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The use of milk progesterone radioimmunossay to assess fertility in the post-partum period of dairy cows Slack, William Leslie Plato 1984

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THE USE OF M I L K PROGESTERONE RADIOIMMUNOASSAY TO A S S E S S F E R T I L I T Y  I N THE POST-PARTUM PERIOD OF DAIRY COWS  By WILLIAM LESLIE PLATO SLACK B.Sc,  The University of Guelph, 1976  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Seience, Faculty of A g r i c u l t u r a l Sciences)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1984 © William L e s l i e Plato Slack, 1984  In  presenting  requirements  this  British  it  freely available  for  that  i n partial  f o r an advanced  of  agree  thesis  Columbia,  I agree  that  f o r reference  permission  scholarly  degree  may  and study.  I  copying  be g r a n t e d  understood  that  f i n a n c i a l gain  or publication  shall  n o t be a l l o w e d  permission.  Department  of  /(/PUSAAJ  The U n i v e r s i t y o f B r i t i s h 1956 Main M a l l Vancouver, Canada V6T 1Y3  C3/81 ) -  Columbia  make  further  of this  by t h e head  o r by h i s o r h e r r e p r e s e n t a t i v e s . copying  University shall  department  for  DE-6  a t the  the Library  f o r extensive  purposes  fulfilment of the  thesis  o f my  I ti s  of this without  thesis my  written  ABSTRACT  The pattern of post-partum and ovarian a c t i v i t y was monitored i n two herds through the use of radioimmunoassay of progesterone i n postmilking s t r i p p i n g s .  The University of B r i t i s h Columbia, South Campus  (UBC) herd was sampled on a twice weekly basis, while the Agriculture Canada Research Station, Agassiz herd was sampled every second day. Sampling began approximately six days post-partum and continued up to 60 days post-conception. The estrous cycle was c l a s s i f i e d into phases based on the concentration of progesterone i n the milk samples.  Progesterone  concentration immediately post-partum and for a varying length of time thereafter remained at basal l e v e l s .  This was c l a s s i f i e d as Phase 0 and  represented the stage of quiescence i n the ovaries post-partum.  Phase 1  of the estrous cycle represented the f o l l i c u l a r stage when the concentration of progesterone was low. Phase 2 represented the stage when the corpus luteum was developing and the concentration of progesterone was r i s i n g .  Phase 3, the f u l l y active corpus luteum stage,  was when the concentration was highest, while Phase 4 represented the regressing corpus luteum stage when the concentration was f a l l i n g . The stage of quiescence, from p a r t u r i t i o n to the i n i t i a t i o n of l u t e a l a c t i v i t y was found to be 21.43 ± 11.84 days f o r 54 animals i n the UBC herd and 19.81 ± 8.85 days f o r 127 animals i n the Agassiz herd. Retained placenta increased the duration of quiescence to 25.62 ± 12.41 days f o r the 12.6% or 16 animals having the condition i n the Agassiz  - iii -  herd.  The 9.3% or f i v e animals having retained placenta i n the UBC herd  had a much shorter stage of quiescence of 10.80  ± 6.42  days.  Two d i s t i n c t types of f i r s t cycles were found, based on the mean concentration of progesterone and the number of days i n the period.  A  normal f i r s t cycle exhibited more l u t e a l a c t i v i t y during Phase 3, and remained i n this Phase longer than did the shortened f i r s t cycle.  This  may be associated with f o l l i c l e l u t e i n i z a t i o n i n the shortened f i r s t cycle rather than true ovulation as i n the normal f i r s t  cycle.  The increased frequency of sampling of the Agassiz herd tended to give a more accurate c l a s s i f i c a t i o n of the phases of the cycle as defined, with more Phase 4 samples being i d e n t i f i e d .  Also days i n  Phase 1 and Phase 2 for both Type 1 (shortened f i r s t cycle), and Type 2 (normal f i r s t c y c l e ) , were approximately half of those observed from the UBC herd. Standard curves of progesterone concentration vs. time for "normal" cycles of various lengths with standard deviations were derived from the pooling of a l l cycles classed as normal f i r s t cycles and normal cycles without a breeding.  These "normal" cycles showed that as the  cycle length increased, so did time spent during Phase 1 when the concentration of progesterone remained below 4 ng/ral milk. Progesterone p r o f i l e s of various animals are included, i l l u s t r a t i n g the wide variety of p r o f i l e s found. of  These include p r o f i l e s  short stages of quiescence, long stages of quiescence, short cycles,  long cycles as well as some abnormalities observed. When used as a test for early detection of pregnancy, a single sample from days 21 to 24 for the UBC herd showed accuracies of 100% i n  - iv -  determining non-pregnancy and 86% i n determining pregnancy.  Test  results for days 21 and 22 for the Agassiz herd were again 100% accurate for determining non-pregnancy and 90.5% pregnancy.  accurate i n determining  The accuracy of the non-pregnant determination can be  increased by including a sample on day of insemination and eliminating those animals inseminated at an obviously incorrect time.  With the two  sample test on the combined UBC data the acuracy of the pregnant diagnosis increased to 91.5%.  On the combined Agassiz data the two  sample test increased the accuracy of the pregnant diagnosis to 93.1%. The use of progesterone pregnancy testing offers a considerable saving i n time i n i d e n t i f y i n g those animals not conceiving to insemination and a reasonably accurate means of early detection of pregnancy. The average number of days from conception to positive palpation was  51.02  ± 13.19  days for 97 animals i n the Agassiz herd and 60.43 ±  23.59 days for the 42 animals i n the UBC  herd.  - v TABLE OF CONTENTS Page ABSTRACT  i i  TABLE OF CONTENTS  v  LIST OF TABLES  viii  LIST OF FIGURES  ix  ACKNOWLEDGEMENTS  x  INTRODUCTION  1  LITERATURE REVIEW  5  A.  5  B.  Factors Influencing F e r t i l i t y 1.  The Bull  5  2.  Handling Frozen Bovine Semen i n the F i e l d  6  3.  The Cow  7  4.  Estrus Detection  8  5.  Timing of Insemination  11  5.1  During Insemination  11  5.2  Post-Insemination  12  6.  Herd Size and Housing  14  7.  Climate and Season  15  8. 9.  Choice of B u l l Pathological Causes of I n f e r t i l i t y  16 17  10.  Nutrition and Reproduction i n Dairy C a t t l e . . . .  18  11.  Ideals and Requirements  20  12.  Pregnancy Diagnosis  21  Milk Progesterone for Monitoring F e r t i l i t y  24  1.  Plasma Progesterone Concentration  24  2.  Milk Progesterone Concentration  24  - vi Page 3.  Milk Progesterone and Pregnancy Diagnosis  25  4.  Milk Progesterone Pregnancy Diagnosis Commercial Operation  26  Further Experiments Using Milk Progesterone Pregnancy Diagnosis  31  5.  6.  Estrus Detection Using Milk Progesterone Assay  41  7.  Embryonic Mortality  46  8.  Milk Progesterone and I n f e r t i l i t y  48  Milk Progesterone to Monitor Hormonal Treatment of S u b f e r t i l i t y Summary  51 56  9.  C.  MATERIALS AND METHODS  58  1.  Milk Sampling  58  2.  Radioimmunoassay.....  58  3.  Animals - General Management Practices  58  3.1  UBC Herd  58  3.2  Agassiz Herd  60  4.  Cycle C l a s s i f i c a t i o n  62  4.1 4.2 4.3  62 65 67  Cycle Phases Types of Cycles Standard Curves  RESULTS AND DISCUSSION  68  1.  Resumption of Luteal A c t i v i t y  68  2.  Cycle Types  72  3.  Standard Curves  89  4.  Progesterone P r o f i l e s  105  - vii Page 5.  6.  Pregnancy Diagnosis  107  5.1 5.2 5.3  113 115 117  UBC Data Agassiz Data Discussion  Estrus Detection and Reproductive  Performance  118  SUMMARY AND CONCLUSIONS  124  BIBLIOGRAPHY  127  - viii LIST OF TABLES Table  Page  1  Recommended Body Weight of Heifers  2  Milk Progesterone Concentrations i n Pregnant & NonPregnant Cows Measured by Rapid Radioimmunoassay....  27  Mean Progesterone Level of Positive Cows from Four East Anglia Herds  29  4  Mean Progesterone Levels of Positive Cows  29  5  Cycle Types  65  6  Phase 1 - UBC Data F o l l i c u l a r Stage  73  7  Phase 2 - UBC Data Developing Corpus Luteum Stage...  74  8  Phase 3 - UBC Data Luteal Stage  75  9  Phase 4 - UBC Data Regressing Corpus Luteum Stage...  76  10  Phase 1 - Agassiz Data F o l l i c u l a r Stage  77  11  Phase 2 - Agassiz Data Developing Corpus Luteum  3  8  Stage  78  12  Phase 3 - Agassiz Data Luteal Stage  79  13  Phase 4 - Agassiz Data Progressing Corpus Luteum Stage  80  14  UBC "Normal" 15 Day Cycles  90  15  UBC "Normal" 18 Day Cycles  91  16  UBC "Normal" 19 Day Cycles  92  17  UBC "Normal" 22 Day Cycles  93  18  Agassiz "Normal" 17 Day Cycles  94  19  Agassiz "Normal" 19 Day Cycles  95  20  Agassiz "Normal" 21 Day Cycles...*  96  21  Agassiz "Normal" 23 Day Cycles  97  22  Pregnancy Test Results (UBC Data)  114  23 24  Pregnancy Test Results (Agassiz Data) Reproductive Performance  116 121  - ix -  LIST OF FIGURES Figure  Page  la  Cycle Phases (Theoretical)  63  lb  Cycle Phases (Derived)  64  2  Cow No. 71008  69  3  Cow No. 73025  70  4  UBC Normal Cycles  98  5  UBC Normal Cycles  99  6  Agassiz Normal Cycles  100  7  Agassiz Normal Cycles  101  8  UBC "Normal" Cycles Combined  103  9  Agassiz "Normal" Cycles Combined  104  10  Cow No. 72007  108  11  Cow No. 73017  109  12  Cow No. 73021  110  13  Cow No. 74004  Ill  14  Cow No. 74016  112  - x -  ACKNOWLEDGEMENTS  I would l i k e to take this opportunity to thank some of the many people whose valuable assistance enabled the successful completion of this t h e s i s .  I am grateful to Dr. J.A. Shelford for his help and  endurance throughout the extended period of time required to complete this work.  I would also l i k e to thank the Department of Animal Science  for allowing me to work on this thesis while employed as herdsman f o r the South Campus dairy herd. Thanks go also to Dr. L.J. Fisher for allowing me access to the information from the experimental herd at the Agriculture Canada Research Station at Agassiz. Additional thanks are due to Mrs. M. Striker for computer programming assistance and to the many people who the completion of this work over the years.  continued to encourage  - 1-  INTRODUCTION  Milk i s the one food for which there seems to be no adequate substitute.  I t constitutes almost the entire diet for the young of a l l  mammals (Hughes and Bennion, 1972). be the mother of mankind.  The dairy cow i s thought by many to  "We rob her of her children that we may rob  her of her milk" (Fussel, 1966). Many authors have indicated that f a i l u r e to breed i s an important reason for discarding animals from herds, p a r t i c u l a r l y i n dairy cattle (Dawson, 1977; Melrose, 1979; Foote, 1979).  Foote (1970) reported  s t e r i l i t y as the second most frequent cause of c u l l i n g dairy cows from New York State herds.  Lineweaver  and Spessard (1975) reported that  reproductive i n e f f i c i e n c y alone accounted for an estimated $540 m i l l i o n loss i n the United States. Reproductive f a i l u r e i n dairy cattle causes economic loss d i r e c t l y as a result of i t s effect on yearly milk production and on surplus calves for sale (Louca and Legates, 1968), and i n d i r e c t l y because the potential selection d i f f e r e n t i a l i s reduced with fewer replacements  (Foote, 1970).  While c u l l i n g for low y i e l d i s part of most  breeders genetic improvement programme, cows culled for reproductive problems decrease the opportunity to select for y i e l d and, therefore, deter genetic improvement.  As well, by increasing the days open and  days dry, the generation i n t e r v a l i s increased and the potential rate of genetic improvement i s decreased over a given period of time (Foley et a l . , 1972).  - 2 -  The optimum calving i n t e r v a l for the dairy cow has been the subject of much study i n the past.  Early conception and short dry  periods are reported to have depressing effects on lactation and milk yields (Smith and Leagates, 1962).  Speiker and Meadows (1967)  recommended twelve month calving i n t e r v a l s .  Louca and Legates (1968)  found during the f i r s t l a c t a t i o n for each additional day open there was a gain of 1.16 kg. milk/day and a decline of 3.58 subsequent  lactations.  and 3.68 kg. for the  Therefore, they recommended that a 13 month  calving i n t e r v a l would be optimal for h e i f e r s , while 12 months for second and later calvings was optimal. ent  Higher producing, more p e r s i s t -  cows, however, may not suffer the production losses observed i n  average cows, when the calving i n t e r v a l i s extended to 13 or 14 months (Foley et a l . ,  1972).  Schneider et a l . (1981) found that cows with an  average calving i n t e r v a l of 13 months produced more fat-corrected milk in 305 day lactation and i n the f i r s t  150 days of the subsequent  l a c t a t i o n than cows with an average calving i n t e r v a l of 12 months. Dawson (1977) reported on a study of the reproductive potential in female c a t t l e discarded as I n f e r t i l e , i n which he found that i n fact many of the animals were f e r t i l e .  These animals had simply reached a  point i n days open, when i t was no longer economical for the producer to continue to attempt to get them bred (Foote, 1970; Bulman and Lamming, 1979 and Dawson, 1977). infertility  The results of Dawson (1977) suggest that  of immature c a t t l e on farms i s often only temporary.  As  s k e l e t a l growth i s not complete i n cows u n t i l 4-5 years of age, Dawson (1977) speculated that early f i r s t calving could be followed by a  - 3 -  suspension of reproductive function, u n t i l the metabolic demands for growth had been s a t i s f i e d .  When mature dairy cows f a i l to breed, the  i n f e r t i l i t y i s more l i k e l y to be permanent, and i s often associated with the a c t i v i t y of pathogens (Dawson, 1977). Karg (1981) gave the following description of f e r t i l i t y : " F e r t i l i t y i s a m u l t i f a c t o r i a l phenomenon where the converses (given i n parenthesis) of each of the following events are major factors i n i n f e r t i l i t y : establishment of c y c l i c i t y during puberty ( a c y c l i a ) ; oestrus, insemination, ovulation ( s i l e n t or unrecognized heat, false timing of AI, delayed ovulation); f e r t i l i z a t i o n , development of the conceptus, s h i f t to maternal conditions, pregnancy (embryonic death, abortion); p a r t u r i t i o n , delivery of the placenta, uterine involution ( s t i l l b i r t h , dystocia, retained membranes, uterine i n f e c t i o n s ) ; and re-establishment of c y c l i c i t y post partum ( a c y c l i a , cysts, e t c . ) . Modulating factors are breed, n u t r i t i o n , housing conditions, season, age, milk y i e l d , suckling, resting time post-partum, regulating disorders, infections, etc."  The discovery that progesterone occurs i n milk and, that i t s concentration reflected the variations of the estrous cycle and the plateau of pregnancy, and l a t e r , the development of a simple and rapid radioimmunoassay for progesterone, has provided a means by which we can assess f e r t i l i t y i n the dairy  cow.  The present study i l l u s t r a t e s the use of progesterone radioimmunoassay of sequential milk samples, a non-invasive technique, to assess f e r t i l i t y i n the post-partum  cow.  The objective of this study was to develop a method of examining and c l a s s i f y i n g the p r o f i l e s of progesterone concentration i n milk, generated by twice a week sampling, and every other day sampling, postpartum to post-conception, of cows located i n two herds.  - 4 -  To this end, the cycle was divided into a series of phases, and based on the duration and l e v e l of progesterone i n each, the cycles were categorized.  A secondary objective of this study was to develop a  standard curve for "normal" cycles, with progesterone concentration means and standard deviations f o r each sampling date. The study also included an examination of the r e l i a b i l i t y of using milk progesterone sampling as a method of early determination of pregnancy.  - 5 -  LITERATURE  REVIEW  The main purpose of this paper i s to review the use of information obtained by milk progesterone radioimraunoasay fertility  i n the post-partum  period of dairy cows.  to assess the  However, as Karg  (1981) states, " F e r t i l i t y i s a m u l t i f a c t o r i a l phenomenon."  It seems  reasonable, therefore, to review some of the many factors influencing the f e r t i l i t y of a dairy cattle population.  This w i l l be followed by a  review of the early work with milk progesterone, and some of the ways i n which i t may  be used to provide a better understanding of f e r t i l i t y i n  dairy c a t t l e .  A.  Factors  1.  The  Influencing  Fertility  Bull  While this review deals primarily with reproduction from the female side, some mention should be made of the male.  When natural  service i s used, i t i s often the b u l l that i s the cause of f a i l u r e of the cow to become pregnant.  The b u l l should be thoroughly examined by a  competent veterinarian prior to inclusion i n any breeding programme. When AI i s used, and semen i s obtained from one of the many stud services, then one can f e e l confident that the b u l l has been thoroughly examined, and meets the minimum standards set out by the breed organizations.  Those bulls which f a i l to f e r t i l i z e or do so  inadequately are eliminated. to  However, a difference of 15% with regard  conception rate after the f i r s t insemination, between the b u l l  displaying the best, and the b u l l showing the poorest f e r t i l i z i n g  - 6 -  capacity, i s a common phenomenon i n AI centres of average size (De K r u i f , 1978).  2.  Handling Frozen Bovine  Semen  In the Field  Most poor f e r t i l i t y results with frozen semen are due to improper handling or deposition of the semen, or both, by the inseminator (Pickett and Berndtson, 1980). Successful storage of frozen semen requires a storage temperature of -130°C or lower at a l l times, for maximum reproductive e f f i c i e n c y . This requirement i s met with l i q u i d nitrogen which has a temperature of -196°C.  Because of the r e l a t i v e l y large surface area, and the  r e l a t i v e l y small volume of extended semen contained i n most straws, the straws are conducive to rapid heat exchange.  Care must be taken to make  any transfer of semen from one storage unit to another as quickly as possible.  Any time the temperature of the semen rises above -130°C,  damage can be expected.  Such damage, due to elevated temperature, i s  additive. Most AI centres monitor the f i r s t service conception rates of each of their inseminators and there are marked differences between inseminators and within inseminators over time (De K r u i f , 1978). Salisbury and Flerchinger (1967) studied the effects of aging i n spermatozoa and found that the f e r t i l i z i n g capacity of the spermatozoa decreased, and an associated increase i n prenatal death of the developing zygotes was noted, as the time from c o l l e c t i o n to insemination increased.  - 7 -  3.  T h e Cow  Swensson and Andersson (1980) reported on a paper by Bar-Anan et a l . (1979) who  R.K.  stated that they found an Indication that a  genetic component i n the ovum or uterus could cause death of the embryo at implantation, which was expressed only as a delay of the following heat period. It i s concluded by nearly a l l investigators that the pregnancy rate i s reduced i n those animals which have calved for the f i r s t time, with the majority of these investigators a t t r i b u t i n g the reduction i n conception rates to increased problems at the time of p a r t u r i t i o n and during puerperium (De K r u i f , 1978).  De Kruif (1978) observed a 5%  increase i n pregnancy rate between primiparous and secundiparous cows. This increase continued up to four years of age, and according to Foley et  al.,  (1972) remains constant from four to six years and then  gradually decreased with advancing age.  This decrease was noted by De  Kruif (1978) as well. Gwasdauskas et a l . (1975) reported f i r s t service conception rates for  various ages of cows as follows:  and older cows 31.9%.  heifers 47.6%, young cows 42.7%  However, age i s not a major cause of i n f e r t i l i t y  i n dairy c a t t l e , as most of the animals i n a production herd w i l l be culled for one reason or another before they reach old age.  What i s  more important i s size of the heifer at f i r s t breeding. Heifers normally reach puberty at six to ten months of age (Noakes, 1979), depending upon the breed.  As noted e a r l i e r , much of the  - 8 -  decrease  i n conception rates following f i r s t calving can be attributed  to problems associated with p a r t u r i t i o n , as the f i r s t calf heifers have greater d i f f i c u l t y than older and larger cows.  Therefore, i t i s of  great importance that the heifer achieve s u f f i c i e n t size prior to f i r s t breeding, to minimize the risks of dystocia. normally fed heifers who  have reached  Delaying the breeding of  the recommended size, beyond  15 months of age, reduces the t o t a l l i f e t i m e production, and, i f the delay i s continued up to three years of age, the heifer may  develop  reproductive disorders.  Table 1 Recommended Body Weight of Heifers At F i r s t Breeding Pounds Holstein Brown Swiss Ayrshire Guernseys Jerseys  4.  Kg  750 750 600 550 500  335 335 270 250 225  Folley et a l .  1972  Post Calving Pounds 1100 1100 1000 950 850 Hafs & Boyd  Kg 500 500 450 425 380 1973  Estrus Detection Estrus detection i s a major problem for a l l dairymen using  artificial  insemination (Whitmore, 1980).  To maintain even production levels throughout the year, dairymen must have cows calving each month, consequently, may  poor estrus detection  give r i s e to a wide variety of f e r t i l i t y management problems within  - 9 -  the herd, such as increased i n t e r v a l between p a r t u r i t i o n and f i r s t insemination, which i n turn leads to a lengthened period from p a r t u r i t i o n to conception (Bozworth et a l . , 1972; Barr, 1975).  Due to  poor estrus detection cows previously inseminated which have not conceived, may pass unnoticed.  This results i n an unduly long i n t e r v a l  between inseminations, again, lengthening the period of time between p a r t u r i t i o n and conception or days open. The signs of estrus can be divided into primary and secondary behavioral t r a i t s (Holstein Friesian Journal, 1979).  The primary and  most important sign of true estrus i s the standing heat, when the animal i s standing firm, with a l l four legs braced, while being mounted by another animal.  Secondary signs of estrus may be mounting of the  cows, swollen vulva, hair over rump r u f f l e d or rubbed o f f , mucus discharge, hyperactivity, i . e . , walking along fence l i n e s , standing while others are lying down, bellowing, clear mucus discharge from vulva, reduced intake and reduced production, and these may be exhibited before, during and after true estrus.  Cows missed, may be noted by  metestrus bleeding (Foote, 1975, Foley et a l . , 1972, Holstein F r i e s i a n Journal, Sept. 1979, Whitmore, 1980). Animals submitted for A.I. on the basis of the primary sign usually have a high pregnancy rate, while those submitted solely on the basis of secondary signs, and those animals which are wrongly submitted for one reason or another (inadequate i d e n t i f i c a t i o n ) have a much lower pregnancy  rate and usually return to estrus (often i r r e g u l a r l y )  increasing the repeat breeder problem, and increasing the services per conception r a t i o (Whitmore, 1980).  - 10 -  De Kruif (1978) reported on papers by Roberts (1971) and Gunnink (1973) that endometritis may  occur i n c i d e n t a l l y i n these animals  because  the resistance of the uterus to bacteria i s lowered during the l u t e a l phase.  This could be one of the reasons for the increase i n repeat  breeder problem cows i n the group of animals mentioned  by Whitmore  (1980). "Silent estrus" and anestrus are often the dairyman's excuses for what i n r e a l i t y are missed heats.  favourite  Zemjanis (1980) stated,  " s i l e n t estrus or quiet ovulation, i s physiological during the immediate post-parturient period, i n both dairy and beef c a t t l e .  Its significance  as the cause of anestrus at the time of planned breeding i s highly questionable."  De Kruif (1978) concluded, " i t i s apparent from several  studies that almost a l l cows i n a herd show signs of estrus but that these are inadequately observed, p a r t i c u l a r l y i n large herds." et a l . , 1975, King et a l . , 1975 and King et a l . , 1976.) observation was  (Hurnik,  Continuous  found to be 100% e f f e c t i v e i n detecting estrus i n a  large dairy herd while casual observation by the herdsman detected only 56% of the same animals (Williamson et a l . , 1972).  It can therefore be  concluded that s i l e n t heat i s a matter of management rather than of cows, and the incidence of s i l e n t estrus can be reduced to a minimum by increasing the number of heat observation periods. There are several types of heat detection aids which have been used to augment direct observation. These include, heat mount detectors, "marker b u l l s " which have been s u r g i c a l l y prepared to prevent  - 11 -  mating and f i t t e d with a halter containing a marking device, testosterone treated cows and steers s i m i l a r l y f i t t e d with a marking device, marking a l l cows with chalk and observing for those with chalk rubbed o f f , e l e c t r i c a l  measurement of vaginal mucus (Gartland et a l . ,  1976), use of pedometers to measure a c t i v i t y of the cow (Kiddy, 1977), and the use of video equipment to assist observation (Foote, 1975). It must be rememberd that these are just aids and i t i s s t i l l the requirement of management that time be spent to properly i d e n t i f y those animals i n estrus for A.I.  5. 5.1  Timing of Insemination During Estrus I t i s p a r t i c u l a r l y important i n a r t i f i c i a l insemination that the  cow be bred at the correct time during the estrus period. When natural breeding i s used, the cow normally w i l l only allow the b u l l to mount when i t i s most opportune.  However, when A.I. i s used, i t i s up to  management to determine when to inseminate the cow. F e r t i l i t y i s not uniformly distributed throughout estrus.  When  inseminated during the early part of estrus, fewer cows w i l l conceive, p a r t i c u l a r l y when the semen i s of i n d i f f e r e n t or i n f e r i o r q u a l i t y . Inseminations after ovulation has occurred w i l l also result i n lower pregnancy rates (Trimberger and Davis, 1943; Boyd, 1970; Deas, 1970; Hafs and Boyd, 1973; de Kruif, 1978).  Lodge (1976) reported that aged  ova can result i n lowered f e r t i l i t y , or, problems associated with the early development of the f e r t i l i z e d ova.  - 12 -  Estrus i n the dairy cow lasts from 12 - 18 hours while ovulation occurs from 4 - 1 6 1979).  hours after estrus (Mather and Rushmer,  The ova remains f e r t i l e for only approximately 6 - 1 0  spermatozoa may  hours and  require up to 6 hours within the cow's reproductive  tract to develop the capacity to f e r t i l i z e the ova (Hafs and Boyd, 1973).  Therefore, the optimal breeding time i s from 10 - 16 hours after  the onset of estrus (Mather and Rushmer, 1979) or between the middle the end of the period of estrus (Trimberger and Davis, 1943; Bane, Swensson and Andersson,  1980).  and 1964;  With once a day heat detection an animal  seen i n standing heat could be beginning estrus or ending, complicating the decision of correct timing of insemination. General recommendations by Hafs and Boyd (1973) and of many A.I. organizations are: 1.  Cows f i r s t noticed i n standing heat i n the morning should be  bred that afternoon; 2.  Cows f i r s t noticed i n standing heat In the afternoon should  be bred before noon the following day.  5.2  Post-Farturn The timing of rebreeding post-partum  studies over the years.  has been the focus of many  Here again we have a management decision.  Are  we concerned with high conception to f i r s t service, or the shortest i n t e r v a l from calving to conception.  Is i t maximal l a c t a t i o n a l y i e l d  that i s the main concern, or i s i t maximum l i f e t i m e production?  The  - 13 -  d e s i r a b i l i t y of a twelve month calving i n t e r v a l has been discussed (see introduction).  To achieve this i n t e r v a l , cows must conceive on the  average by approximately 85 days  post-partum.  Ayalon, et a l . , (1971) suggested breeding prior to 60 days post-partum  that i t was necessary to begin  due to the v a r i a b i l i t y i n the date  of appearance of heat, and waiting u n t i l 60 days post-partum would result i n a much longer i n t e r v a l .  Lowered conception rates have been  reported i n cows inseminated prior to 50 days post-partum al.,  1952;  Touchberry et a l . ,  1959; Whitmore et a l . ,  (Shannon, et  1974, B r i t t ,  1975;  and Whitmore, 1980). Schneider, et a l . , (1981), comparing the effects of early and late breeding, found that i n the group bred early, at f i r s t heat a f t e r 50 days post-partum,  conception occurred by 88 days post-partum,  required 1.5 services per conception. days post-partum  Breeding at f i r s t heat after 80  delayed conception u n t i l 121 days, and  f e r t i l i t y , requiring 1.96  and  decreased  services per conception.  Louca and Legates (1968), pointed out that y i e l d per unit time, milk per day or year, was more important economically than was lactation yield.  total  With shorter i n t e r v a l s , milk production per day of  l i f e was d e f i n i t e l y higher ( B r i t t , 1975).  Averge daily milk production  from beginning of current l a c t a t i o n to day 150 of subsequent l a c t a t i o n (including the dry period) was  the same for both early and late bred  groups (Schneider et. al.,1981).  - 14 -  6.  Herd Size and Housing As the number of cows per farm continues  to increase, there has  been more emphasis placed on the possible effect of size of herd  on  pregnancy rate after f i r s t service and other methods of measuring herd fertility.  It has been concluded  pregnancy rate decreased  by almost a l l investigators that the  with the increased size of the herd (De K r u i f ,  1978). The housing the f i r s t  system has been shown to affect pregnancy rate after  insemination, with the average rate being higher when systems  were adopted i n which the cows had freedom of movement, than i t was i n those systems i n which the cows were restrained i n s t a l l s . difference was 1978).  This  found to disappear during the grazing season (De K r u i f ,  Kiddy (1977) found that freedom of movement increased the  i n t e n s i t y of the signs of estrus.  De Kruif (1978) reported on papers by  Kordts and Gravert (1972) and an e a r l i e r one by himself De Kruif (1977), that freedom of movement also stimulated the onset of ovarian a c t i v i t y after p a r t u r i t i o n , p a r t i c u l a r l y i n primiparous Housing i s also important  animals.  at time of p a r t u r i t i o n , and can have a  great effect on subsequent f e r t i l i t y .  P a r t u r i t i o n and the i n i t i a t i o n of  high milk secretion places great stress on the animal and at this time the dairy cow i s most susceptible to i n f e c t i o n , p a r t i c u l a r l y retained placenta and endometritis (De K r u i f , 1978).  De Kruif (1978) reported  that retained placenta and endometritis would result i n 5 - 10% lower pregnancy rates after the f i r s t insemination and a larger i n t e r v a l between p a r t u r i t i o n and  conception.  - 15 -  7.  Climate and Season High temperature and humidity results i n less marked signs of  estrus and reduced conception rates, as well as increased rates of embryonic death.  Wiersma & Stott (1969) suggested that increased  progesterone secreted from the adrenal i n response to a stressor, caused an imbalance of hormones such that the uterus and embryo became incompatible.  Ulberg & Burfening (1967) studied the effects of  temperature on f e r t i l i t y and found that uterine temperature on day of insemination was inversely related to f e r t i l i t y .  They found that  pregnancy rates declined from 61% to 45% as r e c t a l temperature at 12 hours post-insemination increased 1°C. In a study of the effects of early and late breeding Schneider et a l . (1981), animals were divided into two groups by calving season. Animals calving during the pasture season, May through October were compared to those calving from November through A p r i l . was noted between these groups In onset of estrous. the  No difference  However, animals i n  late bred group required more services per conception and had longer  calving intervals than cows calving i n the non-pasture season.  Cows  calving i n the pasture season produced more 4% fat-corrected milk than cows calving i n the non-pasture season and were s t i l l losing weight between 60 and 80 days post-partum. Other climatological factors affecting reproduction include: solar radiation, atmospheric pressure, p r e c i p i t a t i o n and day length. Gwazdauskas et a l . , (1975) ranked the five most important climatological factors as follows:  - 16 -  1.  Maximum temperature day after insemination,  2.  R a i n f a l l day of insemination,  3.  Maximum temperature day of insemination,  4.  Solar radiation day of insemination  5.  Minimum temperature day after insemination.  The results of insemination vary from month to month and i n temperate regions pregnancy rates are highest i n the spring and decidedly less satisfactory during the autumn and winter months (De K r u i f , 1978).  This seasonal relationship to conception was noted by  many other authors as well (Rosenburg et a l . , 1977; Stott, 1961; Gwazdauskas et a l . , 1975; Marion and Gier, 1968; Thatcher, 1974; Thatcher and Roman-Ponce, 1980).  8.  Choice of B u l l The s i r e chosen to mate with a p a r t i c u l a r female i n the herd  becomes important  i n a number of ways.  F i r s t of a l l , ease of calving  must be taken into consideration. Recently, there has been much interest i n this factor i n the ratings of various sires at many A.I. Units.  If the b u l l chosen to mate with heifers or small cows sires  large calves, this may lead to dystocia, which i n turn may lead to death or severe injury to the dam, retained placenta, endometritis and adhesions, which may be detrimental to subsequent f e r t i l i t y 1966;  (Hansen,  Rasbech, 1967; Williams, 1968; and Foley et a l . , 1972). Secondly, f e r t i l i t y decreases with increased degree of  inbreeding.  However, this may be overcome i n future generations by an  - 17 -  eventual outcross and subsequent Increased hybrid vigor (Foley et a l . , 1972). F i n a l l y , as mentioned e a r l i e r , there i s v a r i a b i l i t y i n the f e r t i l i z a t i o n capabilities  9.  of various s i r e s .  Pathological Causes of I n f e r t i l i t y The pathological causes of reduced reproductive performance can  be divided into three groups:  those that affect only the reproductive  organs; those that affect the reproductive tract and other organs; and those that decrease the general health of the animal and adversely affect  thereby  reproduction.  These would include the veneral disease such as those caused by V i b r i o fetus, Trichomonas fetus and Campylobacter fetus, brucellosis caused by the bacterium Brucella abortus and leptospirosis caused by members of the genus Leptospira. BVD-MO may  Other infections such as IBR-IPV and  lead to reduced f e r t i l i t y .  In most surveys at least 70% of  bovine abortions are of unknown cause (Huck and Lamont, 1979). Also included i n individual pathological conditions are such conditions as anestrus and c y s t i c ovarian disease.  Anestrus, because of  i t s high incidence and resulting losses, i s a most costly problem. 1.  infertility  Zemjanis (1961) c l a s s i f i e d anestrus as follows: Pre-service Anestrus which included a l l animals that had  not  been observed i n heat at the time of planned breeding and included postpartum cows as well as h e i f e r s .  - 18 -  2.  Post-service Anestrus which represented animals that had  f a i l e d to conceive and yet were not observed i n heat. Zemjanis (1980) found that i n herds with averge f e r t i l i t y there was a 12.6% incidence of pre-service anestrus and 30.8% incidence of post-service anestrus. The economic impact of anestrus becomes obvious when one considers that the minimum time lost because of post-service anestrus could be a r b i t r a r i l y set at 42 days, corresponding to two estrous cycles.  Pregnancy diagnosis at 45 days would increase this to three  cycle lengths or 63 days.  Zemjanis (1980) assumed that a minimum of 30  days was lost for each case of pre-service anestrus. Ovarian cysts are a common c l i n i c a l l y of  i n f e r t i l i t y i n dairy cows.  recognized endocrine cause  Surveys indicate that 5% - 10% of dairy  cows are affected during the post-partum and breeding periods (Sequin, 1980).  Zemjanis et a l . , (1961) made over 20,000 genital examinations  from 1955 to 1960 and found abnormalities involving the ovaries represented the majority of detected genital defects (54.3%) and were found i n 7.9% of a l l examinations. was  Cystic degeneration of the ovaries  the most prevalent abnormality diagnosed.  F o l l i c u l a r cysts were  observed i n 4.0% and l u t e a l cysts i n 1.7% of a l l examinations et a l . ,  10.  (Zemjanis  1961).  N u t r i t i o n and  Reproduction  i n Dairy  Cattle  N u t r i t i o n a l deficiencies and imbalances are frequently implicated as the cause of i n f e r t i l i t y i n c a t t l e . (Oxenreider and Wagner, 1971;  - 19 -  Foley at a l . , 1972;  Morrow, 1980a, 1980b; Butler et a l . , 1981).  A  r a t i o n has i t s greatest effect on f e r t i l i t y before puberty, before and a f t e r breeding, and before and after p a r t u r i t i o n (Morrow, 1980b).  When  a ration i s affecting reproduction, i t i s frequently the result of a deficiency of more than one nutrient, making i t d i f f i c u l t  to evaluate  the effects of a s p e c i f i c nutrient on f e r t i l i t y (Morrow, 1980b). Observable signs of deficiency are variable, depending upon the degree of d e f i c i e n c y . Energy i n moderate deficiency reduces f e r t i l i t y , while a severe deficiency results i n anestrus (Morrow, 1980b). f i r s t symptom to appear when a diet Is inadequate  Generally, the  is a decrease i n milk  production, with f e r t i l i t y being affected at a later stage (Morrow, 1980a). The nutrients required for reproduction are those required for many other bodily functions and i t i s doubtful whether there i s any single nutrient required for reproduction that i s not also required for growth and milk production (Hafs and Boyd, 1973). Deficiencies and excesses of a large number of feeds, minerals and trace elements and vitamins have been discussed in the l i t e r a t u r e as a f f e c t i n g reproduction and opinions on their effect d i f f e r markedly (Foley et a l . , 1972;  Morrow, 1980 a & b).  Different l o c a l i t i e s  s o i l s produce s p e c i f i c l o c a l deficiencies and excesses  and  (Morrow, 1980a).  De Kruif (1978) reported on papers by Adler and Trainin, (1960), and Lotthammmer and Ahlswede, (1973), who  suggest  associated with the consumption of phytoestrogens clover, a l f a l f a and sugar beets.  i n f e r t i l i t y may  be  from plants such as,  - 20 -  High milk y i e l d and i t s effect on the energy balance i n the early post-partum dairy cow has also been shown to decrease f e r t i l i t y et a l . , 1981).  (Butler  Ruder et a l . (1981) in a comparison of i n f e c t i o n rates  for protein adequate and protein d e f i c i e n t cows, found an i n f e c t i o n rate of 52.2%  11.  and 48.1%  respectively at 25 days post-partum.  Ideals and Requirements It i s apparent now,  that f e r t i l i t y of a c a t t l e population may  be  affected by an exceptionally large number of factors which often interact so that a correct interpretation of each i n d i v i d u a l factor i s usually  impossible. The various investigations on days to f i r s t estrus, days to  conception, calving i n t e r v a l , non-returns, services per conception and i n t e r v a l from f i r s t service to conception show that these measurements of additive genetic differences In f e r t i l i t y among cows are small. selecting for improved breeding e f f i c i e n c y by these measures has  Thus  little  to offer dairy c a t t l e breeders. De Kruif (1978) l i s t e d the following as " i d e a l " values to s t r i v e for: -  a pregnancy rate of 80% after the f i r s t  insemination  -  an average of 1.3  -  an average i n t e r v a l of 85 days between p a r t u r i t i o n and  inseminations per conception  conception. In actual practice i t w i l l be next to impossible values as one  to obtain these  or several factors w i l l invariably exert an adverse effect  - 21 -  (De Kruif, 1978).  It i s understandable that reproduction does not reach  an optimal l e v e l on a large number of cattle farms, most often due to inadequacies i n management. Melrose (1979) concluded that improved  reproductive e f f i c i e n c y  depends on being able to breed at the correct time, preferably on a pre-determined date, to know they are pregnant and that they w i l l give b i r t h at a stated time and detect a lowered or lowering of f e r t i l i t y to allow c u l l i n g or treatment. Therefore, the producer requires: A.  a r e l i a b l e means of detecting estrus  B.  a proven system f o r estrus and/or ovulation control  C.  a pregnancy diagnostic technique readily applicable under f i e l d conditions and at a date as early as possible postbreeding to allow rebreeding  D.  a f e r t i l i t y control program including access to c l i n i c a l expertise to allow diagnosis of f e r t i l i t y problems (Melrose, 1979).  12.  Pregnancy Diagnosis The main use of pregnancy diagnosis i n the cow i s to enable the '•V  non-pregnant  animal to be recognized as early as possible so that the  time lost as the result of i n f e r t i l i t y may be reduced by the application of suitable treatment (Heap et a l . (1976)). benefits of early accurate assessment 1.  Norman (1982) l i s t e d five  of bovine reproductive rates:  Detection of estrus can be simplified by focussing the manager's attention on those cows l i k e l y to return to heat;  - 22 -  2.  Breeding plans can be used more e f f i c i e n t l y ;  3.  Non-producing cows can be culled early;  4.  F e r t i l i t y testing of bulls i s simplified; and  5.  Diagnosis of reproductive problems i s f a c i l i t a t e d .  Methods employed  such as, observing an animal f o r a return to  service and confirmation of pregnancy through c l i n i c a l diagnosis based on r e c t a l palpation, both lead to the p o s s i b i l i t y of days l o s t and the corresponding increase i n cost of production on the basis that: 1.  Animals which are not pregnant but show no estrus lead the farmer to the erroneous conclusion that they are pregnant. Hoffman et a l . (1976) found this to be the case i n 15.6% of the  2.  t o t a l animal population i n his experiment.  Animals which are pregnant but show signs of estrus lead the farmer to the erroneous conclusion that they are not pregnant, and, i f care i s not taken during the second insemination, the insemination rod passing through the cervix may cause the abortion of the e a r l i e r pregnancy.  During a 21  day period of constant observation seven per cent of pregnant cows exhibited standing estrus (Williamson et a l . , 1972). 3.  The c l i n i c a l diagnosis cannot be performed at an early enough time post-breeding.  Although there have been claims that by r e c t a l palpation at 8 to 19 days after service, and again at 23 to 30 days, i t was possible to diagnose pregnancy or non-pregnancy with 85% accuracy (Ludwick and Rader, 1968), i n practice, the c l i n i c a l examination i s often delayed  - 23 -  u n t i l after the d e f i n i t i v e allantochorionic placenta has formed at about day 45 post-coitum  (Heap et a l . , 1976).  For most dairymen i t i s not  p r a c t i c a l to have a veterinarian come out each time a cow reaches day 45 post-breeding.  Most frequently they would be requested  basis and consequently days post-coitum.  on a monthly  the average r e c t a l examination would occur at 60  Rectal palpation for pregnancy has several  l i m i t a t i o n s , some of which are: 1.  It requires much practice to develop and maintain a r e l i a b l e expertise for palpation and provides i n v a l i d results before f i r s t estrus;  2.  I t requires a f a i r amount of restraint and thus stresses the animal; and,  3.  I t i s an invasive procedure that can result i n r e c t a l perforations, ovarian adhesions,  ruptured corpora lutea, and  abortions (Studer, 1969 and Norman, 1982). A survey on the use of veterinary pregnancy diagnosis i n 767 herds i n England and Wales i n 1969 (Milk Marketing Board, 1969) and of 1692  dairy farms i n 1979 (Newton et a l . , 1982) reveals some interesting  trends.  The population of herd owners using veterinary pregnancy  diagnosis on more than half the herd increased from 9.8 per cent to 14.2 per cent, and the proportion using them on some of the herd increased from 22.8 per cent to 43.8 per cent. service at a l l had decreased  The proportion not using the  from 67.4 per cent to 42 per cent by 1979,  indicating a greater awareness on the part of the dairy farmer f o r increased reproductive e f f i c i e n c y .  - 24 -  B.  Milk  Progesterone  For Monitoring  1.  Plasma Progesterone  Fertility  Concentration  Plasma progesterone measurements have been made for a number of years (Stabenfeldt et a l . , 1961).  A s t r i k i n g difference i n peripheral  plasma progesterone levels between pregnant and non-pregnant cows was observed 19 days after insemination (Shemesh et a l . , 1968). In the cow i t i s essential that the secretory function of the corpus luteum be maintained for the greater part of gestation, since the placenta produces an i n s u f f i c i e n t amount of progesterone to maintain pregnancy i n the absence of the ovary (Heap et a l . , 1976). During gestation, the concentration of plasma progesterone i s similar to that i n the mid-luteal phase of the estrous cycle (Stabenfeldt et a l . ,  1970)  and i t has been used to confirm pregnancy on about day 22 after mating in cows that have not returned to estrus within 21 days of insemination (Robertson and Sarda, 1971).  In the non-pregnant animal, the corpus  luteum regresses on about day 17 or 18, while i n pregnancy, the l u t e o l y t i c property of the uterus i s neutralized, and the corpus luteum survives (Heap et a l . , 1976).  2.  Milk  Progesterone  Concentration  In the early 1970's, i t was established that progesterone could be found i n milk and that i t s concentration there reflected the variations of the estrous cycle and the plateau of pregnancy (Laing and Heap, 1971; Heap et a l . , 1973; Heap et a l . , 1976). Evidence of this hormone was i n t i t i a l l y obtained by combined gas chromatography - mass spectrometry, a technique which provided definitve  - 25 -  i d e n t i f i c a t i o n of a compound behaving l i k e progesterone i n cows milk during pregnancy  (Darling et a l . , 1972).  Laing and Heap (1971) f i r s t  suggested that progesterone concentration i n milk might provide a means of early pregnancy diagnosis i n c a t t l e . In  1973, Heap et a l . , reported on a simple and rapid  radioimmunoa ssay (RIA) which would measure d i r e c t l y the progesterone concentation i n milk.  They used the generic term, "PROGESTAGEN",  in preference to the s p e c i f i c name of the parent steroid, progesterone, since their study showed that the concentration measurements also included an unidentified compound(s), which were not readily separable from progesterone, and were probably closely related to i t . Since the a v a i l a b i l i t y of RIA, milk progesterone levels have become one of the major hormonal parameters used to monitor reproductive e f f i c i e n c y , stimulating extensive laboratory investigations and elaborate f i e l d t r i a l s  (Heap et a l . , 1973; Heap et a l . , 1976, Booth et  a l . , 1976; Bishop et a l . , 1976; Hoffmann et a l . , 1976, Pennington et a l . , 1976;  3.  Milk  Shemesh et a l . , 1978, Foote et a l . ,  Progesterone  and  Pregnancy  1979).  Diagnosis  Milk progesterone levels r e f l e c t ovarian l u t e a l function, not the presence of a conceptus (Norman, 1982).  Heap et a l . (1973) f e l t that a  l i m i t a t i o n of the diagnosis of pregnancy from plasma progesterone concentration lay i n the fact the progesterone values i n the mid-luteal phase of the normal cycle i n the cow resemble those of gestation. therefore sampled, using sequential testing of milk progesterone  They  - 26 -  concentration for diagnosis, at a time when the difference between pregnant and non-pregnant cows i s probably  at i t s greatest (20, 24  and  28 days) and, which also allows for the i n d i v i d u a l variations i n the length of the normal 21 day estrous cycle.  A further sample on day  or about the time of routine c l i n i c a l diagnosis, was  also examined.  Embryonic mortality, the incidence of which ranges from 6.4% 12% (Bulman and Lamming, 1979,  Foote, et a l . , 1979;  60,  -  Holness et a l . ,  1981., B a l l , 1982), would be expected to lower the numbers of animals diagnosed pregnant at 60 days, compared to the number diagnosed pregnant at an e a r l i e r  date.  The results of Heap et a l (1973) given below, show a highly s i g n i f i c a n t difference i n milk concentration of pregnant and non-pregnant cows, suggesting method of pregnancy diagnosis.  that this test may  form the basis of a  Milk samples were taken at the  afternoon  milking and represent a pooling of the f i r s t milk from a l l four quarters.  4.  Milk Progesterone Pregnancy Diagnosis—Commercial  Operation  Following publication of the results of Heap et a l . (1973), Wickham Laboratories Limited, an organization i n the United Kingdom already providing pregnancy diagnostic services i n other classes of l i v e s t o c k , set up f e a s i b i l i t y studies on routine pregnancy diagnosis. The results of these t r i a l s (Bishop et a l . , 1976)  showed they could  predict non-pregnancy with greater than 95% success. pregnancy was more complex and their success rate was lower.  Nevertheless,  as the non-pregnant cow  Prediction of correspondingly  Is more a matter of  - 27 -  Table 2 Milk progesterone concentrations (ng/ml) i n pregnant and non-pregnant cows measured by rapid radioimmunoasay (Method B; antiserum 5-49 No. 6). No. of observations i n parenthesis. Days After Estrus of F e r t i l e Mating  Non-Pregnant  Pregnant  20  3.4 ± 0.69' (29)  7.0 ± 0.76 (37)  < 0.01  24  3.9 ± 1.33 (22)  7.4  ± 0.85 (36)  <  0.05  28  2.8 ± 0.74 (19)  7.6 ± 0.85 (37)  <  0.001  60  1.5 (2)  from Heap et a l . ,  8.7  ± 0.96 (37)  P Value  -  1973.  concern than the pregnant one, the imbalance i s i n the right d i r e c t i o n . Consequently, a commercial al.,  service was launched in June 1974 (Bishop et  1976). Sample bottles and preservative were provided to prospective  c l i e n t s , with the request that the whole milk sample be taken on day 21-23  after insemination. Problems encountered included variable  sampling times from ten days onward, samples of foremilk only, samples bearing l i t t l e r e l a t i o n to the cow specified ( i n some milking systems), samples inadequately mixed with preservative, and over exposure of some samples to sun or heat sources.  Submission forms were redesigned to  include sample data and service date, to a l l e v i a t e some of the problems, and to impress upon the user the fixed relationship between the two.  - 28 -  In  their conclusion, Bishop et a l . (1976) f e l t that the early diagnosis  of non-pregnancy was invaluable, especially as a screen i n larger herds.  They also f e l t that better awareness of the service would  improve  i t s commercial success. The Milk Marketing Board (UK) launched a commercial  testing service on October 1, 1975.  pregnancy  They began by recommending sampling  on day 28, and l a t e r brought this forward to 24 days post-insemination. They also wished to standardize the sampling procedure, r e a l i z i n g that variations i n the butter fat content would result i n variations i n the progesterone content.  Therefore, they asked that samples be taken at  the afternoon milking and be of whole milk (Booth et a l . , After six months, there were 1200 members u t i l i z i n g  1976). the service,  and they found that the service was of p a r t i c u l a r appeal to the owner of larger herds, with 88% of the users having more than the United Kingdom average of 40 cows, and 9% having more than 200 cows (Booth et a l . , 1976). Booth and Holdsworth (1976) reported they were receiving samples at the rate of approximately 7000 a month, and that 85% of the results were dispatched to the farmers within two days of receipt, f i v e per cent of samples were re-assayed for various reasons and the results of these, together with the ten per cent completed but on the same results cards, were returned within four working days of r e c e i p t . In the report they stated that the majority of the farmers used the service primarily for the detection of non-pregnant  cows.  - 29 -  Booth et a l . (1976) also reported on f i e l d t r i a l s conducted.  they  They noted i n the four herds they were monitoring that there  appeared to be somewhat different d i s t r i b u t i o n characteristics progesterone levels of the cows.  of the  These were as follows:  Table 3 Mean progesterone l e v e l of positive cows from four East Anglia Herds sampled 24 days post-breeding Herd  No. of Cows  A B C D  24 15 20 10  Mean Progesterone ng/ml (± S.E.) 12.7 13.0 22.8 19.3  ± ± ± ±  2.6 2.1 2.0 2.6  Booth et a,l . (1976). In a further attempt to determine t y p i c a l l e v e l s , they randomly selected six herds having more than 40 test results, and analysed the progesterone levels of the positive cows. Table 4 Mean progesterone l e v e l of positive cows (March 1976) selected at random sampled 24 days post-breeding Herd 1 2 3 4 5 6 Total Booth et a l . (1976).  No. of Cows 60 51 34 58 43 54 300  Mean Progesterone ng/ml (± S.E.) 23.3 21.6 29.0 27.8 23.9 27.2  ± ± ± ± ± ±  1.3 1.3 2.6 1.7 1.4 1.5  25.3 ± 0.7  - 30 -  They noted Herd No. 3 had a high proportion of both negative (18.8%) and doubtful (10.4%) test results and subsequently discovered that this owner was using one of the new drugs for estrus synchronization. In an attempt to determine what might have caused the extremely low levels of herds A & B of the f i r s t set of t r i a l s reexamined  (Table 3), they  the four herds again.  They found that since herd B was milked on a pipeline system, i t was  therefore impossible to obtain representative samples of whole milk  without the use of cow meters, and a large number of samples submitted had been of foremilk. They could find no explanation for the low levels i n herd A. On the basis of f i e l d  t r i a l s and, with the equipment and  procedures they used, Booth and Holdworth  (1976) found there was a  d i s t i n c t demarcation between positive and negative cows at the l e v e l of 6.5 ng progesterone/ml milk.  They allowed a doubtful area of 1 ng/ml on  either side of this l e v e l and repeat tested a l l doubtful cows (1.2%) at day 42. An interesting aspect brought out in this report, was  the i n i t i a l  apprehension i n the veterinary profession on the announcement of the new milk test for pregnancy.  It was later determined that there was  an  increase i n requests for assistance in treating i n f e r t i l i t y cases at an early stage when i t was s t i l l economical to do so.  Other veterinarians  were found to be using the test as a regular surveillance, which they l a t e r followed with r e c t a l palpation (of particular value to herd where early embryonic/fetal death i s a problem).  They reported that i t i s  - 31 -  also recommended by veterinary surgeons where poor estrus detection i s suspected for which additional samples would be taken on day of insemination.  And f i n a l l y ,  they noted that veterinarians have also used  the test for goats as they are too small for r e c t a l examination.' In concluding their paper Booth and Holdsworth (1976), f e l t that the widespread application of the test to the national herd for the early detection of pregnancy would play a major part i n the reduction of losses due to i n f e r t i l i t y and i n increasing the p r o f i t a b i l i t y of dairy farming.  5.  Further  Experiments  Using  Milk  Progesterone  Pregnancy  Diagnosis  Heap et a l . (1976) continued experiments on the use of milk progesterone concentration as a pregnancy diagnosis. They found that the concentrations were greater i n f u l l milk taken at the afternoon milking than i n f i r s t milk taken at the morning milking. of  the concentration of progesterone  A correlation  i n milk with fat content i n  samples with low progesterone was found but not i n those with high progesterone values.  The success rate of the pregnancy test from a  single milk sample at 21, 24, 28 or 42 days after insemination ranged from 77.5% cows.  to 85.8% for pregnant cows and 85.7% to 100% for non-pregnant  Combining the results of two f i e l d t r i a l s , the highest success  rate was found at 24 days when 142 of 176 pregnant cows (80%) and 25 of 25 non-pregnant  (100%) were correctly diagnosed from either samples of  f i r s t milk from any healthy quarter at morning milking ( F i e l d T r i a l 1), or samples of whole milk collected at mid-day or afternoon milking ( F i e l d T r i a l 2).  - 32 -  Measurements of known amounts of progesterone (0, 5, 10, 20 and 40 ng/ml) added to milk samples with a low endogenous concentration (0 to 4.3 ng/ml), showed that there was a tendency to overestimate low values and underestimate high values (Heap et a l . , 1976) as previously found with a competitive protein binding procedure (Heap et a l . ,  1973).  The milk test of pregnancy has been developed to use r e l a t i v e rather than absolute concentrations of progesterone for the correct i d e n t i f i c a t i o n of pregnant and non-pregnant  animals (Heap et a l . ,  1976).  The fact that progesterone and milk fat concentrations showed a highly s i g n i f i c a n t positive correlation (r = 0.97)  (Hoffmann and Hamburger,  1973) i n cows with an active corpus luteum, led Hoffmann et a l . (1976), to the decision to use only the f a t - r i c h strippings, or, i f this was not possible, the whole milk for assay purposes. Of 133 animals c l a s s i f i e d , Hoffmann et a l . (1976), found the general agreement i n the group of animals diagnosed pregnant was while those diagnosed as non-pregnant  77%,  with values less than 2 ng/ml had  100% agreement. They concluded that the only information obtained which can substitute completely for c l i n i c a l examination i s the exclusion of pregnancy when progesterone i s below 2 ng/ml milk (Hoffmann et a l . , 1976). In order to decrease the range of the questionable c l a s s i f i c a t i o n , Hoffmann et a l . (1976), developed a method of measuring the progesterone i n milk f a t . Changes i n progesterone production during the cycle were well reflected with the range i n values f a l l i n g between 0.15  ng/10  ul milk-fat at estrus and 2.5 ng/10  y l milk fat during the  - 33 -  l u t e a l phase (Hoffmann et a l . ,  1976).  Eastman (1979), also f e l t that the accuracy of the progesterone test could be improved by using a progesterone-in-milk-fat value.  He  used 1.0 ng progesterone/10 y l milk-fat as his discriminatory l e v e l and v e r i f i e d negative diagnosis (less than 1.00 ng progesterone/10 ul milk-fat) by a return to service, and positive diagnosis (greater than 1.00 ng progesterone/10 \il milk-fat) on a 60 day non-return basis.  His  results do not show much improvement i n accuracy however, with positive diagnosis having only 83.3% agreement on 25 of 30 cows, and the negative diagnosis being i n 100% agreement in 24 cows, with samples collected on day 21 after breeding. Pennington et a l . (1976), found the use of extracted or non-extracted milk for progesterone analysis gave similar results for pregnancy diagnosis, and that diagnosis by concentration of progesterone i n milk was not improved by expressing progesterone as progesterone per unit f a t , or progesterone per milking. reduced labour required for the RIA.  Eliminating the extraction step They examined 508 cows and found  that diagnosis of cows as pregnant and non-pregnant  by milk progesterone  on day 21 post breeding had 76% and 98% agreement, respectively, with diagnosis by palpation or return to estrus as v e r i f i c a t i o n of r e s u l t s . Examining the best time to sample for pregnancy post-breeding, Pennington et a l . (1976), found pregnancy diagnosis by progesterone determination i n milk on day 21 or day 23 post-breeding had a greater agreement with diagnosis by palpation than diagnosis by milk progesterone on day 19, 25 or 27 post-breeding.  - 34 -  Pennington et a l . (1976) compared results from the 5 major dairy breeds and determined  that breed of cow did not affect the accuracy of  diagnosis. Zaoral et a l . (1982) studied the effects of sampling time on the accuracy of the milk progesterone  pregnancy test.  They used three  sampling variations, confirming pregnancy with a r e c t a l  examination  60-90 days post-insemination: 1.  Foremilk and whole-milk sample on day 23 for 1691 cows, r e s u l t i n g i n 87.7%  correctly diagnosed non-pregnant,  67.3%  correctly diagnosed pregnant, for an overall agreement of 73.3%. 2.  Sampled on day of insemination or day 1, day 19 and day  No s i g n i f i c a n t difference  23.  i n the proportion of the results i n  agreement i n both tests (progesterone test (PT) vs. c l i n i c a l examination) were found i n the group of non-pregnant cows between sampling v a r i a t i o n (1) and variation (2) or the different of milk sampling i n v a r i a t i o n (2). results (91.9%) was  The highest per cent agreement of  obtained by a single sample on day 23.  pregnant group, the highest per cent agreement (78.7%) was the group sampled a l l three times. any other combination. influenced  This was  In the obtained i n  s i g n i f i c a n t l y higher than  The results of the group of pregnant cows  the overall r e s u l t s , the range being from 62.9%  sample on day 19 to 81.2% 3.  combinations  for one  for a l l three samples.  In variation (3) three samples were again obtained.  This  time however, there were only two sampling days per week (Monday and  Thursday).  - 35 -  The f i r s t sample was taken on day 19 to 22, the second on the following sampling day (day 22 to 25) and the remaining sample the following week (day 29-32). The differences between a l l the combinations i n the group of nonpregnant cows were i n s i g n i f i c a n t , while in the groups of pregnant cows, the single sample showed s i g n i f i c a n t l y poorer agreement than a l l combinations of two samples and i n comparison with the three sample combination.  The range in agreement was from 70.6% to 83.9%.  The significance of the overall results was s i m i l a r .  An  increased number of samplings increased the proportion of correctly PT-determined non-pregnant  cows.  Hence, the proportion of i n c o r r e c t l y  PT-determined cows was reduced in the group of pregnant cows (Zaoral et al.,  1982). These results are contrary to those reported e a r l i e r by Heap et  a l . (1976) and showed that the combination of two samples was more advantageous.  Because v a r i a t i o n (2) and v a r i a t i o n (3) results were  comparable, sampling could be r e s t r i c t e d to two days a week reducing the laborious daily sampling and eliminating errors caused by missed samples. Shemesh et a l . (1978) found similar agreement using milk progesterone to determine pregnancy with 78% accuracy i n predicting pregnancy and 100% accuracy i n predicting non-pregnancy  with an o v e r a l l  accuracy of 88% using afternoon foremilk samples taken on day 24.  They  used a highly s p e c i f i c - a n t i s e r a providing minimal cross-reaction with other metabolites. The average progesterone concentration was 5.1 ng/ml milk and ranged from 1.3 ng/ml to 15.9 ng/ml milk i n l a c t a t i n g  non-pregnant  - 36 -  cows and was  19.7 ng/ml milk and ranged from 7.1 ng/ml to 35.6 ng/ml  milk i n pregnant cows. The results of this study support the opinion of those investigators who  contend that the p r i n c i p l e value of milk progesterone  testing for pregnancy i s to detect those cows which are not pregnant (Shemesh et a l . ,  1978).  The results of positive pregnancy assays from  several countries with varying conditions of management, n u t r i t i o n and l e v e l of production, indicate that with present techniques, positive assays cannot be expected to give an accuracy of more than 80%.  In  p r a c t i c a l terms this means that under present conditions what i s needed are  more e f f i c i e n t methods for estrus detection and milk progesterone  assay w i l l have i t s chief application for early detection of non-pregnant  cows, as well as an aid to c l i n i c a l diagnosis of such  conditions as cystic ovaries, subestrus, anestrus, and for monitoring changes i n progesterone levels before and after gynecological treatment (Shemesh et a l . ,  1978).  Holdsworth et a l . (1979) discussed the problems entailed i n a large scale application of the radioimmunoassay for progesterone.  They  found that the addition of the label immediately after the antiserum, abolishing the twenty minute incubation period, did not appreciably a l t e r the r e s u l t s , the correlation between assays with and without incubation being highly s i g n i f i c a n t (n = 0.93).  They then tested the  e f f e c t s of aging the antiserum/label mixture from one to three hours, and found that levels f e l l throughout the period of the experiment, but the f a l l was not s i g n i f i c a n t .  Use of the premix improved  the rate of  - 37 -  sample through-put s i g n i f i c a n t l y , and removed the necessity for one timed stage. Holdsworth et a l . (1979) attempted to i d e n t i f y sources of v a r i a t i o n , both inter-assay and intra-assay, and concluded that the v a r i a t i o n originates largely from the samples, and the observations of Van der Wiel et a l . (1978) indicate that non-specific binding (NSB) could be implicated.  They found NSB varies from sample to sample  (ranging from 13.9% to 25.6%, mean 21.4%), but were unable to produce any reduction in assay v a r i a t i o n by correction for NSB,  or demonstrate  any a l t e r a t i o n i n NSB by sample treatment (Holdsworth et a l . ,  1979).  They concluded that the importance of assay v a r i a b i l i t y i s largely related to the application of the assay.  If as i n a pregnancy  t e s t , i t s main function i s to distinguish high and low l e v e l s , then greater v a r i a t i o n can be accepted than i f absolute levels are being compared. Stevens et a l . (1981) found that an increase i n temperature, and d i l u t i o n of the milk sample both independently and, i n combination, reduced NSB.  The RIA of Heap et a l . (1973) was modified to account f o r  both increase i n temperature and d i l u t i o n .  Stevens et a l . (1981) also  determined that this refinement i n technique also gave better repeatability. Booth et a l . (1979), reporting on the use of the progesterone test for a pregnancy determination service offered i n the United Kingdom since 1975, found that after three years of operation, more than 100,000 cows per year were being tested and 5.6% of farmers i n England and Wales were using the service.  A sample of large herds using the service found  average accuracy rates of 84.5% and 97.0% respectively for positive and  - 38 -  negative tests. (r = + 0.84)  They found that the highly s i g n i f i c a n t  correlation  between proportion of positive tests i n a herd and the  accuracy rate of these tests, along with an analysis of insemination i n t e r v a l data, were good indicators of herds requiring veterinary investigation (Booth et a l . 1979). In their examination of milk progesterone as a diagnostic a i d , Foote et a l . (1979) found that under f i e l d conditions i t was to use l a s t milk samples, as they tended to be the least  preferable  contaminated,  were highest i n progesterone content and, could be taken after some lapse of time, i f i t was soon discovered that a sample was missed. t o t a l of 315 cows they reported a 98% accuracy for non-pregnant  On a  and 80%  accuracy for pregnant with an overall accuracy of 87% (Foote et a l . , 1979). Laing et a l . (1979) sampled F r i e s i a n cows on days 38 and 46 after service, the sample day chosen so as to cover the known normal v a r i a t i o n i n cycle length, from 17 to 25 days, so that any cow returning to estrus after two cycles should be detected and, because the sample times were later than the end of the main period of embryonic mortality.  They  found the accuracy for positive diagnosis from milk samples to be increased to 95.2%  indicating the inaccuracies caused by  embryonic  mortality had been to some extent overcome (Laing et a l . ,  1979).  Recognizing that the error factor of greater than 20% i n positive pregnancy diagnosis based on milk progesterone may be due to either l u t e a l phase progesterone, or early embryonic Holdsworth,  abortion (Booth and  1976; Heap et a l . , 1976; Hoffmann et a l . , 1976;  Pennington  et a l . , 1976), Shemesh et a l . (1981) used progestin impregnated  vaginal  - 39 -  sponges (PIVS) i n order to diminish the wide spread range i n the time of return to estrus usually seen i n inseminated (Booth and Holdsworth, 1976;  cows which are not pregnant  Heap et a l . 1976;  Hoffmann et a l . 1976;  Pennington et a l . , 1976). PIVS were given six to seven days following insemination  and  removed on day 17 following insemination to 50 I s r a e l i - F r i e s i a n c a t t l e . Ninety-six animals acted as controls. 21-24  Milk samples were obtained on day  after estrus and assayed for progesterone using fat-free milk  while pregnancy was palpation.  confirmed  at six to seven weeks by r e c t a l  In contrast to the control group in which 11 of 52  (21%)  cows were i n c o r r e c t l y diagnosed as pregnant, 100% of 30 cows diagnosed pregnant with PIVS inserted were correctly diagnosed. the test for negative pregnancy was  100%,  The accuracy of  regardless of treatment  (Shemesh et a l . 1981). An interesting aspect of this experiment was f e r t i l i t y rates of the two groups were examined. the control group was  43% and the PIVS group was  discovered when the  The f e r t i l i t y rate for 60%.  Though the sample  is too small to draw any firm conclusions, this indicates a possible b e n e f i c i a l effect on f e r t i l i t y  (Shemesh et a l . , 1981).  The majority of the previously described reports have employed a s p e c i f i e d concentration of progesterone i n milk, or milk f a t , to discriminate between only two categories, pregnant and non-pregnant cows, and have used c l i n i c a l diagnosis, by r e c t a l palpation, as the reference standard, to determine accuracy. s i m i l a r r e s u l t s , namely 76.9% diagnosis, confirmed  Gowan et a l . (1982) found  of p o s i t i v e and 93.8%  of negative  by r e c t a l palpation, or a return to estrus 28 -  150  - 40 -  days after insemination, when 3014 days post-insemination.  cows i n 394 herds were sampled 23  When those animals whose samples on day of  estrus showed a greater than normal concentration of progesterone were excluded,  the accuracy of the pregnant diagnosis increased by 7.1%  84% and the non-pregnant diagnosis increased by 3.1%  to  to 96.9%.  In this experiment dairymen submitting samples were required to record the following information:  date of insemination, diagnosis by  the herd veterinarian after palpation of the uterus, and date of uterine examination, or a record of the date of return to estrus, i f estrus observed after the sample was  taken on day 23.  was  Milk samples were  c o l l e c t e d on day of insemination, and day 23 after insemination, and i n cases where the cows returned to estrus prior to day 23, the samples were not  analysed.  From this information Gowan et a l . (1982) showed an increase i n agreement between the two methods of diagnosing pregnancy as time between insemination and palpation of the uterus increased, observed that the accuracy of diagnosis by palpation of the uterus increases with time during the early stages of gestation, and concluded  that from their  data, the most accurate method currently available to diagnose pregnancy before 50 days of gestation i s concentration of progesterone i n milk collected 23 days  post-Insemination.  Their data also showed that as the concentration of progesterone on day 23 increased, the probability that pregnancy would be  confirmed  by palpation increased u n t i l the maximum agreement between the  two  - 41 -  methods of diagnosing pregnancy was 85% and concentration of progesterone was 9.51  to 10.0 ng/ml.  Assigning a probability  that pregnancy w i l l be v e r i f i e d after 50  days or more of gestation by uterine palpation along with the absolute concentration allows the dairyman to decide how much e f f o r t should be expended on each individual animal for detection of estrus and emphasizes a r e a l i s t i c expectation of the success of a pregnancy diagnostic service (Gowan et a l . , 1982).  6.  Estrus  Detection  Using Milk  Progesterone  Assay  Plasma progesterone concentration returns to a basal l e v e l at parturition  and remains at this l e v e l u n t i l the ovaries begin their  c y c l i c a l function.  Milk progesterone concentration also i s at basal  levels from parturition  u n t i l ovarian a c t i v i t y resumes.  Researchers i n 1979 studied 50 p r o f i l e s and concluded a progesterone value of more than 5 ng/ml from hand-stripped aftermilk was indicative  of l u t e a l a c t i v i t y (vandeWiel et a l . , 1979).  further refined  by B a l l (1982) who  This was  studying more than 3000 milk  progesterone p r o f i l e s used the parameter  of at least two consecutive  values >^ 3.0 ng/ml as the f i r s t progesterone r i s e indicating  luteal  activity. Resumption of l u t e a l a c t i v i t y should occur within 30 days post-partum  (vandeWiel et a l . , 1979).  They found 12% of the animals  they examined took longer than 30 days.  Bulman and Wood (1980) i n an  examination of p r o f i l e s of 533 dairy cows found that 4.9% had not resumed cycling by 50 days post-partum.  - 42 -  Estrus and successful AI are concomitant with a nadir of progesterone l e v e l ; high progesterone concentrations are incompatible with f e r t i l i t y during this time.  Karg (1981) estimated that  progesterone should r i s e 4-5 days after estrus, indicating that ovulation and, In consequence,  corpus luteum formation has occurred, and  suggested that false timing of AI may be even more frequent than indicated by the progesterone assay, due to the fact that the average duration of estrus i s 15-18  hours and standing heat at 9-10  hour i s much  shorter. Shemesh et a l . (1978) reported results of Mylrea (1962) which found that natural service gave better conception rates than insemination, and reports of Gunzler et a l . ,  artificial  (1973) and Appleward  and  Cook (1976) that at least part of the explanation for this difference may  be ascribed to inaccurate diagnosis of estrus as reflected by  progesterone concentrations i n blood and milk of cows presented for insemination. Hoffmann et a l . (1976) stated that the measurement of progesterone i n milk i s an excellent way of identifying those animals which show no behavioural estrus 20 days after insemination and are not pregnant, or those which are inseminated during the active corpus luteum phase and thus cannot conceive.  They found that 22% to 27% of the  samples sent in for routine analysis were calculated to f a l l into these categories. In an experiment, 13% of 299 animals with no chance of conception were i d e n t i f i e d at the time of insemination through analysis of progesterone i n milk (Hoffmann et a l . ,  1976).  - 43 -  Shemesh et a l . (1978) compared the assessment of estrus by progesterone concentration i n milk on day of insemination and assessment of estrus by a veteran herdsman and an experienced inseminator. Accuracy of estrus diagnosis by herdsman, inseminator, and milk progesterone l e v e l were, 84%, 93%, and 96%, respectively (Shemesh et a l . 1978). Sixteen percent of the cows presented for insemination by the herdsman were not i n estrus as judged by milk progesterone l e v e l s . two other studies, between 10% and 20% (Appleward  In  and Cooke, 1976), and  between 14% and 26% (Hoffmann et a l . , 1976), were not i n estrus when inseminated as judged by plasma or milk progesterone respectively.  levels,  Zaoral (1982) sampled cows on day of insemination and  found 14% of the inseminations were performed  outside of estrus.  Foote et a l . (1979), i n an experimental herd where the opportunity for heat detection was poor, found the pregnancy rate for f i r s t service was about 40%.  An analysis of the milk progesterone  records revealed that 98% of the cows were cycling normally, starting at or about 50 days post-partum.  When inseminations were performed  reported estrus which coincided with low progesterone pregnancy rate was  over  at a  values, the  60%.  In a second experimental herd with a good programme of estrus detection and breeding, Foote et a l . (1979) reported that i n 19% of the cycles, estrus was reported when progesterone concentration i n milk was high.  - 44 -  Using defatted milk, samples, McCaughey & Cooper (1980) showed a negative correlation exists between levels of milk progesterone greater than 0.20  ng per ml defatted milk and calving rate.  Eight of 96 cows i n  an experimental herd showed levels of progesterone much above the normal estrus l e v e l (more than 0.30 ng/ml) and f a i l e d to conceive.  Of  1177  milk samples from cows being inseminated i n commercial herds, 91 (7.7%) had levels of milk progesterone i n excess of those observed at normal estrus. The potential value of progesterone assay i n monitoring for nonobserved estrus or sub-estrus i n the post-partum period needs further study, especially i n r e l a t i o n to frequency of sampling, as this would seem to be the most b e n e f i c i a l area for i t s use (Melrose, 1979).  Major  d i f f i c u l t i e s arise i n estrus diagnosis i n up to 20% of cows and i t has been suggested that up to 20% f a i l to conceive due to insemination at the wrong time, emphasizing again the possible use of hormone p r o f i l e s to allow monitoring for such situations (Melrose, 1979). Due  to intensive husbandry  along with increased herd size and  reduced labour force, the requirement of time to reach 80% plus estrus detection would add to the existing unsocial working hours of the cowman.  Melrose (1979) suggested a possible compromise i n which we  monitor i n retrospect, by hormone assay, the e f f i c i e n c y of estrus detection, and when necessary, tighten up the routine.  Where analysis  shows poor estrus detection, there could be a place for aids such as heat mount detectors, to bring out estrus signs more c l e a r l y .  - 45 -  The benefits of recording symptoms rather than simply estrus was reported by Foote et a l . (1979).  They found, with improved management,  only 5% of the c a t t l e reported f o r insemination were i n diestrus as compared to 19% previously (Foote et a l . , 1979). B a l l and Jackson (1979) used a milk progesterone determined estrus as the basis of a double insemination, i n an attempt to reduce the calving i n t e r v a l of those cows which were cycling but not showing behavioural signs.  Cows were observed for estrus s i x times per day  between 5:30 a.m. and 10:00 p.m. and a l l signs were recorded at each observation period. also u t i l i z e d .  Ka-Mar® heat detectors and Delta mate-markers® were  Milk samples were obtained thrice weekly and,  progesterone levels i n fat-free milk were determined.  This information  was used to predict the time of estrus at which insemination was due to within two to three days.  Milk samples were taken daily beginning  five  days before the estrus was due and cows f a i l i n g to exhibit estrus following a drop i n progesterone to below 1.5 ng/ml fat-free milk were Inseminated two and three days after the f a l l . inseminated  Those showing signs were  once at the appropriate time.  Of 55 cows inseminated  on the basis of the f a l l  i n progesterone  concentration, 33 or 60% conceived compared to 86 or 65.2% of the 132 inseminations  on the basis of observed estrus.  The difference i n  conception rate was not s i g n i f i c a n t . Of 3014 cows examined by Gowan et a l . (1982) 352 or 11.7% had higher than normal progesterone concentration on day of insemination. However, an estimate of the frequency with which cows were bred during diestrus was derived from those cows for which the concentration of  - 46 -  progesterone was high and for which uterine palpation or a return to estrus indicated absence of fetus. met  They found only 3.5% of their survey  these q u a l i f i c a t i o n s and, therefore, represented the maximum  frequency with which cows were bred during the estrous cycle when a functional corpus luteum was  7.  present.  Embryonic Mortality Embryonic mortality, s t r i c t l y interpreted, should refer to  f e r t i l i t y losses during the embryonic period, i . e . the period extending from conception to completion of the stage of d i f f e r e n t i a t i o n which, i n the cow,  occurs at approximately 45 days (Committee on Reproductive  Nomenclature, 1972). Using planned slaughter of repeat breeder cows, Ayalon (1981) determined  that the c r i t i c a l period appeared  to be soon after the embryo  enters the uterus, six to seven days after service, when the morula i s developing Into the blastocyst.  Ayalon (1973) has shown that day 7  rather than day 6 i s the c r i t i c a l day on which embryonic death becomes evident. Diskin and Sreenan (1980) investigated 246 beef heifers and determined  early embryonic mortality occurs mainly between days 8 and 16  (Karg, 1981).  Spontaenous embryonic losses afterwards (beyond the  non-return day 21), seem to occur mainly between days 30 and 45 after AI, at the time when the attachment of the placenta to the uterus i s becoming firmly established, B a l l  (1980).  Plasma and milk progesterone concentrations have been used to estimate embryonic l o s s .  When progesterone evaluation on day of  - 47 -  insemination i s included, accuracy of this approach i s increased as you can eliminate those animals with a functional corpus luteum on day of estrus.  However, this method does not overcome the problems associated  with estrus during early pregnancy, estrus i n cows with endometritis, nor w i l l i t detect the majority of embryonic losses, which are occurring before 15 days post-insemination (Ayalon 1981). B a l l (1982) examining over 3000 post-partum p r o f i l e s for cows from 22 herds, determined detectable embryonic loss as those cows having progesterone  levels r i s i n g within eight days of insemination and  remaining high u n t i l at least 25 days after insemination, indicating that pregnancy had been established, followed by a subsequent f a l l i n progesterone  levels indicating that the embryo had been l o s t .  occurred i n only 6.4%  This  of the cows he examined.  Foote et a l . (1979) assumed embryo/fetal mortality for any breeding i n t e r v a l exceeding  28 days up to the 75th day.  In one  experimental herd (herd A previously mentioned), the estimated between 28 days and 75 days was  loss  22.7%, c l e a r l y overestimating the embryo  mortality, as many cows i n estrus before 28 days were missed according to normal c y c l i c patterns i n milk progesterone.  When they  embryo mortality on the basis of any milk progesterone  estimated  cycle longer than  27 days after insemination followed by regular c y c l i c a l patterns the estimated loss was  reduced  to 7.2%  (Foote et a l . , 1979).  Bulman and Lamming (1979) sampled 555 cows twice weekly and  found  that embryonic mortality occurred i n 12% of the animals between 31 and 59 days after insemination.  They compared the mean progesterone  levels  of 57 normal cows and 23 cows i n which embryonic mortality occurred for  - 48 -  the f i r s t 30 days of pregnancy and found no difference.  Following t h i s ,  progesterone levels remained i n the normal range i n the l a t t e r group u n t i l a steep decline to basal levels indicated that embryonic  mortality  had occurred. Holness et a l . (1981) using plasma progesterone concentrations to study f e r t i l i t y , found 11% of 69 cows returned to estrus between 30 and 88 days after insemination. They also found no difference i n mean plasma progesterone concentration from four to 46 days after insemination between cows that returned to estrus between days 30 and 88 after insemination and those that remained pregnant. et  E a r l i e r , Holness  a l . (1977) had found 10% of 90 cows suffered embryonic  mortality  between 26 and 81 days after insemination.  8.  Milk  Progesterone  and  Infertility  Lamming and Bulman (1976) collected twice weekly samples of whole milk from 300 cows i n three commercial herds, from 14 days post-partum u n t i l they were diagnosed pregnant. AI was used.  Signs of estrus were recorded and  Results from the f i r s t 200 cows examined showed the  i n t e r v a l from p a r t u r i t i o n to ovarian a c t i v i t y , as measured by a substantial r i s e i n the milk progesterone l e v e l s , occurred prior to 20 days for 50% of the animals.  Ninety-three per cent had shown ovarian  a c t i v i t y by day 40, allowing at least one cycle prior to an optimal first  insemination date of 65 days post-partum, and 15% produced milk  progesterone  p r o f i l e s suggestive of s u b f e r t i l i t y .  Their data confirmed the problem of " s i l e n t " estrus.  Only 30%  showed estrus at the beginning of the f i r s t cycle, 63% after the f i r s t  - 49 -  cycle and 86% after the t h i r d .  Excluding the f i r s t cycle, where overt  estrus might not have been observed because of a lack of previous progesterone, only 77% of the remaining estrus periods were observed (Lamming and Bulman, 1976). In a large proportion of cows, a small r i s e i n milk progesterone l a s t i n g from six to nine days occurred preceding a f u l l ovarian cycle. Anestrus was determined i n seven per cent of animals showing persistently low levels of progesterone u n t i l after 50 days postpartum.  Two per cent showed a pattern of persistent high milk  progesterone for at least 30 days after a normal estrus and apparent ovulation, suggesting that l u t e o l y s i s did not occur at the normal time of approximately 17 days after ovulation (Lamming and Bulman, 1976). Bulman and Lamming (1979) sampled 37 cows once every 10 days beginning 25 days post-partum u n t i l the establishment of pregnancy on the day of estrus.  and  They determined that these provided s u f f i c i e n t  data to allow an accurate assessment  of herd f e r t i l i t y and management.  Of 125 ovulatory periods covered by sampling, estrus was in 91, giving an estrus detection rate of 73%.  observed  Nine cows had not been  seen i n estrus by 50 days post-partum, f i v e of these were s t i l l  acyclic  and four were cycling but estrus had not been observed. Parameters measured were the overall intervals from calving to f i r s t estrus (43.3 ± 4.51  days, n = 37), f i r s t service (78.7 ± 4.13  days, n = 37), conception (87.1 ± 4.51 rate (1.26 ± 0.08  days, n = 35) and the conception  services per conception, n = 35).  The importance of abnormal patterns of ovarian a c t i v i t y on herd f e r t i l i t y was  examined by analyzing twice weekly milk samples for  - 50 -  progesterone from 533 dairy cows from p a r t u r i t i o n u n t i l the re-establishment of pregnancy (Bulman and Wood, 1980). Based on p r o f i l e analysis, the animals were placed into the following categories: a.  normal - 77.5%*  b.  delayed start to ovarian cycles - 4.9%  c.  cessation of cycles - 5.1%  d.  prolonged l u t e a l a c t i v i t y - 1.9%  e.  s i l e n t estrus - 10.7%  *The incidence of 'normal' cycles was 75% for f i r s t l a c t a t i o n , 80% f o r 2nd to 5th l a c t a t i o n and 65% for 5th to 11th l a c t a t i o n . Animals i n groups "b" to "e" were subdivided into treatment and control groups. 1.  The treatment for groups "b" and "c" were:  a single i n j e c t i o n of 0.5 mg l u t e i n i z i n g hormone-releasing hormone (Hoechst) or,  2.  progesterone-releasing intravaginal device (Abbott Lab. Ltd.) inserted for fourteen days.  For groups "d" and "e," treatment consisted of a single i n j e c t i o n of 0.5 mg cloprostenol (Imperial Chemical Industries). It was determined that none of the treatments reduced calving to conception i n t e r v a l , although the majority responded to treatment. Nearly half (47.8%) resumed c y c l i c ovarian a c t i v i t y within 20 days of calving and this increased to 92.4% by 40 days.  Abnormal ovarian  function of some sort was noted i n 22.5% and had a highly s i g n i f i c a n t i n t e r a c t i o n with age.  - 51 -  9.  Milk  Progesterone  to  Monitor Hormonal  Treatment  of  Subfertllity  In a study by Lamming and Bulman (1976), treatment was  initiated  i n animals i d e n t i f i e d as anestrus by negligible progesterone levels up to 50 days post-partum and consisted of: a.  0.5 mg Gn RH (Hoechst HOE 471) or  b.  a progesterone releasing intravaginal device (PRID Abbotts).  Some animals were l e f t untreated as controls to indicate the extent of spontaneous  recovery.  Cows termed s u b f e r t i l e because of regular l u t e a l a c t i v i t y as shown by progesterone but exhibiting no estrus i n the f o l l i c u l a r phase were treated with prostaglandin (PG) following two consecutive f o l l i c u l a r periods without estrus, " s i l e n t estrus" (Lamming and Bulman, 1976). Of seven animals treated with Gn RH, f i v e responded with c y c l i c a c t i v i t y , although the response was variable, ovulated, and became pregnant.  Generally, animals treated with Gn RH did not show immediate  post-treatment estrus (Lamming and Bulman, 1976). treated with PRID, a l l responded with subsequent  Of seven animals l u t e a l a c t i v i t y , five  showed post-treatment estrus and six became pregnant at the f i r s t or second post-treatment ovulation.  A uniform r i s e i n milk progesterone  following removal of the device was noted, suggesting that a timed insemination might be used.  Since post-treatment f e r t i l e estrus  occurred after PRID i n s e r t i o n , Lamming and Bulman (1976) f e l t that there was a potential to establish an e a r l i e r pregnancy with this device  - 52 -  compared to Gn RH i n j e c t i o n .  Of nine animals treated for " s i l e n t "  estrus with PG, eight showed post-treatment estrus and seven became pregnant after insemination. Bulman and Lamming (1977) collected milk samples from over 300 dairy cows from p a r t u r i t i o n to confirmed pregnancy by milk progesterone test.  A progesterone p r o f i l e was plotted for each animal.  If the  progesterone i n milk of unmated cows remained greater than 13 ng/ml for more than 30 days, the animal was c l a s s i f i e d as having an abnormally long period of l u t e a l a c t i v i t y , and was assigned to either a control, or treatment group which received a single i n j e c t i o n of prostaglandin. Six animals or 1.5% of those surveyed were found to have prolonged l u t e a l a c t i v i t y .  Of the three animals treated, a l l responded  to a single i n j e c t i o n of prostaglandin with complete l u t e o l y s i s .  A  further 7% of cows studied were seen i n estrus between 30 and 70 days after insemination.  It i s possible some of these could have experienced  prolonged l u t e a l a c t i v i t y without being pregnant. One important aspect of this paper i s that Bulman and Lamming (1977) state that prolonged l u t e a l a c t i v i t y can occur i n the absence of any apparent c l i n i c a l abnormality. In another study, the anestrus syndrome was defined as no estrus occurrence by post-partum day 60 (post-partum anestrus), or by 21 to 24 days after insemination, i f a negative early pregnancy test based on milk progesterone concentration was obtained (post-insemination anestrus) and two treatments, gonadatropin releasing hormone (Gn RH) and  - 53 -  prostaglandin  (PGF2 ) analog were tested (Humblot and Thibier, a  1980). In the f i r s t of their experiments, Humblot and Thibier (1980) divided the post-partum  anestrus cows (those not seen i n estrus for 60  days post-partum) into four groups: Group 1 - Control group consisted of 18 non-treated anestrus animals. Group 2 - 131 animals with a functional corpus luteum shown by progesterone values greater than 2 ng/ml milk or 71.5 ng/ml plasma were treated with PGF2 and inseminated a  systematically 72 to 96 hours after i n j e c t i o n . Group 3 - 21 animals with true anestrus with milk progesterone values less than 2 ng/ml or plasma value less than 1.5 ng/ml i n two samples taken at 10 day Intervals, were treated with one i n j e c t i o n of Gn RH and inseminated at the observed estrus. Group 4 - 14 animals with true anestrus were given one i n j e c t i o n of Gn RH and according to ovulation occurrence (as shown by progesterone concentrations i n blood or milk) were assigned to one of the following treatment regimens.  Cows observed i n estrus i n the ten day  period after treatment were inseminated.  Cows not  observed i n estrus and with low progesterone values were treated again with Gn RH.  And f i n a l l y , cows not  observed i n estrus, although ovulation had occurred, were treated with PGF2 . a  - 54 -  Humblot and Thibier (1980) found 29% of the animals showed postpartum anestrus: Control or Group 1 had a mean i n t e r v a l between p a r t u r i t i o n to f i r s t service (1st estrus) of 101 ± 21 days with a conception rate on this service of 44%. Group 2 were bred 27 days before the controls were bred, with a conception rate similar to that of controls.  The mean  i n t e r v a l between treatment and conception was 41 days. Group 3 had a mean i n t e r v a l between p a r t u r i t i o n and insemination of 101 days.  first  The i n t e r v a l between f i r s t Gn RH  i n j e c t i o n and conception was 53 days. Group 4 had a mean i n t e r v a l between f i r s t Gn RH i n j e c t i o n and conception of 34 days. On the whole, more cows i n the treated groups were inseminated within 90 days (42% vs 16%) after p a r t u r i t i o n and conception occurred 17 to 25 days e a r l i e r than i n the control anestrus cows.  Of the 29% of  the cows showing the anestrus syndrome 60 days after p a r t u r i t i o n only 21% did not show ovarian a c t i v i t y . In  the second experiment, Humblot and Thibier (1980) found 71  non-pregnant  cows were anestrus 21 to 23 days after insemination by low  milk progesterone and were not observed i n estrus. Group 1 - Control group of 28 untreated animals. Group 2 - 43 Animals were injected with PGF2 , 10 to 15 days a  after the date of negative early pregnancy diagnosis and were inseminated at 72 and 96 hours post-treatment. The treatment group conceived 24 days e a r l i e r than did the controls.  - 55 -  These authors f e l t that the savings due to the reduction i n mean calving i n t e r v a l would more than cover the cost of the investigations and treatments.  - 56 -  SUMMARY  To summarize the preceding, i t has been shown that the dairyman i s faced with many problems and factors including anatomical, g e n e t i c a l , physiological, pathological and management factors to consider when trying to optimize f e r t i l i t y i n the dairy herd. Modern architectural engineering i n agriculture can modify the environmental  e f f e c t s , feed analysis and ration formulation can  eliminate the n u t r i t i o n a l e f f e c t s , and the many research reports on management strategy can provide the dairyman with reproductive goals to strive for. Through the use of radioimmunoassay of progesterone obtained milk samples the dairyman now  in easily  has a means of early accurate  assessment of bovine reproductive status, the benefits of which as stated by Norman (1982) are: 1.  Detection of estrus can be simplified by focussing the manager's attention on those cows l i k e l y to return to heat;  2.  Breeding plans can be used more e f f i c i e n t l y ;  3.  Non-producing cows can be culled early;  4.  F e r t i l i t y testing of bulls i s simplified;  5.  Diagnosis of reproductive problems i s f a c i l i t a t e d .  Radioimmunoassay of progesterone  and,  from milk samples allows great  quantities of data regarding the reproductivity of dairy animals to be e a s i l y obtained by the researcher. this data needs to be developed.  However, some method of analyzing If the researcher can establish a  - 57 -  "normal" pattern of reproductivlty through this analysis then possibly the radioimmunoassay of progesterone w i l l provide a method of monitoring the effects of some of the preceding factors influencing  fertility.  - 58 -  MATERIALS AND METHODS  The information used i n this study was obtained from two dairy herds, one located at the University of B r i t i s h Columbia South Campus, UBC herd and the other at the Agriculture Canada Research Station at Agassiz, B.C.,  1.  Agassiz herd.  Milk Sampling Post-milking, whole milk strippings were collected i n 200 ml  Whirl Pak® bags, using potassium dichromate LACTAB® as the preservative and stored at 4-10°C u n t i l analysis was performed.  The Agassiz herd was  sampled on an every other day basis while the UBC herd was sampled twice a week with sampling beginning approximately six days post-partum continuing u n t i l approximately 60 days post-conception.  and  Samples from  the Agassiz herd were either mailed, or brought to the University for analysis.  2.  Radioimmunoassay The samples were analysed for milk progesterone by  radioimmunoassay employing a method described by Shelford et a l . (1979) which i s a modification of the radioimmunoassay technique f i r s t described by Heap et a l . (1973).  3. 3.1  Animals - General Management Practices UBC Herd A t o t a l of 53 animals were sampled through 54 lactations between  February 1978 and June 1979.  These animals kept at the south campus  - 59 -  f a c i l i t i e s were placed i n maternity pens six to ten days prior to expected p a r t u r i t i o n and remained post-partum.  there u n t i l five to ten day  The cows were then housed i n a free s t a l l  system with access to a row of 48 Calan Broadbent controlled individual feeders.  confinement  electronically  The animals also had access to an  outside paved exercise area, weather permitting. Feeding took place at approximately 0230 hours and 1300 hours (prior to milking), and consisted of a l f a l f a cubes (fed ad l i b ) , 14% protein textured concentrate ( r a t i o of 1 grain to 3 milk), and beet pulp p e l l e t s (max. 4 kg per day).  A few animals also were fed ad l i b an  experimental ration of corn silage and an 18% protein textured concentrate on a 70 to 30 r a t i o of forage to grain. Cows were moved to a holding area at approximately 0330 hours and 1400 hours and milked i n an i n - l i n e , three s t a l l , high-line parlour, beginning at approximately 0400 hours and 1430 hours. No set times were established for estrus detection, however, the majority of the heats were noted prior to feeding and milking.  The  general practice of monitoring estrus behaviour In the herd was that anyone working with the c a t t l e would note any estrus a c t i v i t y , i . e . mounting, increased a c t i v i t y or vaginal discharge, and inform the herdsman, who would act accordingly. Some animals not noted i n standing heat during the study were bred on the basis of previously predicted heats as determined from milk progesterone analysis. Artificial  insemination was performed by the herdsman at  approximately 1500 hours f o r those animals noted i n heat i n the early  - 60 -  morning hours, and at approxiamtely 0830 hours for those animals noted in heat i n the previous afternoon and evening.  Pregnancy diagnosis by  r e c t a l palpation and post-calving reproductive checks were performed at i r r e g u l a r i n t e r v a l s , 30-60 days apart. When the veterinarian was i n attendance for pregnancy diagnosis, a l l post-partum animals not previously examined were checked to determine the health status of the reproductive t r a c t .  Also, any cows  showing any abnormal reproductive occurrence, nymphomania, anestrus, abnormal vaginal discharge, etc., were examined, and treatment was i n i t i a t e d when any abnormality was diagnosed.  Individual records  consisted of dates of calving, estrus, breeding, treatment of reproductive tract and positive r e c t a l palpation of pregnancy by a veterinarian.  Health data such as retained placental membranes,  m e t r i t i s , milk fever, ketosis, c y s t i c ovaries (when examined by a veterinarian) and mastitis were also noted. Throughout herd.  the sampling period 12 animals were culled from the  Data collected from these animals was used for some of the  calculations i f appropriate. For example, i f early sampling continued up to f i r s t estrus but not u n t i l conception, the data would be used i n c a l c u l a t i o n of days to f i r s t estrus but would not be included i n any pregnancy  test c a l c u l a t i o n s . A  3.2  Agassiz Herd A t o t a l of 110 animals were sampled through 146 lactations from  mid-December 1976 to mid-September 1979.  - 61 -  The Agassiz herd was housed i n a free s t a l l barn.  In contrast to  the UBC herd, the Agassiz herd was pastured from May to October on Orchard Grass with approximately 10% White Clover, using a s t r i p grazing system.  Corn silage and approximately 2 kg. long hay made up the  roughage ration during the winter.  The grain ration was fed i n the  double sawtooth parlour at the rate of 1 kg grain to 3 kg of milk y i e l d i n winter and at the rate of 1 kg grain to 4 kg of milk y i e l d i n the summer.  Depending on other experimental regimes or l e v e l of production,  additional grain was fed i n the parlour. There were set times f o r estrus detection i n the Agassiz herd. An hour i n the late evening, from 2100 to 2200 hours was set aside f o r observation. feeding.  As well, the animals were observed at milking and  Similar to the UBC herd, those animals not noted i n standing  heat during the study, and showing regular heat as predicted by progesterone analysis, may have been bred on days of "expected heat." A l l breeding was by a r t i f i c i a l for  insemination and animals were palpated  pregnancy at approximately two months gestation, along with any  abnormal animals. Individual information was collected from the record system used for  the herd, and was similar to that collected for the UBC herd.  Throughout  the sampling period the animals from which information was  collected were also used for other t r i a l s  and examinations, some of  which involved an early and late breeding regime.  For the early bred  group, animals were inseminated at the f i r s t v i s i b l e heat following 50 days post-partum, while the late bred group was inseminated following 80  - 62 -  days post-partum (Schneider et a l . , 1981).  This may have affected the  results of some of the reproductive s t a t i s t i c s determined f o r the Agassiz herd. Throughout  the sampling period animals were culled from this herd  for normal reasons, i l l n e s s , reproductive problems, etc, as well as a herd reduction. Data collected from these animals was used i f appropriate.  4. 4.1  Cycle C l a s s i f i c a t i o n Cycle Phases The estrous cycle was c l a s s i f i e d into phases based on the  concentration of progesterone i n the milk samples, to aid i n i d e n t i f i c a t i o n of different types of cycles.  Examples of these phases  derived from the following c r i t e r i a are shown i n F i g . l a representing a t h e o r e t i c a l c l a s s i f i c a t i o n and F i g . lb representing the c l a s s i f i c a t i o n as derived from the data. Phase 0:  Represents the stage of quiescence i n the ovaries with  respect to l u t e a l a c t i v i t y immediately post-calving, and l a s t s u n t i l the l a s t sample date with the lowest concentration of progesterone before a r i s e above a concentration of progesterone of 5 ng/ml milk. Phase 1:  Represents the f o l l i c u l a r stage of the ovarian cycle  when the concentration of progesterone i n the sample i s less than 4 ng/ml milk. Phase 2;  Represents the developing corpus luteum stage a f t e r  ovulation when the concentration of progesterone i s r i s i n g from 4 ng/ml  Fig. l a C y c l e Phases  0  (Theoretical)  15  oo a  w 55  o u  H C/5 Id O O Pi  DAYS POSTPARTUM. Phase 0 1 2  Location A + B  B +• C,  C +  3  n + E  4  E * F  Physiological  G H  Stage  Quiescence - F o l l i c u l a r Stage Developing Corpus Luteum Stage L u t e a l Stage R e g r e s s i n g Corpus Luteum Stage  Phase  0 1 2  3 4  Location  Physiological  A B C D E  Quiescence F o l l i c u l a r Stage D e v e l o p i n g Corpus Luteum Stage L u t e a l Stage R e g r e s s i n g Corpus Luteum Stage  + B •>• C, F + G > D, G + H > E •»• F  Stage  - 65 -  milk to 12 ng/ml milk. Phase 3:  Represents the l u t e a l stage when the corpus luteum i s  f u l l y active and producing progesterone  i n concentrations i n excess of  12 ng/ml milk. Phase 4:  Represents the regressing corpus luteum stage and the  maturing of the new f o l l i c l e , when the concentration of progesterone i s declining from 12 ng/ml milk to 4 ng/ml milk.  4.2  Types o f Cycles Once a progesterone  concentration i n d i c a t i v e of a resumption of  l u t e a l a c t i v i t y was established and the cycle was c l a s s i f i e d into d i f f e r e n t phases, a method of c l a s s i f y i n g the cycles into various types was required.  C r i t e r i a for selection for the various types are given  below.  Table 5 Cycle Types Cycle Types 1 2 3 4 5 6 7 8  Descriptive Term Short f i r s t cycle Normal f i r s t cycle Normal cycle, not type 2, not bred Pregnant cycle Early embryonic abortion Atypical cycles, not bred Normal cycle, bred but not conceiving Atypical cycles with a breeding but not conceiving  - 66 -  Upon observing the graphs of progesterone concentration versus time for a number of animals, two d i s t i n c t types of f i r s t cycles were determined. Type 1: quiescence,where  This was defined as the short f i r s t cycle a f t e r the sample concentration of progesterone went from less  than 4 ng/ml milk to greater than 5 ng/ml milk, and returned to less than 4 ng/ml milk i n less than 17 days. In a study of 690 normal cows, Morrow et a l . (1969) found 40% of the cycles were less than 17 or greater than 23 days i n length. Type 2:  This was defined as the normal f i r s t cycle after  quiescence, where the sample concentration of progesterone went from less than 4 ng/ml milk to greater than 12 ng/ml milk, and returned to less than 4 ng/ml milk, i n 17 to 25 days.  This was e s s e n t i a l l y a normal  cycle. Type 3:  This was a normal cycle subsequent  to the f i r s t  cycle,  with the sample concentration r i s i n g from less than 4 ng/ml milk to greater than 12 ng/ml milk, and f a l l i n g to less than 4 ng/ml milk, i n 17-25  days, not including Type 2 cycles.  A further constraint of not  being bred was imposed on these cycles to distinguish them from Type 7 cycles. Type 4:  These cycles represented pregnancy, and the sample  concentration had to remain greater than 12 ng/ml milk for 25 days after breeding. Type 5:  This group represented early embryonic abortion, and the  sample concentration of progesterone had to remain greater than 12 ng/ml  - 67 -  milk f o r 25 days after breeding, similar to Type 4, but  subsequently  f a l l to less than 4 ng/ml milk. Type 6:  This group represented the atypical cycles, with no  breeding. Type 7:  This group was  similar to Type 3, a normal cycle,  however, i t included those animals bred and not conceiving. Type 8:  This group represented those atypical cycles that  included a breeding, not leading to conception. Means, standard deviations, and significance tests, for each cycle phase and cycle type, for both UBC and Agassiz herds were obtained, using the package program UBC  4.3  SPSS.  Standard Curves To develop a standard curve for a normal cycle a l l those cycles  c l a s s i f i e d as Type 2, the f i r s t normal cycles and Type 3, the normal cycles were pooled for each data base. Day one was determined  to be the f i r s t sampling date.  cycles were then grouped by cycle length.  These  Means, standard deviations  and the number of observations for each group were calculated for each sample date i n each group using the UBC Triangular Regression Package.  - 68 -  RESULTS AND DISCUSSION  The results have been presented as the concentration of progesterone i n milk, though i t should be recognized that the values probably over-estimate the true progesterone concentration because of the occurrence of steroid metabolites i n the cow's milk that cross-react with the anti-serum (Heap et a l . , 1976).  Measurements of known amounts  of progesterone with this analysis have also shown a tendency to over-estimate low values and under-estimate high values (Heap et a l . , 1973).  1.  Resumption of Luteal A c t i v i t y Cycling post-calving was determined  to have begun once the  concentration of progesterone i n the sample had reached a l e v e l of more than 5 ng/ml milk.  The time from p a r t u r i t i o n u n t i l the last sample date  with a concentration of progesterone of less than 5 ng/ml milk was called the stage of quiescence. Examples of a normal short quiescence of 20 days, and an abnormally long quiescence of 51 days, are given i n Figs. 2 and 3 respectively. From the Agassiz data there were 127 cases of quiescence examined.  The mean number of days i n quiescence was  with a range of 56 days.  19.81  ± 8.854  S.D.,  A t o t a l of 13% were 30 days or more i n  quiescence. Of the 127, 12.6% placenta.  or 16 animals, were recorded as having retained  They had a mean of 25.62 ± 12.41  S.D.  days i n quiescence,  Days Post»Parturn  DAYS  POST-PARTUM  - 71 -  with a range of 50 days.  Only 18.7% or three animals were longer than  30 days i n quiescence. For the remaining 111, the mean number of days i n the period of quiescence was  18.97  ± 7.95  S.D. with a range of 43 days.  Of these 16%  or 13 animals were 30 days or more i n quiescence. Examination of the UBC data revealed 54 cases of quiescence l a s t i n g for a mean of 21.43  ± 11.84  S.D. days, with a range of 55 days.  Of these, 24% or 13 animals were 30 days or longer i n quiescence. Five cases of retained placenta were noted i n the UBC data.  They  showed a surprisingly short quiescence, with a mean number of days In quiescence of 10.80 Treatment  ± 6.42  S.D.  and a range of 16 days.  for retained placenta i n the UBC herd consisted of an  attempt at manual removal of the placenta i f not too firmly attached, followed by the insertion of a n t i b i o t i c boluses.  Following removal of  placenta, i f any further abnormal discharges from these animals were noted, they received a uterine Infusion of an a n t i b i o t i c solution. For the remaining 49 without retained placenta the mean number of days i n quiescence was 21.86  ± 11.97  S.D.,  with a range of 55 days.  Of  these 25% or 13 animals were 30 days or more i n quiescence. Out of 54 cases of quiescence examined from the UBC data, only two animals or 3.7% remained inactive for more than 50 days.  Only one  of 127 cases of quiescence i n the Agassiz data was greater than 50 days. These results are similar to those found by van de Wiel and co-workers  i n 1979 but do not match those of Bulman and Wood (1980) who  found 4.9% of 533 dairy cows took longer than 50 days post-partum to  - 72 -  resume c y c l i n g . The greatly reduced time i n quiescence for those animals i n the UBC herd treated for retained placenta i s a mystery.  One would expect  that a reproductive problem of this type would extend the time i n quiescence as was the case i n the Agassiz data. One possible explanation for this reduction i s that the frequent manual examination of the uterus, the uterine infusion or the combination may have had a stimulating effect on the ovaries.  2.  Cycle Types By the end of the sequential sampling time period for Agassiz's  every second day sampling and UBC's twice weekly sampling, over 13,000 samples and information were collected, organized and categorized into cycles phases and cycle types as described in Materials and Methods. The number of observations, means, and standard deviations, for progesterone concentration (ng/ml) and days i n the phase, for Phases 1 through 4 of a l l cycle types for the Agassiz data and the UBC data, are given i n Tables 6-13.  They were grouped into homogenous subsets derived  from the Student-Newman-Keuls procedure using the package program UBC SPSS. From the study of many of the p r o f i l e s , i t was apparent there was a great deal of variation i n the f i r s t r i s e of progesterone post-partum and the associated f i r s t cycle.  Many of the p r o f i l e s showed a small  transient r i s e i n progesterone before a normal cycle while others indicated that the f i r s t r i s e continued into a normal type of cycle.  Table 6 Phase 1 - UBC Data F o l l i c u l a r Stage  Cycle Type  Number of Observations  (ng/ml) Mean Progesterone ± St. Dev.  Mean No. of Days ± St. Dev.  1.  Short F i r s t Cycle  18  2.49 ± .37ab  5.83 ± 2 . 7 9  2.  Normal F i r s t Cycle  20  2.32 ± .-35a  6.80 ± 2.80  3.  Normal Cycle Not Type 2 Not Bred  65  2.62 ± .58ab  5.54 ± 2.44ab  4.  Pregnant Cycle  39  2.77 ± .62ab  5.18 ± 2.04ab  5.  Early Embryonic Abortion  2  2.65 ± .21ab  5.00 ± 2.83ab  6.  Atypical Cycle Not Bred  51  2.65 ± .59ab  15.47 ± 24.89b  7.  Normal Cycle Bred not Conceived  27  2.42 ± .65ab  4.93 ± 2.60a  8.  Atypical Cycle With Breeding  12  3.00 ± .66b  T  o  t  a  l  234  2.61 ± .58  M  AB  10.25 ± 8.30ab  7.94 ± 12.56  a,b Denote Homogeneous subsets derived from the Student-Newman-Keuls Procedure at P < 0.05.  Table 7 Phase 2 - UBC Data Developing Corpus Luteum Stage  Cycle Type  Number of Observations  (ng/ml) Mean Progesterone ± St. Dev.  Mean No. of Days ± St. Dev.  1.  Short F i r s t Cycle  17  5.90 ± 1.43  4.47 ± 2.29  2.  Normal F i r s t Cycle  9  7.26 ± 2.03  3.78 ± 1.39  3.  Normal Cycle Not Type 2 Not Bred  45  7.14 ± 2.00  3.73 ± 1.01  4.  Pregnant Cycle  23  6.69 ± 1.99  4.43 ± 2.95  5.  Early Embryonic Abortion  6.  Atypical Cycle Not Bred  31  6.54 ± 2.38  5.58 ± 4.71  7.  Normal Cycle Bred not Conceived  21  7.30 ± 2.46  4.00 ± 1.09  8.  Atypical Cycle With Breeding  10  8.30 ± 2.35  5.10 ± 3.35  6.93 ± 2.15  4.41 + 2.77  Total  1  157  8.70  4.00  Table 8 Phase 3 - UBC Data Luteal Stage  Cycle Type  Number of Observations  (ng/ml) Mean Progesterone ± St. Dev.  Mean No. of Days ± St. Dev.  1.  Short F i r s t Cycle  11  18.78 ± 3.88a  6.18 ± 3.54a  2.  Normal F i r s t Cycle  20  28.20 ± 8.65ab  10.50 ± 3.85a  3.  Normal Cycle Not Type 2 Not Bred  68  32.21 ± 9.46a  13.57 ± 3.03a  4.  Pregnant Cycle  46  42.14 ± 7.59b  80.87 ± 27.64b  5.  Early Embryonic Abortion  37.77 ± 13.55ab  27.00 ± 7.20a  6.  Atypical Cycle Not Bred  58  32.19 ± 12.13a  15.5 ± 13.53a  7.  Normal Cycle Bred not Conceived  27  33.48 ± 9.72a  12.67 ± 2.83a  8.  Atypical Cycle With Breeding  11  34.81 ± 12.19ab  22.36 ± 25.12a  33.47 ± 11.02  26.59 ± 30.21  Total  3  244  a,b Denote Homogeneous subsets derived from the Student-Newman-Keuls Procedure at P < 0.05.  Table 9 Phase 4 - UBC Data Regressing Corpus Luteum Stage  Cycle Type  Number of Observations  (ng/ml) Mean Progesterone ± St. Dev.  Mean No. of Days ± St. Dev.  1.  Short F i r s t Cycle  1  4.60  3.00  2.  Normal F i r s t Cycle  8  6.96 + 2.24  4.75 + 2.87  3.  Normal Cycle Not Type 2 Not Bred  15  6.62 + 2.26  4.47 + 2.26  4.  Pregnant Cycle  3  5.  Early Embryonic Abortion  0  6.  Atypical Cycle Not Bred  7.  Normal Cycle Bred not Conceived  8.  Atypical Cycle With Breeding  Total  + 2.26  3.00 + 1.00  6.92 + 2.49  6.93 + 5.47  7  6.00 + 1.73  3.43 + .53  5  8.70 + 1.32  7.20 + 6.22  7.00 + 2.36  5.56 + 4.36  29  68  10.00  Table 10 Phase 1 - Agassiz Data F o l l i c u l a r Stage  Cycle Type  Number of Observations  (ng/ml) Mean Progesterone ± St. Dev. + .36  Mean No. of Days ± St. Dev. 2.88 + 1.38a  1.  Short F i r s t Cycle  58  2.35  2.  Normal F i r s t Cycle  26  2.34 + .32  3. "Normal Cycle Not Type 2 Not Bred  173  2.35  + .45  5.72 + 1.85a  89  2.39  + .46  5.81 + 1.95a  3.08 + 1.16a  4.  Pregnant Cycle  5.  Early Embryonic Abortion  6.  Atypical Cycle Not Bred  86  2.39  + .66  6.94 + 9.71a  7.  Normal Cycle Bred not Conceived  98  2.43  + 1.12  5.95 + 1.64a  8.  Atypical Cycle With Breeding  68  2.31  + .40  Total  4  602  2.50 + .49  2.37 + .62  7.00 + 2.58ab  16.28  + 30.26b  6.76 + 11.42  a,b Denote Homogeneous subsets derived from the Student-Newman-Keuls Procedure at P < 0.05.  Table 11 Phase 2 - Agassiz Data Developing Corpus Luteum Stage  Cycle Type  Number of Observations  (ng/ml) Mean Progesterone ± St. Dev.  Mean No. of Days ± St. Dev.  1.  Short F i r s t Cycle  65  6.55 + 1.92  3.72 + 2.39  2.  Normal F i r s t Cycle  23  7.96 + 1.99  2.78 + 1.16  3.  Normal»Cycle Not Type 2 Not Bred  154  7.28 + 1.97  2.96  +  1.52  4.  Pregnant Cycle  77  7.00 + 1.89  2.65  +  .99  5.  Early Embryonic Abortion  4  8.65 + 1.29  2.00 + .00  6.  Atypical Cycle Not Bred  75  6.31 + 1.87  3.52 + 2.61  7.  Normal Cycle Bred not Conceived  89  7.06 + 1.91  2.88 + 1.28  8.  Atypical Cycle With Breeding  48  6.84 + 1.85  3.39  +  2.29  535  6.98 + 1.95  3.09  +  1.83  Total  Table 12 Phase 3 - Agassiz Data Luteal Stage  Cycle Type  Number of Observations  (ng/ml) Mean Progesterone ± St. Dev.  1.  Short F i r s t Cycle  55  24.64 + 8.55ab  6.25 + 3.63a  2.  Normal F i r s t Cycle  26  29.38 + 8.84bc  13.54 + 2.79a  3.  Normal Cycle Not Type 2 Not Bred  164  32.05 + 8.69c  12.32 + 2.95a  4.  Pregnant Cycle  96  37.57 + 7 . l i d  50.86 + 39.69b  5.  Early Embryonic Abortion  4  43.15 + 3.32d  37.00 + ll.Olab  6.  Atypical Cycle Not Bred  133  23.25 + 8.93a  9.22 + 12.42a  7.  Normal Cycle Bred not Conceived  107  30.13 + 8.28c  12.74 + 2.97a  8.  Atypical Cycle With Breeding  25.67 + 10.21ab  19.56 + 38.47a  29.42 ± 9.87  17.87 ± 24.96  Total  70  655  Mean No. of Days ± St. Dev.  a,b,c,d Denote Homogeneous subsets derived from the Student-Newman-Keuls Procedure at P < 0.05.  Table 13 Phase 4 - Agassiz Data Regressing Corpus Luteum Stage  Cycle Type  Number of Observations  (ng/ml) Mean Progesterone ± St. Dev.  Mean No. of Days ± St. Dev.  26  7.30 ± 2.21  2.61 ± 1.47a .  Normal F i r s t Cycle  6  6.70 ± 1.38  4.00 ± 4.00a  3.  Normal Cycle Not Type 2 Not Bred  45  7.28 ± 2.49  2.04 ± .29a  4.  Pregnant Cycle  1  8.50  51.00b  5.  Early Embryonic Abortion  1  4.10  2.00a  6.  Atypical Cycle Not Bred  77  8.75 ± 2.21  3.67 ± 10.17a  7.  Normal Cycle Bred not Conceived  35  6.75 ± 2.15  2.26 ± 1.01a  8.  Atypical Cycle With Breeding  47  9.55 ± 2.50  3.00 ± 3.25a  238  8.11 ± 2.51  3.11 ± 6.79  1.  Short F i r s t Cycle  2.  Total  a,b Denote Homogeneous subsets derived from the Student-Newman-Keuls Procedure at P < 0.05.  - 81 -  Robertson (1972) using plasma progesterone assumed that some of the early post-partum progesterone peaks could have been associated with f o l l i c l e l u t e i n i z a t i o n rather than with true ovulation. Caudle et a l . (1982) found that nine of 21 cows i n their study returned to ovarian function with short elevations i n milk progesterone.  Unable to determine i f the source of progesterone was  corpus luteum or luteinized f o l l i c l e s ,  they postulated the control  mechanism for progesterone secretion operated d i f f e r e n t l y during the puerperal period i f a d i f f e r e n t c e l l source i s responsible for progesterone secretion. Examples of these two types of f i r s t cycles can be found i n Figs. 2 and 3.  Figure 2, cow 71008 shows a dramatic i n i t i a l r i s e of  progesterone with the cycle l a s t i n g a normal 21 days. c l a s s i f i e d as Type 2.  This would be  In contrast, F i g . 3, the graph of the  progesterone cycle for cow 73025 i l l u s t r a t e s the short f i r s t cycle Type 1 which may be associated with f o l l i c l e l u t e i n i z a t i o n rather than true ovulation, and may i n fact act as an alternate starter mechanism as the following cycle from day 57 to day 79 appears to be a normal cycle.  A  successful breeding i s noted on day 78 post-partum for this animal and i s i l l u s t r a t e d by a r i s i n g progesterone l e v e l . Examining Table 6, Phase 1, the f o l l i c u l a r stage of the UBC data we find that 18 of 38 cows or 47% returned to ovarian function with a short f i r s t cycle Type 1.  Table 10, Phase 1 of the Agassiz data shows  58 of 84 cows or 69% returned to ovarian function with a Type 1 cycle. Following these f i r s t cycles through the remaining phases of the cycle we find that during Phase 2, the developing corpus luteum stage,  - 82 -  there were only 26 f i r s t cycles c l a s s i f i e d i n the UBC data as shown i n Table 7.  Of these 17 cycles or 65% were c l a s s i f i e d as being Type 1.  Table 11 Phase 2 of the Agassiz data shows 88 f i r s t classifications.  cycle  Of these 65 cycles or close to 74% were i d e n t i f i e d as  being the short f i r s t cycle Type 1 v a r i e t y . Thirty-one f i r s t cycles c l a s s i f i c a t i o n s were i d e n t i f i e d i n Phase 3, the l u t e a l stage from the UBC data as shown i n Table 8. cycles or 35% were i d e n t i f i e d as Type 1.  Of these 11  Table 12 Phase 3 of the  Agassiz data showed 81 f i r s t cycle c l a s s i f i c a t i o n s of which 55 cycles or 68% were c l a s s i f i e d as Type 1. For Phase 4, the regressing corpus luteum stage only one of nine f i r s t cycles or 11% f e l l into the c l a s s i f i c a t i o n for short f i r s t Type 1 i n the UBC data as shown i n Table 9.  cycle  Twenty-six of 32 f i r s t  cycles or 81% of the f i r s t cycles found i n the Agassiz data to be i n Phase 4 were i d e n t i f i e d as being Type 1 as shown i n Table 13. It appears as though the reduced frequency of sampling i n the  UBC  data has led to more of the f i r s t cycles being classed as Type 2 normal f i r s t cycles rather than as Type 1 short f i r s t cycles compared to the results of the Agassiz data.  The results of the Agassiz data show that  the number of animals which return to ovarian function with a short f i r s t cycle out number those that return with a normal length f i r s t cycle. Another trend noted i n the comparison of the results of the UBC sampling regime with that of the Agassiz sampling procedure was that days i n Phase 1 for the Agassiz data was approximately one half that  - 83 -  found f o r the UBC data, with the values being 2.88 ± 1.38 days, n = 58; 3.08 ± 1.16 days, n = 26 vs 5.83 ± 2.79 days, n « 18; 6.80 ± 2.80 days, n = 20 for Agassiz Type 1 and Type 2 vs UBC Type 1 and Type 2 respectively. The length of time i n Phase 2 was also shorter i n the Agassiz data f o r these cycle types.  This trend was not evident during Phase 3  when the number of days i n Phase 3 f o r the Type 2 cycles was less i n the UBC data compared to the Agassiz data. for  Length of time spent i n Phase 4  these cycle types were very similar for both data sets. There were no s i g n i f i c a n t differences noted between cycle Types 1  and cycle Type 2 for days i n phases for both the UBC data and the Agassiz data.  This was also the case for mean progesterone  concentration i n the phase except that during the early stages of progesterone production, Phase 2, the developing corpus luteum stage, the short cycle type tended to reach a lower l e v e l i n both the Agassiz data and the UBC data.  During Phase 3, the l u t e a l stage, the same trend  was noted with the addition that the short f i r s t cycle tended to occupy this period for fewer days.  In fact, days i n Phase 3 was shortest f o r  Type 1 cycles when compared to a l l other types.  I t was expected  from  the c r i t e r i a of selection that Type 1 short f i r s t cycles would be the shortest.  I t i s interesting to note that i t occurs i n the active l u t e a l  stage of the cycle or Phase 3. This tendency of reduced progesterone concentration and reduced days i n the l u t e a l stage tend to further emphasize the postulation by Caudle and co-workers i n 1982, that the control mechanism f o r progesterone secretion operated d i f f e r e n t l y during the puerperal period  - 84 -  i f a d i f f e r e n t c e l l source i s responsible for progesterone secretion. Perhaps the normal f i r s t cycle, Type 2 source i s the corpus luteum, while the short f i r s t cycle, Type 1 source i s luteinized  follicles.  Only a t o t a l of nine cases were i d e n t i f i e d as Phase 4 f i r s t cycles compared to 38 cases of f i r s t cycle Phase 1, 26 cases of f i r s t cycle Phase 2 and 31 cases of f i r s t cycle Phase 3 i n the UBC data. The Agassiz data with every other day sampling showed only a few more Phase 4 first  cycles, with a t o t a l of 32 cases, compared to 84 cases of f i r s t  cycle Phase 1, 88 cases of f i r s t cycle Phase 2 and 81 cases of f i r s t cycle Phase 3. When p r o f i l e s of the animals are examined, we find that frequently there i s a drastic drop i n the progesterone concentration f o r both the sampling regimes, with many samples going d i r e c t l y from a very high l e v e l to a very low l e v e l with no intermediate concentration noted. In a l l , only a t o t a l of 68 Phase 4 observations were made compared to 234 Phase 1 observations In the UBC data.  With the more  frequent sampling of the Agassiz data, s t i l l only 238 Phase 4 observations were made compared to 602 Phase 1 observations. It  i s apparent therefore that the regressing corpus luteum stage  or Phase 4 of the reproductive cycle as defined i n the Materials and Methods i s very precipitous.  Less than half of the cycles examined i n  either the every second day sampling of the Agassiz data or the twice weekly sampling of the UBC data showed samples with concentrations of progesterone i n the range of 12 ng/ml declining to 4 ng/ml. Perhaps a more frequent sampling regime during this phase of the cycle i s required to more accurately determine the beginning of the  - 85 -  regression of the corpus luteum and the maturation of the developing follicle. One would expect that there would be l i t t l e difference noted between cycle Type 2 the normal f i r s t cycle, and Type 3 the normal cycle not bred, as the c r i t e r i a f o r their selection differed only i n that cycle Type 2 was a f i r s t cycle noted.  This was the case with both the  UBC data and the Agassiz data with the two cycle types showing similar means f o r a l l phases of the cycle. Normal cycles with a breeding not resulting i n a conception, Type 7, were compared with cycles with a successful breeding, Type 4.  Phase  1, representing ovulation was similar for both cycle types i n the UBC data as well as the Agassiz data.  There was no s i g n i f i c a n t difference  i n the r i s i n g phase of progesterone production representing the formation of the corpus luteum between the two cycles types i n the two data bases. A s i g n i f i c a n t difference however was found i n Phase 3, representing the f u l l y functional corpus luteum stage.  In both the UBC  data, and the Agassiz data, cycle Type 4 representing pregnancy  reached  a greater concentration of progesterone, and maintained i t longer. Again, this would be expected from the c r i t e r i a of selection, as the requirement f o r cycle Type 4 stated that the concentration of progesterone must remain greater than 12 ng/ml f o r a minimum of 25 days, while the requirements f o r cycle Type 7 were similar to the normal cycle but also include a breeding not leading to conception. Cycles leading to conception, Type 4, were compared to those  - 86 -  cycles leading to early embryonic  abortion, Type 5.  These cycle types  were similar i n c r i t e r i a for selection except that after a minimum of 25 days i n Phase 3 the l u t e a l stage, Type 5 cycles would subsequently show a drop i n progesterone to less than 4 ng/ml. As would be expected, there were few observations i n the Type 5 cycle category representing early embryonic  abortion.  In the UBC data  the three observations noted for cycle Type 5, Phase 3 reached similar concentration of progesterone as Type 4 cycles, but did maintain that l e v e l for a s i g n i f i c a n t l y shorter time.  This difference was not noted  i n the Agassiz data because of the e a r l i e r cut-off date of sampling post-conception.  The Agassiz data tended to cut-off closer to 60 days  post-conception, while i n many cases the UBC cows were sampled longer a f t e r conception. Cycle Types 6 and 8 were used to c l a s s i f y cycles not f i t t i n g  into  the c r i t e r i a of the normal cycles such as Type 2, Type 3, Type 7; the c r i t e r i a of the cycles leading to conception and holding pregnancy, Type 4, and the cycles leading to early embryonic abortion, Type 5, and of course, the c r i t e r i a of the f i r s t short cycle Type 1. Cycle types 6 and 8 were, therefore, the catch a l l for those a t y p i c a l cycles representing reproductive problems, such as cysts of either the corpus luteum or the f o l l i c u l e .  The d i s t i n c t i o n between them  was to i d e n t i f y those atypical cycles which also included a breeding, as represented by Type 8. Phase 3, the f u l l y active l u t e a l stage of the reproductive cycle, showed the greatest number of observations i n both the UBC and the  - 87 -  Agassiz data.  Of a t o t a l of 655 Phase 3 observations i n the Agassiz  data, approximately 31%, or 203 observations were c l a s s i f i e d as Type 6 or Type 8, with 34.48% of the atypical cycles containing a breeding, Type 8.  Of the 244 Phase 3 observations i n the UBC data, 28.28% or 69  observations were c l a s s i f i e d as Type 6 or Type 8.  Of these only 15.94%  contained a breeding. When normal cycles without a breeding, Type 3, were compared to the atypical cycles without a breeding, Type 6, the mean progesterone concentration levels were quite similar for a l l phases except during the l u t e a l stage, Phase 3 of the Agassiz data In which the atypical cycles without a breeding reached a s i g n i f i c a n t l y lower concentration.  The  mean number of days i n the various phases were not s i g n i f i c a n t l y d i f f e r e n t between these two types of cycles. Comparing cycle Type 6 and Type 8, a s i g n i f i c a n t difference was found i n Phase 1 i n the Agassiz data.  Cycle Type 8, the atypical cycles  containing a breeding, remained i n Phase 1 s i g n i f i c a n t l y longer than did cycle Type 6, the remaining atypical cycles.  This would be  representative of those cycles containing a f o l l i c u l a r cyst, which would prevent development of a corpus luteum, and therefore maintain a low progesterone concentration over a longer period of time.  These animals  probably exhibited signs of estrus more frequently than those of Type 6, and therefore would lead to their being bred.  The delay i n ovulation,  or the development of a f o l l i c u l a r cyst could not have been predicted by the herdsman during insemination, and therefore the number of animals i n t h i s category of atypical cycles with breeding i s i n no way a r e f l e c t i o n of the competance of the herdsman or the inseminator.  - 88 -  This difference was not s i g n i f i c a n t i n the UBC data.  There were  no s i g n i f i c a n t differences noted between the two types, Type 6 and Type 8, i n the remaining phases of the Agassiz data, or i n the UBC When the UBC data was  data.  examined the comparison of cycle Type 6,  the remaining atypical cycles with cycle Type 7, the normal cycles with breeding showed that the atypical cycles remained s i g n i f i c a n t l y longer i n the f o l l i c u l a r stage of the cycle.  This difference was  not  s i g n i f i c a n t when the comparison was made between Type 7, the normal cycles with breeding and Type 8, the atypical cycles with breeding, however, the mean number of days in phase 1 was less for Type 7. In the Agassiz data, however this difference was  significant  between Type 7 and Type 8, with Type 7 remaining i n Phase 1, the f o l l i c u l a r stage a s i g n i f i c a n t l y shorter time, while there were no s i g n i f i c a n t differences between Type 6 and Type 7. The c r i t e r i a selected for c l a s s i f y i n g the reproductive cycle into phases on the basis of progesterone concentration, and the c l a s s i f i c a t i o n of cycle types, does provide a means of handling the large amounts of data which can be e a s i l y obtained from milk samples. A modification of the c r i t e r i a for selection of Phase 4, the regressing corpus luteum stage, or a modification of sampling regime or both, may  provide a better picture of what i s happening during this  stage of the reproductive cycle. Since manual examinations  per rectum are very subjective and are  known to affect the a c t i v i t y of the reproductive tract (Bulman and Lamming 1977), the radioimmunoassay of progesterone from sequentially  - 89  -  sampled dairy cows may be used to monitor the f e r t i l i t y of a dairy herd.  3.  Standard Curves Using the cycle c l a s s i f i c a t i o n s described i n Materials and  Methods, cycles c l a s s i f i e d as normal, i . e . , with the sample concentration of progesterone r i s i n g from less than 4 ng per ml milk to greater than 12 ng per ml milk and subsequently declining to less than 4 ng per ml milk i n 17 to 25 days, were grouped according to cycle length, with the f i r s t sample designated as day one.  Means, standard deviations  and the number of observations for each sampling date calculated for each group independently are given In Tables 14 to 21.  P r o f i l e s of mean  progesterone concentration and standard deviations vs. time for each cycle length group are given i n F i g . 4 to 7. Standard deviations for mean progesterone concentration on day one of the cycles, around ovulation were generally smaller than those for  sampling dates l a t e r when the concentration of progesterone was  greater than 5 ng/ml milk, representing l u t e a l a c t i v i t y . There was a great deal of v a r i a t i o n In progesterone concentration during the l u t e a l stage of the "normal" reproductive cycle.  This  increased v a r i a t i o n at higher progesterone concentrations was due mainly to the method of measurement as mentioned  i n the Literature Review.  As noted e a r l i e r i n the discussion of the c l a s s i f i c a t i o n of the cycle into the four phases and subsequently Into various cycle types, here once again we find evidence that with the sampling regimes used there was a lack of sampling during the fourth phase during the stage of  -  90  -  Table 14 UBC "Normal" 15 Day Cycles  Sample Day  Mean Progesterone Concentration ng/ml  Standard Deviation  Number of Observations  1  2.57  0.61  14  4  10.88  10.99  6  5  14.75  8.77  8  8  24.65  16.03  14  11  29.68  14.81  6  12  28.41  10.75  8  15  24.66  13.68  14  -• 9 1 -  Table 15 UBC "Normal" 18 Day Cycles  Sample Day  Mean Progesterone Concentration ng/ml  Standard Deviation  Number of Observations  1  2.41  0.65  27  4  6.27  5.95  27  8  25.13  16.97  26  11  28.29  14.31  26  15  32.36  14.01  25  18  28.70  14.49  27  - 92 -  Table 16 UBC "Normal" 19 Day Cycles  Sample Day  Mean Progesterone Concentration ng/ml  Standard Deviation  Number of Observations  1  2.51  0.43  16  5  5.88  4.80  16  8  20.52  15.71  16  12  35.22  11.10  15  15  33.77  13.79  15  19  26.98  11.07  16  - 93 -  Table 17 UBC "Normal" 22 Day Cycles  Sample Day  Mean Progesterone Concentration ng/ml  Standard Deviation  Number of Observations  1  2.74  0.83  31  4  2.75  0.72  13  5  4.14  2.66  16  8  14.40  11.52  29  11  24.14  14.57  14  12  28.12  14.53  15  15  38.10  12.29  29  18  30.71  13.04  14  19  38.43  10.85  14  22  24.89  14.52  30  - 94 -  Table 18 Agassiz "Normal" 17 Day Cycles  Sample Day  Mean Progesterone Concentration ng/ml  Standard Deviation  Number of Observations  1  2.26  0.63  27  3  4.43  7.35  25  5  9.74  6.05  26  7  16.17  11.13  26  9  24.65  13.61  26  11  29.22  14.88  26  13  31.00  14.71  25  15  32.55  12.54  26  17  28.87  14.83  26  - 95 -  Table 19 Agassiz "Normal" 19 Day Cycles  Sample Day  Mean Progesterone Concentration ng/ml  Standard Deviation  Number of Observations  1  2.82  3.62  74  3  2.67  1.97  71  5  6.14  6.52  69  7  14.18  10.98  71  9  22.53  12.35  68  11  29.77  12.81  71  13  34.86  13.42  68  15  37.76  12.26  71  17  38.49  12.19  70  19  29.14  15.75  71  - 96 -  Table 20 Agassiz "Normal" 21 Day Cycles  Sample Day  Mean Progesterone Concentration ng/ml  Standard Deviation  Number of Observations  1  2.48  0.77  95  3  2.17  1.09  93  5  3.55  3.31  93  7  8.23  7.17  92  9  18.09  12.38  92  11  26.99  13.70  94  13  29.93  13.50  92  15  33.42  12.69  92  17  34.92  12.66  93  19  35.42  12.29  92  21  21.37  13.98  93  - 97 -  Table 21 Agassiz "Normal" 23 Day Cycles  Sample Day  Mean Progesterone Concentration ng/ml  Standard Deviation  Number of Observations  1  2.75  0.67  43  3  2.18  0.55  42  5  3.09  1.96  43  7  7.01  5.22  43  9  15.14  8.74  42  11  24.60  10.31  42  13  30.70  12.45  43  15  33.02  12.89  43  17  36.19  12.55  42  19  35.86  12.59  42  21  33.79  12.82  42  23  18.07  13.87  43  DAYS  DAYS  DAYS  -  1 0 1  im/8u 3N0>J3IS300tfd  -  - 102 -  the regressing corpus luteum and developing f o l l i c l e when the progesterone concentration should be f a l l i n g from a value of 12 ng/ml milk or greater to a concentration of progesterone of around 4 ng/ml milk. This f a l l from a high concentration, representing l u t e a l a c t i v i t y to a very low value indicative of ovulation therefore must be very precipitous. With the c l a s s i f i c a t i o n of the cycles as defined, we find that i n those cycles of longer duration, such as the UBC  "Normal" 22 day cycles,  F i g . 5, and the Agassiz "Normal" 23 day cycles, F i g . 7, the p r o f i l e of the "Normal" cycle does not necessarily begin with day one representing the nadir of progesterone concentration and consequently  ovulation.  When the data for normal cycles of various lengths are combined for each data base as i n F i g . 8 and 9,  we find that the p r o f i l e s are  quite s i m i l a r , with graphs of increasing cycle length being shifted s l i g h t l y to the r i g h t . Much of the increase in cycle length may Increased  be attributed to  time spent at a low concentration of progesterone.  of this period may  Duration  be correlated to cycle length and serve as a method  of predicting cycle length. It i s known that ovulation occurs during the nadir of progesterone production however, pinpointing ovulation by progesterone analysis seems u n l i k e l y . Timing of ovulation during this stage of low progesterone concentration may  provide some answers to the repeat  breeder cow problem often observed.  It i s possible these repeat breeder  DAYS  DAYS  -  105  -  cows remain i n phase one longer than those cows which conceive readily making timing of insemination d i f f i c u l t .  This stage of the reproductive  cycle deserves further examination. When the various p r o f i l e s for the "normal" reproductive cycle were superimposed  one upon another i t was found that the slopes for the  r i s e i n progesterone concentration from approximately 4 ng/ml milk to the peak did not d i f f e r markedly between any of the cycle lengths nor between the UBC data and the Agassiz data. Longer cycles did tend to remain longer at a greater concentration of progesterone.  4.  Progesterone P r o f i l e s Many graphs of the cycles were produced to f a c i l i t a t e monitoring  the reproductive status of the animals being sampled.  As soon as  possible the results of the radioimmunoassay were forwarded to both farms, so that the herdsman would have a better understanding of the status of the animals, and could more accurately predict upcoming estrus periods. As has been noted, some animals not having been observed i n estrus were mated on the basis of previously predicted ovulations and cycle lengths. Many of these animals conceived, unfortunately accurate records of this aspect were not maintained and therefore proof cannot be offered. A great variety of types of cycles were noted and examples of these follow.  -  106  -  Animals exhibiting a delayed start to ovarian cycles such as cow 73025 F i g . 3 are easily missed as most dairymen do not concern themselves with the reproduction of the dairy cow u n t i l approximately 50 to 60 days post-partum when they feel that the animal should be ready to breed to maintain a yearly calving i n t e r v a l .  They may be included  with any cows to be palpated at this time i f records of early detected heats are kept and i t i s noted that they have not had a heat recorded, or i f some other abnormality i s noted.  Palpation of the ovaries i s an  extremely d e l i c a t e and somewhat subjective technique. of  Through the use  the progesterone p r o f i l e s we can i d e n t i f y these problem animals and  i n conjunction with palpation, treatment can be undertaken to shorten the period of ovarian i n a c t i v i t y . Short f i r s t cycles are noted i n the graphs for cows 72007, F i g . 10; 73017, F i g . 11; 73021, F i g . 12, and i s speculated from the graph f o r 74004, F i g . 13.  The short f i r s t cycle shown i n F i g . 11; 73017 may  be  due to treatment stimulating the reproductive t r a c t . The graph for cow 71008, F i g . 2, i l l u s t r a t e s a normal f i r s t and second cycle followed by a successful breeding.  cycle  This animal shows  the i d e a l reproductive performance with a calving i n t e r v a l well under 365 days. The graph for cow 72007, F i g . 10, shows a short f i r s t  cycle  followed by the development of a corpus luteum cyst on the right ovary. This graph also shows the immediate response to treatment of prostaglandin with a return to cycling behaviour. Treatment  for reproductive problems i s not always successful.  This i s i l l u s t r a t e d by the graph for cow 73021, F i g . 12.  This graph  - 107 -  shows a normal cycle followed by the growth of f o l l i c u l a r cysts on both the right and l e f t ovaries.  These did not respond to manual expression.  Estrus was detected two days after treatment with ECP® ( e s t r a d i o l cypionate) and was associated with an unsuccessful breeding.  This was  followed 40 days l a t e r with an i n e f f e c t i v e treatment of prostaglandin following palpation which detected an inactive l e f t ovary and a c y s t i c right ovary.  This animal continued  was subsequently  to exhibit signs of nymphomania and  slaughtered.  The graph of cows 74004 F i g . 13 and 73017, F i g . 11 show possible early embryonic abortion.  Cow 74004 F i g . 13 i l l u s t r a t e s a breeding  followed by high progesterone values for a period of about 40 days followed by a precipitous drop and subsequent estrus. Fig.  Cow 73017,  11, shows high values following breeding for 30 days and a  subsequent drop i n value. A short quiescence  i s i l l u s t r a t e d i n graphs for cows 71008, F i g .  2; 72007, F i g . 10; and 74016, F i g . 14.  Abnormally long cycles are  i l l u s t r a t e d i n the graph of cow 74016, F i g . 14.  This animal exhibits an  abnormally long cycle length of 28 days i l l u s t r a t i n g the v a r i a b i l i t y between animals.  5.  Pregnancy Diagnosis Keeping i n mind the constraints of the analysis as stated by Heap  et a l . (1973) and Heap et a l . (1976) a progesterone concentration i n d i c a t i v e of the l u t e a l stage or Phase 3 of the reproductive cycle, 12 ng/ml milk, was used as a cut-off value for the pregnancy test.  Fig. 11 Cow No. 73017  Bred Day 147 Vet Check Pos. Preg. Day 219 or Day 72 Post AI  10  15  20  25  30  35  40  45  50  55  60  65  70  75  DAYS POSTPARTUM  80  85  90  95  100  105 110  115  120  125  Cow No. 73021  Became Nymphomaniac and was Slaughtered  (cont.)  C y s t i c Right Inactive Left Treated 5 cc Lutaly.se Estrus and Al  Fig. 12 Cow No. 73021  1  145 ~ 150 155~lto" 1*65 "?70" 180 185 190 DAYS POSTPARTUM  25 2015  C y s t i c Ovaries I Palpated and [Express Left 'and Right  Ketosis  ia  10  15  20  25  3D  —I35  i  40  45  50  >  55  60  65  70  75  DAYS POSTPARTUM  80  85  90  95  100  ll 0 l  115  120  125  Fig. C o w  Progesterone L e v e l s Remained E l e v a t e d Vet Check Pos. Preg. Day 48 Post AI  13 N o .  7 4 0 0 4  DAYS POSTPARTUM  - 113 -  Those values above 12.0 ng progesterone per ml milk s i g n i f i e d a pregnancy while those below were diagnosed open.  5.1  UBC Data The results of the progesterone pregnancy  test for UBC data are  given i n Table 22. There was a t o t a l of 28 day 21 values from the UBC data of which 64.3% or 18 animals were diagnosed pregnant.  Of these, three animals  were i n c o r r e c t l y diagnosed pregnant, for a success rate of 83.4% for positive diagnosis. Of the 10 animals diagnosed open, a l l were correct. If the value at breeding i s considered, two of the three cases diagnosed i n c o r r e c t l y positive would have been rejected as having been bred at the wrong time.  With a two sample test, that i s a sample on day  of insemination as well as a sample on day 21, the success rate for positive diagnosis rises to 93.8%.  Also, one of the cases diagnosed  open had a value at breeding of 11.5 ng progesterone per ml which would have served as an early warning of an i n c o r r e c t l y timed breeding. For those tested for pregnancy with a sample collected at 22 days post-breeding, the test proved once again 100% correct i n predicting six cases open. 83.4%  of 18 cases.  The pregnant diagnosis was again correct i n  Incorrect timing of Insemination would have  eliminated one of the three incorrectly diagnosed pregnant cases giving a two sample test success rate of 88.2% for positive diagnosis. There were 13 values for day 23, nine of which were correctly diagnosed pregnant and four were correctly diagnosed open.  The day 24  Table 22 Pregnancy Test Results (UBC Data)  Positive Correct  Day 21  Negative Correct  Positive Incorrect  Value at Breeding  Value at Test Day  Value at Breeding  Value at Test Day  Value at Breeding  (15) 2.76 ± .69  (15) 43.06 ± 8.40  (10) 3.79 ± 2.68  (10) 3.57 ± 1.33  (3) 16.96 ± 17.74  (3) 37.66 ± 8.81  (6) 3.06 ± 1.35  (3) 15.43 ± 17.37  (3) 26.33 ± 6.85  (1) 2.6  (1) 22.0  +  Value at Test Day  (9)* 2.93 ± 0.80 Day 22  (15) 2.56 ± .61  (15) 39.67 ± 10.30  (6) 10.36 ± 17.63  x  (5)* 2.44 ± .67 Day 23  (9) 2.81 ± .76  (9) 40.10 ± 14.04  Day 24  (4) 3.37 ± 1.78  (4) 49.25 ± 1.29  Note:  (4) 2.35 ± .56 (1) 4.5  (4) 2.35 ± .49 (1) 2.1  Number of observation in brackets ( ). Value given i s mean ng progesterone/ml post milk stripping ± S.D. *0ne sample with a high value omitted +0ne sample value 42.0 ng/ml included, x One sample value 40.0 ng/ml included.  - 115 -  test proved 80% accurate i n diagnosing correctly four of f i v e cases pregnant  and one case was  correctly diagnosed open.  Combining a l l the values for the UBC data we find that 50 cases were diagnosed pregnant with an accuracy of 86.0%. increases this accuracy to 91.5%.  A two sample test  Although there were a t o t a l of seven  incorrect positive diagnosis of which only three can be explained by improper  timing of insemination, further examination of the UBC  data  shows that three animals also f i t the c r i t e r i a set down for cycle Type 5 i n d i c a t i v e of early embryonic abortion.  With the c r i t e r i a for cycle  Type 5 requiring the concentration to remain greater than 12 ng progesterone per ml. milk, the remaining incorrect positive diagnosis would have remained high longer than normal yet not long enough to f i t into the Type 5 category.  5.2  Agassiz Data The results of the progesterone pregnancy test for the Agassiz  data are given i n Table 23. As the Agassiz herd was  sampled every other day, pregnancy  testing was done for days 21 and 22 post-insemination.  From  75, day 21 values, 70.7% or 53 cases were diagnosed pregnant with a success rate of 90.56%.  There was an accuracy of 100% i n the diagnosis  of 22 cases open. From 74 day 22 values, 70.3% or 52 cases were diagnosed with a success rate of 90.38%. accurate i n 22 cases.  pregnant  The diagnosis of open again proved  100%  Table 23 Pregnancy Test Results (Agassiz Data)  Positive Correct Value at Test Day  Value at Breeding Day 21  (48) 1.95 ± .52  (48) 38.97 ± 10.85  Negative Correct  Positive Incorrect  Value at Breeding  Value at Test Day  Value at Breeding  Value at Test Day  (22) 2.14 ± 1.11  (22) 2.98 ± 1.89  (5) 2.66 ± 1.24  29.76 ± 14.37  (5)  (21)* 1.92 ± 0.5 Day 22  Note:  (47) 2.29 ± .59  (47) 35.87 ± 10.95  (22) 2.51 ± 1.46 (20) 2.08 ± 0.59  (22) 2.95 ± 2.08 (21) 2.55 ± 1.03**  Number of observation in brackets ( ). Value given i s mean ng progesterone /ml post milk strippings. *0ne high value omitted. **0ne sample near the cutoff value of 12.0 ng/ml omitted.  (5) 13.7 ± 14.48  (5) 31.4 ± 10.27  - 117 -  Of the t o t a l 10 cases of incorrect pregnant diagnosis i n the Agassiz herd, 30% or three animals were bred at an obviously incorrect time.  With a two sample test and the removal of these values, the  accuracy of the pregnant diagnosis for the combined day 21 and day 22 values rises to 93.1%.  Further examination of the Agassiz data shows  that four animals also f i t the c r i t e r i a set down for cycle Type 5, i n d i c a t i v e of early embryonic  5.3  Pregnancy  Diagnosis  abortion.  Discussion  These results closely correspond to those found by Booth and Holdsworth (1976) who reported on a commercial service with non-pregnant  test results close to 100% and an error rate of  approximately 10% i n pregnant test results due mainly they f e l t to early f e t a l death, to those reported by Eastman (1979) who  reported an  accuracy rate for pregnancy of 83.3% for 30 cows, and to those reported by Foote et a l . (1979) who non-pregnant  reported an accuracy rate of 98% for 126  animals and 80% for 180 predicted p o s i t i v e .  Bulman and Lamming (1977) through the use of sequential sampling of the progesterone concentration i n milk, showed that prolonged l u t e a l a c t i v i t y could occur i n the cow i n the absence of any apparent abnormality. unaccounted  clinical  It i s , therefore, possible that some of the remaining for false pregnancies could be attributed to these animals  experiencing persistent l u t e a l a c t i v i t y without being pregnant. The radioimmunoassay of progesterone i n milk provides a very r e l i a b l e means of detecting non-pregnancy and a reasonably accurate  - 118 -  means of detecting pregnancy with a single sample taken on day 21 to day 24 post-insemination. The addition of a sample on day of breeding increases the accuracy of the pregnant diagnosis to greater than 90%. This could provide the producer the a b i l i t y to rebreed the non-pregnant cow at an e a r l i e r date reducing the cost of production associated with increased days open.  Those animals i n c o r r e c t l y diagnosed pregnant as  well as those animals correctly diagnosed pregnant which subsequently experienced early embryonic  abortion may be found i f a r e c t a l palpation  for pregnancy i s performed at approximately 60 days post-insemination.  6.  Estrus Detection And Reproductive Performance Combining both UBC and Agassiz data for an assessment  of estrus  detection and assigning a value of less than 5 ng progesterone per ml post-milk strippings as a correct value for estrus, from a t o t a l of 220 cases only nine cases or 4.1% were i n c o r r e c t l y detected i n estrus. McCaughey and Cooper (1980), studied 1177 milk samples taken from cows being inseminated and found 91 animals or 7.7% had levels of milk progesterone i n excess of those observed at normal estrus. Separating the two herds we find near average accuracy i n heat detection with the UBC data showing s i x incorrect timing out of 71 cases for 8.5% incorrect timing of insemination.  It must be noted  that one of the incorrect timing of insemination cases did result i n a pregnancy.  This lowers the true incorrect timing of insemination to  f i v e from 71 cases, or 7.0%. The Agassiz data shows remarkable accuracy i n timing of insemination with only three of 149 cases or 2.0% being performed at a  - 119 -  time when the milk sample concentration of progesterone was greater than 5 ng/ml. The number of days from p a r t u r i t i o n u n t i l the f i r s t detected estrus, or i n the case of an animal with no early estruses noted, u n t i l the f i r s t breeding, was compared to the number of days from p a r t u r i t i o n u n t i l the f i r s t progesterone predicted estrus.  Values are l i s t e d i n  Table 6. Days to f i r s t progesterone detected estrus was determined as the number of days from p a r t u r i t i o n u n t i l a r i s e i n progesterone concentration of greater than 5 ng/ml milk and back to less than 4 ng/ml milk. The mean, standard deviation, and range, i n days post-partum f o r f i r s t v i s u a l l y detected estrus for 42 animals i n the UBC herd was 49.79 ± 21.67  days, with a range of 90 days.  This compares to 41.0 ±  15.14  days, and a range of 73 days, for f i r s t progesterone predicted estrus for 40 animals.  There are less animals i n the progesterone predicted  estrus group due to the fact that some animals lacked early sampling. A greater difference was noted i n the Agassiz data when comparing these t e s t s .  The mean, standard deviation, and range, i n days post-  partum for f i r s t v i s u a l l y detected estrus for 112 animals was 53.02 ± 29.14  days, with a range 175 days, compared to 34.34 ± 11.67  days with a  range of 66 days, for f i r s t progesterone predicted estrus for 101 animals. Nearly one f u l l heat, or 18.68  days for every animal was being  missed i n the Agassiz herd when these two values were compared.  Looking  - 120 -  at the range i n values, we find the p o s s i b i l i t y that one animal  was  cycling for up to (175-66) 109 days without being noticed. The mean, standard deviation, and range, i n days to f i r s t service, for 42 animals i n the UBC herd was 74.86 ± 22.21  days, with a  range of 87 days, compared to 82 ± 23.94 days, with a range of 152 days for 112 animals in the Agassiz herd. Agassiz herd may  The greater number of days for the  r e f l e c t the imposed breeding regime.  Further reproductive performance data i s given i n Table 24.  When  viewing reproductive performance results for both these herds, the fact that both herds are used for various experimental procedures must be taken into account.  For example, during the period of sampling for this  data base, the Agassiz herd was also being used for another study involving early and late breeding regimes. d r a s t i c a l l y reduced i n number.  As well the herd was  being  With respect to the UBC herd, normal  c u l l i n g and breeding times were employed i n conjunction with some feeding t r i a l s .  These factors must be considered i n judging the results  of any reproductive performance tests.  Only animals with the most  complete data available were used i n a r r i v i n g at these values. Comparing the results l i s t e d i n Table 24 with those of De Kruif (1978), as mentioned i n the l i t e r a t u r e review, we find that the mean number of days open for the UBC herd of 96.48 days comes close to De Kruif's (1978) ideal of an average i n t e r v a l of 85 days between p a r t u r i t i o n and conception.  This allows for an average calving i n t e r v a l  for the UBC data of s l i g h t l y over a year.  Four animals i n the UBC  herd  had days open ranging from 165 - 184 days giving them calving intervals  Table 24 Reproductive Performance  Agassiz  UBC  Mean Value ± Std. Dev. (Range)  No. Cases  Mean Value ± Std. Dev. (Range)  No. Cases  111.75 ± 47.83 (246)  112  96.48 ± 34.95 (136)  42  Days to F i r s t Service  82.32 ± 23.94 (152)  112  74.86 ± 22/21 (87)  42  Days to f i r s t Progesterone Detected Estrus  34.34 ± 11.67 (66)  101  41.0 ± 15.14 (73)  40  Days to F i r s t Visually Detected Estrus (or F i r s t Breeding)  53.02 ± 29.14 (175)  112  49.79 ± 21.67 (90)  42  Days from Conception to Positive Vet Check  52.01 ± 13.19 (74)  97  Indicator Days Open  60.43 ± 23.59 (96)  42  - 122 -  of nearly 15 months.  These animals would have to exhibit great  persistency i n this l a c t a t i o n or extremely high production to warrant keeping them for the predictably long dry period of 140-159 days, i f they were milked for only the regular 305 day production record. Days open for the Agassiz herd was 111.75 days. 13 months.  greater at a mean l e v e l of  This would be reflected i n a calving i n t e r v a l of close to  Again this may  r e f l e c t the restraint of a specified breeding  regime enforced upon this herd. Similar to the UBC herd 19 animals in the Agassiz herd show number of days open greater than 150 days, and even as high as 277 days.  Days open should be included i n the c r i t e r i a for c u l l i n g for the  most e f f i c i e n t and economical management strategy. Neither the UBC herd, nor the Agassiz herd, compare well with De K r u i f s (1978) ideal of 80% conception after the f i r s t insemination. Only 25 of the 42 animals i n the UBC data, to have a positive veterinary check for pregnancy, conceived to a f i r s t breeding. only 59.5%  pregnant after f i r s t service.  This represents  One additional animal  conceived to a double breeding with f i r s t and second breedings only one day apart.  Including this animal only increases the percent pregnant  after f i r s t service to 61.9%. With respect to the Agassiz herd, of the 97 animals to receive a positive veterinary check for pregnancy, only 48 animals or 49.48% conceived to f i r s t service.  If an additional seven animals who were  bred a second time within six days of f i r s t service, and conceived, are included, this brings the pregnancy rate to f i r s t service to only  - 123 -  56.70%.  These multiple services, within s i x days, may be explained by a  controlled breeding system following hormonal treatment, or possibly inaccurate i n i t i a l heat detection. Regarding  the number of services per conception, De Kruif i n 1978  stated that an i d e a l number to s t r i v e conception.  for would be 1.3 services per  In this respect, both the UBC, and the Agassiz herds,  required more services per conception but were similar with values of 1.79 services per conception on 42 conceptions and 1.88 services per conception on 97 conceptions, respectively. Both of these herds showed examples of cows being bred up to eight times, and are reflected i n those animals with extended number of days open. The mean, standard deviation, and range, i n days from conception u n t i l a positive palpation was 60.43 ± 23.59 days, with a range of 96 days f o r 42 animals i n the UBC herd.  The Agassiz herd received  confirmation of pregnancy by veterinary palpation e a r l i e r , 52.01 ± 13.91 days, with a range of 74 days.  - 124 -  SUMMARY AND CONCLUSIONS  The length of time spent i n quiescence post-partum can greatly a f f e c t reproductive parameters such as days to f i r s t estrus, which i n turn may affect days to f i r s t breeding and ultimately calving I n t e r v a l . Progesterone analysis and the interpretation of i t s concentration over time provides an effective non-invasive technique to monitor this Important aspect of f e r t i l i t y . By establishing a normal period of quiescence for a herd the use of  progesterone radioimmunoassay of sequential milk samples may provide  the researcher with a means of assessing the effects of various treatments and diets on reproductive a c t i v i t y , without the invasive technique of r e c t a l palpation which has been shown to affect the reproductive t r a c t . Because two different herds have been sampled under two d i f f e r e n t sampling regimes no d e f i n i t e conclusion can be drawn from the comparisons  of the r e s u l t s .  Differences noted between the two data sets  may be due to sampling technique or possibly to factors associated with the p a r t i c u l a r herd. For  a comparison of sampling regimes, one herd should be sampled  d a i l y and from this data set different sampling regimes can be compared. The c l a s s i f i c a t i o n of the cycle as described did not adequately cover the fourth phase of the estrus cycle when the corpus luteum regresses as a new f o l l i c l e matures, and led to the standard curves f o r the "normal" 22 and 23 day cycles not necessarily beginning at the nadir of  progesterone concentration.  - 125 -  The combination of the various "normal" cycles showed that much of the increase i n cycle length occurs during the f i r s t phase of the cycle when the concentration of progesterone was less than 4 ng/ml milk.  As the cycles lengthen out, the time spent i n phase one  increases. The slopes for the r i s e i n progesterone concentration over time for the "normal" cycles did not d i f f e r markedly between any of the cycle lengths nor between the UBC data and the Agassiz data. The progesterone p r o f i l e s produced for each of the animals provided the herdsman with a means of predicting estrus, monitoring reproductive treatments and reducing the number of animals that must be observed for estrus through the early i d e n t i f i c a t i o n of pregnant animals. Due to the number of incorrect positive pregnancy determination and the number of these which could be i d e n t i f i e d as having been bred at an incorrect time, i t would be this researchers recommendation that when progesterone radioimmunoassay i s used i n a pregnancy determination role a sample be taken at breeding to be compared with the sample taken on day 21. Examination of the p r o f i l e s of progesterone concentration vs. time of a l l the animals sampled shows that sampling twice per week was adequate  for determining abnormal cycles.  The increased number of short  f i r s t cycle c l a s s i f i c a t i o n s found i n the Agassiz data compared to the UBC data shows that accuracy i n determination of reproductive status increases with the frequency of sampling.  The limited number of  - 126 -  c l a s s i f i c a t i o n s i d e n t i f i e d i n Phase 4 suggests that d a i l y sampling at least i n this phase of the cycle may be required to accurately monitor the regressing corpus luteum  stage.  It i s therefore this researchers radioimmunoassay  opinion that the  of progesterone from milk samples collected i n a  sequential manner and the reproductive p r o f i l e s derived from them, may provide  the producer a useful means of meeting the needs of improved  reproductive  e f f i c i e n c y as stated by Melrose (1979).  - 127 BIBLIOGRAPHY Abraham, G.E., R. Swerdloff, D. Tulchinsky and W.D. Odell. 1971. Radioimmunoassay of plasma progesterone. J . C l i n . Endocr. 32: 619-24. Adler, J.H. and D. 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