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Effects of human chorionic gonadotropin administration at various times following breeding on corpus.. 1990

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EFFECTS OF HUMAN CHORIONIC GONADOTROPIN ADMINISTRATION AT VARIOUS TIMES FOLLOWING BREEDING ON CORPUS LUTEUM NUMBER, DIAMETER, PROGESTERONE PROFILES AND PREGNANCY RATES IN DAIRY CATTLE. by PHARAOH COLLINS SIANANGAMA B.Sc. Agr. (Hons.), University of Alexandria, Egypt, 1986. A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1990 @ Pharaoh C o l l i n s Sianangama, 1990 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 of A M . t v i A c t - <S(M,gN3Cje The University of British Columbia Vancouver, Canada Date Q^ypfeErg- 7, DE-6 (2/88) ABSTRACT Corpus luteum (CL) dysfunction has been implicated among various factors predisposing early embryonic mortality i n c a t t l e . Two experiments were conducted to evaluate the e f f i c a c y of using human chorionic gonadotropin (hCG) given eit h e r at the time of breeding (day 0) , day 7 or 14 post breeding, i n reducing that component of early embryonic mortality caused by CL dysfunction. The aims of experiment 1 were to investigate the effectiveness of using hCG, i n inducing the development of accessory CL, t h e i r formation and growth, and the e f f e c t of such treatments on the function of both the induced and spontaneous CL. Thirty-four l a c t a t i n g Holstein cows were randomly assigned to one of four treatments. A single intramuscular i n j e c t i o n of 1000 IU of hCG was given either at the time of breeding (day 0, n=8), day 7 (n=9) or 14 (n=9) post breeding or no hCG given (control, n=8). A real-time ultrasound machine was used to study f o l l i c u l a r dynamics and CL growth. The CL and antral f o l l i c l e diameter was determined using a b u i l t - i n system of cal i b r a t e d c a l l i p e r s . Ultrasound scanning was carr i e d out on days 7, 9, 11, 14, 16, 18, 21, 28, 35 and 42 post breeding. Blood and milk samples, for progesterone (P4) determination using radioimmunoassay, were co l l e c t e d on days coincident with ultrasonography. Diameter of the CL i s presented as the sum of the diameter of a l l l u t e a l t i s s u e i n each animal. Differences i n CL diameter, milk and plasma P4 were analyzed using the General Linear Models Procedures while pregnancy data were analyzed using Chi-Square analysis i n S t a t i s t i c a l Analysis Systems (SAS, version 6.3). a i Based on the day 7 ultrasound scanning, the incidence of twin ovulations was higher among cows treated on day 0 (3/8) compared to control cows (1/8) and day 7 (1/9). Accessory CL were detected in 7/9 of the day 7-treated cows compared to 4/9 among the day-14 treated cows. Least squares means (LSMeans) for CL diameter were s i g n i f i c a n t l y higher (P<0.001) among cows treated with hCG compared to control cows s t a r t i n g at day 7 continually u n t i l day 42. Plasma P4 p r o f i l e s were s i g n i f i c a n t l y higher (P<0.05), at days 18, 35 and 42, i n cows treated on day 7 or 14 compared to control cows. The f i r s t detectable differences (P<0.05) between hCG treated and control cows, i n milk P4 occurred at day 21 and persisted u n t i l day 42. Pregnancy rates were highest among cows treated with hCG on day 7 where 6 of the 9 cows were diagnosed pregnant. Corresponding pregnancy rates for day 0, 14 or control cows, were 4/8, 5/9 and 3/8, respectively. In the second experiment, two t r i a l s were conducted at two d i f f e r e n t farms to investigate the e f f i c a c y of using hCG to increase milk P4 and pregnancy rates. In t r i a l one, 79 l a c t a t i n g Holstein cows were exposed to the treatment protocol described i n experiment 1. In addition to the milk sample c o l l e c t i o n schedule given i n experiment 1, a sample was c o l l e c t e d on day 0. Milk samples were stored at 4°C and l a t e r transported to the UBC laboratories for P4 analysis. LSMeans for milk P4 concentrations were d i f f e r e n t only at days 16 and 18 post breeding. Pregnancy rates were improved (P<0.01) by hCG treatments. The respective pregnancy rates for cows receiving hCG on day 0 (n=20), 7 (n=20), 14 (n=20) or control (n=19) were 25, 35, 35 and 21 %. i i In the second t r i a l , 121 l a c t a t i n g Holstein cows were randomly assigned to treatments as described e a r l i e r . Weekly milk samples were c o l l e c t e d from each animal and assayed for P4 as described above. LSMeans for milk P4 were s i g n i f i c a n t l y d i f f e r e n t (P<0.05) among groups s t a r t i n g at day 14 u n t i l day 4 2 post breeding. hCG increased pregnancy rates over control cows. The pregnancy rates for cows treated on day 0, 7, 14 and control were 31, 50, 41 and 26 %, respectively. In conclusion, t h i s study revealed that treatment with hCG induced accessory CL development, increased P4 production and improved pregnancy rates. I t i s evident, too, that treatment with hCG on day 7 post breeding may have greater p o t e n t i a l for improving pregnancy rates not only i n dairy and beef c a t t l e but equally b e n e f i c i a l to the embryo transfer programmes. Increased pregnancy rates confirm the hypothesis that CL dysfunction does cost the live s t o c k industry appreciable losses i n embryos. i i i TABLE OF CONTENTS page ABSTRACT i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES v i i i LIST OF PLATES ix LIST OF APPENDICES X ACKNOWLEDGEMENTS x i 1. INTRODUCTION 1 2. LITERATURE REVIEW 3 2.1. Extent and Timing Of Embryonic Mortality 3 2.2. Factors Predisposing Early Embryonic Mortality 5 2.3. Role Of Progesterone In Gestation 11 2.4. Methods For Reducing Early Embryonic Mortality 12 2.4.1. Hormone Replacement Therapy 13 2.4.2. Uterine Infusions 13 2.4.3. Use Of Gonadotropins 15 2.4.4. Use Of Gonadotropin-Releasing Hormone 17 2.5. F o l l i c u l a r Dynamics In The Cow 19 3. OBJECTIVES AND RATIONALE 2 0 3.1. OBJECTIVES 21 3.1.1. Experiment One 21 3.1.2. Experiment Two 21 3.2. RATIONALE 21 3.2.1. Day 0 (Oestrus) 21 3.2.2. Day 7 Post Oestrus 22 3.2.3. Day 14 Post Oestrus 22 4. Experiment one: E f f e c t of hCG Administration At Various Times Following Breeding on CL Number, Diameter and Progesterone P r o f i l e s i n Dairy Cattle 23 4.1. MATERIALS AND METHODS 2 3 4.1.1. Animals: General Management Practices 23 4.1.2. Treatments 24 4.1.3. Ultrasound Examination 24 4.1.4. Blood and Milk Sample C o l l e c t i o n 29 iv 4.1.5. Radioimmunoassay (RIA) 29 4.1.6. Radioimmunoassay K i t Validation 31 4.1.7. Pregnancy Diagnosis 3 2 4.1.8. S t a t i s t i c a l Analysis 32 5. RESULTS 33 5.1. Induction Of Accessory Corpora Lutea 3 3 5.2. Growth Of The Corpora Lutea 40 5.3. Progesterone P r o f i l e s 44 5.3.1. Plasma Progesterone 44 5.3.2. Milk Progesterone 47 5.3.3. E f f e c t Of hCG On F e r t i l i t y 51 6. Experiment two: E f f e c t of hCG Administration At Various Times Following Breeding on Progesterone P r o f i l e s and Pregnancy Rates i n Dairy Cattle 53 6.1. MATERIALS AND METHODS: FARM # 1 53 6.1.1. Animals: General Management Practices 53 6.1.2. Treatments 55 6.1.3. Milk Sample C o l l e c t i o n 55 6.1.4. Pregnancy Diagnosis 56 6.1.5. S t a t i s t i c a l Analysis 56 6.1.6. RESULTS : FARM # 1 56 6.1.7. Milk Progesterone P r o f i l e s 56 6.1.8. Pregnancy Rates 60 6.1.9. E f f e c t Of hCG on Cycle Length and Exhibition Of Oestrus 61 6.2. MATERIALS AND METHODS: FARM #2 64 6.2.1. Animals: General Management Practices 64 6.2.2. Treatments 66 6.2.3. Milk Sample C o l l e c t i o n 66 6.2.4. Pregnancy Diagnosis 66 6.2.5. S t a t i s t i c a l Analysis 66 6.2.6. RESULTS : FARM # 2 67 6.2.6.1. Milk Progesterone P r o f i l e s 67 6.2.6.2. Pregnancy Rates 71 6.2.6.3. E f f e c t Of hCG on Cycle Length and Exhibition Of Oestrus In Dairy Cows 71 v 7. DISCUSSION 74 7.1. Induction Of Accessory Corpora Lutea 74 7.2. Growth Of Corpora Lutea 77 7.3. Progesterone P r o f i l e s 78 7.4. Pregnancy Rates 81 7.5. E f f e c t Of hCG on Cycle Length and Exhibition Of Oestrus In Dairy Cows 87 8. SUMMARY AND CONCLUSIONS 93 9. BIBLIOGRAPHY 95 v i LIST OF TABLES Table Page 1. O v u l a t o r y R e s p o n s e O f Cows T r e a t e d W i t h hCG E i t h e r A t T h e T i m e O f B r e e d i n g , Day 7 o r 14 P o s t B r e e d i n g I n C o m p a r i s o n T o C o n t r o l Cows 3 6 2 . E f f e c t O f Human C h o r i o n i c G o n a d o t r o p i n T r e a t m e n t G i v e n E i t h e r A t T h e T i m e O f B r e e d i n g , Day 7 o r 14 P o s t B r e e d i n g , On C o r p u s L u t e u m D i a m e t e r I n H o l s t e i n D a i r y Cows (UBC) 42 3 . E f f e c t O f Human C h o r i o n i c G o n a d o t r o p i n T r e a t m e n t G i v e n E i t h e r A t T h e T i m e O f B r e e d i n g , Day 7 o r 14 P o s t B r e e d i n g , On P l a s m a P r o g e s t e r o n e C o n c e n t r a t i o n s I n H o l s t e i n D a i r y Cows (UBC) 45 4 . E f f e c t O f Human C h o r i o n i c G o n a d o t r o p i n T r e a t m e n t G i v e n E i t h e r A t T h e T i m e O f B r e e d i n g , Day 7 o r 14 P o s t B r e e d i n g , On M i l k P r o g e s t e r o n e C o n c e n t r a t i o n s I n H o l s t e i n D a i r y Cows (UBC) 49 5 . E f f e c t O f Human C h o r i o n i c G o n a d o t r o p i n T r e a t m e n t G i v e n E i t h e r A t T h e T i m e O f B r e e d i n g , Day 7 o r 14 P o s t B r e e d i n g , On M i l k P r o g e s t e r o n e C o n c e n t r a t i o n s I n H o l s t e i n D a i r y Cows ( F a r m # 1) 58 6 E f f e c t o f Human C h o r i o n i c G o n a d o t r o p i n T r e a t m e n t G i v e n E i t h e r A t T h e T i m e o f B r e e d i n g , Day 7 o r 14 P o s t B r e e d i n g , On P r e g n a n c y R a t e s I n H o l s t e i n D a i r y Cows 60 7 . E f f e c t O f Human C h o r i o n i c G o n a d o t r o p i n T r e a t m e n t G i v e n E i t h e r A t T h e T i m e O f B r e e d i n g , Day 7 o r 14 P o s t B r e e d i n g , On M i l k P r o g e s t e r o n e C o n c e n t r a t i o n s I n H o l s t e i n D a i r y Cows ( F a r m # 2) 69 v i i LIST OF FIGURES Figure Page 1. E f f e c t s Of Human Chorionic Gonadotropin On The Growth Of The CL In Dairy Cattle (UBC) 43 2. E f f e c t Of Human Chorionic Gonadotropin On Plasma Progesterone Concentrations In Dairy Cattle (UBC) 46 3. E f f e c t Of Human Chorionic Gonadotropin On Milk Progesterone Concentrations In Dairy Cattle (UBC) 50 4. E f f e c t Of Human Chorionic Gonadotropin On Milk Progesterone Concentrations In Dairy Cattle (Farm # 1) 59 5. E f f e c t Of Human Chorionic Gonadotropin On The Length Of The Oestrous Cycle In Dairy Cattle 63 6. E f f e c t Of Human Chorionic Gonadotropin On Exhibition Of Oestrus In Dairy Cattle 63 7. E f f e c t Of Human Chorionic Gonadotropin On Milk Progesterone Concentrations In Dairy Cattle (Farm # 2) 70 8. E f f e c t Of Human Chorionic Gonadotropin On The Length Of The Oestrous Cycle In Dairy Cattle. 7 3 9. E f f e c t Of Human Chorionic Gonadotropin On Exhibition Of Oestrus In Dairy Cattle 73 v i i i LIST OF PLATES P late Page 1. Ultrasound image of a uterine horn and ovary of a cow (85022). Note CL (echogenic) on rig h t ovary (arrow on r i g h t screen) and an embryo proper (echogenic) enclosed within embryonic v e s i c l e s (arrow on the l e f t screen) 28 2. (a) Twin ovulations (arrows on rig h t screen) i n animal # 86011 treated with HCG on day 0. No apparent difference between the two CL 37 (b) Spontaneous twin ovulations (arrow on l e f t screen) in animal # 83010 37 3. (a) Cystic f o l l i c l e (arrow on l e f t screen) i n animal # 86016 treated with HCG on day 0 38 (b) Cystic f o l l i c l e (arrow on l e f t screen) i n animal # 85001 (control) 38 4. (a) Day 28 images of rig h t ovary (right screen) and righ t uterine horn ( l e f t screen). Note both CL present (arrows on rig h t screen), and the embryo proper (arrow on l e f t screen) 39 (b) Day 70 image of rig h t ovary (right screen) and a section of the rig h t uterine horn. Note both CL are s t i l l present (arrows on rig h t screen) 39 i x LIST OF APPENDICES Appendix Page 1. Analysis of variance for Corpus Luteum diameter among animals receiving hCG either on day 0, 7, or 14 post breeding i n comparison to control dairy cows UBC South Campus I l l 2. Analysis of variance for Plasma Progesterone concentration among animals receiving hCG either on day 0, 7, or 14 post breeding i n comparison to control dairy cows at UBC South Campus 112 3. Analysis of variance for Milk Progesterone concentration among animals receiving hCG either on day 0, 7 or 14 post breeding i n comparison to control dairy cows at UBC South Campus 113 4. Analysis of variance for Milk Progesterone concentration among animals receiving hCG eithe r on day 0, 7 or 14 post breeding i n comparison to control dairy cows at Farm # 1 114 5. Analysis of variance for Milk Progesterone concentration among animals receiving hCG either on day 0, 7 or 14 post breeding i n comparison to control dairy cows at Farm #2 115 6a. Number of animals (n) used i n the s t a t i s t i c a l analyses at each respective time following breeding at the UBC South Campus 116 6b. Number of animals (n) used i n the s t a t i s t i c a l analyses at each respective time following breeding on Farm # 1 116 7. Number of animals (n) used i n the s t a t i s t i c a l analyses at each respective time following breeding on Farm #2 117 x ACKNOWLEDGEMENTS I wish to a v a i l myself t h i s opportunity to express my most sincere gratitude to my supervisor, Dr. R. Rajamahendran, for the academic guidance that I came to know as a student under his supervision. His s p i r i t of understanding and the encouragement that I received from him, w i l l always be remembered. My special thanks are also due to the National Council for S c i e n t i f i c Research (Zambia) for providing the funds that made i t possible for me to pursue further studies. The thesis research project was conducted using monies from the Dean's Fund, and t h i s support i s greatly acknowledged. I remain indebted, too, to the management at the collaborating farms for making i t possible for me to conduct my thesis project at the respective farms. The tremendous amount of assistance rendered by the technicians and milkers at the South Campus (UBC) and the collaborating farms, i s greatly appreciated. Thanks also go to the summer students, Marina and Joanne, for help with the radioimmunoassays. Various other people that I have had the pleasure of int e r a c t i n g with during my stay at the UBC deserve my most sincere thanks i n a special way. Notable among these were colleagues i n the Department of Animal Science and members of the Zambian Community in Vancouver. Lastly, but not least, I wish to thank my family for the tremendous amount of support, perseverance, s a c r i f i c e and understanding, a l l of which virtues were never wanting during the course of my studies. x i This thesis i s dedicated to the l a t e MUNYAMA S. SYANANGAMA (R.I.P.) You are greatly missed. x i i 1. I N T R O D U C T I O N In mammals, the establishment and maintenance of early pregnancy depends on progesterone (P4) which i s secreted by the corpus luteum (CL). This support must continue beyond the normal duration of an oestrous cycle. In c a t t l e , r e s u l t s obtained with either natural mating or a r t i f i c i a l insemination (AI) f a l l short of the expected pregnancy rates. Hammond, (1921) suggested that the d i s p a r i t y between the expected and observed pregnancy rates may be due to the demise of embryos or fetuses p r i o r to p a r t u r i t i o n . Several workers have attempted to elucidate the causative factors predisposing losses i n reproductive e f f i c i e n c y . Heap, (1985) wrote "...we are far from understanding which one of these factors i s the main causative agent predisposing the demise of embryos early i n gestation..." A considerable number of animals f a i l i n g to maintain t h e i r pregnancies tend to have low P4 concentrations (Bulman and Lamming, 1978). I t has been suggested that i n these cases, the CL may not be functioning at an optimal l e v e l . Hence considerable attention has focused on the e f f e c t s of an inadequate CL function (Randel et a l . , 1971; Erb et a l . , 1976; Bulman and Lamming, 1978). Equally, research e f f o r t s have gone into finding solutions to overcome such factors as an inadequate CL function (Hansel et a l . , 1976; Wagner et a l . , 1973 ; McDermott et a l . , 1986). Consequently, a number of options to resolve t h i s problem have been proposed. Unfortunately, r e s u l t s obtained with any of the various treatment regimen developed thus far, have not conclusively demonstrated that such measures are successful i n improving reproductive e f f i c i e n c y in c a t t l e . 1 The purpose of t h i s study was to attempt to reduce that component of embryonic mortality that may be attributed to an inadequate CL function. To achieve t h i s , i t was postulated that supplementing endogenous P4 production can be achieved much more e f f i c i e n t l y by inducing the development of accessory CL. I t was desired that suitable f o l l i c l e s be present during the times we proposed to administer the treatments. Taylor and Rajamahendran (1990) reported that in the Holstein, as i n other breeds of c a t t l e , waves of f o l l i c u l a r growth that are a common feature i n c y c l i c cows, are also present i n early pregnancy. At the l e v e l of ultrasound scanning, no differences could be found between the dominant f o l l i c l e of the l u t e a l phase and that present during the f o l l i c u l a r phase. The dominant f o l l i c l e of the l u t e a l phase possess receptors to gonadotropins and have been shown to bind the same (Ireland and Roche, 1983). I t was hypothesised that the dominant f o l l i c l e present during early pregnancy i s capable of being induced to ovulate. Under normal physiological conditions, such ovulations are brought about by a surge of the gonadotropin l u t e i n i z i n g hormone (LH). I t was, therefore, presumed that i f animals were challenged with an ovulatory dose of Human Chorionic Gonadotropin (hCG), the dominant f o l l i c l e would ovulate. Giving the same dose at the time of breeding was taken to be an assurance that the dominant f o l l i c l e present at oestrus would ovulate. Being a luteotropin, i t seemed reasonable to expect that giving hCG would augment the function of the spontaneous CL. 2 2 . LITERATURE REVIEW In c a t t l e , acceptable l e v e l s of production dic t a t e that cows in t h e i r reproductive l i f e conceive every 12-13 months. This demands that cows be rebred and est a b l i s h pregnancy by 60 days post partum (Nakao et a l . , 1983). A va r i e t y of hurdles, however, stand i n between the e f f o r t s of farmers and the r e a l i z a t i o n of these goals. F i r s t among the obstacles to successful reproduction i s the f a i l u r e of some f o l l i c l e s to ovulate. The incidence of t h i s problem i s not known. I t has been estimated that f e r t i l i z a t i o n rates in the cow range from 88-90 % (Linares, 1982; Sreenan and Diskin, 1985). 2 . 1 . Extent And Timing Of Embryonic M o r t a l i t y Pregnancy rates at 60 days post breeding are much lower than the f e r t i l i z a t i o n rates quoted above. These range from 20-84.5 % (Boyd et a l . , 1969; Diskin and Sreenan, 1980; Roche et a l . , 1981). It has been postulated that t h i s d i s p a r i t y between the expected and observed l e v e l of f e r t i l i t y stems mainly from the loss of embryos or fetuses p r i o r to term (Hammond, 1921; Corner, 192 3). Embryonic mortality alluded to i n t h i s thesis, s t r i c t l y interpreted, refers exclusively to losses i n embryos that are experienced during the period extending from conception to the completion of the stage of d i f f e r e n t i a t i o n (Committee on Reproductive Nomenclature, 1972). This stage of development i n the cow i s taken to occur around day 45 post breeding. A recent survey undertaken i n the Fraser Valley of B r i t i s h Columbia over the past two years estimated a 48.3 % pregnancy rate. Assuming a 10 % rate of f e r t i l i z a t i o n f a i l u r e that has been reported to occur i n the cow 3 (Sreenan and Diskin, 1985) t h i s implied a 41.7 % incidence of early embryonic mortality (Rajamahendran et a l . , 1990). This study u t i l i z e d low milk P4 concentrations between days 19 to 21 following breeding as being i n d i c a t i v e of a return to oestrus. I t should be appreciated, therefore, that such an estimate has a tendency to over estimate the actual l e v e l of early embryonic mortality. Such a conclusion i s based on the fact that the milk P4 method does not eliminate losses incurred due to anovulation or f e r t i l i z a t i o n f a i l u r e (Moore, 1985). Embryonic mortality has for long been known to occur i n almost a l l mammalian species (Boyd, 1965; Hanly, 1961; Short, 1979). The extent of t h i s wastage i s greatly influenced by the time at which losses i n embryos are estimated. Counting CL has been used as one means of estimating ovulation rates and based on which embryonic losses are also assessed (Ayalon, 1978). U t i l i z i n g such a method, Ayalon (1978) estimated the incidence of embryonic mortality to range from 25-40 %. Boyd et a l . , (1969) reported an 8 % loss by day 25 post breeding, while Roche et a l . , (1981) estimated the loss at 23 % also taking the estimates at day 25 post breeding. At day 42 post breeding, Ayalon et a l . , (1978) as well as Diskin and Sreenan, (1980) found the incidence to be 2 0 and 4 2 %, respectively. On the other hand, a 5-8 % loss i n embryos was reported by Boyd et a l . , (1969) who took the measurement at 42 days following breeding. Although i t has been suggested that most embryos die between conception and 25-30 days of gestation (Robinson, 1951; Moore et a l . , 1960; Perry and Rowlands, 1962 ; Roche et a l . , 1981; Sreenan and Diskin, 1983) , evidence generated over the l a s t 5-6 years indicates that 75-80 % of these losses take place between days 15- 4 18 post breeding (Sreenan and Diskin, 1985) i e at the time of placental attachment (King et a l . , 1982). The majority of cows losing t h e i r pregnancies p r i o r to day 16 return to oestrus at day 21 with no i n d i c a t i o n of the fact that they were pregnant. It has been shown that animals losing t h e i r pregnancies on or a f t e r day 16, have a s l i g h t extension i n the oestrous cycle length (Northey and French, 1980; Heyman et a l . , 1984). This difference due to the fact that day 14-16 has been established to be the time of maternal recognition of pregnancy i n the cow (Thatcher et a l . , 1989). This loss in embryos early i n gestation, has been reported to cost the beef and dairy industries of the United Kingdom alone amounts well i n excess of £ 300 m i l l i o n per annum ( F l i n t et a l . , 1990). This does not seem to be anywhere near what Bishop (1964) referred to as "...losses i n embryos...at a low b i o l o g i c a l cost...". 2 . 2 . Factors Predisposing E a r l y Embryonic M o r t a l i t y Embryonic mortality was postulated to be nature's way of eliminating impaired genotypes early in gestation (Bishop, 1964). These genetic abnormalities could either be of maternal or paternal o r i g i n , and tend to favour the elimination of any embryos bearing grossly abnormal chromosome complements (Bishop, 1964; Berepubo and Long, 1983). The extent of the losses incurred cannot be accounted for i n f u l l s o l e l y by genetic abnormalities (Edey, 1969; Gayerie, 1983; Moore, 1985). Various other factors have since been postulated to be involved i n the demise of embryos. It has been reported that i n c a t t l e and sheep, twinning rates are lower i n u n i l a t e r a l double ovulaters compared to b i l a t e r a l 5 double ovulaters (Hafez, 1976; White et a l . , 1981; Woods and Ginther, 1983). This indicates that multiple embryos contained in the same horn ( i p s i l a t e r a l ) are more l i a b l e to be l o s t compared to those contained in separate horns (c o n t r a l a t e r a l ) . S i m i l a r l y , in the human being occasionally, twins embryos do not r e s u l t in the b i r t h of baby twins. This has been attributed to the disappearance of one of the p a i r (Landy et a l . , 1982). The pronghorn antelope and South African long-eared elephant shrew are two animal species that ovulate and f e r t i l i z e several ova but only give b i r t h to a s u b s t a n t i a l l y reduced number of o f f - s p r i n g (Mossman and Duke, 1973) . The presence of pathogens in the uterus has also been shown to increase the basal l e v e l of embryonic mortality (Bouters, 1985). The predisposing factors for some of these uterine infections could conditions such as retained placentae (Erb et a l . , 1958) or prolapse of the reproductive t r a c t (Woodward and Quesenberry, 1956). Retained placentae i n dairy c a t t l e can depress pregnancy rates by as much as 40 % (Erb et a l . , 1958; Patterson et a l . , 1981). I t has also been reported that spontaneous asynchrony between the embryo and the uterus (Rowson and Moor, 1966; Wilmut et a l . , 1985; Willingham et a l . , 1986), may also lead to the termination of a pregnancy. The same authors suggested that the phenomenon of asynchrony between the embryo and the uterine milieu could develop spontaneously. Adverse environmental conditions such as high ambient temperature have been reported to increase the incidence of embryonic mortality by acting d i r e c t l y on the embryo ( A l l i s t o n et a l . , 1965; Edey, 1979). A l t e r n a t i v e l y , high ambient temperature may 6 also increase early embryonic mortality by a l t e r i n g the hormonal status of the dam (Thatcher et a l . , 1974). However, for t h i s deleterious factor to take e f f e c t , the body temperature of the dam must increase (Ulberg and Burfening, 1967). An i n t r i n s i c development programme has been postulated to e x i s t i n the bovine embryo. The programme assumes that embryos, in the f i r s t 3-4 days of t h e i r existence, continue growing oblivious of maternal or environmental control. Disruption of t h i s i n t r i n s i c programme for development (Johnson, 1979) has been associated with f a i l u r e of the embryo's a b i l i t y to communicate with the dam (Bazer and F i r s t , 1983) . This, i n turn, has been implicated i n the loss of embryos early i n gestation. The loss of communication with the dam i s indispensable for normal establishment and maintenance of pregnancy. The process involves the elaboration of factors that either serve as a n t i l u t e o l y s i n s or as luteotropins. The l a t t e r group of products are associated with augmentation of CL synthesis and secretion of P4. The q u a l i t y of the feed that an animal i s dependent on has been shown to influence f e r t i l i t y . N utrition, however, exerts i t s e f f e c t s on f e r t i l i t y mostly p r i o r to puberty, around the period of breeding and at p a r t u r i t i o n (Morrow, 1980). Hence elucidating the mechanism by which n u t r i t i o n influences reproduction becomes very d i f f i c u l t . Excess dietary protein has been shown to increase the incidence of embryonic mortality (Rattray, 1977). Short and Bellows (1971) found that fewer heifers were bred, fewer pregnancies established and s t i l l fewer pregnancies maintained during early pregnancy when animals were at lower rates of gain. Anderson and Melamphy in 1972, reported that higher energy l e v e l s i n the diet 7 resulted i n concomitant increase i n both ovulation rates and embryonic mortality. Conversely, Dyck and Strain (1979) could not show any such e f f e c t i n the g i l t . Severe undernutrition and stress (Edey, 1969) have also been shown to increase the incidence of embryonic mortality. Deficiency i n dietary constituents such as minerals (e.g. selenium) has been associated with reproductive f a i l u r e . Inadequate selenium i n the d i e t predisposes the retention of placentae at p a r t u r i t i o n (Trinder and Renton, 1973) . This i n turn, renders the uterus susceptible to i n f e c t i o n which i n turn reduces the chances of an animal establishing and maintaining i t s pregnancy (Bouters, 1985). The necessity for the CL i n the establishment and maintenance of early pregnancy i n domestic animals i s well documented (Henricks et a l . , 1970; Randel et a l . , 1971; Erb et a l . , 1976; Bulman and Lamming, 1978). The endocrine function of the ovary was discovered from s u r g i c a l procedures involving ovariectomy or luteectomy. In either case provision of P4 from an alt e r n a t i v e source was found to maintain pregnancies. Conducting the same experiments but without P4 replacement resulted i n the interruption of pregnancy (Fraenkel, 1903; Amorosso and Perry, 1977). The requirement for t h i s endocrine gland (CL) i n the cow, sow, goat and ewe has been established (Hansel, 1988, review). Given the above facts and observations made regarding the high prevalence of low P4 p r o f i l e s among animals which f a i l to maintain t h e i r pregnancies, a possible contribution of a dysfunctional CL as a causal agent has attracted considerable attention. This l u t e a l phase i n s u f f i c i e n c y i s defined as a l u t e a l phase of normal duration but showing reduced secretion of P4 by the CL of 8 a s p e c i f i c oestrous cycle (diZerega and Hodgen, 1981). It has been reported that there are, indeed, low P4 l e v e l s during early and/or la t e l u t e a l phase of the oestrous cycle i n n o n - f e r t i l e breedings. This phenomenon i s suggestive of an inadequate CL function which has been suspected to occur i n c a t t l e (Henricks et a l . , 1970; Randel et a l . , 1971; Erb et a l . , 1976; Bulman and Lamming, 1978; Lukaszewska and Hansel, 1980; Wilmut et a l . , 1985). Low l e v e l s of c i r c u l a t i n g P4 have been associated with i n f e r t i l i t y i n various species of li v e s t o c k (Bulman and Lamming, 1978). Attempts have been made to t r y and associate concentrations of P4 preceding a p a r t i c u l a r AI (Corah et a l . , 1974; Folman et a l . , 1973) with the incidence of unsuccessful pregnancies. Folman et a l . , (1973) found a p o s i t i v e c o r r e l a t i o n between P4 l e v e l s at 2, 3, 5, 7, 8, 11, 12 and 15 days preceding a p a r t i c u l a r AI and the p r e v a i l i n g conception rates. Similar r e s u l t s were also reported by Holness et a l . , (1977) who found higher milk P4 concentrations on days 7 to 9 p r i o r to a f e r t i l e insemination. Rosenberg et a l . , (1977), on the other hand, found that P4 l e v e l s only reached peak near the time of AI among animals conceiving to that insemination. Contrary to these observations, however, Bulman and Lamming, (197 8) found no re l a t i o n s h i p between pre-service P4 concentrations and conception rates. It has been postulated that such low l e v e l s of P4 lead to the impairment (decrease) i n the rate at which accelerated transport of the embryo into the uterus takes place (Day and Polge, 19 68; Hunter, 1980). Once exposed to such P4 d e f i c i e n t environments, embryos f a i l to develop to the blastocyst stage i n the pig (Murray et a l . , 1971). Si m i l a r l y , i n the mouse (Kirby, 1962), the rabbit 9 (Adams, 1958), the rat (Alden, 1942) and sheep (Wintenberger-Torres and Flechon, 1974) , i t has been reported that the c a p a b i l i t y of these blastocysts to sustain subsequent growth i s impaired. This phenomenon i s observed despite the fact that morulae w i l l develop into the blastocyst stage i n the aforementioned species. Available evidence indicates that there exists a divergence in peripheral plasma P4 concentrations i n pregnant versus n o n - f e r t i l e inseminations or sham-inseminated c y c l i n g cows and that t h i s difference i s evident as early as day 6 through day 14-18 (Henricks et a l . , 1970; Lukaszewska et a l . , 1979; Hansel, 1981). Using rates of return to oestrus, Fonseca et a l . , (1983) found that conception rates at f i r s t AI postpartum increased i n proportion to peripheral concentration of P4 at 12 days p r i o r to that insemination. It has also been reported that mean concentrations of plasma P4, at 48-34 hours p r i o r to the pre-ovulatory LH surge, were higher i n cows that conceived than i n n o n - f e r t i l e inseminations (Erb et a l . , 1976). The same authors also observed that i n animals that conceived, P4 concentrations were higher at 6 days following AI. This c l e a r l y points to an e f f e c t of a dysfunctional CL wherever P4 l e v e l s were low. It i s not clear, however, which single factor i s responsible for the losses i n embryos early i n gestation (Heap, 1985). I t could just be that these factors vary depending on the l o c a l i t y , the l e v e l of management and the environment p r e v a i l i n g at any one given l o c a l i t y . 10 2.3 . Role Of Progesterone In Gestat ion The establishment and maintenance of early pregnancy i n most mammals has been demonstrated to be dependent on P4 o r i g i n a t i n g from the ovary (Randel et a l . , 1971; Erb et a l . , 1976; Bulman and Lamming, 1978) . In mid-gestation (second one h a l f ) , the involvement of the placenta i n P4 production (luteoplacental s h i f t ) ensures the maintenance of pregnancy i n most domestic animals except the sow where the presence of an intact and f u l l y functional CL i s required throughout gestation (Csapo, 1969). The l e v e l s of the hormone must also remain r e l a t i v e l y elevated in order for pregnancy to be maintained (Robinson et a l . , 1989; Heap, 1972) . P4 i s also essential for the establishment and maintenance of a quiescent uterus during pregnancy, thus permitting implantation to continue. The secretions of the endometrial secretory granules and hence the uterine milieu are a l l regulated by P4 ( F l i n t et a l . , 1990). Such a mode of uterine milieu control occurring p r i o r to oestrus, serves to prevent the establishment of an environment h o s t i l e to the embryo (Moore, 1985). In addition to the d i r e c t e f f e c t that P4 exerts on pregnancy, treatment with P4 p r i o r to breeding was found to ra i s e the e f f i c i e n c y of oestrus expression i n cows from 54 to 71 % (Stevenson et a l . , 1989). I t i s also essential for the accelerated transport of embryos from the oviduct into the uterus (Day and Polge, 1968; Hunter, 1980). P4 i s involved i n the regulation of the secretions of the anterior p i t u i t a r y gonadotropin, LH (Robinson et a l . , 1989; Ireland and Roche, 1982; Goodman and Karsch, 1980). LH, on the other hand, i s essential not only for the optimal function of CL, 11 but also the growth and maturation of ovarian f o l l i c l e s . These f o l l i c l e s have, i n turn, been suspected of playing a role i n CL function. Lukaszewska and Hansel (1980) showed that both P4 and Oestradiol-176 (E2) were higher i n pregnant compared to c y c l i c non- pregnant animals. While l e v e l s of P4 were high between days 10-18, le v e l s of E 2, on the other hand, were high between days 6-16 post oestrus. This was assumed to implicate ovarian f o l l i c l e s i n CL function. 2 . 4 . Methods For Reducing Early Embryonic M o r t a l i t y The majority of hypotheses developed for the purpose of addressing the problems imposed by an inadequate CL have mostly evolved around t r y i n g the following options, v i z ; (i) since the assumption i s that the CL i s not functioning optimally, exogenous P4 has been provided i n order to supplement endogenous P4 secretion. ( i i ) b e l i e v i n g that a bovine embryo does influence the secretory capacity of the CL, various laboratories have appropriately concentrated on infusing into the uterus of the cow either viable day 16 embryos or homogenates of embryonic v e s i c l e s , ( i i i ) believing, also that CL function i s dependent on continued support from the anterior p i t u i t a r y secretory products, namely LH, gonadotropins, e.g. human chorionic gonadotropin (hCG) have duly been u t i l i z e d , (iv) since the p i t u i t a r y i s stimulated by secretions o r i g i n a t i n g from higher centres (hypothalamus), an a l t e r n a t i v e rationale has involved the use of gonadotropin releasing hormone (GnRH). 12 2.A.l. Hormone Replacement Therapy Exogenous administration of P4 i n ovariectomized animals has been shown to maintain pregnancy to term (Fraenkel, 1903; Amorosso and Perry, 1977; F l i n t et a l . , 1990). This c l e a r l y emphasised the i n d i s p e n s a b i l i t y of the hormone P4 i n gestation. For t h i s reason, P4 has been administered i n farm animals i n an attempt to t r y and increase c i r c u l a t i n g l e v e l s of P4 such that l e v e l s are elevated to those c h a r a c t e r i s t i c of mid-luteal phase l e v e l s . The use of P4 replacement regimen has, however, had variable e f f e c t s . In studies u t i l i z i n g intact animals, peripheral l e v e l s of P4 were increased i n some t r i a l s (Northey et a l . , 1985; Robinson et a l . , 1989). Conversely, there were also no detectable differences in P4 concentrations between cows given PRIDs either between days 5-12 or days 10-17 post AI. Despite the absence of s i g n i f i c a n t differences at 8 days post AI, there was observed a trend towards higher P4 values i n cows treated with the PRIDs. Endogenous P4 production was attenuated i n animals receiving the treatment between days 10-17 (Robinson et a l . , 1989). However, such an e f f e c t of P4 treatment was not evident i n the t r i a l s of Loy et a l . , (1960) or Zimbelman et a l . , (1959). It was against such a background that Walton et a l . , (1989) concluded that exogenous P4 supplementation may not be a f r u i t f u l approach for overcoming l u t e a l inadequacy i n the cow. 2 . 4 . 2 . Uterine Infusions In the bovine, only embryos i n excess of or equal to 16 days of age have been shown to be luteotropic. Hence one vi a b l e option for extending the duration of the l u t e a l phase and enhancing CL 13 function i n the cow has been the use of embryonic v e s i c l e s or products elaborated by embryos. Proof of the luteotropic e f f e c t of the bovine embryo has since been documented (Betteridge et a l . , 1980). These authors transferred v i a b l e day 16 embryos into r e c i p i e n t cows that had been synchronized to be 16 days post oestrus on the day of transfer. The r e s u l t was maintenance of CL function and establishment of normal pregnancy. S i m i l a r l y , i t was shown that removing day 17-19 embryos r e s u l t s i n the extension of the CL l i f e s p a n (Northey and French, 1980). The same r e s u l t could not be shown i n unmated control or i n animals from which embryos were removed p r i o r to day 15 post breeding. This confirmed e a r l i e r opinions s t a t i n g that day 16 embryos secrete products which influence the e f f e c t of l u t e o l y t i c agent on the CL and augment endogenous P4 secretion. These secretory products can either be a n t i - l u t e o l y t i c or luteotrophic i n function. Uterine infusions of day 16 embryonic v e s i c l e s have since been demonstrated to extend the oestrous cycle i n the cow (Betteridge et a l . , 1980; Northey and French, 1980; Heyman et a l . , 1984). Conceptually, t h i s enables an embryo that i s possibly lagging in development, and hence p o t e n t i a l l y incapable of preventing l u t e o l y s i s , to continue growing u n t i l i t i s capable of secreting a n t i - l u t e o l y t i c or luteotrophic products which, i n turn, permit the establishment of pregnancy (MacCracken et a l . , 1984). Both homogenates and aqueous extracts of the day 16-18 blastocysts have been shown to increase net P4 production by dispersed l u t e a l c e l l s i n culture (Beal et a l . , 1981). The same products could also be deemed as a means for amplifying the signal (bovine trophoblastic protein 1 or bTP-1 i n short) elaborated by 14 the embryo for the purpose of a l e r t i n g the dam of the presence of a conceptus in-utero (Thatcher et a l . , 1989). Clearly, although t h i s method has been shown to increase the duration of the l u t e a l phase and an increase i n the synthesis and secretion of P4, i t s application i n the f i e l d pauses technical problems of i t s own. 2.4.3. Use Of Gonadotropins Al t e r n a t i v e l y , luteotrophic substances such as hCG have been used i n an attempt to augment the function of spontaneously derived CL (C h r i s t i e et a l . , 1979; Holness, et a l . , 1982; Santoz-Valadez et a l . , 1982; Sreenan et a l . , 1979; Veenhuizen et a l . , 1972; Wagner et a l . , 1973). hCG i t s e l f , i s a glycoprotein whose beta sub-unit bears 90 % homology with the corresponding sub-unit of LH. I t i s t h i s beta sub-unit that imparts these hormones with t h e i r respective b i o l o g i c a l a c t i v i t y . Due to t h i s homology, hCG has been shown to mimics the actions and ef f e c t s of LH ( F l i n t et a l . , 1990). Although hCG has been shown to improve l u t e a l function, and to increase peripheral P4 l e v e l s , i t s e f f e c t on pregnancy rates remains controversial (Diskin and Sreenan, 1985). Some reports indicate no b e n e f i c i a l e f f e c t on pregnancy rates r e s u l t i n g from using hCG (Holness et a l . , 1982; Sreenan et a l . , 1979; Wiltbank et a l . , 1961), other workers have shown only a s l i g h t increase in pregnancy rates among treated animals (Santoz-Valadez, et al.', 1982) or no b e n e f i c i a l e f f e c t at a l l (Looney et a l . , 1984). Walton et a l . , (1989) treated cows with hCG (1500 IU) on day 5 post insemination and reported a s i g n i f i c a n t difference i n both plasma and milk P4 p r o f i l e s between hCG treated and control cows. 15 These s i g n i f i c a n t differences were noted a f t e r day 8 for plasma and at days 18 and 2 0 for milk samples. Treatment with hCG as a method of increasing peripheral l e v e l s of P4 was found to be b e n e f i c i a l i n t h i s case. They, however, noted a 3-day extension i n the oestrous cycle length i n n o n - f e r t i l e inseminations. Wiltbank et. a l . , (1961) used 1000 IU of hCG given as d a i l y intramuscular injections s t a r t i n g at day 14 u n t i l 34. They reported a 6 % improvement in pregnancy rates i n cows receiving the treatment. Wagner et a l . (1973) gave 2000 IU of hCG on day 3 and reported an 11 % improvement i n pregnancy rates. Santos-Valadez et a l . , (1982) administered 5000 IU of hCG on day 15 and reported an 11 % increase i n pregnancy rates. In a s i m i l a r experiment, Massey et a l . (1983) administered the same dose 7 days a f t e r embryo transfer and found a 5.7 % improvement in pregnancy rates. Greve and Lehn-Jensen (1982) treated cows with 1500 IU on alternate days s t a r t i n g on day 13 u n t i l day 35 post breeding. Although not s i g n i f i c a n t , hCG treatments increased pregnancy rates by 6 %. Echternkamp and Maurer, (1983) administered 1000 IU of hCG on day 0 and found a 10.2 % reduction i n pregnancy rates. When the treatment was given to twice open cows, a 5.5 % loss in pregnancy rates was detected among cows, but not heifers, treated i n a s i m i l a r manner. Sreenan and Diskin, (1983) administered 1500 IU of hCG between day 10 and 2 0 post oestrus in two t r i a l s . They reported a decrease (11 %) i n one t r i a l , and increase (11 %) i n another. McDermont et a l . , (1986) reported giving, i n one instance, 3300 IU of hCG to cows of low f e r t i l i t y at day 15. They found hCG could lead to a s l i g h t (5.3 %) improvement i n pregnancy rates. In another instance, they administered 3 000 IU of hCG to animals of 16 high f e r t i l i t y . They found a s l i g h t (6.4 %) decrease i n pregnancy rates amongst animals receiving hCG. While i t i s clear that the re s u l t s obtained with the use of hCG i n cows have been very variable, i t i s also quite apparent that we do not seem to be consistent with respect to the dose of hCG administered to animals. The frequency of administering hCG has been as variable as the timing of administering these treatments. One r e a l i s e s and appreciates that the main rationale behind the administration of hCG, at least u n t i l now, has mainly been due to the luteotropic e f f e c t of hCG. 2.4.4. Use of Gonadotropin Releasing Hormone P i t u i t a r y function i s under the control of the hypothalamic decapeptide gonadotropin releasing hormone (GnRH) (Henderson, 1979). Once GnRH i s released or administered exogenously, i t causes the secretion of the gonadotropins from the anterior p i t u i t a r y (Kesler et a l . , 1978). This e f f e c t has, however, been shown to be limit e d to s e l e c t i v e l y inducing the secretion of LH within approximately 2 hours of exposure to GnRH (Cantley et a l . , 1975). Administration of GnRH has been used with the aim of inducing the release of LH from the anterior p i t u i t a r y . Hence, GnRH has also been used i n attempts at improving pregnancy rates i n c a t t l e (Lee et a l . , 1985). It i s apparent, however, that the use of GnRH has produced very variable r e s u l t s (Lewis et a l . , 1990). Nakao et a l . , (1983) administered 100 uq intramuscularly to cows at the f i r s t breeding post partum. They found benefit from using GnRH at breeding on f e r t i l i t y , more so in areas where f e r t i l i t y was already low. In concurrence with these findings, Phatak et a l . , (1986) 17 found a 9.3 % increase i n conception rates due to 111 IU of GnRH treatments administered immediately a f t e r the fourth breeding. Similar r e s u l t s were also reported by Lee et a l . , (1983) but d i f f e r e n t from those reported by Pennington et a l . , (1985). Echternkamp and Maurer, (1983) administered GnRH on day 0 (day of breeding) and found a 3 0 % reduction i n pregnancy rates among twice-open cows treated with GnRH on day 0. A series of four experiments conducted by Macmillan et a l . , (1986) revealed that a single i n j e c t i o n with GnRH given between days 1-13 induced d i f f e r e n t responses i n re c i p i e n t animals depending on the time the treatment was given. Animals receiving 5 jug/hr of GnRH on days 1-6 af t e r the f i r s t oestrus lead to a reduction i n pregnancy rates. When treatments were administered between days 7-10 and increasing the dose of GnRH to 10 ng, GnRH had no e f f e c t on pregnancy rates. Both dosages when given a f t e r day 6, however, resulted i n increased the number of animals showing' extended cycles. This e f f e c t was more pronounced i n animals receiving the treatments between days 11-13 af t e r the f i r s t oestrus post partum. Treatments at a dose of 10 /zg rendered between days 11-13 also lead to increases i n pregnancy rates i n both f i r s t and second inseminations. Lee et a l . , (1985) reported t r e a t i n g 24 dairy cows with 100 ug of GnRH intramuscularly on day 0. Such a treatment lead to s i g n i f i c a n t increases in P4 concentrations during the 4 days a f t e r breeding. Pregnancy rates, however, were not influenced by the GnRH treatment. Examining the l i t e r a t u r e pertaining to the use of GnRH i n c a t t l e leaves one to draw one or two deductions. I t appears that GnRH has been used mostly around oestrus with the aim of 18 synchronizing ovulations. This has found wider application among animals returning for breeding more than 3 times. Such cows have been termed "repeat breeders". Despite showing s l i g h t increases i n pregnancy rates following the use of GnRH, res u l t s obtained to date s t i l l remain inconsistent. 2.5. F o l l i c u l a r dynamics i n the cow It was postulated by Rajakoski i n 1960 that ovarian f o l l i c l e s i n the cow e x i s t as waves of continuous f o l l i c u l a r growth. Other workers (Choudry, 1968; Savio et a l . , 1988; S i r o i s and Fortune, 1988) have since shown that f o l l i c l e s do indeed e x i s t as continuous waves of (2 or 3) f o l l i c u l a r growth. I t appears, on one hand, that 80-81 % of h e i f e r s tend to have 3 waves of f o l l i c u l a r growth while pluriparous cows, on the other hand, normally exhibit 2 waves of f o l l i c u l a r growth (Pierson and Ginther, 1986). Investigations c a r r i e d out i n the UBC laboratories using ultrasound scanning confirmed the existence of waves of f o l l i c u l a r growth, leaning towards 2 as being the norm i n both c y c l i c non- pregnant and pregnant dairy cows. A few cows w i l l show 3 waves of f o l l i c u l a r growth, but these tend to have extended cycles (Taylor and Rajamahendran, 1990). These waves of f o l l i c u l a r growth that have been reported to ex i s t i n the non-pregnant cow, have also been determined to be present during early pregnancy (Taylor and Rajamahendran, 1990) . These authors also determined that these waves culminate i n the emergence of at least one preovulatory-size f o l l i c l e at days 7 and on day 0. Another pre-ovulatory size f o l l i c l e may occasionally appear 14 days post breeding but does not seem to reach maximal size and instead regresses paving the way for 19 a t h i r d wave which appears about day 18-19 (Taylor and Rajamahendran, 1990). The f o l l i c l e ranges from 10-30 mm i n diameter. Using ultrasonography, no v i s u a l differences were found when the preovulatory-size f o l l i c l e of the l u t e a l phase and that found at oestrus were compared. F o l l i c l e s have been reported to acquire receptors for LH as they enter the antral stage of development (Ireland, 1987). Ireland and Roche (1983) have i n fact reported that the f o l l i c l e s present during the oestrous cycle of the cow, have receptors for LH, being maximal on day 7 and a second peak occurs around days 12 to 14 post oestrus. Similar r e s u l t s were reported by Bennett et a l . , (1989), whose i n - v i t r o and in-vivo studies revealed a greater response to hCG treatment at days 3-7 (early l u t e a l phase) compared to days 8- 13 (mid to l a t e l u t e a l phase). The presence of f o l l i c l e s during the l u t e a l phase and early pregnancy and f u l l y equipped with receptors to LH, affords us with a t o o l that might have potential for supplementing P4 l e v e l s i n the dairy cow. The advent of ultrasound scanning i n the early 1970's presented us with an equally powerful aid with which to conduct studies i n reproductive physiology. This permits us to v i s u a l i z e the reproductive t r a c t and to obtain real-time or dynamic images of the same (Pierson et a l . , 1988). This makes i t possible to cl o s e l y follow ovarian dynamics. 3. O B J E C T I V E S AND RAT IONALE This study was, therefore, aimed at evaluating the e f f i c a c y of using hCG for inducing CL development, increasing the s i z e of the spontaneously derived CL and t h e i r c a p a b i l i t y to synthesize and secrete P4. It was intended also to investigate the e f f e c t of such increased P4 concentrations on pregnancy rates i n the Holstein breed of dairy c a t t l e . 3 .1 . OBJECTIVES 3 . 1 . 1 . Experiment one The objectives for experiment one, of t h i s study, were to determine i f giving hCG either at the time of breeding (oestrus=day 0), 7 or 14 days post breeding would; (a) Increase the number of CL, (b) Increase the diameter of the spontaneous CL, (c) Increase plasma and milk P4 l e v e l s 3 .1 .2 . Experiment two The objectives of experiment two were to; (1) increase milk P4 l e v e l s i n hCG treated animals. (2) reduce the extent of embryonic mortality by reducing that which i s caused by CL dysfunction. 3.2 . RATIONALE The aspirations borne during the implementation of t h i s experiment, were as follows; 3 . 2 . 1 . Day 0 (oestrus) (i) Giving hCG at the time of breeding would ensure ovulation of the dominant f o l l i c l e present at that time. ( i i ) hCG given at the time of breeding would possibly influence the formation, growth and function of the CL. 21 3 .2 .2 . Day 7 post oestrus (i) Treatment with hCG at 7 days post breeding would attempt to ovulate the dominant f o l l i c l e present on that day. ( i i ) To increase both the size and function of the spontaneously derived CL. 3 .2 .3 . Day 14 post oestrus (i) hCG was administered on day 14 post breeding so as to ovulate the dominant f o l l i c l e present at t h i s stage of the lu t e a l phase. . ( i i ) I t was also intended that hCG i n j e c t i o n given at day 14 post breeding would augment a possibly waning endogenous P4 production. This was deemed to be a c r i t i c a l time i n the l i f e span of the CL as t h i s represents the time when the CL begins i t s exposure to the influence of l u t e o l y t i c prostaglandins of uterine o r i g i n . I t also represents the timing of signal transmission from the embryo to the dam, thus i n i t i a t i n g the process of maternal recognition of pregnancy. Supplementing P4 production at t h i s time would, conceptually, allow the embryo some extra time for s i g n a l l i n g i t s presence. 22 4. Experiment One: E f f e c t Of hCG Administration At Various Times Following Breeding On CL Number, Diameter And Progesterone P r o f i l e s In Dairy C a t t l e . 4.1. MATERIALS AND METHODS 4.1.1. Animals : General Management Practices When t h i s experiment was conducted, the UBC farm at South Campus had almost 100 Holstein dairy cows, 47 of which were milking. The cows were housed i n free s t a l l barns. Cows seen in oestrus between September, 1989 and May, 1990 were included i n the t r i a l . Cows used i n the experiment ranged from 3-8 years of age and were maintained on a die t consisting of a dairy textured concentrate, a l f a l f a cubes (both 16 % protein), chopped grass (11 % protein). The dairy concentrate rati o n contained grains (corn, barley, oats), soybean and mineral supplements. In addition to grain mixtures, l a c t a t i n g cows were allowed free access to an iodized cobalt s a l t block l i c k . A l l milking cows were fed on average a production ration of 1 part of feed for every 3-4 kg of milk produced. The feed to milk r a t i o was dependent on the stage of l a c t a t i o n that each cow was at. Dry cows, on the other hand, were allocated d a i l y 10 kg of hay and 12 kg of a l f a l f a cubes. The cows were examined a f t e r p a r t u r i t i o n by a veterinarian from the Animal Care Centre. The f i r s t breeding post partum were conducted after day 60. Only cows confirmed to have resumed c y c l i n g were used in the experiment. During the period of conducting the experiment, the average milk production was 7802 kg, with 2.90 % protein and 3.20 % butter f a t . 23 Although detection of oestrus was conducted by most of the farm workers, a greater number of heat checks were done by the respective milkers on duty. Animals were checked for oestrus at least twice d a i l y , between 02:00 to 04:00 and between 14:00 and 17:00 hours. Oestrus detection was based on observing for cows attempting to mount and standing to be mounted by other cows, presence of vaginal discharge, restlessness, and feeding habits. An attempt was also made to v e r i f y the occurrence of unobserved oestrus by noting whether or not there was metoestrus bleeding. When detected, t h i s was assumed to indicate that the animal had been i n oestrus within the l a s t 2-3 days. The day of oestrus was designated as day 0 of the oestrous cycle. A l l cows seen in standing heat were bred approximately 12 hrs l a t e r . A l l breedings were ca r r i e d out by farm technicians trained i n a r t i f i c i a l insemination (AI). 4.1.2. Treatments At unsynchronized oestrus, t h i r t y four l a c t a t i n g dairy cows were randomly assigned to one of four treatment groups. A single intramuscular i n j e c t i o n containing 1000 IU of hCG (1 ml of APLR, hCG, Ayerst Laboratories, Montreal, Canada) was administered either on day 0, (n=8) , day 7 (n=9), 14 (n=9) post breeding, or no hCG treatment (control, n=8). 4.1.3. Ultrasound Examination A r e a l time ultrasound scanning device (Ultrasound scanner, model LS 300, Tokyo Keiki Company Limited, Tokyo, Japan) equipped with a 5 Mega Hertz (MHz) probe was used to monitor ovarian 24 dynamics during the time of investigation. The 5 MHz transducer was chosen due to i t s greater resolving power. This c a p a b i l i t y made i t more suitable for examining deeply situated structures such as ovaries. Pierson et a l . , (1988) described development of real-time ultrasound scanning i n the la t e 1970's as the most profound technological advance i n the f i e l d of large animal research and c l i n i c a l reproduction since the introduction of transrectal palpation and radioimmunoassay (RIA) of c i r c u l a t i n g hormones. Thus, we now have a non-invasive method for v i s u a l i z i n g the reproductive organs and t h e i r payload-the conceptus". At each ultrasound evaluation, the rectum was evacuated of a l l faecal material and a quick note was made of the uterine tone and the location of the ovaries was established by palpating per rectum. The procedure of ultrasound examination was that described by Squires et a l . , (1988). During ultrasound examination, internal structures i n the body of cows f a l l under two categories. The probe emitted sound waves. The c h a r a c t e r i s t i c s of s p e c i f i c tissues determined what proportion of the sound beam were r e f l e c t e d . Images of the r e f l e c t e d portions registered on the screen as shades of grey, extending from black to white. A l l f l u i d - f i l l e d body structures being non-echogenic (absorb ultrasound waves emitted by the probe) appeared black. F o l l i c l e s for example were v i s u a l i z e d as black, roughly c i r c u l a r ultrasound images (plate 1). On the other hand, dense tissues such as bone or cervix appeared white. Tissues in between these extremities were seen as d i f f e r e n t shades of grey, depending upon t h e i r degree of echogenicity (Pierson et a l . , 1988). In order to keep a permanent record and to f a c i l i t a t e the measurement of the structures of interest, once the optimum scan was obtained, these were frozen on the screen. The dimensions of CL and f o l l i c l e s measuring more than 3 mm i n diameter were obtained. Measurements were taken at the widest poles using a b u i l t - i n system of c a l i b r a t e d c a l l i p e r s on the ultrasound machine. Information on in d i v i d u a l cows was recorded on the frozen screen/images using an alpha-numeric key board. Such data included the date and time of scanning, the cow i d e n t i f i c a t i o n number, number of days post AI. Additional data pertaining to the measurements of the structures of i n t e r e s t were recorded on the same frozen screen. Noted, too, was the tone and contents (where present), of the uterus. Hard copies of the frozen images of the CL and f o l l i c l e s more than 3 mm were obtained using a Mitsubishi Video copy processor (Model number P60U, Mitsubishi E l e c t r i c Sales America Inc., Cypress, C a l i f o r n i a , USA) connected to the ultrasound scanner. Ovulations r e s u l t i n g from treatment with hCG were v e r i f i e d by observing for an acute disappearance of one or more of the dominant or pre-ovulatory si z e (antral) f o l l i c l e s present at the time of administering the treatment. The emergence of an induced CL was characterized by l u t e a l tissue appearing on a s i t e previously occupied by an antral f o l l i c l e . The growth of such induced CL was followed by determining the diameter of the "new" l u t e a l tissue at the widest poles. This, i n some cases, was only possible u n t i l the induced CL reached the size of the spontaneous CL. Due to t h i s compounding nature of the CL dimensions, a l l diametrical measurements were summed up to y i e l d a t o t a l diameter of the l u t e a l t i s s u e i n each experimental animal. Cows developing accessory CL were palpated per rectum between days 75-80 to determine the fate 2 6 of the induced CL and/or the persistence of c y s t i c structures observed following treatment with hCG. The functional capacity of induced CL could not be established from t h i s in-vivo study. I t was, however, hoped that increased le v e l s of c i r c u l a t i n g P4 would be i n d i c a t i v e of an additive e f f e c t of an increase i n spontaneous CL production of P4 and P4 o r i g i n a t i n g from the induced CL. The compounding nature of the e f f e c t of hCG on P4 production from both the induced and spontaneous CL, were f u l l y appreciated. I t was not possible to separate the two sources of P4. 27 I I • o *z . 5 MHz D i s t a n c e : R r e i : C i r c : Pase 1 3 5 DO V S P O S T Pi I P l a t e 1. Ultrasound image of a u t e r i n e horn and ovary of a cow (85022). Note CL (echogenic) on r i g h t ovary (arrow on r i g h t screen) and an embryo proper (echogenic) enclosed w i t h i n embryonic v e s i c l e s (arrow on the l e f t screen). 2 8 4 .1 .4 . Blood and M i l k Sample C o l l e c t i o n At the time of ultrasound evaluation, each animal was bled v i a the coccygeal vein/artery. 10 ml of blood was col l e c t e d in vacutainer tubes (Becton Dickinson, Vacutainer Systems, Rutherford, New Jersey, USA) containing sodium heparin. A l l blood samples were centrifuged within an hour of c o l l e c t i o n and plasma separated. Blood samples were co l l e c t e d either immediately p r i o r to or after the afternoon milking (15:00 hours) at which time bulk milk samples were co l l e c t e d . A l l milk samples were co l l e c t e d i n 13 dram snap-top sample v i a l s (Capital V i a l Corporation, Fonda, New York, USA vi a Systems Plus). A potassium dichromate preservative tablet (J.R. Dairy Laboratories, Burnaby, B.C., Canada) was dissolved i n each milk sample. Both milk and plasma samples were frozen soon afte r c o l l e c t i o n and only thawed when assaying for P4. Ultrasound scanning, milk and blood sample c o l l e c t i o n were ca r r i e d out on days 7, 9, 11, 14, 16, 18, 21, 28, 35 and 42 post AI or u n t i l the f i r s t observed oestrus occurring p r i o r to day 42. A l l control cows were only scanned and sampled at weekly in t e r v a l s s t a r t i n g on day 7 post breeding. 4 .1 .5 . Radioimmunoassay (RIA) The Coat-A-Count R P4 RIA k i t (Coat-A-CountR Progesterone k i t , Diagnostic Products Corporation, Los Angeles, C a l i f o r n i a , USA) procedure used for P4 analysis was a solid-phase RIA. Over a fixed period of time, r a d i o - l a b e l l e d P4 (buffered 1 2 5I-P 4) competes for binding s i t e s with native P4 i n samples. The Coat-A-Count R k i t s consisted of polypropylene tubes coated with antibodies to P4, iodinated P4 ( 1 2 5I-P 4 or tracer) , human serum-based standards having 29 P4 values ranging from 0.1 to 4 0.0 ng/ml (in ready-to-use l i q u i d form). The coat-A-count R k i t P4 antiserum used was of a very high s p e c i f i c i t y for P4 and low cross r e a c t i v i t y (<10 %) to other steroids which might have been present i n the samples. Three groups of blood samples were obtained from cows either observed to be i n oestrus, early dioestrus (days 3-5) or late dioestrus (days 12-14). Samples belonging to each type were pooled together and aliquoted into several tubes. Values from these tubes were used to calculate the inter-assay c o e f f i c i e n t of v a r i a t i o n (CV). Intra-assay CV, on the other hand, were calculated using the duplicate samples of each standard within an assay. While the intra-assay CV ranged from 1.36 to 7.89, the inter-assay CV for the oestrual, early and late dioestrus samples were 10.3, 11.0 and 9.86, respectively. At each assay an aliquot of - 100 /zl of standard, was transferred into the appropriate antibody (Ab) coated tubes (labelled A l , A2, B1,....G1 and G2). Tubes containing standards A through G corresponded to P4 concentrations of 0.00, 0.1, 0.5, 2, 10, 20 and 40 ng/ml, respectively. A 100 yitl aliquot from each sample was transferred into antibody coated tubes and numbered 19....n, where n was the number of the l a s t tube i n any given assay. One m i l l i l i t r e (1 ml) of the tracer ( 1 2 5I-P 4) was added into each tube, including two pairs of p l a i n polypropylene tubes. These four p l a i n tubes were included i n order to determine the t o t a l a c t i v i t y of the tracer (2 tubes), and amount of non-specific binding (2 tubes). After adding the tracer, the tubes were incubated for 3 hrs. After incubation, the tracer was decanted from a l l tubes, except the t o t a l count tubes, and any v i s i b l e moisture 30 above the 1 ml l i n e was wiped out from a l l decanted tubes using cotton swabs (Q-tips R) . The amount of r a d i o a c t i v i t y (counts per minute = CPM) bound to antibody i n the tubes was quantified i n a gamma counter. Using S t a t i s t i c a l Analysis System (SAS), l o g i t - l o g transformation of the standards was performed so as to y i e l d a standard curve which was used to derive the concentrations of P4 for each respective sample. 4.1.6. Radioimmunoassay K i t V a l i d a t i o n . The concentrations of P4 i n milk and plasma were quantified as described above. While blood plasma has been the main b i o l o g i c a l material u t i l i z e d i n the assessment of the reproductive status of livestock, the discovery i n the 1970's that progestagens were detectable i n milk (Darling et a l . , 1972) pointed to the potential use of such a material i n routine diagnostic procedures. I t was proposed that progestagen concentrations i n milk are one means of diagnosing early pregnancy (Laing and Heap, 1971). A simple and rapid RIA for quantifying "progestagens" i n milk soon followed (Heap et a l . , 1973). RIA i n the present experiment was performed using commercially available k i t s . These k i t s have since been validated (Gowan and Etches, 1979; Srikandakumar et a l . , 1986; Robinson et a l . , 1989) and found to be a r e l i a b l e t o o l for monitoring l u t e a l function in the cow as well as other domestic cows (Srikandakumar et a l . , 1986). Preliminary data from research undertaken i n the Department of Animal Science using the Coat-A- Count" P4 RIA k i t indicated that P4 l e v e l s lower than 1.00 ng/ml, in both plasma or milk samples, were i n d i c a t i v e of the presence of a non-functional CL or complete absence of one (Sianangama and Rajamahendran, unpublished r e s u l t s , 1989). Such observations were confirmed using ultrasound scanning. The findings regarding the k i t are i n agreement with those obtained by other laboratories (Srikandakumar et a l . , 1986; Gowan and Etches, 1986). A P4 concentration of 1.00 ng/ml was, therefore, used to discriminate between f e r t i l e versus n o n - f e r t i l e inseminations. 4 .1 .7 . Pregnancy Diagnosis Establishment of pregnancy was v e r i f i e d using ultrasonongraphy. Detection of embryonic v e s i c l e s was always possible by day 21 post AI. The embryo proper, on the other hand, was consistently i d e n t i f i a b l e on day 28 post AI. Cows not returning to oestrus by day 4 2 were palpated per rectum to determine whether they were pregnant or not. The pregnancy check done at day 60 was carr i e d out by a veterinarian. 4.1 .8 . S t a t i s t i c a l Analys i s Data for milk and plasma P4 and CL diameter were analyzed using a s p l i t - p l o t experimental design. The lea s t square analysis of variance procedure i n the General Linear Models, S t a t i s t i c a l Analysis System (SAS Inst i t u t e , Inc., SAS User's Guide: S t a t i s t i c s , 1987, Version 6.3, Edition) was used to tes t for differences among treatments. The model used was as follows: Y j j k = U + A,- + B j(A.) + Ck + A, X Ck + E i j k where Y = n t h observation, U = population mean, A. = treatment, Bj (A1-) = cow nested within treatment, Ck = sample number or number of days post breeding (AI) , A- X Ck = treatment 32 i n t e r a c t i o n with number of days post AI, E... = experimental I J K (residual) error. The cow(treatment) mean square was used as error term to test treatment mean square. The other e f f e c t s ((cow(treatment), sample number and time int e r a c t i o n with treatment), were tested using the residual mean square. Due to v a r i a b i l i t y i n the frequency of sampling, each data set was analyzed i n two batches (sampling days 7, 14, 21, 28, 35 and 42 and days 9, 11, 16, and 18). Contingency tables i n Chi-square analysis was used to t e s t for treatment e f f e c t on pregnancy rates. Unless stated otherwise, a l l comparisons were made at the 5 % l e v e l of significance (P<0.05). 5. RESULTS 5.1. Induction Of Accessory Corpora Lutea Results of the responsiveness of cows to hCG treatment are summarized i n table 1. Ultrasound scanning of the ovaries revealed that 1/8 control cows had a spontaneous twin ovulation (plate 2b) and 1/8 f a i l e d to ovulate (plate 3b). The other 6 had normal CL. The t o t a l number of CL i n the control group ranged from 0 to 2. Among cows treated on day 0, 3/8 were found to have twin ovulations (plate 2a) . At the l e v e l of the ultrasound machine i t was not possible, i n two of the three cows, to esta b l i s h whether any one of the twin CL were induced by the treatment. Both CL had the t y p i c a l day 7 appearance, as described above. One of the two CL i n the t h i r d cow was at y p i c a l of that r e s u l t i n g from a spontaneous ovulation. I t was small i n appearance (9 mm) . This p a r t i c u l a r induced CL, however, regressed by day 21 post oestrus. The spontaneous CL persisted u n t i l day 42 post breeding. One cow did 33 not ovulate and proceeded to develop a c y s t i c f o l l i c l e . This f o l l i c l e appeared l u t e i n i z e d as evidenced by a layer of fine l u t e a l - l i k e grainy area circumscribing the dark f o l l i c u l a r f l u i d (plate 3a) . For t h i s reason, the P4 p r o f i l e s r e s u l t i n g from the l u t e i n i z e d structure, i n t h i s cow were not used i n the subsequent s t a t i s t i c a l analysis. Of inte r e s t , however, was the fact that the concentrations of P4 i n t h i s p a r t i c u l a r cow were comparable to those of CL-bearing cows i n the control group. The t o t a l number of CL per cow among cows treated with hCG on day 0, therefore, ranged from 0 to 2. Amongst cows treated with hCG on day 7 post AI, the dominant f o l l i c l e present at the time of administering the treatment ranged in s i z e from 13 to 22 mm i n diameter. The second largest f o l l i c l e , on the other hand, measured 9 to 21 mm diameter. The orientation of the induced CL developed following treatment with hCG did not seem to be influenced by the p o s i t i o n of the spontaneous CL. The dominant f o l l i c l e was the ovulatory f o l l i c l e i n 5/9 cows treated on day 7 post AI. This responsive dominant f o l l i c l e was positioned either c o n t r a l a t e r a l or i p s i l a t e r a l (plate 4a) with respect to the spontaneous CL and there were 2 cases of each orientation observed. Plate 4b c l e a r l y authenticates the persistence of an induced CL u n t i l day 70 when an additional ultrasound evaluation was conducted in cows developing accessory CL. The persistent f o l l i c l e s that appeared either l u t e i n i z e d or showed no obvious signs of l u t e i n i z a t i o n following treatment with hCG on day 7 post AI, were a l l found to have regressed by day 64 post AI. In the f i f t h cow, the dominant f o l l i c l e on one ovary and the second largest f o l l i c l e on the other ovary, ovulated. This p a r t i c u l a r cow had double 34 spontaneous ovulations and these were, therefore, additional two CL induced by the treatment. In 2/9 cows, the second largest f o l l i c l e located i p s i l a t e r a l to the CL resulted i n an induced ovulation. The largest f o l l i c l e was present a f t e r the emergence of the induced CL but subsequently regressed. Thus, seven out of the nine cows treated on day 7 post AI developed accessory CL. The number of CL per cow among cows receiving treatment with hCG on day 7 post breeding, ranged from 1 to 4. Among cows treated on day 14 post AI, 4/9 were found to have accessory CL. The dominant f o l l i c l e present when treatments were administered ranged i n size from 9 to 27 mm. In 3/4 of these cows a dominant f o l l i c l e , located cont r a l a t e r a l to the CL, developed an accessory CL. Of these three cases, two were dominant f o l l i c l e s , while the t h i r d was the second largest f o l l i c l e . The only induced ovulation positioned i p s i l a t e r a l to the spontaneous CL was a 9 mm, t h i r d largest but the only growing f o l l i c l e observed i n the fourth cow. Both CL had regressed by day 2 9 post oestrus. One cow treated on day 14 post breeding was found to have an "apparently" non- l u t e i n i z e d f o l l i c l e p e r s i s t i n g following the hCG treatment. This f o l l i c l e was shown to have regressed by day 75 post breeding. The t o t a l number of CL, per cow i n t h i s treatment group, ranged in number from 1 to 2. 35 Table 1. Ovulatory Response Of Cows Treated With Human Chorionic Gonadotropin Either At The Time Of Breeding, Day 7 or 14 Post Breeding In Comparison To Control Cows. Treatment Group Day 0 Day 7 Day 14 Control Total Number Of Cows Treated (n) 8 9 9 8 34 Number of Cows Developing Accessory Corpora Lutea 3 7 4 1 15 Number Of Corpora Lutea (Range)/cow 0 - 2 1 - 4 1 - 2 0 - 2 0 - 4 36 14:48 I P : 86311 Probe : 5MHz D i i t i n c e : Rre*: Circ: P *9e 2 06 DfiVS POST 01 Twin ovulations (arrows on right screen) in animal # 86011 treated with HCG on day 0. No apparent difference between the two CL. Spontaneous twin ovulations (arrow on l e f t screen) in animal # 83010. Plate 3. (a) Cystic f o l l i c l e (arrow on l e f t screen) in animal # 86016 treated with HCG on day 0. (b) Cystic f o l l i c l e (arrow on l e f t screen) in animal # 85001 (control). 38 Nou' 1 7 / 8 9 1 2 : 0 0 I D : 87005 Probe: 5MHz Distance: R r e a . : C i r c : Page 1 70 DfiVS P O S T fiI RieHTWfT^TlNE HORN *> ~ - R m i T . 0UftRV<3; 31X16 MM C L .32X19 MM C L - R I B H T ^ ' o v R R Y A B O T H C L S T I L L 2 P R E S E N T Plate 4. (a) Day 28 images of right ovary (right screen) and right uterine horn ( l e f t screen). Note both CL present (arrows on right screen), and the embryo proper (arrow on l e f t screen). (b) Day 70 image of right ovary (right screen) and a section of the right uterine horn. Note both CL are s t i l l present (arrows on right screen). 39 5.2. Growth of The Corpora Lutea P r o f i l e s of CL diameter and LSMeans are presented in figure 1 and table 2, respectively. Results of the analysis of variance of the CL diameter are presented i n appendix l a . Diameter increased s i g n i f i c a n t l y (P<0.02) with hCG treatment for day 7, 14, 21, 28, 35 and 42 values. Figure 1 show higher CL diameter p r o f i l e s among cows receiving hCG treatment either on day 0, 7 or 14 post AI compared to the control cows. Overall LSMeans for CL diameter were higher among day 7 treated cows (50.22±2.08 mm) compared to day 0 treated (35.94+2.64 mm), day 14 treated (35.45±2.15 mm) and control cows (29.13± 2.60 mm). The o v e r a l l e f f e c t of the sampling periods on CL diameter was not s i g n i f i c a n t . However, o v e r a l l LSMeans for CL diameter increased from 34.14±2.23 to 39.96±2.28 mm around which l e v e l they remained u n t i l day 42. For samples co l l e c t e d on days 9, 11, 16 and 18 post AI the s i g n i f i c a n t i n t e r a c t i o n between sampling period and treatment made i t i s d i f f i c u l t to separate the e f f e c t of these two e f f e c t s . The v a r i a b i l i t y between individual cows receiving hCG was also high. Nonetheless, at day 9 post AI, cows treated on day 0 had higher LSMeans for CL diameter than any of the other groups of cows treated subsequently. The LSMeans for the three groups were 45.14±3.10, 38.97±2.98 and 34.5614.56 mm, for day 0, 7 and 14 treated cows, respectively. By day 11 post AI, LSMeans for CL diameter were, however, s i g n i f i c a n t l y higher (P<0.01) i n cows treated either on day 0 (47.43±3.10 mm) or 7 days post AI (49.47±2.98 mm) compared to cows receiving t h e i r treatment on day 14 post AI (34.52±3.35 mm). 40 At day 16 post AI, both groups of cows receiving treatment eithe r on day 0 (42.70±3.46 mm) or 7 post AI (58.75±2.95 mm) had s i g n i f i c a n t l y higher (P<0.05 and P<0.01), respectively) least squares mean values for CL diameter compared to cows receiving treatment on day 14 post AI (33.62±3.06 mm). However, LSMeans for CL diameter of cows treated on day 7 post AI were s i g n i f i c a n t l y higher (P<0.01) than cows treated on day 0. At 18 days post AI, cows treated on day 7 (57.67±2.73 mm) s t i l l had s i g n i f i c a n t l y higher (P<0.01) LSMeans for the CL diameter compared to both groups of cows treated either on day 0 (39.04±3.46 mm) or 14 days post AI (43.05±2.98 mm). There were no s i g n i f i c a n t differences between LSMeans for the CL diameter of cows receiving treatment on day 0 when compared to those treated on day 14 post AI. 41 Table 2. E f f e c t Of Human Chorionic Gonadotropin Treatment Given Either At The Time Of Breeding, Day 7 or 14 Post Breeding, On Corpus Luteum Diameter* In Holstein Dairy Cows. Days Post Treatment Group a p c Breeding Day 0 Day 7 Day 14 Control 7 42 . 14±4 . 53a 30 . 7 5 ± 4 .30 b 31 .8914 . 00b 28 . 3 0 ± 5 . 02b 9 45. 14±3 . 10a 38 . 9 7 ± 2 .98 b 34 . 56±4 . 56b 11 47 . 43±3 . 10a 49 . 4 7 ± 2 .98 a 34 . 52±3 .3 5b 14 35. 8 6 ± 4 . 54b 55 . 00±4 . 3 0a 33 . 63±4 . 67b 32 . 57±4 .54 b 16 42. 70±3 . 46a 58 . 75±2 .95 b 33 . 62±3 . 06a 18 39 . 03±3 . 46a 57 . 67±2 .73 b 43 . 05±2 .98 a 21 33 . 8 0 ± 5 . 74b 54 .2014 . 36a 33 . 03±4 . 3 6b 30 . 00±4 .54 b 28 34 . 80±5 . 7 4 bc 52 . , 2 3 ± 4 . ,75a 44 . . 0 9 ± 5 . . 22 a b 26. 0 8 ± 5 . 62c 35 36. 74±7 . 49 a b 54 . , 2 2 ± 5 . . 19a 35. , 7 6 ± 5 . . 21b 27 . , 4 0 ± 7 . ,47b 42 31. 8 8 ± 6 . 4 5b 53 . 2 5 ± 5 .73 a 33 .2715 .74 b 30 .4017 .47 b a,b,cNumbers with d i f f e r e n t l e t t e r superscripts within row d i f f e r at the 5 percent l e v e l of confidence (P<0.05.). Values are presented as least squares means for CL diameter. 42 F i g u r e 1. E f f e c t s O f H u m a n C h o r i o n i c G o n a d o t r o p i n On The Growth Of The CL In Dai ry Cat t le (UBC) 70 i r 0 10 20 30 40 50 Number Of Days Post Breeding T R E A T M E N T Day 0 — D a y 7 Day 14 "-B~- Control 5.3. Progesterone P r o f i l e s 5.3.1. Plasma Progesterone P r o f i l e s , LSMeans and analysis of variance on plasma P4 concentrations are presented i n figure 2, table 3 and appendix 2, respectively. Administration of hCG did not lead to s i g n i f i c a n t increase i n P4 at days 7, 14, 21, 28, 35 and 42 post AI. However, there was a trend for higher p r o f i l e s among cows treated at either day 7 (6.0610.40 ng/ml) and day 14 (4.7310.43 ng/ml) i n comparison to cows treated on day 0 (3.9210.62 ng/ml) or control (3.8410.46 ng/ml). P4 concentrations were s i g n i f i c a n t l y (P<0.01) influenced by the sampling period. The response of cows receiving hCG was highly variable. Analysis of samples c o l l e c t e d at days 9, 11, 16 or 18 post AI shows no e f f e c t of hCG on progesterone p r o f i l e s . High in d i v i d u a l (P<0.01) variations among cows receiving hCG are indicated during t h i s sampling period. Overall P4 increased with time post AI. Levels rose from 4.03± 0.53 ng/ml at day 9 to a high of 6.6210.54 ng/ml on day 16 but decline thereafter. By day 18 post AI, both day 7 and 14 treated cows had s i g n i f i c a n t l y higher plasma P4 concentrations compared to cows treated on day 0. The respective LSMeans for plasma P4 concentrations were 3.5111.05, 5.97+0.90 and 6.57+0.90 for cows treated on day 0, day 7 or 14 post AI, i n that order. At day 35, LSMeans for plasma P4 concentrations of cows treated on day 7 (9.4911.02 ng/ml) were not d i f f e r e n t (P<0.05) from those for cows treated on day 0 (4.2912.58 ng/ml) but were d i f f e r e n t (P<0.02) from those of cows either treated on day 14 (5.6111.03 ng/ml) or receiving no treatment with hCG (5.72+1.27 ng/ml). Forty two days AI, no differences could be detected i n the LSMeans for plasma P4 among cows treated on day 7 (7.1811.02 ng/ml) compared to those receiving t h e i r treatment on day 14 post AI (4.5011.13 ng/ml). Cows treated on day 7, however, had LSMeans for plasma P4 concentrations higher than those treated on day 0 (4.2111.13 ng/ml) and the control group (1.46+1.27 ng/ml). Table 3. E f f e c t Of Human Chorionic Gonadotropin Treatment Given Either At The Time Of Breeding, Day 7 or 14 Post Breeding, On Plasma Progesterone Concentrations* In Holstein Dairy Cows. Days Post Treatment Group a , b Breeding Day 0 Day 7 Day 14 Control 7 2 .8610. 89 2 .40+0.79 2.9010.85 2.60+0.84 9 3 .62+0. 94 4 .12+0.83 4 . 37 + 0.97 11 4 .85+0. 94 5 .9010.83 5.23+0.83 14 4 .40+0. 85 6 .27+0.85 4.9010.79 4.96+0.84 16 5 .27+1. 05 7 .38+0.90 7.2010.83 18 3 .5111. 05a 5. .9710.90ab 6.57+0.90b 21 4 .08+1. 13 5 .42+0.93 5.26+0.93 3 . 3810.84 28 3 .68+1. 13 5 .57+0.93 5.2011.13 4.92+1.12 35 4 .29+2. 58 a b 9 . 4911.02a 5.61+1.03b 5 .72+1.27b 42 4 .2111. 13a 7 . 18 + 1.02b 4.50 + 1. 13 a b 1 .47+1.27a a,bNumbers with d i f f e r e n t l e t t e r superscripts within a row d i f f e r at leas t at the 5 % (P<0.05) l e v e l of confidence. Values are presented as least squares means for Plasma P4. 4 5 F i g u r e 2 . E f f e c t O f H u m a n C h o r i o n i c G o n a d o t r o p i n On P l a s m a P 4 C o n c e n t r a t i o n s In Dai ry Catt le (UBC) 10 0 10 20 30 40 50 N u m b e r Of Days Post Breed ing T R E A T M E N T S — Day 0 — D a y 7 Day 14 5.3.2. Milk Progesterone The p r o f i l e s , LSMeans and analysis of variance on P4 concentrations are presented i n figure 3, table 4 and appendix 3, respectively. Treatment with hCG did not show s i g n i f i c a n t P4 increase i n milk samples c o l l e c t e d on days 7, 14, 21, 28, 35 and 42. Sampling period showed s i g n i f i c a n t contribution to P4 increases. However, individual animal v a r i a b i l i t y was also highly s i g n i f i c a n t (P<0.01). Treatments with hCG lead to s i g n i f i c a n t increase i n milk P4 of samples c o l l e c t e d on days 9, 11, 16 and 18. Overall LSMeans during t h i s period were 10.97+0.94, 12.2210.77 and 24.68+2.25 ng/ml for cows treated either on day 0, 7 or 14. Although the response of cows to hCG treatment was highly s i g n i f i c a n t , there did not seem to be an apparent e f f e c t of sampling period. At day 21 post AI, the LSMeans for milk P4 concentrations among cows receiving treatment with hCG on day 7 (12.60+2.03 ng/ml) were s i g n i f i c a n t l y higher compared to those of cows in the control group (7.19+1.98 ng/ml). These were, however, not d i f f e r e n t from those of cows treated e i t h e r on day 0 (9.22+2.68 ng/ml) or on day 14 post AI (10.4112.03 ng/ml). By 28 days post AI, a l l cows receiving treatment with hCG had s i g n i f i c a n t l y higher milk P4 concentrations compared to the control group cows (4.94+2.38 ng/ml). There were no detectable differences among cows receiving treatment with hCG. The LSMeans for milk P4 of cows treated at day 0, 7 and 14 were 15.97 + 2.68, 12.53 + 2.21 and 13.17+2.42 ng/ml, respectively. At day 35, cows treated on day 14 (17.76+2.68 ng/ml) had higher LSMeans for milk P4 compared to cows not receiving treatment with hCG (8.7212.98 ng/ml). The day 14 treated cows, however, had LSMeans for milk P4 not d i f f e r e n t from 47 those of cows treated either on day 0 (12.68±3.02 ng/ml) or at 7 days post AI (14.86+2.41 ng/ml). Cows receiving hCG either on day 0 or those treated on day 7 post AI, were also not d i f f e r e n t from the control group. Forty two days post AI, cows treated on day 0 (10.7412.68 ng/ml) and those treated at day 7 post AI (15.2512.41 ng/ml) both had higher LSMeans for milk P4 concentrations compared to the control group (2.8312.99 ng/ml). Cows receiving hCG at 14 days post AI (9.4113.01 ng/ml) had P4 concentrations not d i f f e r e n t from those of cows i n the other treatment groups. 48 Table 4. E f f e c t Of Human Chorionic Gonadotropin Treatment Given Either At The Time Of Breeding, Day 7 or 14 Post Breeding, On Milk Progesterone Concentrations* In Holstein Dairy Cows. Days Post Treatment Group a , b Breeding Day 0 Day 7 Day 14 Control 7 5.26+2.32 10.6611.87 5.9411.87 5.8411.20 9 8.8911.89 10.8711.50 18.98+5.04 11 11.3911.70 11.2611.50 26.31+5.04 14 9.8012.12 11.94+1.87 10.2311.87 7.93+1.98 16 11.6011.91 13.24+1.62 24.6513.19 18 12.0011.19 13.5211.50 28.78+3.19 21 9.92 + 2.68ab 12.6012.03a 10.41 + 2.03ab 7.19 + 1.98b 28 15.97+2.68b 12.53+2.21b 13.1712.42b 4.94+2.38a 35 12.6813.02ab 14.86 + 2.41ab 17.76 + 2.68a 8.72 + 2.99b 42 10.75 + 2.68a 15.2512.41a 9.14 + 3.01ab 2.8312.996 a,bNumbers with d i f f e r e n t l e t t e r superscripts within each row d i f f e r at the 5.00 % l e v e l of confidence (P<0.05.). Values are presented as least squares means for milk P4. 49 l i r e 3 . E f f e c t O f H u m a n C h o r i o n i c G o n a d o t r o p i n On M i l k P 4 Concen t r a t i ons In Dai ry Cat t le (UBC) 20 r- 15 - 10 - / j f r ^ 5 - ^ 10 20 30 40 N u m b e r Of Days Post Breeding T R E A T M E N T S ~ D a y 0 1 Day 7 * Day 14 C o n t r o l 5.3.3. E f f e c t Of hCG On F e r t i l i t y Three parameters were be used to evaluate the e f f e c t of hCG on f e r t i l i t y , v i z ; pregnancy rates, oestrous cycle length in non- f e r t i l e inseminations and c a p a b i l i t y to show oestrus i n cows showing low P4. Treatment with HCG on day 0 did not seem to extend the cycle length i n the cow. Examination of P4 p r o f i l e s of cows that did not conceive shows that P4 concentrations were better at evaluating the e f f e c t of hCG on cycle length compared to reports on when in d i v i d u a l cows were actually observed i n oestrus. Four cows treated with HCG on day 0 had low P4 l e v e l s at one time or another between days 14 and 42 post AI. One animal had low (0.02 ng/ml) P4 both i n milk and plasma by day 14 post AI. The P4 p r o f i l e of t h i s cow was i n agreement with the data on the growth and regression of the CL which was confirmed to have regressed at the ultrasound scanning conducted on day 14 post AI. The animal continued cycling every 21 days as determined by i t s subsequent return to both low P4 and regression of the CL which were a l l detected on day 35 post the f i r s t oestrus. A second cow had low P4 values on day 18 and was subsequently observed in oestrus on day 21. In a t h i r d case, the cow had low P4 on day 21 but oestrus was not detected u n t i l day 42 post AI. The fourth and l a s t of the inseminated but non-pregnant cows i n t h i s group had low P4 on day 2 8 but only observed in oestrus on day 3 5 post AI. Three cows receiving hCG on day 7 post AI returned to oestrus. While two of them had low P4 concentrations by day 18 and detected in heat on day 21, the t h i r d cow only showed a decline i n P4 concentrations on day 28 post AI. This l a s t cow was observed in oestrus on day 42 post AI. 51 Of the four cows not conceiving i n the day 14 treatment group, three of them had low P4 concentrations by day 21 post AI. These were only observed i n oestrus on either day 21, 3 5 and 4 2 post AI. The fourth cow showed low P4 concentrations and was observed to be in oestrus on day 35 post AI. Five cows in the control group were observed to have low P4 concentrations at one time or another between days 21 and 4 2 post AI. Ultrasound evaluation at day 7 post AI revealed that one of these f i v e cows was actually c y s t i c . The remaining four cows were found to have low P4 concentrations either on days 21, 28, 35 and 42 post AI. While the cow that was observed to be i n oestrus on day 21 post AI was bred l a t e r that day, the other three went undetected and were only reported to have been bred on day 4 2 a f t e r the f i r s t AI. I t was noteworthy that more pregnancies were established among cows receiving hCG. The respective pregnancy rates for day 0, 7, or 14 hCG treated and control cows were 4/8, 7/9, 4/9 and 3/8, respectively. This experiment established that the dominant f o l l i c l e present during the l u t e a l phase i n the cow, i s capable of being induced to ovulate and proceeds to form a structure not d i f f e r e n t from the spontaneous CL. This set a premise for i t s use as a method for supplementing endogenous P4 production. The experiment also set a premise for an b e n e f i c i a l e f f e c t of hCG on pregnancy rates. 52 6. Experiment Two : E f f e c t Of hCG Administration At Various Times Following Breeding On Progesterone P r o f i l e s And Pregnancy Rates In Dairy C a t t l e . To evaluate the e f f e c t of hCG treatments on P4 p r o f i l e s and pregnancy rates, two f i e l d stations collaborated i n the implementation of t h i s experiment. These were a research farm in the Fraser Valley (farm # 1) and a research farm on Vancouver Island (farm # 2), both stations are within B r i t i s h Columbia. 6.1. MATERIALS AND METHODS: Farm # 1 6.1.1. Animals : General Management Practices A l l cows presented for AI s t a r t i n g from and including August, 1989 were included i n the t r i a l . The t r i a l was concluded in February, 1990. The cows u t i l i z e d i n the experiment were part of the 230 Holstein cows constituting the herd on the farm. During the time t h i s experiment was conducted, average milk production record i s 84 03 kg with 3.02 % butter f a t and 2.72 % protein. Cows were normally allowed 77 days of a dry period p r i o r to p a r t u r i t i o n . The calving i n t e r v a l i n t h i s herd was 12.7 months. The experiment commenced i n August, 1989. This month was part of a period (April to October) during the summer when cows were maintained on pasture. This pasture consisted of a mixture of clovers, orchard grass and rye. During t h i s period, the cows were also provided with grain for both maintenance and production. The c r i t e r i a used i n deriving the production rat i o n was on a 1 part of grain to each 4 kg of milk produced. Five kg of t h i s portion of the feed was presented to the animal i n the milking parlour, s p l i t i n 53 equal portions given to the animal at each milking time. The balance was given to each animal i n the barn. During the winter months (November to March) cows are mostly fed a d i e t consisting of grass si l a g e and corn s i l a g e . These were provided to the animal at a r a t i o of 1:2, i n that order. In addition, the cows were allowed free access to l o c a l hay. This hay was made from the clovers and grasses from the pasture mentioned above. The cows were also allocated with t h e i r f u l l amount of grain feed as outlined above. In a l l , the rati o n was formulated to provide the cows with a 18 % protein i n the d i e t . I t i s noteworthy that a l l cows on the farm received neither copper nor selenium supplement i n t h e i r d i e t . I t has been common practice on the farm to provide selenium to these cows as intramuscular inj e c t i o n s given some 3 months following calving. The farm i s part of a private herd health programme through which a veterinarian from the programme pays regular v i s i t s to the farm. This was undertaken every 3 weeks. Cows i n the herd were normally examined by the veterinarian shortly a f t e r p a r t u r i t i o n , and any that were found to be lagging i n accomplishing uterine involution were given a t e t r a c y c l i n e uterine infusion at approximately 3 0 days post partum. Most cows were reported to have exhibited a " f a l s e " oestrus approximately 5 days following p a r t u r i t i o n . This was ignored and any subsequent demonstration of the same phenomenon was recorded. A l l cows were subject to c e r t i f i c a t i o n by the veterinarian as being either f i t for AI or requiring further veterinarian attention. Cows thus declared to be ready for AI were bred by day 60 post partum. Oestrus detection was normally done by the herdsmen early i n the morning (05:00 to 06:00 54 hours) and p r i o r to and at the afternoon milking (15:00 to 16:00 hours). Heat checks, each l a s t i n g 5 to 15 minutes, were conducted during the course of the day. Observation for oestrus were as indicated under materials and methods of experiment one. A l l cows were bred using AI. An e f f o r t was made to t r y and ensure the cows were bred by AI approximately 12 hrs following a reported standing oestrus. Enforcement of s t r i c t abidance to t h i s rule was, unfortunately, compromised. This was due to the fact that the farm was dependent on the services of technicians based i n Chilliwack, a short drive away from the station. Despite the rather short proximity to the research station, the technician in-charge of the AI programme also catered for other farms i n the v a l l e y . At the e a r l i e s t , the technician only arrived to render h i s services at 10:30 hours each morning and at 15:00 hours i n the afternoon. This was, however, standard practice for most commercial dairy farms served by the same technicians. 6.1.2. Treatments Seventy-nine l a c t a t i n g dairy cows were randomly assigned to receive treatment with hCG i n one of four treatment groups. A single intramuscular i n j e c t i o n of hCG 1000 IU (1 ml of APLR) was administered either at the time of AI (Oestrus=D0, n=20), day 7 (n=20), 14 (n=2 0) post AI, or no hCG treatment given (control, n=19). 6.1.3. Milk Sample C o l l e c t i o n Milk sample c o l l e c t i o n was c a r r i e d out on days 0, 7, 9, 11, 14, 16, 18, 21, 28, 35 and 42 post AI or u n t i l the f i r s t observed 55 oestrus. A l l samples were stored at 4°C and transported to the laboratories at UBC. Quantification of P4 was ca r r i e d out using a Coat-a-Count R s o l i d phase RIA k i t described e a r l i e r . 6.1.4. Pregnancy Diagnosis A l l cows not returning to oestrus p r i o r to day 60 were examined to v e r i f y t h e i r pregnancy status. Pregnancy diagnosis was conducted at day 60 using the r e c t a l palpation technique performed by a veterinarian from the herd health programme. 6.1.5. S t a t i s t i c a l Analysis The s t a t i s t i c a l model used and the e f f e c t s included i n the model are given e a r l i e r . Due to v a r i a b i l i t y i n the frequency of sampling, each data set was analyzed i n two batches (sampling days 0-18 and days 21-42). Contingency tables i n Chi-square analysis were used to te s t for treatment e f f e c t on pregnancy rates. Unless stated otherwise, a l l comparisons were made at the 5 % l e v e l of si g n i f i c a n c e (P<0.05). 6.1.6. RESULTS : Farm # 1 6.1.7. Milk Progesterone P r o f i l e s The p r o f i l e s , LSMeans and analysis of variance on milk P4 concentrations are presented i n figure 4, table 5 and appendix 4, respectively. Although treatment with hCG did not increase milk PA p r o f i l e s , there was a trend for higher p r o f i l e s among cows receiving the treatment. The respective o v e r a l l LSMeans for days 0 to 18 were 6.53+0.57, 9.80+0.44, 7.57+0.56 and 6.70+0.51 ng/ml for cows treated on day 0, 7, 14 or those receiving none. LSMeans for 56 milk P4 concentration, however, tended to be higher i n cows receiving hCG at various times following AI. Despite having higher p r o f i l e s amongst cows receiving hCG, there were no detectable differences u n t i l day 16 post AI. LSMeans for milk P4 concentrations were s i g n i f i c a n t l y higher i n cows given hCG treatment on day 7 post AI (17.42±1.15 ng/ml) compared to cows treated ei t h e r on day 0 (7.9911.40 ng/ml), 14 days post AI (11.19±1.33 ng/ml) and the control group (9.18±1.36 ng/ml). By day 18, cows treated on day 7 (12.7 6±1.2 0 ng/ml) had LSMeans not d i f f e r e n t from those of cows receiving treatment on day 14 (10.71±1.27 ng/ml). The day 0 treated cows, whose LSMeans for milk P4 (8.65±1.59 ng/ml) were not d i f f e r e n t from those of cows treated on day 14, were also not d i f f e r e n t from those of cows not receiving any injec t i o n s with hCG (6.3511.62 ng/ml). Cows treated on day 7 and those treated on day 14 both had LSMeans for milk P4 concentrations greater than those of the control group. 57 Table 5. E f f e c t Of Human Chorionic Gonadotropin Treatment Given Either At The Time Of Breeding, Day 7 or 14 Post Breeding, On Milk Progesterone Concentrations* In Holstein Dairy Cows. Days Post Treatment Group a , b' c Breeding Day 0 Day 7 Day 14 Control 0 7 9 11 14 16 18 21 28 35 42 0.7811.40 5.3711.60 6.8411.50 8.1111.50 7.9311.40 7.99il.40 a 8.6511.59a 6.2912.08 8.4613.78 3.4313.83 3.3713.83 0.7611.25 5.9311.11 9.5511.11 11.3211.11 10.9011.15 -0.1511.37 5.1011.40 7.0711.34 9.2411.34 9.8211.27 ab 17.4211.15° 11.1911.33 12.7611.20° 10.71il.27 a b 10.5111.44 6.6312.02 8.4912.47 9.0412.92 9.4711.74 6.1212.13 9.8213.15 8.1712.82 0.7911.29 4.4211.29 7.4611.29 8.9811.23 9.7011.23 9.18il.36 a 6. 3511. 62a° 9.1211.99 3 .2212.31 6.8312.80 4.7513.16 a,b,cNumbers with d i f f e r e n t l e t t e r superscripts within each row are d i f f e r e n t at least at the 5.00 percent l e v e l of confidence (p<0.05). Values are presented as least squares means for milk P,. 5 8 F i g u r e 4. E f f e c t O f Human C h o r i o n i c G o n a d o t r o p i n On M i l k P4 C o n c e n t r a t i o n s In Dai ry Cat t le ( F a r m ff 1) p 4 C o n c n f m 1 20 15 10 0 -5 0 i r 10 20 30 40 Number Of Days Post Breeding Day 0 T R E A T M E N T S - Day 7 - * ~ Day 14 " B " Control 50 CT) LO 6.1.8. E f f e c t Of hCG on Pregnancy Rates Treatment with hCG lead to s i g n i f i c a n t (P<0.01) increase in pregnancy rates (table 6). Pregnancy diagnosis at day 60 conducted by palpation per rectum showed that out of 20 cows treated i n each of the day 0, 7 and 14 treatment groups, 25.00, 35.00, and 35.00 percent, i n that order, were diagnosed pregnant. There was a 21.05 percent pregnancy rate among 19 cows cons t i t u t i n g the control group. Table 6. E f f e c t of Human Chorionic Gonadotropin Treatment Given Either At The Time Of Breeding, Day 7 Or 14 Post Breeding, On Pregnancy Rates* In Holstein Dairy Cows. Treatment Day 0 Day 7 Day 14 Control Total S i t e farm # 1 5/20 7/20 7/20 4/19 79 (%) (25) (35) (35) (21) farm # 2 9/29 16/32 12/29 8/31 121 (%) (31) (50) (41) (26) Grand Total 14/49 23/52 19/49 12/50 200 (%) (29) (44) (39) (24) hCG s i g n i f i c a n t l y increased (P<0 .01) pregnancy rates in treatments given on either day 7 or 14 post breeding. 60 6.1 .9 . E f f e c t Of hCG On Cycle Length and E x h i b i t i o n Of Oestrus Levels of P4 concentrations were used to determine the time when cows returned to oestrus. Based on P4 p r o f i l e s , 13 cows treated with hCG on day 0 were found to have low P4 l e v e l s at various times ranging from 14 to 42 days post AI (figure 5) some of which were also observed to be i n oestrus by day 42 post oestrus (figure 6) . There was one case of low P4 by day 14 post AI and these were subsequently low again on day 3 5 post AI. The cow was, however, not detected u n t i l discontinuation at day 42 post AI. Four cows had low P4 l e v e l s , two on days 16 and the other two on day 18 post AI. The remaining seven cows had low P4 levels either on day 21 (n=5) , 23 (n=l) or 28 (n=l) . Based on the P4 l e v e l s , i t appeared that the cow with low P4 l e v e l s on day 2 3 was c y s t i c . One cow had high P4 l e v e l s u n t i l day 42 post AI but was reportedly rebred on day 56 post AI. Four cows were rebred on day 21, one on day 24, one on day 26, one on day 3 0 and another on day 39 post AI. The remaining cows s t i l l had high P4 l e v e l s on day 42 post AI. There i s no data to indicate when these cows returned to oestrus. Among cows treated on day 7 post AI, 14 were found to have low P4 between day 21 and 42. Four were found to have low P4 l e v e l s on day 21 post AI, 3 were rebred the same day. The other cows was rebred on day 28. Four more cows were reported to have been rebred by day 28. No data i s currently available on the remainder of the cows. Twelve cows treated on day 14 post AI were found to have low P4 concentrations at various times a f t e r AI. Out of these cows, 6 had low P4 either on day 18 or 21 post AI. While four of these were rebred on schedule (day 21), the other two went undetected u n t i l a 61 subsequent oestrus on day 4 2 post AI when one of them was rebred. Two cows had low P4 concentrations on day 24 and were observed to be i n oestrus on the same day. Hence they were both rebred on day 24 post AI. Another two cows had low P4 l e v e l s on day 28 post AI. One of them was rebred on day 3 0 while the other was rebred on day 50 post AI. One cow was found to have consistently low P4 concentrations way into l a t e dioestrus and was, therefore, assumed to have been c y s t i c . No complete data for P4 concentrations was available for the l a s t cow. Among the cows not receiving treatment with hCG, two had r e l a t i v e l y short oestrous cycles. One had low P4 concentrations on day 14 post AI and these subsequently rose and remained high u n t i l the l a s t recorded sample on day 35 post AI. The other cow that had low P4 concentrations on day 16 was rebred on day 18 post AI. Normally P4 concentrations begin to f a l l s t a r t i n g on day 18 post AI and h i t a nadir on day 21 (day of oestrus). Such was the case in f i v e cows and a l l of them were rebred by day 21 post AI. One cow had low P4 concentrations on day 2 4 and was subsequently rebred on that day. Two other cows were found to have low P4 p r o f i l e s on day 28 post AI. Both, however, did not show any signs of oestrus u n t i l day 53 when one i s reported to have been rebred. No data i s available for the other cow. On day 35, P4 l e v e l s were low i n one cow although the cow was reported to have been bred again on day 60. No information regarding i t s reproductive performance during the interim period was immediately available. The l a s t cow had low P4 l e v e l s on day 42 post AI but did not show any decline in P4 values during the intervening period. 6 2 F i g u r e 5. Ef fec t OF H u m a n C h o r i o n i c G o n a d o t r o p i n On Oestrous Cycle Length In Dairy Cattle (Farm § 1) 22 or less 23-28 29-35 36 or more Number Of Days To Low progestrone Level In Milk • 1 Day 0 Day 7 E l Day 1 4 H I Control F i g u r e 6. Ef fec t Of H u m a n C h o r i o n i c G o n a d o t r o p i n On Exhibit ion Of Oestrus In Dairy Cattle (Farm # 1) 22 or Less 23-28 29-35 36 or More N u m b e r Of Days To Observed Oestrus Day 0 Day 7 I Day 1 4 i M C o n t r o l 63 6.2. MATERIALS AND METHODS: Farm # 2 6 .2 .1 . Animals: General Management Prac t i ces The cows included i n t h i s t r i a l were part of the dairy herd of Holstein cows on farm # 2. In t o t a l , there were 340 Holstein cows, 150 of which were l a c t a t i n g when t h i s experiment was conducted. Twenty-five to 3 0 cows were i n the dry herd. Cows included i n the t r i a l were those presented for AI between September 18, 1989 and January, 1990. Cows were milked twice d a i l y at 03:00 and 14:00 in a herring bone milking parlour. These cows produced, on average, 8436 kg during the period t h i s experiment was conducted. The average % fat and protein were 3.20 and 2.96, respectively. During the f i r s t 2 months of the t r i a l , the cows were on pasture and concentrates for t h e i r n u t r i t i o n . The pasture consisted of orchard grass while the concentrate portion of the feed (16 % protein) was made up of 3 kg of barley mash and grain. Each animal was allocated 4 kg per day to meet i t s maintenance requirements. Extra grain, at a r a t i o of approximately 1 kg of grain to 4 kg of milk produced, was given to each animal i n order to meet the requirements for production. This concentrate r a t i o n also contained mineral supplement but did not include copper, excluded due to a t o x i c i t y problem experienced by the farm. I t was standard practice on the farm to give each animal an intramuscular i n j e c t i o n of selenium at the time of drying o f f . Dry cows were not provided with any mineral supplements. Af t e r October 1989, the cows were fed based on the above mentioned concentrate plus si l a g e . The sil a g e was made up of a 60 % corn to 40 % grass s i l a g e that together delivered 11.7 % protein. In addition, approximately 2 kg of hay (19.2 % protein) was given 64 to each cow. A l l feed was provided to the animal i n the barn. Although the farm belongs to a herd health programme, i t was not common practice to have cows examined p r i o r to the f i r s t AI post partum. A veterinary evaluation was only resorted to i f an animal f a i l e d to exhibit c y c l i c i t y well beyond 60 days following p a r t u r i t i o n . The f i r s t AI post partum normally took place around 60 days following calving, but could range from 45 to 85 days. Most cows bred during the t r i a l period f e l l between the 45 to 60 day period. Rarely was an animal v e r i f i e d to have resumed c y c l i c i t y following p a r t u r i t i o n . Emphasis was mainly on observing for oestrus at around day 60 post partum. There were no set times for oestrus detection but t h i s exercise involved almost every cow hand available on the farm. This entailed that at most times during the day, there was at least one person checking cows for heat. Heat detection, as outlined under materials and methods of experiment 1, was mostly conducted at the time of c o l l e c t i n g the animal i n preparation for milking. This took place at 02:30 and 13:30 hrs every day. A l l AI were conducted by a trained farm technician. The timing of AI was based on observing for standing oestrus. Cows were bred 12 hrs following standing oestrus. Occasionally, timed inseminations, involving the use the l u t e o l y s i n PGF2a, were used for problem cows f a i l i n g to exhibit oestrus at expected times. AI was conducted 72 hrs following the administration of the l u t e o l y s i n . 65 6.2.2 . Treatments One hundred and twenty-one l a c t a t i n g dairy cows were randomly assigned to receive treatment with hCG i n one of four treatment groups. A single intramuscular i n j e c t i o n of 1000 IU of hCG (1 ml of APLR) was administered eit h e r on day 0 (n=29) , day 7 (n=32) , 14 (n=29) post AI, or no treatment (control, n=31). Although t h i s many cows were involved i n the study, milk samples were only c o l l e c t e d from 23, 27, 24 and 2 6 cows receiving hCG either on day 0, 7, 14 post AI or not receiving any (control), i n that order. 6.2 .3 . Mi lk Sample C o l l e c t i o n Weekly milk samples were c o l l e c t e d s t a r t i n g on day 0 u n t i l day 42 post AI or u n t i l the f i r s t observed oestrus, whichever came f i r s t . A l l samples were stored at 4°C and transported to the laboratories at UBC. Quantification of P4 was ca r r i e d out as described under materials and methods of experiment 1. 6.2.4. Pregnancy Diagnosis A l l cows not returning to oestrus p r i o r to day 60 were examined to v e r i f y t h e i r pregnancy status. Pregnancy diagnosis was conducted at day 60 using the r e c t a l palpation technique by a veterinarian from the area herd health programme. 6.2 .5 . S t a t i s t i c a l Analys i s Data for milk P4 concentrations were analyzed using the procedures stated under experiment 1. Due to the homogeneity of the sampling periods (frequency), the data was analyzed i n one batch. 66 6.2 .6 . RESULTS: Farm # 2 6 . 2 . 6 . 1 . M i l k Progesterone P r o f i l e s The p r o f i l e s , LSMeans and analysis of variance on P4 l e v e l s are presented i n figure 7, table 7 and appendix 5, respectively. Overall, hCG s i g n i f i c a n t l y increased (P<0.01) milk P4 l e v e l s i n cows receiving the treatment at either 7 (13.01±0.56 ng/ml) or 14 (10.65±0.58 ng/ml) days post AI. Cows receiving the treatment on day 0, however, had P4 l e v e l s not d i f f e r e n t from those of the control group. Their milk P4 l e v e l s were 7.54±0.74 and 8.39±0.73 ng/ml, respectively. P4 Levels i n milk also tended to increase with time. S i g n i f i c a n t interactions between treatment and time made i t d i f f i c u l t to ascribe the s i g n i f i c a n t increase i n P4 to either treatment or merely time (sample number). Nonetheless, there were no differences between LSMeans for milk P4 l e v e l s at oestrus when cows were randomly assigned to receive hCG at the respective time. This lack of divergence between means persisted u n t i l day 7 post AI. At 14 days post AI, cows receiving treatment at day 7 had s i g n i f i c a n t l y (P<0.05) higher P4 l e v e l s (12.38±1.27 ng/ml) compared to cows that received treatment at oestrus (8.65±1.36 ng/ml). There were no detectable differences between cows treated either on day 14 (10.46±1.27 ng/ml) or those not receiving treatment with hCG (11.09±1.37 ng/ml) when compared to the day 7 treated cows. Comparisons made at day 21, however, show both day 7 (13.75±1.27 ng/ml) and day 14 (10.31±1.31 ng/ml) treated groups of cows as having s i g n i f i c a n t l y higher (P<0.01) le v e l s of P4 when compared to ei t h e r control group cows (5.7411.36 ng/ml) or those treated on day 0 (4.0511.40 ng/ml). 67 At day 28 post AI, cows treated on either day 7 (14.22±1.44 ng/ml) or day 14 (13.59+1.47 ng/ml) both s t i l l had s i g n i f i c a n t l y higher (P<0.01) P4 le v e l s than those treated on day 0 (6.29+1.94 ng/ml) . Although these cows also had le v e l s of P4 tending to be higher than the control group (10.70+2.14 ng/ml), no s i g n i f i c a n t differences, however, could be detected between t h e i r respective treatment LSMeans for milk P4 l e v e l s . At day 35, milk P4 remained elevated i n cows treated on either day 7 or 14 (17.67 + 1.59 and 18.77+1.63 ng/ml, respectively) when compared to l e v e l s of the same hormone i n cows either not receiving (9.7912.27 ng/ml) or those administered with hCG on day 0 (9.55+2.04 ng/ml). Forty-two days post AI, cows treated on day 7 (26.78 + 1.72 ng/ml) had s i g n i f i c a n t l y higher (P<0.01)1) P4 l e v e l s than any other treatment group. Corresponding P4 l e v e l s , i n cows either not receiving hCG or those administered with the hormone on day 0 or at day 14, were 14.4212.44, 17.34+2.46, and 15.92+1.63 ng/ml, i n that order. 6 8 Table 7 . E f f e c t Of Human Chorionic Gonadotropin Treatment Given Either At The Time Of Breeding, Day 7 or 14 Post Breeding, On Milk Progesterone Concentrations* In Holstein Dairy Cows. Days Post Treatment Group3'b Breeding Day 0 Day 7 Day 14 Control 0 1 . 19 + 1 . 74 - 0 , . 0 1 1 1 . . 5 3 0 . 68 + 1 . 3 6 1 . 0 9 1 1 . 5 2 7 5 . 7 2 1 1 . 5 5 6, . 2 6 1 1 . . 2 7 4 . 7 9 1 1 . 2 7 5 . 9 2 + 1 . 4 2 14 8 . 6 5 + 1 . 3 6 a 1 2 . 3 8 ± 1 . 2 7 b 1 0 . 4 6 + 1 . 2 ? a b 1 1 . . 09 + 1 . 3 7 a b 2 1 4 . 05 + 1 . 4 0 a 13 . 7 5 ± 1 . 2 7 b 1 0 . 3 1 1 1 . 3 1 b 5 . . 7 4 1 1 . 3 7 a 28 6 . 29 + 1 . 9 4 a 14 . 2 2 1 1 . 4 4 b 13 . 5 9 1 1 . 4 7 b 10 . 7 0 1 2 . 1 4 a b 3 5 9 . 5 5 1 2 . 0 4 a 17 . 67 + 1 . 5 9 b 18 . 77 + 1 . 6 2 b 9 . 7 9 + 2 . 2 7 a 42 17 . 34 + 2 . 4 6 a 2 6 . 78 + 1 . 7 2 b 1 5 . 9 2 1 1 . 6 3 a 14 . 4 2 1 2 . 4 4 a a,b'Numbers with d i f f e r e n t l e t t e r superscripts within each row are d i f f e r e n t at least at the 5.00 percent l e v e l of confidence (P<0.05). Values are presented as least squares means for milk P,. 6 9 F i g u r e 7 . E f f e c t O f H u m a n C h o r i o n i c G o n a d o t r o p i n On M i l k P4 C o n c e n t r a t i o n s In Dai ry Catt le ( F a r m § 2 ) 30 i 0 10 20 30 40 50 N u m b e r Of Days Post Breeding T R E A T M E N T S Day 0 — D a y 7 Day 14 C o n t r o l 6.2.6.2. E f f e c t Of hCG On Pregnancy Rates Pregnancy rates were s i g n i f i c a n t l y improved by treatment with hCG given either on day 0, day 7 or 14 post AI (table 6). Out of twenty-nine cows receiving treatment on day 0, 31.00 % (n=9) were found to be pregnant using the r e c t a l palpation technique undertaken at 60 days post AI. The respective percentage of cows conceiving to the inseminations done p r i o r to treatment with hCG on either day 7 or 14 post AI were 50.00 (n=16) and 41.40 (n=12). The corresponding values for the control group were a 25.80 % (n=8). 6.2.6.3. E f f e c t Of hCG on Cycle Length and Ex h i b i t i o n Of Oestrus For e f f e c t s of hCG on oestrous cycle length i n n o n - f e r t i l e inseminations re f e r to figure 8 and 9. Nine cows were found to have low P4 l e v e l s on day 21 post AI. One cow had low P4 on day 24 post AI was observed i n oestrus the same day. Two cows were found to have low P4 p r o f i l e s on day 14 post AI. Both were not detected u n t i l day 42 post AI. Two cases of cows that had low P4 l e v e l s on day 35 and 3 8 were recorded. Only the l a t t e r was detected i n oestrus on day 3 8 post AI. Among cows receiving hCG on day 7 post AI, 9 cows had low P4 l e v e l s p r i o r to day 42 post AI. One cow had a short cycle whose duration was 14 days. I t i s not clear whether t h i s cow returned to oestrus or not. Three cows had low P4 l e v e l s by day 21 post AI. While one of them was rebred on day 21, the other two were rebred on day 24 post AI. One cow had low P4 l e v e l s on day 26, another on day 28, each one of them being rebred on the day when they showed low P4 l e v e l s . Two cows had high P4 l e v e l s u n t i l 3 3 and 3 5 days post AI, the former being rebred on the day i t s P4 l e v e l s were low the l a t t e r was rebred 40 days post AI. The l a s t cow had high P4 l e v e l s u n t i l sampling ceased at day 42 post AI. Amongst cows treated on day 14 post AI, 10 cows showed low P4 l e v e l s . P4 p r o f i l e s of two cows did not decline u n t i l day 42. There was one case of a short cycle l a s t i n g 14 days and was rebred on the same day. Five cows showed low P4 l e v e l s on day 21 post AI. Three of these 5 cows, however, had high P4 l e v e l s u n t i l day 42 post AI. While the fourth cow was rebred on day 24, the l a s t one was rebred on day 38 post AI. Two cows were found to have low P4 l e v e l s on days 2 5 and 2 6 post AI. Each one was rebred on the respective day when P4 l e v e l s were low. The l a s t four cows a l l had P4 levels dropping to oestrus l e v e l s on day 28 post AI. Only one cow was rebred on the day coinciding with the low P4 l e v e l s . The other three s t i l l had high P4 l e v e l s at day 42 when sample c o l l e c t i o n was terminated. P4 l e v e l s were.low in 15 cows i n the control group. Eleven of these cows had low le v e l s of P4 on day 21 post AI. However only 6 were rebred by day 22 post AI. Two cows were rebred before day 30 post AI, one on day 25 and the other on day 28. The remaining 4 were not observed i n oestrus u n t i l day 42 when sample c o l l e c t i o n ceased. Of the remaining 3 cows, one had low P4 p r o f i l e s on day 2 5 and another on day 28. Both were rebred on day 28 post AI. For the l a s t cow, l e v e l s of P4 were low on day 38, and was rebred on the same day. 72 Figure 8. Effect Of Human Chorionic Gonadotropin On Oestrous Cycle Length In Dairy Cattle (Farm # 2). 14 - i 22 or Less 23-28 29-35 36 or More N u m b e r Of Days To Low P r o g e s t e r o n e Levels I n M i l k •I Day 0 B ^ l Day 7 111 Day 14 H H C o n t r o l Figure 9. Effect Of Human Chorionic Gonadotropin On The Exhibition Of Oestrus In Dairy Cattle (Farm # 2). 22 or Less 23-28 29-35 36 or More N u m b e r Of Days To Observed Oestrus • i Day 0 Day 7 I I Day 14 H H C o n t r o l 73 7. DISCUSSION Techniques for reducing losses i n reproductive e f f i c i e n c y i n liv e s t o c k have eluded mankind for time immemorial. Much ground has been covered i n of elucidating, f i r s t what forces predispose the losses i n reproduction and secondly finding ways and means for overcoming compromised f e r t i l i t y . Although we are far from understanding the agents causing the demise of embryos (Heap, 1985), i t i s becoming increasingly c l e a r that some factors predisposing early embryonic mortality are susceptible to manipulation at one point or another. The data to under discussion i s j u st one example. The purpose of conducting t h i s experiment was to develop a viable method of supplementing endogenous P4 concentrations. I t was intended to emulate nature i n exemplified by the mare wherein "new" CL are developed some 45 to 60 days post oestrus (Hafez, 1976). These accessory CL are developed when the spontaneous CL begins to exhibit diminished functional e f f i c i e n c y . 7.1. Induction Of Accessory Corpora Lutea One of the aims of the f i r s t experiment was to determine i f giving intramuscular injections of hCG, at various times following breeding, would induce the development of accessory CL. Treatment with hCG increased the number of CL i n cows treated on day 0. Such a finding i s corroborated by re s u l t s obtained by Price and Webb, (198 9). The report of these authors i s the only one that documents v i s u a l i z i n g , with the aid of a laparoscope, the e f f e c t s of treatments such as hCG given during the l u t e a l phase i n the bovine. The increased number of ovulations observed i n the group of animals treated on day 0 could be attributed to the e f f e c t of hCG for two reasons; f i r s t l y , the incidence of twin ovulations i n c a t t l e i s low (Price and Webb, (1989) , secondly the ultrasound scanning conducted on day 7 showed that i n one cow, the appearance of one CL was not t y p i c a l of that found at t h i s stage of the oestrous cycle. The greatest po t e n t i a l to be r e a l i s e d from using hCG on day 0 l i e s in i t s c a p a b i l i t y to synchronize ovulations at oestrus and possibly increase the frequency or number of these ovulations (Wagner et a l . , 1973). The present study shows that 7/9 of the cows treated on day 7 developed accessory CL. Similar r e s u l t s were reported by Price and Webb, (1989). These authors found that 83 % of hei f e r s receiving hCG given during the early l u t e a l phase (days 4 to 7 post oestrus) developed accessory CL. The ovulatory pot e n t i a l of the dominant f o l l i c l e was the subject of a recent report by Savio et a l . , (1990). The dominant f o l l i c l e was the ovulatory one i n almost 93 % (n=40) of the hei f e r s following treatment with a l u t e o l y s i n (PGF2a or PGF2a analogue) given on day 7 post oestrus. S i m i l a r l y , Walton et a l . , (1989) evaluated the ovaries of beef h e i f e r s treated with hCG on day 5.5 and slaughtered on day 12. The spontaneous CL was accompanied by a "new" 4-day old CL. However, only 50 % of the animals treated on day 14 responded by developing accessory CL as a consequence of the day 14 treatments with hCG. Similar results were reported by Price and Webb, (1989). In t h e i r experiment, 66 % of the animals receiving the hCG treatment between days 14-16 post oestrus developed accessory CL. McDermont et a l . , (1986) also found a higher frequency of accessory CL i n animals given hCG on day 15 post oestrus. 75 de Los Santos-Valadez, (1982) gave hCG on day 15 post oestrus and found 25 % of the animals receiving hCG had palpable accessory CL. When animals were examined at caesarian section, Greve and Lehn-Jensen (1982) found that hCG given every other day st a r t i n g from day 13 u n t i l day 35 had stimulated the ovaries, the number of accessory CL ranged from 2 to 5. Wiltbank et a l . , (1961) also found 1 to 3 accessory CL i n 67 % of the pregnant h e i f e r s treated with d a i l y i n j e c t i o n s of hCG s t a r t i n g on day 14 u n t i l day 34. The fa c t that only 7/9 of the cows developed accessory CL indicates that a f a i r number of f o l l i c l e s f a i l e d to respond to the gonadotropin. The reduced response, and the v a r i a t i o n from other values quoted i n other experiments, could be due to a vari e t y of reasons. Variations i n accessory CL development could be found i n postulations that i n the cow and sheep (Webb et a l . , 1978; England and Webb, 1979; Webb and England, 1979) the binding of hCG to the f o l l i c u l a r wall i s very variable and the binding c a p a b i l i t y i s only lim i t e d to one or two large antral f o l l i c l e ( s ) . Mertz et a l . , (1981) presented evidence, based on f o l l i c u l a r f l u i d content of P4 and/or oestradiol (E2) , ind i c a t i n g that the large day-6 f o l l i c l e s i n the cow were less l i k e l y to be a t r e t i c . The above authors' observations were i n agreement with postulations of Rajakoski (1960) and both the i n - v i t r o and in-vivo studies of Bennett et a l . , (1989). The l a t t e r authors found greater response to hCG treatment when t h i s was administered during the early l u t e a l phase (days 3- 7), a phase when receptors to LH/hCG were reported to be maximal. It i s possible, too, that cows show a diminished response to such treatments (de Los Santos-Valadez et a l . , 1982) or that day 7 could have been premature to administered the treatments. Even though 76 these f o l l i c l e s were growing, they probably s t i l l did not have the f u l l complement of receptors (Lavoir and Fortune, 199 0). S t i l l l ess animals developed accessory CL from treatments given at l a t e to mid-dioestrus (day 14-15 i n the present experiment). I t i s possible that most of the dominant f o l l i c l e s present during dioestrus might have been a t r e t i c or regressing (Rajakoski, 1960, Mertz et a l . , 1981). Evidence supplied by Ireland and Roche (1982, 83) indicates that once ovarian f o l l i c l e s become a t r e t i c , t h e i r capacity to bind LH/hCG diminishes dramatically. A reduction i n the number of receptors during dioestrus has been documented (Ireland and Roche (1983), i n d i c a t i n g that less receptors were available at the time we report the l e a s t response (day 14) . Lavoir and Fortune (1990) have also recently reported that there appears to be congruence between the attainment of morphological dominance and functional dominance. The two phenomena coincide during the f i r s t wave of f o l l i c u l a r growth (early plateau phase), t h i s coincidence i s l o s t as the f o l l i c l e s proceed in development (late plateau phase). Solely using ultrasound, i t i s not easy to detect these differences. I t i s apparent from the foregoing that f o l l i c l e s do respond to ovulatory doses of gonadotropins. That responsiveness seems to be li m i t e d to a window such as the one i d e n t i f i e d i n t h i s experiment (day 7 post oestrus). 7.2. Growth of Corpora Lutea The e f f e c t of hCG on the formation, growth and function of the CL was c l e a r l y demonstrated by comparisons between hCG-treated versus control animals. Treatment with hCG lead to s i g n i f i c a n t 77 increase i n CL diameter. This premise of the e f f e c t of hCG on the CL was sustained u n t i l day 42. I n i t i a l l i n e a r increase was evident and t h i s was taken to be in d i c a t i v e of the response of spontaneous CL to the luteotropic e f f e c t of hCG. The growth of these induced CL was superimposed on the e f f e c t of hCG on the spontaneous CL. The CL which were induced by the treatment grew to an extent that at one point i t was not possible to dis t i n g u i s h between spontaneous versus induced CL. Such a d i f f i c u l t y was also experienced by Price and Webb, (1989). No data i s presently available i n the l i t e r a t u r e to warrant any comparisons on the formation and growth of the CL. Most data examined i s only li m i t e d to c i t i n g the development of accessory CL but lack biometrical measurements of these structures. Results obtained from t h i s study provide evidence that the induced CL are maintained for a considerable period of time during gestation. The only short coming of the experiment i s that i t did not follow the fate of these induced CL and t h e i r functional c a p a b i l i t y remains undetermined. 7.3. Progesterone P r o f i l e s Elevated P4 concentrations i n female animals, have been shown to be indispensable for the establishment and maintenance of pregnancy (Hammond, 1927; Estergreen et a l . , 1968). I t has been shown that such l e v e l s must be maintained at a higher than basal l e v e l (Staples and Hansel, 1961), f a i l i n g which pregnancy i s negated (Bulmann and Lamming, 1978). Levels of P4 i n an oestrous cycle preceding (Folman et al.,1973; Fonseca et a l . , 1983) or proceeding a p a r t i c u l a r insemination (Erb et a l . , 1976; Lukaszewska 78 and Hansel, 1980; Thomson et a l . , 1980) have been shown to be associated with p r e v a i l i n g l e v e l s of f e r t i l i t y . Differences i n plasma P4 concentrations between hCG treated versus control cows i n the current study were present only on days 18, 35 and 42 post breeding. Similar r e s u l t s were reported by McDermott et a l . , 1986, de Los Santos-Valadez et a l . , 1982) Interesting enough, r e s u l t s reported by de Los Santos-Valadez et a l . , (1982) indicate that t h i s difference was only evident in he i f e r s and not i n cows. Responses such as t h i s one lead to the supposition that animals may d i f f e r i n t h e i r c a p a b i l i t y to respond to gonadotropins depending on t h e i r age status. Bennett et a l . , (1989) also reported s i g n i f i c a n t differences i n plasma P4 concentrations s t a r t i n g at day 4 u n t i l day 8 post oestrus. Conversely, Morris et a l . , (1987) suggested that there are no differences between P4 concentrations among cows having single ovulations compared to those having twin ovulations. This assertion lead Price and Webb, (1989) to believe that c o l l e c t i o n of blood for P4 concentration determination may not be worthwhile. I t could be argued, however that these induced structures are functional. If such i s the case, then i t seems reasonable to assume that peripheral l e v e l s of P4 should be d i f f e r e n t at some point i n time. Whether or not t h i s lack of divergence i s due to a non-functional state of induced CL, i s yet to be established. Results obtained from t h i s study seem to indicate that these accessory CL are functional. This lack of agreement regarding differences between animals bearing twin versus those carrying only the spontaneous CL, may be attributed to the use of peripheral blood. 79 The e a r l i e s t detectable differences i n milk P4 occurred on day 14 post breeding among animals on farm # 2. Similar differences were observed also i n the animals at the South Campus of UBC, although differences i n t h i s case were only evident s t a r t i n g from day 21 post breeding and persisted u n t i l day 42 post breeding. The P4 concentrations of the animals on farm # 1 were d i f f e r e n t between hCG-treated versus control cows only on days 16-18 post breeding. The present experiment detected a tendency towards lower milk P4 concentrations among animals receiving hCG on day 0. Such a finding i s i n agreement with r e s u l t s reported by Echternkamp and Maurer (1983). There i s no immediate explanation for t h i s inconsistency i n the variations of P4 p r o f i l e s i n these groups. I t i s improbable that the f a i l u r e to es t a b l i s h pregnancy i n a majority of animals on farm # 1 might have contributed to lowering the e f f e c t of hCG on P4 concentrations (Lewis et a l . , 1990). There are speculations, however, to the e f f e c t that d i f f e r e n t animal populations and, indeed, d i f f e r e n t batches of the hormone administered w i l l i n some way influence the response by animals receiving the treatment (de Los Santos-Valadez et a l . , 1982). It i s possible that differences i n P4 concentrations could have been s i g n i f i c a n t at the utero-ovarian vein. Such a conclusion i s supported by the work of Ireland and Roche (1983) who f a i l e d to detect any changes i n serum P4 concentrations despite showing several f o l d increases i n f o l l i c u l a r f l u i d content of P4. This fact merely underscores the suggestion that hormones i n peripheral c i r c u l a t i o n are greatly d i l u t e d . The lack of s i g n i f i c a n t increases i n P4 concentrations among cows treated on day 0 may have been due to the reduction i n the s i z e and number of small and large l u t e a l 80 c e l l s , respectively (sheep, A l i l a and Hansel, 1984; pig, Wiesak, 1989). Such treatments with hCG given i n late-dioestrus, were reported to induce morphological changes of the l u t e a l c e l l s . The reduction i n the small l u t e a l c e l l s could have contributed to the lack of s i g n i f i c a n t increases i n P4 concentrations reported i n the present study. This was, however, i n contrast with the results reported by Bennett et a l . , (1989) who found no such morphological changes when animals received hCG l a t e r i n the oestrous cycle. I t i s possible the lack of s i g n i f i c a n t increases i n P4 concentrations i n animals receiving hCG might been mediated v i a reductions i n LH receptors seen between days 10 and 14 post oestrus (Mee et a l . , 1990). These re s u l t s were done using CL preparations from animals receiving hCG either at oestrus or 2 days post oestrus. Similar suggestions were proposed by Lucy and Stevenson (1986). It should be appreciated, however, that P4 concentrations i n animals tend to be very variable. This v a r i a b i l i t y has been attributed to differences i n development rates of CL and the o s c i l l a t o r y nature of P4 secretion during the l u t e a l phase (Stubbings and Walton, 1986). 7.4. Pregnancy Rates Estimating the f i n a n c i a l costs that are imposed by losses in pregnancy i s a very d i f f i c u l t task to accomplish, amounting to US $1.40 m i l l i o n per annum i n the US (Roberts et a l . , 1990). F l i n t et a l . , (1990) on the other hand, estimated such losses as being in excess of £300 m i l l i o n s t e r l i n g per annum for the dairy and beef industry i n the United Kingdom. 81 The use of hCG i n the current experiment s i g n i f i c a n t l y increased pregnancy rates, more so when given on day 7 or 14. Such a finding i s corroborated by some reports (Hansel et a l . , 1976; Echternkamp and Maurer, 1983 i n h e i f e r s ; Babbler and Hoffman, 1974; Wagner et a l . , 1973; de Los Santos-Valadez et a l . , 1982; McDermott et a l . , 1986 i n animals with compromised f e r t i l i t y ) . Reports of no s i g n i f i c a n t e f f e c t of the treatment are also documented (Hansel et a l . , 1976 i n beef c a t t l e ; Looney et a l , 1984). Conversely, evidence of a negative e f f e c t on pregnancy has also been advanced (Hansel et a l . , 1960 i n dairy c a t t l e ; Ecternkamp and Maurer, 1983 i n cows; McDermott et a l . , 1986 i n c a t t l e of high f e r t i l i t y ) . Although the pregnancy rates i n the control group of the UBC South Campus herd was comparable with those obtained i n an e a r l i e r study (Rajamahendran et a l . , 1989), corresponding rates for the two farms involved i n experiment two were very low. Providing a concise breakdown of the reasons for such low rate of f e r t i l i t y i s an insamountable task. Some of the losses i n f e r t i l i t y could be attributed to wrong timing of inseminations, more so on farm # 1 where 2 3 % of the inseminations were ca r r i e d out when P4 concentrations were above a 1.00 ng/ml l e v e l and were either 3-5 hrs premature or overdue. These observations are incompatible with those observed i n the t r i a l conducted on farm # 2 where only 7 % of the inseminations were ca r r i e d out when P4 concentrations were s t i l l above the 1.00 ng/ml l e v e l . Previous reports indicate that t h i s i s a problem f a i r l y common to many a dairyman. Appleward and Cooke, (1976) reported that based on plasma P4 concentrations, 10 to 20 % of the animals presented for insemination were not ready for insemination. On the other 82 hand, Hoffmann et a l . , (1976) based t h e i r study on milk P4 concentrations and found that between 14 and 26 % of the animals were not i n oestrus at the time they were bred. I t was, however, reported by Foote et a l . , (1979) that 19 % of animals do show oestrus even though P4 concentrations i n milk are high. I t i s not cle a r as to how many of these are i n true oestrus. We note from t h i s study that on farm # 1, 3/18 of the animals inseminated when P4 concentrations were high conceived. Animals inseminated when ovulation has already occurred have been reported to exhibit compromised f e r t i l i t y (Trimberger and Davis, 1943; Boyd, 1970; de Kruif, 1978). One reason for t h i s depression i n f e r t i l i t y i s that the ova are over aged or old (Lodge, 1976) by the time the animals are presented for AI. This has been associated with impairment i n the development of the early embryo. An optimal time for conducting AI has since been defined (Hafs and Boyd, 1973). This was s i m p l i f i e d by these authors to be; "....that a l l animals observed i n oestrus i n the morning should be bred that afternoon, while those observed i n the evening to be standing to be mounted by other cows should be bred the following morning...." I t i s plausible that some of the losses i n pregnancy rates on farm # 2 could have been due to the f i r s t inseminations being done too early r e l a t i v e to p a r t u r i t i o n . I t could be possible, too, that the f a i l u r e to ensure that an animal was observed to have resumed c y c l i c i t y p r i o r to breeding could have increased the incidence of animals f a i l i n g to conceive. This i s drawn from the fact that animals seen i n oestrus by day 60 were bred i r r e s p e c t i v e of whether these cows were detected i n heat since p a r t u r i t i o n . The f i r s t heat 83 exhibited following p a r t u r i t i o n i s usually followed by a l u t e a l phase of diminished duration. The meagre pregnancy rates recorded i n the control groups of the second experiment are only comparable to those obtained by (Monty and Wolf, 1974). While the adverse environmental conditions was one possible reason advanced i n an attempt to explain such low rates of f e r t i l i t y , i t i s probable that the change i n the feeding systems undertaken when the current experiment was underway may have played a part i n influencing these f e r t i l i t y rates. The change involved a switch from an open grazing system based on a pasture consisting of mixture of grasses (timothy and rye) and clovers (red) to a winter schedule of ind i v i d u a l feeding system. Inherent i n t h i s change i n the n u t r i t i o n a l q u a l i t y of the feed that the animals received was a possible change i n the protein content of the feed. Rattray, (1977) reported that excess dietary protein was associated with increases i n the basal rate of loss in embryos. Since nutrients required for reproduction are generally the same nutrients required for growth and milk production (Hafs and Boyd, 1973), i t i s not easy to accept t h i s as an explanation for the reduction i n f e r t i l i t y . No report i s available i n d i c t i n g that animals i n the study experienced loss i n milk production. Conversely, indications are that production remained constant over the years. The denial of elements such as selenium and copper to the animals on farm # 1, may have compromised f e r t i l i t y . The use of selenium to improve f e r t i l i t y rates i n c a t t l e i s s t i l l unconvincing. Its major benefit i s the reduction i n the incidence of retained placentae (Ammerman and M i l l e r , 1975; Trinder and 84 Renton, 1973) or development of a muscular condition (white muscle disease) among calves borne of cows d e f i c i e n t i n selenium (Kincaid and Hodgson (1989), more so when the deficiency i s severe (Weiss et a l . , (1990). Less severe cases cause m e t r i t i s (Harrison et a l . , 1984) . Deprivation of such other elements as copper, vitamin A and Iodine, have also been implicated i n the incidence of retained placenta i n c a t t l e (Eger et a l . , 1985). Information available at t h i s moment indicate no change i n the rate of retained placentae or, indeed, white muscle disease. On the contrary, i n a herd of 230 on farm # 1, only 2-3 cases are normally experienced each year. It should be r e a l i s e d , however, that the metabolism of these minerals in-vivo, i s greatly influenced by how other elements are metabolised and vice versa. I t might s t i l l be possible that the e f f e c t of inadequate selenium and/or copper could have been effected v i a changes i n another element. Wagner et a l . , (1973) suggested that the main benefit of administering hCG at the time or just p r i o r to breeding mainly l i e s i n i t s a b i l i t y to influence the timing rather than increasing ovulation rates i n treated animals. Results from t h i s study and information available i n the l i t e r a t u r e seems to support t h i s view. Greater p o t e n t i a l for increasing pregnancy rates i n c a t t l e seems to be the use of hCG on day 7 post oestrus. The response i n t h i s group of animals c l e a r l y shows supe r i o r i t y not only i n developing accessory CL but also t h e i r capacity to secrete P4. I t i s , no doubt, due to t h i s that we also experienced the greatest increase i n pregnancy rates among the same group of animals. The 2 4 % increase i s the greater than any s i t e d i n the l i t e r a t u r e . Premise for t h i s response was provided by the detailed study (experiment 85 one) conducted at the South Campus of UBC where we found a 40 % increase i n animals treated on day 7 post oestrus compared to the control group. Hence, the contention that the use of hCG may only be b e n e f i c i a l i n cases of established i n f e r t i l i t y (Diskin and Sreenan, 1985). This should be approached with caution, because the current study has established the appropriate time for giving such treatments and the discussion on P4 p r o f i l e s / c y c l e length have shown the adverse e f f e c t s of rendering these treatments at certain times r e l a t i v e to oestrus. I t must be resolved, however, what the dosage of hCG and the frequency of administering the hormone should be. I t i s not clea r whether the route of administering the drug play any ro l e i n manipulating the responsiveness of animals. There seems to be much promise i n r e a l i s i n g better responses with use of hCG to improve f e r t i l i t y i f only we can get evaluate contribution of each one of these factors that are so variable r i g h t now. Diskin and Sreenan (1985) emphasized that the use of hCG to improve f e r t i l i t y , was not l i k e l y to produce conclusive results u n t i l we a t t a i n a better understanding of the endocrine processes involved i n l u t e o l y s i s and the establishment of pregnancy. The same problem can be adequately answered by obtaining a thorough understanding of f o l l i c u l a r dynamics i n the cow. Such again seems to be the advantage of the present study. The question, however, as to whether the f o l l i c l e s are present during the l u t e a l phase in order to ensure optimal function of the CL, i s yet to be answered. It might j u s t be that the f o l l i c l e s are present i n order to ensure l u t e o l y s i s i n the event of a pregnancy f a i l i n g to es t a b l i s h . 86 This study has also i d e n t i f i e d the fact that pregnancy rates can be improved upon through treatments with hCG given on day 7 post oestrus. Whether the r e s u l t s obtained from t h i s study can not be obtained i f the animals with a high l e v e l of f e r t i l i t y were used, i s another question that remains unanswered. 7.5. Cycle Length And Expression Of Oestrus Treatment with hCG has received variable r e s u l t s i n several aspects of reproductive physiology i n the cow. Notable among these, and equally unresolved, i s the e f f e c t of hCG on the length of the oestrous cycle i n non-pregnant animals. Included for discussion, i s the e f f e c t of hCG on the psychic expression of oestrus. S i l e n t oestrus i s one of the scapegoats that most dairymen use i n an attempt to explain the high incidence of cows returning for service. I t has, however, been stated that several cows in the herd do show signs of oestrus but due to inadequate observations, these go unnoticed (de Kruif, 1978). Animals observed continuously approached a 100 % rate of being detected i n standing oestrus (Williamson et a l . , 1972). Casual observation, on the other hand, were only capable of detecting 50 % of cows in oestrus. Such observations lead one to conclude that most of what we have termed as " s i l e n t oestrus" may r e a l l y be a r e f l e c t i o n of the inadequacy reproductive management rather than an i n d i c a t i o n of physiological f a i l u r e on the part of the cow to express oestrus. I t would be i n t e r e s t i n g to f i n d out through continuous observation of animals prone to exh i b i t i n g s i l e n t oestrus, i f indeed the phenomenon exists at proportions we have ascribed to i t . Several animals included in the two experiments showed low P4 concentrations at one time or 87 another between days 21 and 4 2 post oestrus. For most of these animals, at le a s t one cycle was not detected. The proportion of hCG-treated cows with low peripheral P4 and detected i n oestrus never exceeded 50 %, which was r e l a t i v e l y lower than the 78 % detection rate for control cows. This trend towards an increase i n the number of cows not returning for service by day 21 was s i m i l a r to rates reported by Macmillan et a l . , (1986) who used a GnRH agonist (buserelin). They noted a s l i g h t increase i n the number of animals not returning to oestrus at day 21 post oestrus. A s l i g h t l y longer (8-10 day) extension of the cycle was experienced by cows treated on day 14. Similar r e s u l t s have been documented (de Los Santos-Valadez et a l . , 1982; Macmillan et a l . , 1986; Morris et a l . , 1976; Wiltbank et a l . , 1961; Eduvie and Seguin 1982; Seguin et a l . , 1977). Conversely, Helmer and B r i t t , (1986) who found no difference i n days to f i r s t expressed oestrus following hCG treatment. This extension ranged from 2 to 8 days. Indications from the present experiment are that the functional capacity of the CL of both the treated and control cows ceased at approximately the same time. There was, however, an impairment i n the expression of oestrus as shown by the low number of animals a c t u a l l y observed i n oestrus. This trend leads to contentions that hCG increased cycle length. Clearly, the P4 concentrations corroborated the data on the diameter of the CL which showed regression of the CL at times corresponding to a decline i n P4 p r o f i l e s . The f u n c t i o n a l i t y of CL cease on schedule and yet for some reason standing oestrus i s suppressed, t h i s points to an area that does not seem to have adequate answers available to 88 date. The mechanism by which cows f a i l to exhibit oestrus c a l l for further research i n t h i s area. The suppression of oestrus a f t e r day 21 merely allows a predicament to surface. Two factors have become confounded. Based on P4 p r o f i l e s , i t was not possible to determine whether early embryonic mortality occurred i n animals showing extended cycles. I t could be that the extension i n the length cycle was due to the hCG treatment. Treatment with P4, which r e s u l t i n higher P4 concentrations, increases the rate of a t r e s i a i n bovine ovarian f o l l i c l e s (Maracek et a l . , 1977). I t should be borne i n mind that the concentrations of P4 at the ovarian vein/artery l e v e l are much higher (Ireland and Roche, 1983) . This i s a viable cause of a t r e s i a i n these f o l l i c l e s . This suggested mode of action for P4 does not agree with e a r l i e r reports i n d i c a t i n g that P4 treatments p r i o r to oestrus increased the rate of detecting animals i n heat (Stevenson et a l . , 1989). If such an e f f e c t was detected, i t i s reasonable to assume that the f o l l i c l e s present were oestrogen active, hence, non-atretic. There exi s t s a p o s s i b i l i t y , however, that the timing and amount of P4 administered may be the l i m i t i n g factor i n determining when at r e s i a w i l l set i n . The increase i n P4, although not that s i g n i f i c a n t l y improved at the peripheral l e v e l , may have altered the P 4:E 2 r a t i o (Roberson et a l . , 1989). This r a t i o of P 4:E 2 represents the balance at which the two steroids either i n concert or i n d i v i d u a l l y , act to modulate p i t u i t a r y function (Stumpf et a l . , 1988). P4 alone may be the negative feedback on the p i t u i t a r y and t h i s may explain the increase i n LH concentrations i n animals receiving sub-normal l e v e l s of P4 i n the experiments of Roberson et a l . , (1989). These 89 increases i n the gonadotropin may have merely been due to the removal of an a l t e r n a t i v e negative feedback mechanism or i g i n a t i n g from the ovary, possibly a l t e r a t i o n s i n the s t e r o i d balance due to l u t e i n i z a t i o n of f o l l i c l e s . This increase i n gonadotropin lev e l s (LH) most l i k e l y may have been accompanied by concurrent increases in i n h i b i n (Padmanabhan et a l . , 1984). Increased l e v e l s of inhibin may modulate reproductive physiology by preventing the secretion of FSH from the anterior p i t u i t a r y . This, which i n turn, could have lead to f a i l u r e i n the development of receptors for LH. This mode of action i s supported by r e s u l t s obtained by Padmanabhan et a l . , (1984). LH increased i n concomitance with increases i n f o l l i c u l a r f l u i d content of i n h i b i n . No c o l l a t e r a l increases were reported for f o l l i c l e stimulating hormone. This, further provides another possible causative agent for the process of l u t e i n i z a t i o n . Hernandez-Ledezma et a l . , (1982) concluded that GnRH-induced LH release appeared to stimulate l u t e i n i z a t i o n and possibly influenced the steroidogenic pathways of these f o l l i c l e s . I t i s possible, too, that the change i n the s t e r o i d balance i n the f o l l i c l e s seems to be the f a i l u r e of oestrogen-inactive (atretic) f o l l i c l e s to convert aromatize androgens. This lead to increases i n the l e v e l of androgens i n c i r c u l a t i o n , hence, i n d i r e c t l y increased the rate of a t r e s i a (Ireland and Roche, 1983). Cystic f o l l i c l e s exhibit extensive thickening of the thecal layer while granulosa c e l l s may or may not be present (Hernandez-Ledezma et a l . , 1982). Due to these morphological a l t e r a t i o n s i n c y s t i c f o l l i c l e s , the content of f o l l i c u l a r f l u i d varies f r o m t h a t c h a r a c t e r i s t i c of normal f o l l i c l e s . This difference i s due to the lack of granulosa c e l l s which are indispensable for the aromatization of androgens. Kesler et a l . , (1980) postulated that oestrogens produced by c y s t i c f o l l i c l e s are sequestered within the f o l l i c l e . Whether the l u t e i n i z a t i o n of f o l l i c l e s observed during experiment one also p r e c i p i t a t e such functional changes i s not clear. Farookhi, (1980) suggested that androgen treatments block FSH- induced increases i n LH receptors i n the r a t . Conceptually, although large antral f o l l i c l e s are present during the l u t e a l phase of the bovine, i t i s possible that P4 or androgens may block the a b i l i t y of these f o l l i c l e s to respond to gonadotropins p r i o r to the gonadotropin surge. Similar observations were alluded to by Ireland and Roche (1983). No data on oestrogen concentrations for animals used i n t h i s study are available. Based on the f o l l i c u l a r dynamics of cows examined, the dominant f o l l i c l e present on day 14 was more l i k e l y a remnant of the day 7 dominant f o l l i c l e . I f such was the case, then i t i s possible that the f o l l i c l e s could have been regressing. The induction of accessory CL during l a t e dioestrus might have deprived the cows of the one f o l l i c l e that could have secreted s u f f i c i e n t oestrogen to i n i t i a t e the cascade of events leading to l u t e o l y s i s . L uteinization of f o l l i c l e s i s tantamount to destruction of the same. Fogwell et a l . , (1985) showed that cauterization of f o l l i c l e s extended the length of the cycle. Another hypothesis suggests that hCG i n j e c t i o n s may i n h i b i t oestrogen production by causing the depletion of the aromatizable substrates (Dieleman et a l . , 1983) or even barring the transfer of thecal androgens to the granulosa c e l l s (Evans et a l . , 1981). It has also been proposed that hCG may decrease the aromatizing c a p a b i l i t y of the granulosa membrane (Fortune and Hansel, 1979). If these hypotheses hold true, then i t would be reasonable to assume that extensions i n cycle length that have been reported, may be due to the e f f e c t of hCG either acting d i r e c t l y on the f o l l i c l e or on i t s c a p a b i l i t y to synthesize steroids. These hypotheses are corroborated by evidence supplied by Howard and B r i t t , (1989) who reported that the presence of oestrogens i s esse n t i a l for increasing the s e n s i t i v i t y of the CL to prostaglandins. The above mentioned authors also noted no apparent changes i n PGF2a secretion. Is i t possible that the e f f e c t of hCG on cycle length i s mediated v i a morphological changes i n the various types of c e l l s making up the CL (Wiesak, 1989). Treatments administered during early dioestrus (days 3-7) post oestrus reduce the size of the small l u t e a l c e l l s while decreasing, concomitantly, the number of large l u t e a l c e l l s (Bennett et a l . , 1989). Such an e f f e c t on the number of the large l u t e a l c e l l s would p o t e n t i a l l y attenuate the e f f e c t of PGF2a. These c e l l s possess receptors to PGF2a as a re s u l t reducing t h e i r numbers w i l l jeopardise the process of l u t e o l y s i s . But do these morphological changes take place i n induced CL? Evidence of such responses i s a subject that awaits future research. I t may be due to these various factors that there appears to be lack of unanimity on the e f f e c t of hCG on cycle length i n the bovine. This extension varies depending on the timing, the dose used and the frequency of administering the treatments. 92 8. SUMMARY AND CONCLUSIONS This study has contributed to the understanding of CL function i n the Holstein dairy cow. We have demonstrated, with the aid of information available i n the l i t e r a t u r e , that the dominant f o l l i c l e present during the l u t e a l phase of a cow i s capable of ovulating. This observation supports the res u l t s obtained by Price and Webb, (1989). While the technique employed to v e r i f y the e f f e c t of hCG by Price and Webb, (1989) did not allow them to d i f f e r e n t i a t e the induced structures from the spontaneous ones, use of an ultrasound machine i n the present study f a c i l i t a t e d our c a p a b i l i t y to i d e n t i f y the two types of CL. That technique and timing available for inducing the development of accessory CL repaid our e f f o r t s i n two ways. F i r s t , P4 concentrations were increased i n animals treated with hCG and one can only assume that the magnitude of the increaments were much more pronounced at the utero-ovarian vein. Animals showing the best response also showed increase even in pregnancy rates. This was more so among treatments rendered on day 7 post oestrus, a time i d e n t i f i e d as an appropriate window for supplementing endogenous P4 production. Based on t h i s type of response, we derive second conclusion. The i n i t i a l assumption was that low P4 concentrations which were i n d i c a t i v e of CL dysfunction, accounted for a considerable proportion of the embryos which perished during the f i r s t 30 days of gestation. Such increases as we have shown confirm the fact that d e f i c i e n c i e s i n P4 are, indeed, a legitimate cause of losses i n f e r t i l i t y and that when the appropriate therapeutic measure i s given at the correct time, we can minimize the incidence of such losses. 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Analysis of variance for Corpus Luteum diameter among animals receiving hCG either on day 0, 7, or 14 post breeding i n comparison to control dairy cows at UBC South Campus. (a) Dependent Variable : CL Diameter Source of Sum of Mean F Value Inference Variance df Squares Square Model 51 29049.60 569.60 3.86 ** Treatment 3 7017.54 2339.18 3.95 * Cow(trt) 28 16587.28 592.40 4.01 ** Sample 5 568.38 113.68 0.77 ns Sam X T r t 15 2899.25 193.28 1.31 ns Error 95 14019.06 147.57 Total 146 43068.67 N.B.: Values used in the above analysis were on data obtained on days 7, 14, 21, 28, 35 and 42, i n c l u s i v e . * = S i g n i f i c a n t at P<0.05 ** = S i g n i f i c a n t at P<0.01 ns = Not S i g i n i f i c a n t P<0.05 R2 = 67.44 % (b) Dependent Variable : CL Diameter Source of Sum of Mean F Value Inference Variance df Squares Square Model 33 15682.66 475.23 7 . 07 ** Treatment 2 2642.78 1321.39 3 . 29 * Cow(trt) 22 8836.99 401.68 5 .98 ** Sample 3 471.79 157.26 2 . 34 ns Sam X T r t 6 1788.60 298 .1,0 4 . 44 ** Error 52 3494.65 67.2*0 Total 85 19177.30 N.B.: Values used i n the above analysis were on data obtained on days 9, 11, 16 and 18, in c l u s i v e . * = S i g n i f i c a n t at P<0.05 ** = S i g n i f i c a n t at P<0.01 ns = Not S i g n i f i c a n t P<0.05 R2 = 81.78 % Appendix 2. Analysis of variance for Plasma Progesterone concentration among animals receiving hCG either on day 0, 7, or 14 post breeding i n comparison to control dairy cows at UBC South Campus. (a) Dependent Variable : Plasma Progesterone Source of Sum of Mean F Value Inference Variance df Squares Square Model 52 1092.88 21. 02 3 . 75 ** Treatment 3 91. 05 30.35 1.55 ns Cow(trt) 29 567.82 19.58 3.50 ** Sample 5 142.01 28.40 5. 07 ** Sam X Trt 15 96.56 6.44 1.15 ns Error 97 543.36 5. 60 Total 149 1636.24 N.B.: Values used i n the above analysis were on data obtained on days 7, 14, 21, 2.8, 35 and 42, i n c l u s i v e . * = S i g n i f i c a n t at P<0.05 ** = S i g n i f i c a n t at P<0.01 ns = Not S i g n i f i c a n t P<0.05 R2 = 66.79 % (b) Dependent Variable : Plasma Progesterone Source of Sum of Mean Variance df Squares Square F Value Inference Model 33 585.84 17.75 2.87 ** Treatment 2 40.90 20.45 1. 05 ns Cow(trt) 22 427.22 19.42 3.14 ** Sample 3 72.42 24 .14 3.91 ** Sam X T r t 6 17.94 2.99 0.48 ns Error 59 364.67 6. 18 Total 92 950.51 N.B.: Values used i n the above analysis were on data obtained on days 9, 11, 16 and 18, in c l u s i v e . * = S i g n i f i c a n t at P<0.05 *'* = S i g n i f i c a n t at P<0.01 ns = Not S i g n i f i c a n t P<0.05 R2 = 61.63 % 112 Appendix 3. Analysis of variance for Milk Progesterone concentration among animals receiving hCG either on day 0, 7, or 14 post breeding i n comparison to control dairy cows at UBC South Campus. (a) Dependent Variable : Milk Progesterone Source of Sum of Mean F Value Inference Variance df Squares Square Model 52 5682.37 109.28 3 . 49 ** Treatment 3 750.83 250.28 2 . 02 ns Cow(trt) 29 3589.13 123.76 3 .95 ** Sample 5 535.66 107.13 3 .42 ** Sam X T r t 15 451.41 30. 09 0 .96 ns Error 103 3229.54 31.35 Total 155 8911.91 N.B.: Values used in the above analysis were on data obtained on days 7, 14, 21, 28, 35 and 42, i n c l u s i v e . * = S i g n i f i c a n t at P<0.05 ** = S i g n i f i c a n t at P<0.01 ns = Not S i g n i f i c a n t P<0.05 Rz = 63.76 % (b) Dependent Variable : Milk Progesterone Source of Sum of Mean Variance df Squares Square F Value Inference Model 26 2381.75 91. 61 4 . 50 ** Treatment 2 650.08 325.04 3 .71 * Cow(trt) 15 1315.80 87.72 4 .31 ** Sample 3 105.28 35. 09 1 .73 ns Sam X T r t 6 43 . 64 7 .27 0 . 36 ns Error 39 793.11 20.34 Total 65 3174.87 N.B.: Values used i n the above analysis were on data obtained on days 9, 11, 16 and 18, in c l u s i v e . * = S i g n i f i c a n t at P<0.05 ** = S i g n i f i c a n t at P<0.01 ns = Not S i g n i f i c a n t P<0.05 R2 = 75.02 % Appendix 4. Analysis of variance for Milk Progesterone concentration among animals receiving hCG either on day 0, 7, or 14 post breeding i n comparison to control dairy cows at Farm # 1. (a) Dependent Variable : Milk Progesterone Source of Sum of Mean F Value Inference Variance df Squares Square Model 7 6 9188.83 120.91 6 . 15 ** Treatment 3 603.18 201.06 2 .27 ns Cow(trt) 49 4339.70 88 . 57 4 . 50 ** Sample 6 3593.73 598.96 30 • 45 ** Sam X T r t 18 521.41 28 . 97 1 .47 ns Error 257 5055.91 19. 67 Total 3 33 14244.74 N.B.: Values used i n the above analysis were on data obtained on days 0, 7,9 , 11, 14, 16 and 18, i n c l u s i v e . * = S i g n i f i c a n t at P<0.05 ** = S i g n i f i c a n t at P<0.01 ns = Not S i g n i f i c a n t P<0.05 R = 64.51 % (b) Dependent Variable : Milk Progesterone Source of Sum of Mean Variance df Squares Square F Value Inference Model 53 4486.58 84 . 65 2 .80 ** Treatment 3 102.55 34 .18 0 . 35 ns Cow(trt) 38 3697.47 97.30 3 .22 ** Sample 3 101.84 33.95 1 . 12 ns Sam X T r t 9 115.34 12.82 0 .42 ns Error 50 1510.02 30.20 Total 103 5996.59 N.B.: Values used in the above analysis were on data obtained on days 21, 28, 35 and 42, inc l u s i v e . * = S i g n i f i c a n t at P<0.05 ** = S i g n i f i c a n t at P<0.01 ns = Not S i g n i f i c a n t P<0.05 R = 74.82 % 114 Appendix 5. Analysis of variance for Milk Progesterone concentration among animals receiving hCG either on day 0, 7, or 14 post breeding i n comparison to control dairy cows at Farm # 2. (a) Dependent Variable : Milk Progesterone Source of Sum of Mean Variance df Squares Square F Value Inference Model 111 27961.38 251.90 7.12 ** Treatment 3 1570.95 523.65 5.05 ** Cow(trt) 84 8712.28 103.72 2.93 ** Sample 6 8263.46 1377.24 38.93 ** Sam X T r t 18 1953,64 108.54 3.07 ** Error 344 12169.66 35. 38 Total 455 40131.03 N.B.: Values used i n the above analysis were on data obtained on days 0, 7, 9, 11, 14, 16 and 18, i n c l u s i v e . * = S i g n i f i c a n t at P<0.05 ** = S i g n i f i c a n t at P<0.01 ns = Not S i g n i f i c a n t P<0.05 R2 = 69.68 % 115 Appendix 6a. Number of artimals (n) used i n the s t a t i s t i c a l analyses at each respective time following breeding at the UBC South Campus Farm. Treatment Day 0 Day 7 Day 14 Control Time Post AI 7 7 9. 9 7 9 7 9 9 7 11 7 9 9 7 14 6 •9 9 7 16 6 9 9 7 18 5 7 9 7 21 4 7 6 3 28 3 6 6 3 35 3 6 5 3 42 3 6 5 3 Appendix 6b. Number of animals (n) used i n the s t a t i s t i c a l analyses at each respective time following breeding on Farm # 1. Treatment Day 0 Day 7 Day 14 Control Time Post AI 0 19 20 20 19 7 19 20 20 19 9 19 20 20 19 11 19 20 20 19 14 18 20 20 18 16 16 20 20 17 18 14 20 20 17 21 9 14 14 12 28 7 14 10 9 35 7 14 9 8 42 7 14 9 7 116 Appendix 7. Number of animals (n) used i n the s t a t i s t i c a l analyses at each respective time following breeding on Farm # 2. Treatment Day 0 Day 7 Day 14 Control Time Post AI 0 23 27 24 26 7 23 27 24 26 14 21 26 23 26 21 12 23 18 15 28 12 21 12 13 35 10 19 12 13 42 10 19 12 12 117

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