of calf birth weights Statistical Analysis of the length of the post-partum interval Statistical Analysis of the progesterone concentration of the first CL Statistical Analysis of the number of corpora lutea before ova recovery Statistical Analysis of the number of embryos recovered  147 148 150 151 152 153 154 155 156 157 158 159  Appendix VI: MINERAL CONCENTRATIONS Summary of Mineral Analysis  160 161  132  APPENDIX I: RATION FORMULTION Ration formulation was done using the Nutrition Module of  Cowchips which was  developed by Alberta Agriculture, Food and Rural Development (1993). Adjustments to the ration were made on a monthly basis and any time a new batch of hay was introduced. Adjustments were also made during 3 critical developmental periods: growth, the last trimester of pregnancy and lactation.  133  Example Ration Formulation  PEN 4 - 100 PPM MOLY + 200 PPM COPPER RATION REPORT  BEEF RATION BALANCER by ALBERTA AGRICULTURE  J a n 20,1996  HIGH MOLY 20/1/1996 C a t t l e D e s c r i p t i o n : 550 l b HED HEIFER 1 ADG Number of Head : 1 Nuaber of Days : 1 LBS/HEAD /DAY 10.000 3.500 0.080  FEED TYPE BRENDA HAY 95 BARLEY 6RAIN PUT CUSTOM 13 TOTAL FEED COST / LB GAIN  I OF TOTAL COST/HEAD I RATION /DAY 8 73.64 N/A J 25.77 0.29 I 0.59 N/A I 100 I  13.580  $ 0.29 $ 0.29  NUTRIENTS - RECOMMENDED AND SUPPLIED  RECOMMENDED / DAY SUPPLIED / DAY  DH INTAKE DE PROTEIN CALCIUM (LB) MCAL (Kq> (a) 14.2 18.3 0.58 19 11.9 * 14.9 * 0.53 47 Cu •a 64 35 *  RECOMMENDED / DAY SUPPLIED / DAY *  'tit  =  PHOS. (a) 14 18  SALT VIT A Ca/P IU (a) Ratio 17 27500 4 * 27223 * 2.57  COST j /HD/DAY 0.29  Mn  Zn  Se  Mg  K  S  Na  SQ  tq  aq  Q  q  q  q  258  322 423  1.3 2.3  10 8*  42 89  10 8*  * Nutrient not within reconoended range  THIS RATION IS INTENDED AS A GUIDELINE R e s p o n s i b i l i t y for i n t e r p r e t a t i o n of t h i s r a t i o n r e s t s with the user.  100  PPM MOLY 200 PPM Cu  220 g C u S 0 /40 kg mix = 2,750 mg C u S o Cu i n t a k e = 688 + 35 = 722 mg Cu / d a y 4  4  = 688 mg Cu  DMI 5.4 kg = 134 mg /kg Cu Mo i n t a k e 4.0 k g x 90 mg / k g = 360 mg Mo / d a y Cu:Mo 2:1  6 3•  134  APPENDIX II: WEIGHTS The mean weights for each treatment groups are presented. Statistical analysis was done randomly throughout the study and no differences between the groups was observed. The heifers were no weighed in the later stages of pregnancy since calf weight was an uncontrollable variable affecting any comparison between the TR groups. Weights were not measures after July 16/97 since the scale was broken and was not fixed before the end of the study.  Mean weight (kg) for each group throughout the study period. Date Nov 15/95 Nov 23/ 95 Nov 30/95 Dec 7/ 95 Dec 20/95 Dec 28/95 Jan 4/96 Jan 11/96 Jan 18/96 Jan 30/ 96 Feb 15/96 Feb 29/96 Mar 7/96 Mar 14/96 Mar 21/96 Mar 28/96 Apr 4/96 Apr 11/96 Apr 25/96 May 2/96 May 16/96 May 22/96 May 30/96 June6/96 June19/96 July 3/ 96 July 17/96 Aug 1/96 Aug 13/96 Aug 29/96 Sept 13/96 Sept 27/96 Oct 11/96 Oct 25/96 Nov 8/96 Nov 22/96 Dec 18/96 Jan 14/97 Feb 5/97 Feb 19/97 Apr 30/97 May 14/97 June 25/97 July 16/97  TR1 230 230 229 225 237 239 243 247 246 262 272 279 286 289 296 300 303 308 316 317 332 336 345 347 357 360 370 386 391 399 407 411 405 425 430 434 449 470 486 503 512 521 507 broken scale  TR2 232 232 230 229 233 243 245 248 251 265 270 278 284 291 296 296 302 307 310 316 326 334 339 346 349 356 364 381 385 396 408 410 410 425 431 434 451 471 488 501 546 547 499 511  TR3 209 228 226 223 233 238 240 245 250 253 267 271 276 284 291 290 295 299 304 314 320 322 331 335 340 349 350 368 367 378 387 393 396 405 416 427 437 460 475 478 478 487 480 496  TR4 234 233 232 230 236 241 243 247 252 251 267 275 278 283 285 293 301 307 317 318 330 340 348 349 361 367 372 388 394 400 405 411 414 428 437 435 452 477 486 496 506 512 465 499  TR5 227 225 228 223 238 238 232 234 230 235 255 265 269 275 278 281 287 296 304 308 316 324 328 334 337 345 347 357 358 365 372 374 378 396 404 408 423 450 461 474 491 495 470 489  136  APPENDIX III: PUBERTY Puberty assessment began after the initiation of the treatment diets. Age at puberty attainment, progesterone concentration from the first CL, as well as the prepubertal concentrations of LH and FSH were compared. There were no significant differences between the treatment groups.  137  Statistical analysis of age at puberty attainment (months). TR1 10 10 10 10 10 10 10 10 11 12 12 12  TR2 10 10 10 10 11 11 11 11 11 11 12 12  TR aroup TR1 TR2 TR3 TR4 TR5  Mean 10.6 10.8 10.6 10.0 11.0  TR3 8 10 10 10 10 10 10 11 12 12 12 12  TR4 8 8 10 10 10 10 10 10 11 11 12  SD 0.90 0.72 1.24 1.18 0.85  Analysis of Variance: df 4 54 58  TR E  TOT F(calculated) F(4,54)  =  _  SS 6.53 53.5 60.03  MS 1.63  1 -65  2.54  • therefore there is no significant difference between the treatments  TR5 10 10 10 10 11 11 11 11 12 12 12 12  138  Statistical analysis of weight at puberty attainment (kg). TR1 338 333 312 296 303 278 356 307 273 308 366  TR a roup TR1 TR2 TR3 TR4 TR5  TR2 332 284 268 261 291 348 316 341 307 295 307  Mean 306.4 304.6 301.9 297.1 308.67  TR3 271 339 325 319 294 268 280 336 318 320 267 286  TR4 287 235 311 334 297 266 265 283 356 337  SD 28.06 28.36 27.08 37.67 32.78  Analysis of Variance: df 4 51 55  TR E TOT '"(calculated) ^"(4,54)  =  SS 1113.13 50957.09 52070.21  MS 278.28 999.16  -0.28 2.55  • therefore there is no significant difference between the treatments  TR5 301 325 284 287 312 297 304 265 388 337 326 278  Statistical analysis of progesterone concentrations of the first corpus luteum. TR1 4.690 2.395 5.932 I. 038 2.376 4.141 6.788 II. 489 9.186 1.079  TR a roup TR1 TR2 TR3 TR4 TR5  TR2 7.433 2.024 7.197 7.536 7.918 5.826 6.496 5.991 5.008 8.767 4.248  Mean 4.90 6.24 4.97 5.32 6.29  TR3 1.993 1.610 2.286 6.005 7.847 5.576 10.777 5.113 3.709 5.695 4.049  TR4 7.928 6.491 1.324 3.398 2.663 7.013 7.909 5.872  SD 3.48 1.88 2.72 2.53 3.69  Analysis of Variance: df 4 42 46  TR E TOT F (calculated) r  "(4,42)  =  SS 17.18 344.20 361.38  MS 4.29 8.20  0.52  =  2.59  • therefore there is no significant difference between the treatments  TR5 2.141 11.836 3.360 4.734 4.673 6.586 10.704  140  Statistical analysis of prepubertal luteinizing hormone concentrations (ng/ml). TR1 0.37 0.04 0.15 0.43 0.17 0.28  TR2 0.14 0.11 0.16 0.21 0.09 0.11  TR arouo TR1 TR2 TR3 TR4 TR5  Mean 0.24 0.14 0.13 0.23 0.22  TR3 0.26 0.15 0.14 0.11 0.09 0.05  TR4 0.22 0.04 0.46 0.20 0.16 0.27  SD 0.16 0.04 0.07 0.14 0.10  Analysis of Variance: df 4 25 29  TR E TOT  '"(calculated) F(4, ) = 25  -  1  SS 0.07 0.29 0.36  MS 0.016 0.012  -38  2.76  • therefore there are no significant differences between the treatments  TR5 0.27 0.18 0.17 0.38 0.09 0.22  141  Statistical analysis of prepubertal follicle stimulating hormone concentrations (ng/ml). TR1 0.69 0.31 0.27 0.44 0.51 0.38  TR group TR1 TR2 TR3 TR4 TR5  TR2 0.44 0.64 0.28 0.18 0.25 0.39  Mean 0.43 0.36 0.45 0.32 0.46  TR3 0.53 0.42 0.51 0.19 0.64 0.39  TR4 0.39 0.18 0.49 0.16 0.43 0.28  SD 0.15 0.17 0.15 0.14 0.13  Analysis of Variance: df 4 25 29  TR E TOT  F(4,25) -  SS 0.09 0.54 0.63  MS 0.21 0.22  2.76  • therefore there are no significant differences between the treatments  TR5 0.58 0.35 0.53 0.37 0.32 0.60  142  APPENDIX IV: ESTROUS CYCLE After a synchronized estrus, 6 heifers from each treatment groups were used to compare estrous cycle parameters. There was no significant difference in the diameter of the dominant follicle, the diameter of the CL or in the corresponding P 4 concentrations. Only statistically significant difference was in the length of the cycle although this did not appear to be due to the dietary treatments.  143  Statistical analysis of the diameter (mm) of the dominant follicle. TR1 18.0 10.5 11.7 16.2 12.2 12.4  TR2 14.7 13.1 14.5 15.9 17.4  TR aroup TR1 TR2 TR3 TR4 TR5  Mean 13.5 14.4 15.9 17.1 15.0  TR3 18.5 14.4 15.3 14.1 18.5 14.7  TR4 17.7 18.9 17.3 15.2 16.4  TR5 18.3 12.9 13.3 12.5 18.7 15.0  SD 2.67 0.97 1.86 1.24 2.67  Analysis of Variance: df 4 23 27  TR E TOT f" (calculated) F(4,23)  =  =  SS 38.29 119.72 158.01  MS 9.57 5.21  1 -8  2.8  • therefore there are no significant differences between the treatment groups.  Statistical analysis of the diameter (mm of the corpus luteum. TR1 23.9 27.3 29.4 27.6 23.5 24.5  TR2 18.3 27.8 28.5 25.6 24.2  TR group TR1 TR2 TR3 TR4 TR5  Mean 26.0 24.9 24.7 25.2 24.5  TR3 24.6 25.3 23.9 27.4 24.0 23.1  TR4 27.4 23.9 29.5 23.0 21.5 26.0  TR5 21.6 29.4 29.6 10.8 31.1  SD 2.19 3.63 1.38 2.71 8.26  Analysis of Variance: df 4 23 27  TR E TOT f~ (calculated) F(4,23)  =  SS 8.23 559.9 448.1  MS 2.06 19.12  02.8  • therefore there are no significant differences between the treatment groups.  145  Statistical analysis of the progesterone concentrations (ng/ml) on day 13 of the estrous cycle. TR1 .918 7.066 5.055 4.647 4.662 3.291 TR group TR1 TR2 TR3 TR4 TR5  TR2 3.795 4.165 8.736 3.331 4.774 3.401 Mean 7.8 4.3 4.7 5.1 7.5  TR3 2.895 9.317 7.121 3.891 1.901 5.227  TR4 9.00 4.439 3.948 0.304 19.152 8.252  TR5 7.598 8.529 6.052 8.044 7.731 9.125  SD 1.04 2.05 2.05 2.77 6.52  Analysis of Variance: df 4 25 29  TR E TOT '"(calculated) '"(4,23)  -  =  1  SS 67.15 298.10 365.25  MS 16.79 11.92  -41  2.8  • therefore there are no significant differences between the treatment groups.  146  Statistical analysis estrous cycle length (days) during the estrous cycle assessment. TR1 21 20 21 21  TR a roup TR1 TR2 TR3 TR4 TR5  TR2 20 21 21 20 19  Mean 20.8 20.2 19.7 18.3 20.5  TR3 19 19 19 20 20 21  TR4 16 19 19 19  SD 0.43 0.75 0.75 1.30 1.12  Analysis of Variance: df 4 18 22  TR E TOT F(calculated) ~ f" (4,18)  =  SS 15.98 18.63 34.61  MS 3.995 1.035  3.86  2.93  therefore there are significant differences between the treatment groups TR4 is significantly shorter than TR1  TR5 22 21 20 19  147  APPENDIX V: FERTILITY. POST-PARTUM AND SUPEROVULATION After estrus synchronization , the heifers were bred through artificial insemination. Each heifer was given a maximum of 3 attempt to conceive,. There was no significant difference after 1, 2 and 3 inseminations and there was no difference in the number of inseminations required for pregnancy to occur.  There was also no significant  differences in gestations length, calf birth weight, post-partum interval or P  4  concentrations from the first post-partum CL. Once all the heifers were cycling again, they were superovulated and superovulatory response, ova recovery and embryo quality was compared with no significant differences between the treatment groups.  1 Animal # iDate of Red Kamar |TR 1 (1 mg/kg Mo, 16 mg/kg Cu) |BGrl: 1 9453 I July 17 (am) | 82033 I July 17 (am) I July 17 (am) 1 July 18 (am) I 8923 I July 19 (am) I July 17 (pm) iBGrll: 1 9445 I did not turn red | July 20 (pm) | 9449 I fell off | 9457 I did not turn red I partial red July 20 I 9430 I did not turn red  CD  sI  co  s  co  S  S3  cn  CO  s  o  I  CN > 3 —}  CD  CM  a «E SoD «S E a ~» < o r• ra  K •  CO  < •  CO  co  3  •  CO  s  CM  3  QC  I  eg  CM >L 3  u  "5"  red kamar red kamar red kamar red kamar  S  4 (30-40 mg/kg Mo. 60-80 mg/kg Cu) I I BGrI: | I 81021 I July 18 (am) I July 18/19 I I 9432 I July 18 (am) I July 18/19 I I 87019 | July 16 (am) I July 16/17/18 I I 9024 I did not turn red I July 18/19 I I 9426 I July 17 (pm) j July 18/19 j  CO •  in 3  >r o> ca  CM a> ty-  •  •  •  3  CO TT •  cn s  CD CM  3 3 3  CM  I | j I j  Not Pregnant Not Pregnant Confirmed Confirmed Confirmed | I I  9025  9420  3  -  c-  •  co CM  S  CM > 3 3 —>~1  o CMCM •  Pregnant Pregnant Pregnant  I I I I I j  Pregnant Confirmed Not Pregnant Confirmed Not Pregnant Not Pregnant I |  I I | j j  | I  I Not Pregnant J Confirmed I Not Pregnant j Not Pregnant | Confirmed Not Pregnant  I Not Pregnant | Confirmed | Confirmed I Not pregnant I| Confirmed j Pregnant  9457  s  | | I I  Pregnant Pregnant  Not Pregnant Pregnant not done Pregnant  -  I j I Aug 7th | I  I Aug 6/7 I I Aug 6/7 j | Aug 7th | | (  •  red kamar red kamar red kamar red kamar  •  standing standing red kamar red kamar red kamar  • Pregnant Pregnant Pregnant  s  |  |  9445 9449 9440  | | |  81021 9432  9422  9022  | |  |  Not Pregnant Not Pregnant  Pregnant  Pregnant  J  9459  I  Pregnant Not Pregnant Pregnant  I!  9419  Pregnant  Pregnant I  (  Pregnant Pregnant Pregnant I I |  86006  Pregnant  |  5 3  ITR  •  c~  Mounting I Aug 7th red kamar | red kamar | I Aug 7th red kamar | |  cco  | 90099 |  y-  3 8  s  |TR 3 (6-6 mg/kg Mo, 6-8 mg/kg Cu) I I BGrI: I July 16 (am) I July 16/17/18 I I 9421 j July 17 (am) | July 18/19 I July 17 (pm) I July 18/19 I | 9461 | July 17 (pm) I July 18719 | July 17 (am) I July 17/18 I I 9459 I did not turm red I July 18/19 I JBGrll: | July 22 (am) I July 22/23 I July 22 (am) I July 22/23 | I 9022 I July 23 (am) j July 22/23 I partial July 22 j July 20 (pm) I Jury 21 (am) | July 21/22 |  | | | I I  |  8923  |  3  July 21/22 July 22/23 July 22/23 July 22/23  8  i Not Pregnant | Pregnant I Confimied  |  Pregnant  Pregnant Pregnant  | j  I Not Pregnant | Confirmed | Not Pregnant I Not Pregnant I Not Pregnant | Confirmed  cC  1  Pregnant  |  Pregnant  Pregnant Pregnant  |Resynchronized? 1135 day preg check |(AI Sept 18/19) !  CO  I I I I II I  CN >. 3  co  July 17/18 July 18/19 July 17/18 July 18/19 July 17/18 July 18/19  | Aug 6/7  •  m  July 22 (am) July 20 (pm) July21(pm) July 22 (am) July 22 (am) July 21 (pm)  >• 3  c- c- c-  I | I I II I  •  |  I I c-  July 17 (am) July 17 (pm) July 16 (am) July 17 (pm) July 17 (am) July 18 (am)  •  Aug 8th  {Animals pregnant from 2nd Al  5  I I | 9435 I I I 9408 I I 9452 I JBGrll: I | 9436 |I | 9023 | I | 9025 | I | 9438 | I I 67022 jI  red kamar red kamar red kamar red kamar  I | | |  July 22/23 July 22/23 July 22/23 July 21/22  j j j I  I  j Confirmed | Pregnant | Not Pregnant I Confirmed | Not Pregnant | Confirmed  Pregnant  •  I  j 35 day preg check  126 day preg check  [2nd Al  CD  |BGrl:  red kamar Standing heat  |Return to heat?  I July 18/19 July 17/18 I July 18/19 July 18/19 July 18/19  to  | j | I |  |Date of Al  JBreeding summary for each treatment group.  mucus discharge - Nov 14th  |  I  I j  !  rebred - Pregnant  I  embryonic mortality - rebred • not pregnant] |  I | |  I  Comments  148  •• •  July 22/23 _j July 21/22 I July 22/23 I July 22/23 1 July 22/23  3  3  3  S  oco  at  co US  CO  's  OCO *  CB  E  o"  s  ca caE  w> or  V-  3  m m  3 •  (D lO  3 c*-  •  •  •  r»-  <N  3  o  •  Pregnant Pregnant j j  | | | | I  Not Pregnant Not Pregnant Confirmed Confirmed Pregnant  9455  9009 9444  3  red kamar red kamar  | j j j  Not Pregnant Confirmed Not Pregnant Confirmed Not Pregnant Not Pregnant  9460 9450  CM  • • •  3 3  m I  •  • •  s  CO  eS  8  O cn n  c  i  CM •*  3  BGrI = synchronization by two prostaglandin injections BGr2 = synchronization by GnRH followed bv prostaglandin  | |  • c*-  co Ul  6006 |  in •  s  60008 |  1 • CD  red kamar  cPregnant Pregnant  j  |  3  red kamar  p- o Pregnant Pregnant  | ] |  Pregnant Not Pregnant Pregnant Confirmed Pregnant Not Pregnant  88002  CM  Spares (50-60 mg/kg Mo, 8-10 mg/kg Cu) BGrI: I did not turn red I July 16/19 I red * date unknown j July 17/18 | 84027 I red - date unknown J July 18/19 red - date unknown I July 18/19 I  • •  Aug 6/7  | | | | | |  Not Pregnant Confirmed Not Pregnant Not Pregnant Not Pregnant Not Pregnant 3  red kamar  j "» co  July 22/23 Jury 22/23 July 22/23 July 22/23 . July 22/23 July 21/22 ;  •  I  •  •  July 18/19 ! July 18/19 July 17/18 July 18/19 July 18/19 July 18/19  Pregnant O"  1 I July 18 (am) red - date unknown I July 17(am) I July 17 (pm) July 18 (am) July 17 (am) BGrll: I did not turn red I 9443 I July 22 (am) I 9458 I July 21 (pm) I 8924 I July 21 (am) did not turn red July 21 (am)  | Aug 7tfi | | | Aug 6/7 I •  mounting red kamar red kamar occ  BGrl: 9437 i 87004 i 9423 9433  1 I I I j 1 o|  did not turn red July 20 (am) partial July 22 July 22 (am) July 22 (am) July 22 (am) CO  60009  BGf Ii: 1 86002 1 R03 I  | |  |  Pregnant Pregnant  Pregnant Pregnant Not Pregnant Pregnant  Not Pregnant  Not Pregnant  Pregnant Pregnant Not Pregnant Pregnant  | I  ] I  | I  Rebred - Pregnant  Rebred - Pregnant  |  |  embryonic mortality - not pregnant |  149  •  Statistical analysis of the number of pregnant heifers after 1 insemination (arcsine transformation). TR1 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0 0 0 0 0 0  TR2 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0 0 0  TR3 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0 0 0 0 0 0 0  TR4 1 (90) 1 (90) 1 (90) 1 (90) 0 0 0 0 0 0 0  TR5 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0 0 0 0 0 0  1 = pregnant 0 = not pregnant TR arouD TR1 TR2 TR3 TR4 TR5  Total Number of Preanant Heifers 6/12 9/12 5/12 4/11 6/12  Analvsis of Variance: df 4 54 58  TR E TOT F(calculated) F(4,54)  =  _  1  SS 8372.5 111068.2 119440.7  MS 2093.12 2056.82  -02  2.54  therefore there are no significant differences between the treatment groups.  151  Statistical analysis of the number of pregnant heifers after 2 inseminations (arcsine transformation). TR2 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0  TR1 1 (9G) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90)  TR3 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0 0  TR4 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0 0 0  TR5 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0 0 0  1 = pregnant 0 = not pregnant TR arouD TR1 TR2 TR3 TR4 TR5  Total Number of Preanant Heifers 12/12 11/12 10/12 7/11 9/12  Analvsis of Variance: df 4 54 58  TR E TOT ' (calculated) f"(4,54)  =  SS 7503.01 59768.18 67271.19  MS 1875.75 1106.82  '• 2.54  • therefore there are no significant differences between the treatment groups.  152  Statistical analysis of the number of pregnant heifers after 3 inseminations (arcsine transformation) TR1 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90)  TR2 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90)  TR3 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0  TR4 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0 0  TR5 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 1 (90) 0  1 = pregnant 0 = not pregnant TR group TR1 TR2 TR3 TR4 TR5  Total Number of Pregnant Heifers 12/12 12/12 11/12 9/11 11/12  Analysis of Variance: df 4 54 58  TR E TOT ^"(calculated) ^"(4,54)  -  -  ^  SS 2098.84 28104.55 30203.39  MS 524.71 520.45  -01  2.54  • therefore there are no significant differences between the treatment groups.  153  Statistical analysis of the number of inseminations required for pregnancy to occur. TR1 1 1 2 1 2 1 2 1 2 2 2 1  TR aroup TR1 TR2 TR3 TR4 TR5  TR2  Mean 1.5 1.3 1.6 1.8 1.6  TR3 2 1 3 1 2 1 1 2 1 2 2  TR4 3 1 1 1 2 1 2 3 2  TR5 1 3 1  SD 0.52 0.65 0.67 0.83 0.81  Analysis of Variance: df TR E TOT F (calculated) F(4,50) =  4 50 54  SS 1.11 24.73 25.84  MS 0.28 0.48  0.58  =  2.56  • therefore there are no significant differences between the treatment groups.  1 1 2 1 2 1 3 2  154  Statistical analysis of the gestation length* (days). TR1 282 287 280 283 254 287 282 277 289 287 278 265  TR2 285 285 285 280 287 279 278 286 274 290  TR3 281 278 289 281 277 282 288 283 276 286  TR4 292 280 282 278 276 275 276 287  TR5 284 282 288 285 286 281 288 282 282 282  includes calves born both dead and alive TR qroup TR1 TR2 TR3 TR4 TR5  Mean 279.25 283.33 282.10 280.75 2.84.00  SD 10.23 4.68 4.48 6.02 2.62  Analysis of Variance: df 4 47 51  TR E TOT F(calculated)  F 4 47) (  =  1  SS 163.99 2053.31 1889.32  MS 41 40.2  -02  = 2.57  • therefore there are no significant differences between the treatment groups.  155  Statistical analysis of calf birth weights TR1 40 48 35 38 36 39 30 37 37 57 30  TR2 42 37 43 38 38 36 33 37 41 49  Mean 38.8 39.4 38.7 36.2 41.6  TR a roup TR1 TR2 TR3 TR4 TR5  TR3 45 32 40 38 31 44 45 45 29 38  TR4 24 36 35 37 34 30 56 38  TR5 46 52 42 40 45 36 37 40 35 43  SD 7.76 4.27 6.22 9.18 5.19  Analysis of Variance: df 4 45 49  TR E TOT F(calculated) = F  (4,45) =  SS 130.63 2014.19 2144.82  MS 32.66 44.76  0-73  2.58  • therefore there are no significant differences between the treatment groups.  V  156  Statistical analysis of the length of the post-partum period (days). TR1 49 56 63 51 49 37 50 54  TR qroup TR1 TR2 TR3 TR4 TR5  TR2 53 61 47 52 54 69 52 53 59 52 55 32  Mean 51.13 53.25 49.70 50.17 54.00  TR3 56 45 49 35 51 54 34 63 67 43  TR4 56 48 71 45 46 35  TR5 61 67 65 68 57 41 33 40  SD 7.40 8.74 10.88 12.22 13.89  Analysis of Variance: df 4 39 43  TR E TOT '"(calculated) F 4,39) = (  SS 127.67 4386.06 4513.73  MS 31.92 112.46  -0.28 2-61  therefore there are no significant differences between the treatment groups.  157  Statistical analysis of the comparison of post-partum progesterone levels (ng/ml) (from the first CL)  TR1 5.960 5.362 1.354 8.236 7.832 7.068 3.109 8.392  TR2 4.231 5.511 4.772 5.510 6.934 4.824 3.184 5.762 5.874 3.970 6.867  Mean 5.91 5.22 4.69 4.36 3.32  TR aroup TR1 TR2 TR3 TR4 TR5  TR3 2.840 5.319 4.408 5.599 6.018 1.734 5.627 5.934  TR4 6.503 1.542 5.771 2.158 5.830  TR5 3.385 1.371 1.555 4.210 6.362 4.329 2.058  SD 2.550 1.166 1.588 2.320 1.806  Analysis of Variance: df 4 34 38  TR E TOT '"(calculated) F<4,34) =  SS 28.20 117.85 146.06  MS 7.05 3.47  -2.03 2.65  • therefore there are no significant differences between the treatment groups.  158  Statistical analysis of the mean number of corpora lutea before ova recovery for heifers in Trial III TR1 13 14 13  TR2 + TR3 12 11 14 10  TR4 + TR5 7 12 5 12 6 13 6  TR1 - control TR2 + TR3 - low Mo TR4 + TR5 - high Mo  TR group TR1 TR2+TR3 TR4 + TR5  Mean 13.3 11.8 8.7  SD 0.58 1.71 3.45  Analysis of Variance: df 2 11 13  TR E TOT F (calculated) F  (2,11)  =  SS 52.58 80.85 133.43  MS 26.29 7.35  3.58  =  3.98  • therefore there are no significant differences between the treatments.  159  Statistical analysis of the mean number of embryos recovered from heifers in Trial III TR1 0 1 0  TR2 + TR3 8 6 4  TR4 + TR5 3 5 1 1 0 1 7 3  TR1 - control TR2 + TR3 - low Mo TR4 + TR5 - high Mo  TR group TR1 TR2 + TR3 TR4 + TR5  Mean 0.33 4.75 2.87  SD 0.58 2.99 2.48  Analysis of Variance: df TR E TOT  2 11 13  f" (calculated) F  (2,11)  =  SS 33.44 64.27 97.71  MS 16.72 5.84  2.86  =  3.98  • therefore there are no significant differences between the treatments  160  APPENDIX VI: MINERAL CONCENTRATIONS Liver biopsies were done three times during the course of the study to monitor the levels of Mo and Cu within the heifers. Plasma samples from the same time were also analyzed. There were significant differences between the treatment groups but these appeared to reflect the intake levels of the mineral. It is unlikely that there were any interactions between Mo and Cu.  161  Summary of Mineral Analysis  Dec '95 - Liver Mo (mg/kg DM)  TR1 1.78 0.27 0.33 1.96 2 1.1 1.29 1.24 0.77 0.9  TR E Total  TR2 1.78 0.96 0.56 0.39 2.11 1.6 0.66 0.31 0.84  TR3 2.49 0.97 0.9 0.66 0.64 0.52 1.49 1.35 0.41 0.34 0.31 0.2  TR4 1.12 2.92 0.85 0.47 0.65 2.7 0.71 2.4 2.64 1.74  TR5 2.95 1.97 2.21 3.27 2.45 0.5 2.27 2.84 2.43  df 4 45 49  SS 12.46 26.25 38.71  MS 3.12 0.58  F 5.38  mean SD TR1 1.164 0.6198064 TR2 1.0233333 0.6500192 TR3 0.8566667 0.6552215 TR4 1.62 0.9691921 TR5 2.3211111 0.7943778  F4,45=2.58  there are significant differences between the treatments  Dec '95 - Plasma Mo (mg/L)  TR1 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03  TR2 0.03 0.03 0.03 0.05 0.03 0.03 0.03 0.03 0.05 0.03 0.03 0.03  TR3 0.05 0.03 0.03 0.03 0.03 0.05 0.03 0.03 0.03 0.05 0.03  TR4 0.03 0.04 0.03 0.03 0.05 0.05 0.03 0.03 0.03 0.03 0.03  TR5 0.03 0.03 0.03 0.03 0.05 0.03 0.03 0.03 0.03 0.03 0.03 0.03  TR E Total  df 4 53 57  SS 0.0002 0.0026 0.0028  MS 0.00005 0.0005  F 0.1  TR1 TR2 TR3 TR4 TR5  mean SD 0.03 0 0.0333333 0.007785 0.0354545 0.009342 0.0345455 0.008202 0.0316667 0.0057735  F4,53=2.55  there are no significant differences between the treatments  162  Apr '96 - Liver Mo (mg/kg DM) TR1  TR2  TR3  TR4  TR5  2.36  3.75  3.8  6.51  20.37  TR1  3.01  3.36  1.06  4.27  23.11  3.51  1.94  4.05  6.02  17.11  2.51  2.25  3.72  6.59  2.92  1.84  3.37  3.47  3.69  5.61  9.66  2.61  4.16  4.57  10.6  3.01  4.09  5.48  18.17  2.28  4.33  5.84  21.59  3.08  4.27  5.92  17.49  3.21  5.02  5.07  20.13  '  7.69  mean  S D  2.9063636  0.4197445  TR2  2.805  0.8912631  TR3  3.9527273  1.0943117  18.93  TR4  6.1472727  1.5108547  12.27  TR5  17.930909  3.7272147  17.47  3.5  df  SS  MS  F  TR  4  1726.21  431.55  141.96  E  46  139.79  3.04  Total  50  1866  F4.46=2.58 there are  significant  differences  between  the treatments  Apr '96 - Plasma Mo (mg/L) TR1  TR2  TR3  TR4  TR5  0.03  0.03  0.03  1.56  8.7  TR1  0.0333333  0.007785  0.03  0.03  0.03  0.69  12  TR2  0.03  0  0.03  0.03  0.03  0.72  4.2  TR3  0.03  0  0.03  0.03  0.03  1.49  7.8  TR4  0.6672727  0.5214996  0.05  0.03  0.03  0.03  6.3  TR5  7.85  2.4832164  0.03  0.03  0.03  0.51  6.6  0.03  0.03  0.03  0.06  8.1  0.03  0.03  0.03  0.45  4.2  0.05  0.03  0.03  0.54  6.3  0.03  0.03  0.03  1.08  10.2  0.03  0.03  0.03  0.21  8.7  0.03  0.03  mean  11.1  df  SS  MS  F  TR  4  562.8  140.7  105.79  E  53  70.54  1.33  Total  57  633.34  F4.53=2.55 there are  significant  differences  between  the treatments  S D  163  Mar ' 9 7 - Liver Mo (mg/kg DM) TR1  TR2  TR3  TR4  TR5  2.58  6.34  6.75  19.26  20.91  TR1  3.0872727  1.1482778  mean  S_D_  3.16  6.04  5.37  17  11.74  TR2  8.0881818  1.2034934  2.89  8.76  6.57  18.02  13.18  TR3  8.177  1.763028  3.91  8.45  6.86  19.35  21.17  TR4  19.45375  3.1452637  1.94  9.5  10.24  26.72  17.96  TR5  15.974444  4.0275958  4.16  8.08  8.4  20.07  10.71 13.87  3.92  9.03  8.24  18.35  4.41  9.58  8.9  16.86  3.7  8.35  10.75  2.82  7.96  9.69  0.47  6.88  14.47 19.76  df  SS  MS  F  TR  4  1637.65  409.41  76.1  E  44  236.75  5.38  total  48  1874.4  F4,44=2.58 there are  significant  differences  between  the treatments  Mar ' 9 7 - Plasma Mo (mg/L) TR1  TR2  TR3  TR4  TR5  mean  SJQ  0.39  3  3.6  2.34  6.6  TR1  0.134  0.1192756  0.03  2.52  2.76  4.5  4.8  TR2  3.522  1.0643496  0.05  4.8  2.73  9.3  8.7  TR3  3.411  0.6990064  0.03  3.6  2.91  6.3  8.4  TR4  6.4425  2.5441572  0.03  5.7  3  10.2  7.8  TR5  6.3  1.8384291  0.09  3  3.3  7.5  5.4  0.12  3.3  2.91  5.4  3.6  0.15  3.3  3.9  6  5.4  0.18  3.9  4.2  5.4  0.27  2.1  4.8  6.9  df  SS  MS  F  TR  4  254.88  63.72  32.35  E  43  84.79  1.97  Total  47  339.67  F4,43=2.58 there are  significant  differences  between  the treatments  164  Dec "95 - Liver Cu (mg/kg TR1 TR2 94.08 121.68 92.23 110.83 34.7 82.49 87.01 103.4 79.09 61.8 71.54 120.26 99.19 102.94 129.59 63.27 111.78 38.96 64.07  TR E Total  df 4 45 49  DM) TR3 35.13 46.18 49.64 27.7 61.05 107.62 94.08 84.94 71.14 63.36 53.62 53.04  SS 5493.91 28236.09 33730  Dec "95 - Plasma Cu (mg/L) TR1 TR2 TR3 0.78 0.5 0.93 1.08 0.72 0.75 0.87 0.96 0.69 0.87 0.75 1.26 0.96 0.66 1.17 0.72 0.55 0.9 0.69 0.66 0.78 0.6 0.72 1.11 0.45 0.54 0.9 0.78 0.81 0.65 0.72 0.57 0.66 0.87 0.96  TR E Total  df 4 53 57  SS 0.54 0.73 1.27  TR4 53.25 112.31 82.93 45.12 83.33 85.92 53.25 90.61 20.56 66.55  MS 1373.48 627.47  TR4 0.75 0.4 0.51 0.72 0.45 0.45 0.81 0.69 0.57 0.72 0.69  MS 0.135 0.014  TR5 54.6 49.22 84.56 98.93 61.14 81.85 66.85 89.86 56.96  F  2.19  TR5 0.63 0.75 0.87 0.51 0.45 0.75 0.4 0.69 0.72 0.84 0.54 0.66 F  9.64  TR1 TR2 TR3 TR4 TR5  mean 88.188 87.447778 62.291667 69.383 71.552222  SD 27.372105 28.248576 23.690927 26.761866 17.640607  FM5=2.58  there are no significant differences between the treatments  mean SD TR1 0.8875 0.1574296 TR2 0.7725 0.1658655 TR3 0.6972727 0.2078505 TR4 0.6145455 0.1425737 TR5 0.6508333 0.149451  F4.53=2.55  there are significant differences between the treatments  165  Apr '96 - Liver Cu (mg/kg DM)  TR1 163.26 126.18 125.46 31.99 174.11 100.41 178.17 262.67 188.01 320.2 240.86  TR E Total  TR2 25 89.69 19.38 19.38 0 42.89 184.5  df 4 46 50  TR3 0 0 3.28 2.6 0 12.14 5.2 8.07 12.68 1.22 6.51 1.95  TR4 180.36 292.68 280.26 150.16 208.62 85.15 190.55 303.64 176.47 199.7 233.56  SS MS 571931.1 142982.78 196628.41 4274.53 768559.51  TR5 425.16 130.55 304.92 256.41 435.19 301.37 332.58 225.93 285.52 280.94 246.39  F 33.45  TR1 TR2 TR3 TR4 TR5  mean 173.75636 54.405714 4.4708333 209.19545 293.17818  SD 80.162745 64.005722 4.3763368 65.380646 86.060289  F4,46=2.58  there are significant differences between the treatments  Apr '96 • Plasma Cu (mg/L)  TR1 0.9 0.6 0.75 0.55 1.02 0.65 0.84 0.54 0.66 0.48 0.48 0.99  TR2 0.57 0.48 0.78 0.6 0.69 0.45 0.6 0.39 0.75 0.45 0.81 0.6  TR3 0.15 0.15 0.15 0.27 0.36 0.21 0.15 0.39 0.27 0.15 0.15  TR4 0.81 0.96 1.08 0.99 0.9 0.75 0.85 0.87 1.05 0.93 0.66  TR5 0.75 1.08 1.11 0.57 0.54 0.78 0.81 0.81 1.56 1.05 0.87 0.9  TR E Total  df 5 53 57  SS 3.16 3.17 6.33  MS 0.79 0.06  F .13.17  mean SD 0.1924247 TR1 0.705 TR2 0.5975 0.1386379 TR3 0.2181818 0.0910844 TR4 0.8954545 0.1260447 TR5 0.9025 0.2741972  F4,53=2.55  there are significant differences between the treatments  166  Mar '97 - Liver Cu (mg/kg DM)  TR1 202.36 245.99 132.96 210.02 202.46 260.45 181.53 254.65 216.71 209.68 65.67  TR2 212.69 179.5 105.86 295.8 249.9 124.59 116.62 170.39 158.55 101.28 234.53  TR3 20.69 23.34 11.37 36.96 62.35 43.88 65.91 83.6 13.24 22.85  TR4 208.43 259.72 165.39 223.75 219.55 198.07 232.42 219.22  TR5 259.58 158.84 349.78 257.73 205.84 257.73 179.36 223.57 168.47  TR E Total  df 4 44 48  SS 222257.65 149967.31 372224.96  MS 55564.41 3408.35  F 16.3  TR1 TR2 TR3 TR4 TR5  mean 198.40727 177.24636 38.419 215.81875 228.98889  56.650684 64.488739 24.835995 27.265853 59.989662  F4.44=2.58  there are significant differences between the treatments  Mar '97 - Plasma Cu (mg/L)  TR1 0.87 0.84 0.65 0.87 0.87 0.93 0.99 0.87 0.75 0.81  TR2 0.9 0.69 0.96 0.75 0.81 0.6 0.75 0.84 0.84 0.6  TR3 0.66 0.78 0.75 0.72 0.84 0.75 0.69 0.69 0.66 0.72  TR4 0.69 0.45 0.87 0.84 0.72 0.84 0.93 0.93  TR5 0.6 1.08 0.99 0.78 0.84 0.97 0.99 0.75 0.99 0.72  TR E Total  df 4 43 47  SS 0.13 0.63 0.76  MS 0.03 0.015  F 2.28  TR1 TR2 TR3 TR4 TR5  mean 0.845 0.774 0.726 0.78375 0.871  F4,43=2.58  there are no significant differences between the treatments  SD 0.0937194 0.1198332 0.0562139 0.1607071 0.1549516  

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