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OVERDUE FINES: 25¢ per dey per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records EFFICACY OF AN EQUINE PITUITARY EXTRACT T0 SUPEROVULATE CONS By Myron Lindie Danner A DISSERTATION Submitted to Michigan State University in partial fquiTTment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of AnimaT Science 1981 “JPIMZLU ABSTRACT EFFICACY OF AN EQUINE PITUITARY EXTRACT TO SUPEROVULATE CONS By Myron Lindle Danner Three trials utilizing sixty-one animals were conducted to study the efficacy of a commercially available equine pituitary extract (trade name--Pitropin; Biological Specialties, Middletown, Wisconsin) for inducing multiple ovulations in beef cows. Variables included: dose of equine pituitary extract (EPE), addition of HCG, number of days for injections and number of injections per day. A comparison of EPE with pituitary FSH and LH of domestic animal origin (FSH-LH) was made. Estrous cycles of cows within each trial were synchronized with 2 injec- tions of prostaglandin F2o (PGFZG) given ll days apart. Gonadotropin injections began on day l2 of the cycle (O= day of estrus) followed 72 hr later by PGFZa' Multiple inseminations were performed starting l2 hr after onset of estrus. Cows were slaughtered 7 days postestrus and reproductive tracts were removed for study. EPE was a very potent stimulus for follicular growth and ovulation although variation between animals was high. The range in number of ovulations for EPE treated cows was 0 to 96. Addition of HCG to EPE treatments was contraindicated. To be effective, a total dose of 750 Fevold-Hisaw Rat Units of EPE administered over at least 3 consecu- tive days was necessary. Seventy-five percent of cows receiving this Myron Lindle Danner treatment responded with 2 5 ovulations. Once daily injections of EPE were adequate. FSH-LH decreased the number of unovulated follicles 2 10 mm (0.8 vs 3.l) and increased the number of embryos recovered (ll.2 vs 6.2) over EPE treated cows. Blood samples were taken during the gonadotropin treatment period and the relationship between serum concentration of progesterone and estradiol l7B and the resulting ovarian response was determined. Cows having 3 3 ovulations were classified as a poor ovulatory response while those having 2 5 ovulations were classified as a good ovulatory response. High progesterone level during the period from initiation of gonadotropin injection to PGan injection was associated with a good ovulatory response. No relationship between progesterone level following PGFZa injection or estradiol l7B concentration during the gonadotropin treatment and the resulting ovulatory response was observed. A positive correlation between the progesterone level on day 7 postestrus and the number of corpora lutea present was observed. ACKNOWLEDGMENTS The author is grateful to the members of his Graduate Committee: Dr. w. D. Oxender, Veterinary Medicine; Dr. R. L. Fogwell, Dairy Science; Dr. w. R. Dukelow, Dr. D. R. Hawkins, Dr. H. D. Ritchie, and Dr. R. H. Nelson, Animal Husbandry. Their guidance in planning and conducting this investigation and critique of this dissertation is deeply appreciated. Sincere appreciation is expressed to the Animal Husbandry Department, Dr. R. H. Nelson, Chairman, for providing facilities and research animals with which to work. The excellence of both facilities and cattle enhance the value of this dissertation. Appreciation is extended to the Dairy Science Department, Dr. H. D. Hafs, Chairman, for the use of facilities and equipment to complete hormone assays. Gratitude is expressed to Dr. R. D. Baker, American Embryos, Middleville, Michigan, for assistance in evaluation of embryos; to Dr. w. T. Magee and Dr. J. L. Gill for guidance with statistical analysis; to the graduate students of the Animal Husbandry and Dairy Science Departments for assistance in data collection; to the per— sonnel of the Beef Cattle Research Center for care of experimental animals; and to Mrs. Grace Rutherford for her excellent typing of this manuscript. Sincere thanks are expressed to the author's wife, Kathy, for her help and support throughout the graduate program. Special appreciation is also extended to the author's parents. TABLE OF CONTENTS Page LIST OF TABLES ......................... v LIST OF FIGURES ....................... . vii INTRODUCTION .......................... l LITERATURE REVIEW ....................... 3 Superovulation in Cows .................. 3 Nature of Gonadotropin .................. 5 Dose-Response Relationship ................ 8 Batch of Gonadotropin and Method of Administration . . . . 9 Time ofEstrousCycle to Initiate Gonadotropin Treatment . ll Interval Between Gonadotropin Treatment and Estrus . . . . l2 Population of Ovarian Follicles ............. l4 Endocrine Environment During Superovulation ....... l6 Immunological Response .................. 19 Breed Differences .................... 2l Seasonal Effects ..................... 22 Nutritional Effects ................... 23 Summary ......................... 24 MATERIALS AND METHODS ..................... 27 Experimental Animals . .................. 27 Feeding ........................ . 28 Products Used . ..................... 29 Preliminary Treatment and Allotment ........... 29 Experimental Design ................... 30 Blood Collection Procedures ............... 33 Necropsy Procedures ................... 37 Ovarian and Embryonic Data Collection .......... 37 Quantification of Progesterone .............. 39 Quantification of Estradiol-l78 ............. 4l Data Calculations and Statistical Analysis ........ 44 RESULTS . . .......................... 45 Estrus Synchronization .................. 45 Trial l ....................... . . 47 Trial 2 ......................... Trial 3 ......................... Hormonal Profiles .................... Embryo Recovery and Condition of Embryos ......... DISCUSSION ........................... SUMMARY ............................ APPENDIX ............................ LITERATURE CITED ........................ Table TO. 12. A.l A.2 A.3 A.4 A.5 A.6 LIST OF TABLES Effects of Dose Level of Porcine FSH on Ovulation, Recovery and Fertilization Rates . . . . . Progesterone Intra- and Inter-Assay Coefficients of Variation ...... . . . . . . . . . . . . . . Estradiol l7B Intra- and Inter- Assay Coefficients of Variation . . . . ...... Estrous Synchronization of Experimental Animals Ovarian Response--Trial l Ovarian Response for 3 Day EPE Treatments—-Trial 2 . Ovarian Response for Low Dose EPE--lX--3 Days vs Low Dose EPE--lX--2 Days--Trial 2 . . . . Ovarian Response for 3 Day EPE Treatments vs FSH-LH--Trial 2 . . . . . . . . . . . . Ovarian Response-—Trial 3 ..... Ovulatory Response of Experimental Animals . . Percentage of Potential Embryos Recovered Occurrence of Normal Embryos . . . Manufacturer's Description of EPE Manufacturer's Bioassay of EPE . Manufacturer's Description of HCG Manufacturer's Analysis of FSH . . . Manufacturer's Analysis of LH . . . . ..... Estrous Synchronization of Experimental Animals (Individual Data) . . . . . . . . . . . . . Page 4l 43 46 48 50 51 52 53 54 6O 6l 69 7O 71 72 73 75 Table A.7 A.8 A.9 Individual Ovarian and Embryo Data for Trial 1 ..... Individual Ovarian and Embryo Data for Trial 2 ..... Individual Ovarian and Embryo Data for Trial 3 ..... vi Page 75 77 79 Figure 1. LIST OF FIGURES The relative FSH and LH potency of the pituitary in the cow, ewe, sow, and mare .......... Experimental design for Trial 1 ...... Experimental design for Trial 2 . . . . ....... Experimental design for Trial 3 ......... Blood sampling schedule for Trials l and 2 ...... Progesterone profiles during the period from initiation of gonadotropin injection to PGFZa injection . . . ...... Progesterone profiles during the period from l2 to 48 hrs after PGFZQ injection . . . . . . . . . . Estradiol l7B concentrations during the period of gonadotropin injection with regression lines Relationship between number of corpora lutea and serum progesterone concentration on day 7 postestrus . . . . . . . . ...... . ...... Page 32 34 35 36 55 56 58 59 INTRODUCTION Early developments in embryo transfer techniques were stimulated by the increased financial gains through multiplying exotic breeds of cattle. Present interest in embryo transfer is stimulated by newly developed techniques which can be used for genetic improvement by progressive cattle breeders. Recent improvement of nonsurgical techniques for collection of embryos has decreased chances of causing sterility in genetically valuable donor cows. In combination with superovulation, nonsurgical collection techniques make it possible to obtain 5 to 25 embryos from a single collection. This procedure dramatically increases the number of offspring which can be obtained from females genetically superior for production of meat or milk. Furthermore, potential exists for the import and export of embryos rather than live animals reducing transportation costs and the risk of spreading diseases. Artificial insemination (A.I.) has made leading sires available to all breeders at a reasonable cost. Given total use of superior sires available through A.I., considerable genetic progress can be realized over use of average natural service sires. However, only 50 percent of the genotype for any calf is from the sire. This leaves 50 percent of the genotype contributed by the dam and consequently, a heretofore untapped resource for genetic improvement of livestock. Embryo transfer techniques may increase the potential for genetic improvement from superior females by decreasing the generation interval (ova can be collected from young females) and increasing the number of calves per female. Gordon (T975), in a review of embryo transfer in cattle, concluded that superovulation must be regarded as a major problem blocking progress in expanding the use of embryo transfer. Extreme variation and lack of repeatability in superovulatory responses of donor females are just two problems cited by numerous investigators. Improved methods for superovulation in donor females are necessary to increase the use of embryo transfer. The purpose of this dissertation research was to study superovulation in cows using equine pituitary gonadotropin extract (EPE). Specific objectives in this study were: l. To determine the dosage and treatment schedule for administration of EPE to give optimal superovulation response in beef cows. 2. To determine the relative efficacy of EPE and pituitary extracts of domestic animal origin to induce multiple ovulations. 3. To determine relationships between serum hormone concentrations (progesterone and estradiol l7B) throughout the gonadotropin treatment period and the resulting ovarian response. LITERATURE REVIEW Superovulation in Cows The ultimate objective of superovulation is to increase the number of normal fertile eggs or embryos per donor. The basic prin- ciple is to stimulate extensive follicular development through intramuscular or subcutaneous administration of a preparation with follicle—stimulating hormone (FSH) activity at levels in excess of normal endogenous levels. Preparations utilized with FSH activity include pregnant mares' serum gonadotropin (PMSG) and cattle, sheep, swine or horse pituitary extracts. In addition, a preparation is occasionally injected intravenously or intramuscularly to help assure ovulation. Such preparations include pituitary luteinizing hormone (LH) or human chorionic gonadotropin (HCG) In a review of superovulation, Gordon (T975) summarized data of 436 sexually mature cattle in six studies. Cows had been treated with both pituitary extracts and PMSG. He found that the average number of eggs released was 18.l i8.l. Of these, 9.8: 5.l (54%) were recovered and 4.3i:0.9 (44% of eggs recovered) were considered to be fertilized. In 1974, Graham reported the results from seven embryo transfer units. Of the donors treated, 73.5% responded to the gonadotropin treatment, yielding an average of 8.2 ova. Of the collected ova, 65.5% were fertilized, giving an average of 5.3 per donor that were considered transferable. Of those, only 2.2 per donor resulted in pregnancies. The number of recipient pregnancies from one recovery attempt ranged from O to 32. In a more recent review, Betteridge (l977) summarized data from 1,343 donors in l9 studies. The range in the average number of ovulations per flushed donor from all studies was from 3.5 to l8.5 with a mean of l0.2. The number of embryos and unfertilized ova recovered varied from 2.6 to l2.3 with a mean of 6.2 (6l% of ovula- tions). The range in number of fertile embryos recovered was from 2.0 to 9.6 with a mean of 4.7 (76% of eggs recovered). In studying superovulation, it becomes obvious that large variation exists in the number of eggs released, recovered, and fertilized. A number of factors which have been suggested as. possible sources of variation will be discussed. These are: Nature of gonadotropin, Dose-response relationship, Batch of gonadotropin and method of administration, Time of estrous cycle to initiate gonadotropin treatment, Interval between gonadotropin treatment and estrus, Population of ovarian follicles, Endocrine environment during superovulation, Immunological response, Breed differences, Seasonal effects, and Nutritional effects. Nature of Gonadotropin In l939, Fevold showed that among the mare, the sow, the ewe, and the cow, the pituitary of the cow is lowest in FSH, and the pituitary of the mare is nearly ten times richer in FSH than the pituitary of either the sow or the ewe. The relative FSH and LH levels in these species are shown in Figure l. Among the four animals mentioned above, the ewe pituitary is the highest in LH, followed by that of the sow, the mare, and the cow, in that order. Although critical data comparing pituitary extracts from different species are lacking, cattle, sheep, pig, and horse pituitary extracts have all been used with success to superovulate cattle (Gordon, l975). Because of their short half-lives, pituitary extracts have to be administered daily or twice daily for periods of up to five days. Another commonly used gonadrotropin has been PMSG which has a longer half—life. With PMSG, the normal treatment has been to administer a single dose usually between l500 and 3500 I.U. Limited data comparing pituitary extracts to PMSG do exist. In sheep there is firm evidence that a crude horse pituitary extract of FSH (HAP) can be superior to PMSG in achieving effective high superovulation (Moore and Shelton, l962, l974; Shelton and Moore, l967). However in cattle this has not been confirmed. Dowling (l949) observed an average ovulation rate of l2.0 for PMSG treated cows and 6.5 for HAP but recovered a higher number of fertilized eggs with HAP. Gordon (l975) reported an average ovulation rate of l9.3 with PMSG and 7.3 with HAP. eshus (\xoxn-s OvulationCnoms 0‘3 1‘ \ 2 O A 3 8 fig offgtik AfiXeT \ A‘gie‘r \ Be’fioxe Be‘pte 5 occu‘mme 'tho‘uen‘t'-’§¢—-R 0.1- e. — Durationo‘g\\ X8 36 \ 72 \\AT: Si\e1\\. Meo’ts Figure l. The relative FSH and LH potency of the pituitary in the cow, ewe, sow, and mare. (Taken from Salisbury and VanDemark, 1961. ) Comparing PMSG to HAP, Moore (l975a) found both to be equally effective in superovulating mature cows. In heifers there was little difference in ovulatory response due to PMSG or HAP, however HAP resulted in three to four times as many large unovulated follicles. Moore concluded that due to availability and need for fewer injections, PMSG provided a simpler treatment to obtain fertilized cow eggs than HAP. Pituitary follicle stimulating hormone of domestic animal origin (FSH) has been more extensively studied than HAP. FSH is available commercially and marketed in 50 mg lots according to an Armour standard. It is quite common to use a treatment consisting of a 5:l mixture of FSH and pituitary luteinizing hormone (LH) (Elsden §t_§l,, l976). Elsden §t_al, (1978) compared FSH—LH to PMSG and observed more corpora lutea, ova and pregnancies in cows treated with FSH-LH than in cows treated with PMSG. Mean numbers of corpora lutea, ova and pregnancies were 6.2, 2.0 and l.2 for PMSG and ll.4, 7.9 and 4.2 for FSH-LH, respectively. Hasler (l978) observed no significant differences in numbers of corpora lutea, ova recovered, fertilized ova or pregnancy rate following transfer of fertilized ova after treatment of cows with FSH-LH or PMSG. However, in cows considered to be infertile, those treated with FSH-LH produced more corpora lutea and fertilized ova than cows treated with PMSG. Number of corpora lutea were ll.9: 9.6 vs 4.9: 2.4 while number of fertilized ova were 4.4i 8.0 vs 0.6: 0.9 for FSH-LH and PMSG, respectively. In an investigation comparing efficacy of PMSG to FSH for production of twins, Laster (1973) indicated that FSH may have greater potential to induce high numbers of ovulations than PMSG. Dose-Response Relationship A definite dose-response relationship has been demonstrated for PMSG and pituitary extracts. Sreenan and Beehan (1976) injected three levels of PMSG and observed the mean ovulation response. PMSG dose levels (IU) were 1500, 2000 and 2500, which resulted in 7.8: 1.4, 12.1: 2.3 and 13.1: 3.9 ovulations, respectively. Percentage of ova recovered was 65, 56 and 51 for the three levels, respectively. There was a tendency for a lower proportion of ova recovered at the high level of PMSG. Moore (1975a) gave PMSG or horse pituitary extract (HAP) at three dose levels. There was a significant linear effect of PMSG and HAP on the numbers of CL and numbers of follicles. Percentage of eggs recovered decreased as dose of gonadotropin increased. However, the fertilization rate was consistent across treatments. Combined results of eight studies utilizing repeated injections of FSH are presented in Table l (Bellows §t_a1:, 1969). As dose level increased, ovulation rate increased, as did the range in number of ovulations per heifer. Ova recovery and fertilization rates were lower at the higher dose levels. Gordon (1975) observed that the higher the PMSG level, the greater the variability in response. He reported that at PMSG dose levels of 1000 IU and even 2000 IU, some proportion of cattle may not TABLE 1. EFFECTS OF DOSE LEVEL OF PORCINE FSH 0N OVULATION, RECOVERY AND FERTILIZATION RATES Total Ovulations Ova Dosea Number —————-—————-—— . _ b (mg eq) An1mals Mean Range Recovered Fert1l1zed % % 3.1 8 1.1 1-2 88 93 6.2 18 2.2 1-6 98 95 10.0 81 2.5 0-8 --C -—C 12.5 13 6.5 1-14 79 75 25.0 11 14.0 1—32 48 79 50.0 4 17.8 13-25 58 52 aMAP fed 180 mg/day for 11 days, first MAP feeding==day 1; 5 mg estradiol injected day 2; gonadotropin injected 2X/day on days 8, 9, 10, 11 and 12. bIncludes all cleaved ova. CData not available. be induced to superovulate. However at 3000 IU, practically all cattle can be expected to release additional eggs, but the variation may be enormous. Gordon found that response to 3000 IU PMSG varied from one to 112 ovulations. The work of Newcomb _t.al. (1979) supports a dose-response relationship. Batch of Gonadotropin and Method of Administration It has been suggested that the superovulatory effect of PMSG can vary with the particular batch employed. Stewart t 1. (1976), using rat testis radioreceptor assays to measure FSH and LH activity, showed the FSH:LH ratio to remain constant (about 1:5) in unextracted serum from six different mares throughout the period between days 40 and 80 of gestation. They also tested six batches of commercially available PMSG and found no significant difference in the FSH:LH ratios. Further, using the same assayed batches to superovulate cattle and sheep, they found no significant variation in mean ovu— lation rates between groups. They concluded that variation between animals in response to PMSG is unlikely to be due to differences in the FSH:LH ratio of the preparation used. Similarly, using rat testis radioreceptor assays, Newcomb _t_al. (1979) found no significant differences in the FSH:LH ratio of three different batches of PMSG. When administered to heifers, no differences were found in ovarian response from the three batches. Gordon (1975) also showed little difference in ovarian response of cows from two different batches of PMSG. In contrast, Humphrey gt al. (1979) observed FSH activity was significantly higher in serum of pregnant mares at 60 and 90 days of gestation when compared to days 45 or 120. In Shorthorn cows, high FSH/LH ratio PMSG induced more cows to ovulate than low FSH/LH ratio PMSG (88% vs 50%). Addition of HCG to medium ratio PMSG reduced ovulatory success to 37%. They concluded that both high and low FSH/LH ratio PMSG could induce follicular activity; however prep- arations with high FSH/LH PMSG were more conducive to the induction of ovulation. The way in which PMSG is administered to the cow on day 16 of the cycle can markedly affect the ovulatory response. When a dose of 2500 IU was given in low volume (2.5 ml) by intramuscular injection, the superovulatory effect was substantially greater than when the same dose was given in a 25 ml volume subcutaneously (Gordon, 1975). Time of Estrous Cycle to Initiate Gonadotropin Treatment Most early superovulation treatments were initiated on day 16 of the cow's cycle to coincide with the follicular phase. However, the development of PGF20c and progestogens for controlling the time of ovulation has opened new possibilities of beginning superovulation earlier in the cycle. The ability to control the time of ovulation has numerous advantages in a commercial embryo transfer program. Elsden et_al, (1974) compared responses of cows treated with PMSG during the mid-luteal phase of the cycle followed by PGF a 48 hr 2 later to response of cows given PMSG on day 16 of the cycle. All cows given PMSG followed by PGan ovulated with the mean ovulation rate being 13.2: 1.9. Of cows treated with PMSG alone on day 16, 50% responded with 8.0: 1.5 ovulations, 20% did not ovulate but had cystic follicles and 30% did not respond. These results suggest that the use of PMSG together with PGF a was superior to PMSG alone 2 in terms of the proportion of animals ovulating and the higher ovu- lation rates achieved in the animals which responded. In agreement are Seidel gt_ 1. (1975) and Nelson et_al, (1976) who observed animals brought into estrus by PGan treatment after PMSG had higher superovulation rates than those treated with PMSG on day 16 of the natural cycle. Ford and Stormshak (1978) investigated gonadotropin—induced follicular development and ovulation during the three-day period after the cow had ovulated spontaneously. Treatment of heifers with PMSG failed to stimulate follicular growth during metestrus, as determined by palpation. Phillippo and Rowson (1975) compared the ovulatory response of cows whose treatment was begun during different days of the cycle. Cows were grouped into the following periods: days 3—7, 8—12, and 13—16. The percentage responding with three or more ovulations were 37.5, 77.6 and 55.5, respectively. Responses of cows treated prior to day 8 were considerably lower. Sreenan (1976) also demonstrated that treatment initiated during the mid-luteal phase (days 8-12) gave higher ovulation rates and yields of embryos than treatment begun earlier. Newcomb §t_a1, (1979) injected cows with PMSG from days 9-12 and observed no systematic effect of day on response. They concluded that when PMSG is administered during the mid-luteal phase, after day 8, there is no significant effect of day of treatment on response. Interval Between Gonadotropin Treatment and Estrus It has been reported that following the use of PMSG during the follicular phase of the cycle, a definite relationship exists between the mean percentage of follicles ovulating and the time interval separating PMSG and estrus. Gengenbach §t_al, (1978), utilizing various combinations of PGan and PMSG, grouped animals according to ovulation rates and observed significant differences in the interval to estrus. Heifers with the highest ovulation rates tended to have the longest interval to estrus. Two heifers which did not show estrus until 120 and 144 hrs after treatment had 16 and 19 ovulations, respectively. Sreenan and Beehan (1976) also reported that the longer the interval, the higher the proportion of total ovarian response that is represented as ovulations. Animals with an interval from PMSG to estrus of 3 days had the lowest percentage of ovulations with 59%. Those with an interval of 4 days ovulated 80% while animals with an interval of 5-7 days ovulated 97% of stimulated follicles. Using various combinations of PMSG and PGFZa’ Henricks and Hill (1978) recorded the days from PMSG to estrus and the number of ovulations. The treatment group having the least days from PMSG to estrus, 2.7, also had the fewest ovulations, 2.3. Treatment groups averaging 4.2 and 5.3 days from PMSG to estrus produced 5.7 and 4.6 ovulations, respectively. In contrast, Lopez-Barbella $3.21: (1979) found an increasing interval from PMSG to observed estrus coincided with a decrease in ovulation rate (72 hrs, 5.50: 1.29 CL vs 97 to 144 hrs, 0.67: 0.82 CL). Furthermore, Moore (1975a)observed the time elapsing between treatment with PMSG or HAP and the onset of estrus in 140 mature cows. No apparent effect upon ovarian response was seen. However, mean number of corpora lutea was 2.9 while large unovulated follicles averaged 3.7. This is a very poor response and could suggest subfertility of experimental animals. Using PMSG followed by HCG, Hafez et_al, (1963) observed that the longer the interval from PMSG to HCG injection, the greater the percentage of follicles that ovulated. They concluded that at least 5 days should elapse between PMSG and HCG injections for a high ovulation percentage. Betteridge (1977) reviewed work covering superovulation of prepuberal calves. The most successful treatment regimen consisted of inserting vaginal sponges impregnated with 60 mg fluorogestone acetate (FGA) at the time of PMSG treatment and leaving them in place for 4 days. The 4 days of FGA blocked ovulation until day 6. Begin- ning 41 hrs after sponge withdrawal, 75.3% of 93 calves averaged 13.9 ovulations each and they were grouped within a 20-hr ovulatory period. Without FGA the time span over which ovulations occurred was prolonged. Population of Ovarian Follicles During fetal life in cattle, the definitive stock of oocytes is constituted which will be used during the entire sexual life. At the end of fetal life, follicle growth cycles succeed one another, causing constant formation of graafian follicles which disappear by atresia. It is only following puberty that regular estrous cycles and ovulation commence. However, follicular growth and atresia continue to take place throughout the reproductive life cycle of the cow. 15 Sreenan and Beehan (1976) have suggested that changes in the ovarian population of follicles could affect the response to super- ovulation. Rajakoski (1960) characterized changes taking place in the ovarian follicular system during one cycle in sexually mature heifers. He observed that during the bovine sexual cycle, follicles 2 5 mm diameter go through two growth phases. The first of these occurs during the third and fourth days of the cycle and gives an increased number of medium-sized follicles and a single large fol- licle which undergoes atresia during the eleventh and twelfth days of the cycle. A second similar growth wave appears between the twelfth and fourteenth days of the cycle and leads to the development of a large follicle which attains maturity during the first and second days of the subsequent cycle and then ovulates. Rajakoski (1960), however, stated that there was tremendous variation due to individual differences. In a similar study Cahill gt a1. (1979) studied ovarian follicular populations in two breeds of ewes which differed in their ovulation rates. Mean ovulation rate for Romanov ewes was 3.1 while Ile-de-France averaged 1.4 ovulations. 'The researchers observed half as many small follicles but 1.5 to 2.0 times more large follicles in the ovaries of the Romanov ewes compared to those of Ile-de-France ewes. They concluded that the higher ovulation rate in the Romanov ewe is due to the greater number of large follicles available to be stimulated for ovulation. Follicles 2 5 mm diameter generally are held to be responsive to gonadotropin stimulation. Changes in the population of these follicles as demonstrated by Rajakoski (1960) could very likely explain some of the variation in individual response to superovulation. Endocrine Environment During Superovulation Betteridge (1977) has described knowledge of the endocrinology of superovulation as fragmentary and disputed. Understanding hormonal interrelations in cows with induced multiple ovulations may provide a key to obtaining greater precision in response. Numerous researchers have shown circulating estrogen levels rise tremendously and at estrus may be three to four times higher in superovulated compared to untreated cattle (Lemon and Saumande, 1972; Henricks gt_al,, 1973; Hallford, Turman, Wetteman and Pope, 1975). Booth et_al: (1975) found a temporary decline in circulating estrogen levels followed by a secondary rise around days 5 and 6 post estrus when levels were eight times those found in normally cycling animals. They fell to normal levels by day 12. Spilman et_al, (1973) also observed secondary peaks of estrogen in superovulated calves. Estrogen concentrations have been positively correlated with the subsequent number of CL (Henricks et_al,, 1973; Henricks and Hill, 1978). However it is also possible, as observed by Booth t al. (1975), that the high estrogen levels can be due to large numbers of unovulated follicles > 15 mm diameter. Using prepuberal calves, Spilman §t_al, (1973) found circulating estrogen levels are greatly elevated before ovulation. Levels were well correlated with the degree of follicular development but not with the number of ovulations. Gengenbach et_al. (1978) reported no relationship between estradiol concentrations prior to estrus and the number of CL formed. In rabbits it is known that estrogen can accelerate the transport of eggs in the oviduct (Harper and Change, 1971). It therefore seems likely that, in the superovulated cow, the high levels of estrogen which occur after ovulation, could modify the motility of the oviduct and uterus causing premature transport of eggs into the uterus or expulsion into the vagina. It is also possible that high levels of estrogen may bring about premature shedding of the zona pellucida, which would lead to a subsequent degeneration of the egg (Dickman, 1969). A steep rise in the plasma progesterone of superovulated heifers after day 2 has been found by numerous workers (Booth §t_§1,, 1975; Spilman gt_al,, 1973; Gengenbach et_al,, 1978; Henricks and Hill, 1978; Henricks et_al,, 1973). Levels as high as 60-100 ng/ml have 1. (1979) observed a coefficient been reported. Lopez—Barbella gt of correlation of 0.62 between number of CL and plasma progesterone level 13 days after estrus. Gengenbach §t_al, (1978) conducted a study to determine if differences in plasma progesterone concentration at the time of PMSG administration affected the variability of ovulatory response to PMSG. They reported that duration of increased plasma progesterone concentrations, particularly following the highest dose of PMSG, seemed to be more important in determining ovarian response than changes in progesterone prior to PMSG treatment. Five of eight heifers treated with 2000 IU of PMSG having low progesterone con- centrations before the end of the 84 hr post-treatment period, averaged 1.6 corpora lutea and 4.6 large follicles and returned to estrus 51.4 hrs after treatment. The heifers with higher progesterone concentrations during this period averaged 12.3 corpora lutea and three large follicles and returned to estrus after 120 hrs. Avery §t_al. (1962) pretreated cows with progesterone for 10 days prior to superovulating them with FSH. Cows receiving pretreatment with progesterone produced an average of 7.9 more ovulations than cows not receiving prior progesterone treatment. Lopez-Barbella gt El, (1979) grouped cows according to number of corpora lutea (CL): 0 to 1; 2 to 3; and greater than 3. LH, progesterone and estrogen changes with time were similar in all groups following PMSG treatment, although progesterone and estrogen concentrations were higher in cows with a larger number of CL. In a similar manner, Solti _t_al. (1978) studied the plasma progesterone level at the time of PMSG administration and the subsequent number of corpora lutea. No correlation was found. Spillman et_al, (1973) state that PMSG leads to release of endogenous LH within 24 to 48 hrs of injection. This was not the experience of others (Henricks et_al,, 1973; Lemon and Saumande, 1974; Saumande and Pelletier, 1975; Hallford, Turman, Wetteman and Pope, 1975) who describe no LH peaks before the one coinciding with estrus. Hallford et_al, (1979) failed to detect a relationship between plasma LH and reproductive criteria after PMSG treatment. Betteridge (1977) in reviewing superovulation of prepuberal calves found no evidence that PMSG alone induced an immediate release of endogenous LH into the circulation. Instead, it resulted in a short-lived peak of 3 to 4 ng/ml for up to 8 hrs between 108 and 132 hrs after PMSG which is considered insufficient to bring about ovulation. The use of FGA improved results by leading to a much greater LH release for 8 to 16 hrs with peak values of 11 to 72 ng/ml 12 to 20 hrs after FGA withdrawal. This matches LH release in normally cycling adult cows and was sufficient to lead to multiple ovulation some 20 hrs later. There was also an FSH peak coincident with the major LH peak. Immunological Response Seidel et_al, (1978) have discussed the possibility of immunological response to repeated injections of gonadotropins which may limit the number of times a donor may be superovulated. Both PMSG and FSH are proteins and, therefore, potential inducers of anaphylaxis. This antigenicity also implies that repeated injec- tions may stimulate the production of antigonadotropins which may inhibit subsequent responses or perhaps even interfere with endogenous gonadotropins. 20 Jainudeen et_al. (1966) studied the use of repeated injections of gonadotropins to superovulate cows. Multiple ovulations were obtained in cows after the first PMSG injection. A larger dose of PMSG injected 5 to 7 months later also produced a similar ovulatory response; however, the same dose of PMSG repeatedly injected at subsequent estrous cycles failed to stimulate the ovaries. Using immature intact female rats they assayed antigonadotropic activity in the blood serum of treated cows. They found the level of anti- gonadotropic activity was low prior to the second PMSG injection but increased with successive treatments and attained maximal values 16 days after the fourth PMSG injection. Antigonadotropins in PMSG— treated cows inhibited the follicular stimulating properties of PMSG, but had no adverse affect on follicular development and ovulation resulting from endogenously secreted gonadotropins. They concluded that repeated therapeutic doses of PMSG failed to induce multiple ovulations in the cow and the failure was due to the presence of antigonadotropins. These results are in accord with the findings of Willet et_al, (1953) and Havez et_al, (1964). Similarly, Hallford e__a1, (1979) observed three of six cows previously treated with PMSG failed to ovulate and none ovulated more than one egg when PMSG was administered on day 17 or days 5 and 17 of the previous cycle. Newcomb §t_al, (1979) also observed fewer ovulations but no reduction in the number of follicles after a second PMSG treatment to cows. The mean interval between the two treatments was 51 days. 21 Turman gt_gl, (1978) conducted a study to determine if PMSG injections given one year may adversely affect the superovulatory response of cows to PMSG injections the following year. Treatment with PMSG the previous year reduced the superovulatory response of cows to PMSG. Cows that had never been previously treated had a significantly greater ovulation rate (5.3 vs 1.8), a wider range in ovulations (1-16 vs 0-5) with more cows ovulating four or more eggs (45% vs 9%) than did cows that had been previously treated. Seidel gt_al, (1978) have not encountered any difficulties with immunological response but concede that data on this problem are scarce. They suggest that the possibility of endocrine pathology may be reduced if the donor cow is allowed to carry a pregnancy after several superovulation treatments. Breed Differences There is evidence of a breed response relationship, with beef breeds showing a greater sensitivity to PMSG than dairy breeds. Sreenan and Beehan (1976) have reported higher mean ovulating responses in Hereford and Angus beef cattle as compared with Freisians, all treated with a standard dose of PMSG (3000 IU) in the follicular phase of the cycle. Likewise, Mariana et_a1, (1970) reported a higher ovulation response in Charolais than in Freisian cattle following 1600 IU PMSG. Further breed differences in responsiveness to PMSG are recorded by Shea §t_gl. (1976), 178 Simmental, 79 Limousin, 54 Chianina and 93 Maine Anjou donors averaged 15.2, 13.6, 12.4 22 and 9.9 ovulations, respectively. The Maine-Anjou ovulation rate was significantly (Ps .05) poorer than for other breeds. Seasonal Effects Gordon (1975) presented evidence that seasonal fluctuations in ovarian response to PMSG did exist. Using 3000 IU PMSG on day 16 of the cycle, he observed the highest average number of ovulations (17.9) and total follicle development (CLi-large follicles, 26.8) in the period from February to April. Remaining periods of the year were similar to each other with responses of: November to January, 10.0 ovulations and 17.5 CLi-large follicles; May to July, 9.5 ovulations and 15.0 CLi-large follicles; and August to September, 8.9 ovulations and 12.2 CLi-large follicles. Subsequent data have not supported the existence of sea50na1 variations very well, however. In a study of 582 cows, data reported by Shea 23.91: (1976) showed similar responses in all quarters of the year. Sreenan and Beehan (unpublished, reported in Betteridge, 1977) also observed nearly equal ovulation responses during different seasons of the year. Rajokoski (1960) observed the total number of follicles< 5 mm diameter varied for the seasons with the lowest mean total, 73.1 per heifer, during the autumn and a distinct increase during the winter and spring to 118.5 and 116.5 per heifer, respectively. However, there was no systematic seasonal variation in the number of follicles with a diameter2 5 mm. 23 Nutritional Effects Nutrition is known to have dramatic effects upon reproductive processes in cattle. Inadequate rations for growing heifers result in both reduced body weight and increased age at puberty (Wiltbank et al., 1969). Restricted diets can also increase the post-partum interval in mature cows (Dunn gt_al,, 1969). Ovulation rate in sheep is known to be affected by nutrition (Ensminger, 1970). It is widely held that flushing, the practice of feeding ewes more generously during the period of 2 to 8 weeks imme- diately prior to breeding, will result in a 15 to 20% increase in the lamb crop. Level of nutrition has also been demonstrated to affect the ovarian response to PMSG in ewes (Allen and Lamming, 1961). Similarly in swine, gilts on a high energy ration have a greater number of ovulations than those on a low energy ration (Sorenson et_al., 1961; Self 23.91:, 1955; Zimmerman §t_al,, 1960). However it has also been shown that a higher percentage of live embryos at 40 days follow- ing breeding was obtained on gilts fed the low energy ration (Sorenson _t__l,, 1961). A study was undertaken by Staigmiller et_al, (1979) to examine the effects of undernutrition on ovarian response to exogenous gonadotropin. Mature cows were fed a high or low level (130% and 70% of NRC requirements for TDN) ration for 92 days prior to being super— ovulated with FSH. The number of large follicles and CL at 3 days post estrus was correlated with estimated body condition, being higher in cows with more condition. However, neither ovulation rate nor fertilization rate differed between cows in the high or low TDN groups. 24 Summary A major factor which limits success of embryo transfer is large variation in numbers of ovulations after treatments imposed to cause superovulation. The most widely used gonadotropin has been PMSG. A batch-to-batch variation has been suggested to exist but evidence to support this is not conclusive. Prior to the development of PGFZQ, PMSG was administered as a single injection on day 16 of the natural cycle. However, since then it has been shown that initiation of gonadotropin treatment during the mid-luteal phase, after day 8, of the cycle followed by PGFZa 48 to 72 hrs later gives superior results to treatments begun earlier or during the follicular phase of the cycle. This appears to be the treatment of choice. Recently there has been much interest in the use of pituitary extracts. Limited studies comparing PMSG to FSH have suggested that FSH may be superior to PMSG for induction of multiple ovulations. A dose-response relationship has been shown to exist. As dose levels of gonadotropin increase, ovulation rate increases. However the variation from animal to animal also increases. Furthermore, as ovulation rate increases, recovery and fertilization rates tend to decrease. An optimum response has been described by Seidel et_al, (1978) as one in which each ovary has five to ten ovulations. A relationship has been shown to exist between the mean percentage of follicles ovulating and the time interval separating PMSG injection and estrus. A time period of 5 days has been suggested as optimal to allow follicular maturation prior to the LH surge. High 25 progesterone levels are important during the first 3 days of gonadotropin treatment to block LH release and prevent premature ovulations. An immediate decrease of progesterone is then necessary to insure rapid onset of estrus and endogenous release of LH. Estrogen concentration has been shown to be correlated with ovarian response, however extremely high levels have been suggested to be detrimental to egg transport and fertilization. Progesterone concentration several days following estrus has been positively correlated with ovulation rate. Differences in populations of ovarian follicles at the time of initial gonadotropin treatment could explain much of the variation in response to superovulation. However, at present, methods of deter- mining populations of ovarian follicles are impossible, and as a consequence, little can be done to incorporate knowledge of their status into a superovulation regimen. Development of an immunological response to repeated injections of gonadotropins has been demonstrated. It has been suggested that this problem may be overcome by allowing a cow to carry a pregnancy after several superovulation treatments. Further variation in superovulation response can result due to breed differences, although this is probably not a major factor. A seasonal variation in response has been suggested but evidence to support this is inconclusive. Ovulation rate in sheep and swine can be increased with elevated energy intake. Even though this relationship has not been 26 shown in cattle; past experience with the effects of nutrition on reproduction dictates that properly balanced rations adequate in TDN be fed. Numerous factors have been shown to affect the response to superovulation but individual variation always remains. At the present time, conditions can be defined to optimize the ovarian response but precise control over ovulation rate is not possible. MATERIALS AND METHODS Experimental Animals The cows used in Trial 1 originated from the Lake City Experiment Station breeding project. Cattle in this herd are of four types: Herefords—-unse1ected or selected for growth rate, Charolais x Hereford x Angus, and Holstein x Hereford x Angus. This herd is managed on a spring calving basis. Twenty-seven cows diagnosed as non-pregnant at the end of the breeding season in 1978 with anatom- ically normal reproductive organs ranging in age from 2 to 9 years were selected for Trial 1. Five were heifers which had failed to conceive and 22 were parous cows 5 to 8 months post—partum. All animals utilized were in good body condition and had been vaccinated for brucellosis, leptospirosis, vibriosis and IBR. Charolais crossbred virgin heifers originating from northern Michigan were purchased in April 1979 at approximately 12 months of age for use in Trial 2. They were in thin body condition and many had not reached puberty at the time of their purchase. All had been calfhood vaccinated and upon arrival were vaccinated for leptospirosis, vibriosis and IBR. Serum progesterone concentrations were monitored until 26 heifers were found to be cycling. Reproductive organs of the heifers were palpated to eliminate any with genital abnormalities. These heifers were approximately 16 months of age at the start of the experiment. 27 28 In Trial 3, eight Charolais crossbred virgin heifers were used. They had previously been involved in a nutrition study at the MSU Beef Cattle Research Center. Age, origin and vaccination history were unknown. Feeding All cattle were housed at the Beef Cattle Research Center (BCRC). Daily feeding and management were performed by BCRC personnel. Cattle on Trial 1 were fed a 100% corn silage ration. The silage had been treated with anhydrous ammonia at the time of ensiling and was balanced for calcium, phosphorous and salt with a mineral supplement. Vitamins A and D were provided. The ration contained 10.9% crude protein (DM basis). Additionally, cows received 200 mg monensin per head per day. For 3 months prior to the start of Trial 2, Charolais cross heifers received a 60% corn silage, 40% concentrate ration containing 13.3% crude protein (DM basis). One week before starting the exper- iment, the ration was increased to 85% concentrate, 15% corn silage (13.0% crude protein, DM basis). The rations were balanced for calcium, phosphorous and salt. Vitamins A and D were provided. All heifers received 200 mg monensin per head daily. Heifers in Trial 3 were fed a 60% corn silage, 40% concentrate ration containing 11.3% crude protein (DM basis). The ration was balanced for calcium, phosphorous, salt and Vitamins A and D were provided. No monensin was fed. 29 Products Used Equine pituitary extract (EPE) was obtained from Biological Specialties, Middleton, Wisconsin and originated from one lot. The manufacturer's description is given in Appendix Table A1. Further, a bioassay (Fevold and Hisaw, 1934) is completed by the manufacturer for each lot processed. The equivalent of 125 Fevold—Hisaw Rat Units of gonadotropic hormones are packaged per 5 m1 vial. The results of the bioassay for Lot No. 1803 are presented in Appendix Table A2. Human Chorionic Gonadotropin (HCG) was also obtained in a single lot from Biological Specialties. The manufacturer's description is given in Appendix Table A3. Pituitary FSH and LH of domestic animal origin were purchased from Reheis Chemical Company, Kankakee, Illinois. The manufacturer's analysis for FSH used in this experiment is given in Appendix Table A4, while analysis for LH is reported in Appendix Table A5. Prostaglandin an (Lytalyse<:)) was obtained through the courtesy of Dr. James Lauderdale, The Upjohn Company, Kalamazoo, Michigan. A dose of 25 mg of PGFZQ was injected intramuscularly. EPE, HCG and FSH-LH are packaged in sterile, lyophilized form. Reconstitution was completed no more than 2 hr prior to administration. Preliminary Treatment and Allotment Estrous cycles of cattle within each trial were synchronized using two injections of prostaglandin an (PGFZQ) given 11 days apart. At the time of the second PGFZQ injection, a blood sample was taken from each animal in Trials 1 and 2 for determination of serum 3O progesterone concentration. Cattle in all trials were then observed for estrus three times daily during the 6 day period following injection of PGFZa' Cattle in Trial 1 were blocked by age and randomly allotted to treatment groups. Cattle in Trials 2 and 3 were randomly allotted to treatments. Due to the large number of animals in Trials 1 and 2, equal numbers from each treatment were grouped for slaughter on each of three successive days. This was necessary since a maximum of 10 reproductive tracts could be processed in one day. Cattle in Trial 3 were slaughtered on the same day. Slaughter dates were as follows: Trial 1: November 7, 8, 9, 1978 Trial 2: August 13, 14, 15, 1979 Trial 3: October 31, 1979. Experimental Design In order to establish a starting point for dosage of equine pituitary extract (EPE), a preliminary study was conducted. Two cows treated with 750 Fevold-Hisaw Rat Units of EPE responded with 2 to 4 ovulations based 0n rectal palpation. Since one of the objectives of Trial 1 was to find the upper limit of a workable dose range for EPE, dosages were set higher than perceived necessary. Total doses of EPE were: 2250 and 4500 Fevold-Hisaw Rat Units. Trial 1 consisted of four groups of cows given the low or high dose of EPE with or without HCG. A fifth group served as controls. Daily doses of EPE (Fevold-Hisaw Rat Units) and HCG (IU) were divided 31 into two equal amounts and injected subcutaneously at 12 hr intervals for five days beginning on day 12 postestrus (0 = day of estrus). Daily doses were: low EPE = 750, 500, 500, 250, 250; high EPE = 1500, 1000, 1000, 500, 500; and HCG = 0, 1000, 1000, 500, 500 for days 12, 13, 14, 15 and 16, respectively. Prostaglandin F was injected intramuscularly at 60 hr in 2a control and 72 hr in EPE-treated cows, after initiation of gonadotropin injections on day 12. Two, three, and one straws of frozen semen were inseminated 12, 24, and 36 hrs after estrus was first detected but starting no later than 60 hrs after prostaglandin F2u was injected. Cows were slaughtered seven days postestrus and reproductive tracts were removed for study. The experimental design for Trial 1 is shown graphically in Figure 2. Trial 2 consisted of four groups of heifers (n= 4/group) receiving a low or high dose of EPE for 3 days injected subcutaneously once daily (1X) or divided equally and administered at 12 hr intervals (2X). A fifth group received the low dose for 2 days injected once daily (n= 5). A sixth group received a 5:1 mixture of follicle stimulating hormone and luteinizing hormone (FSH-LH) of domestic animal origin, injected in equal daily doses at 12 hr intervals for 5 days (n= 5). Injections began on day 12 of the estrous cycle. Daily doses of EPE (Fevold-Hisaw Rat Units) were: low EPE--3 days= 375, 250, 125; high EPE--3 days= 750, 500, 250; and low EPE--2 days= 375, 375. The dose rate of FSH-LH was based on mg of FSH (Armour standard) and was 32 .F megp Low :mwmwu Fmpcmeecwaxm .m mezmwu com com 0mm ww com com omw mr 000w. 000w oom qr v m::.coo .m m our + mammmoucm::.e 000v oooF oom Mr 8 mam omen 2m:2.m o oomw omk NF 0 00+*.+ mam mmou 304 .N ~D_VOOT* cm_I 30; Nae . 523$ 335.296:QO m mam 3,8 23 F 89800 d... 358:3; .30 cm mwnwwwmw Nw o m. qr- _ _ a A _ — _ _ 4 mo 07 av V AV 9 V 009 eaves $0 09.. $0 $0 &MY AWW. AU & AU AU 0 Theorem; TIIIIlm~EoEoc>wllli .cmEEmC >8 m 33 given subcutaneously twice daily as follows: 5, 5, 4, 4, 3, 3, 2, 2, 2, 2 mg. Prostaglandin an was injected 72 hr after initiation of gona- dotropin injections on day 12. Insemination and necropsy procedures were the same as those used in Trial 1. The experimental design for Trial 2 is shown graphically in Figure 3. Trial 3 consisted of 2 groups of heifers receiving equal total doses of EPE administered at constant or variable daily doses. EPE was injected once daily for 5 days beginning on day 12 of the estrous cycle. Daily doses of EPE (Fevold—Hisaw Rat Units) were: constant EPE= 250, 250, 250, 250, 250 (n= 4); and variable EPE= 375, 250, 125, 125, 375 (n= 4). Syncro-Mate-B1 was administered on day 11 postestrus, 24 hr prior to initiation of gonadotropin treatments. Implants were removed 72 hr following the initial EPE injection. Insemination and necropsy procedures were the same as those described for Trial 1. The experimental design for Trial 3 is shown in Figure 4. Blood Collection Procedures In Trials 1 and 2, 20 ml blood samples were taken for quanti— fication of progesterone and estradiol-17B. All samples were taken from the jugular vein using an 18 gauge needle and 20 ml disposable syringe. Blood was transferred to 16 x 100 mm disposable culture 1Syncro-Mate-B is a product of Searle Agriculture, Inc., which consists of an implant containing 6 mg of norgestomet, placed subcutaneously on the back of the ear, and an intramuscular injection of 3 mg norgestomet and 6 mg estradiol valerate. .N Fave» Low :mwmmu _mpcmevcmaxm .m wczmwd v o o o. or v o o o 3 m 0235:. tmv 1.71m". .0 m 0 0mm mg. 3 m «>2. u.x7mam omen 26.. .m 98 . .mam 3% :9: .4 o m5 com one 2 a a a xu e 2% 9x795 3% 29: .n 2 m5 one Sn 3 a ecu...” .xubam $8 23 a u “95:9. €3.23 “€3.29: €3.23 N8 . EE: Em zam_:.u_o>a$mam a 2% n .meam 3% 23 r mommmoo 1:1 95.53:. >3 E 3:33 a? o m. 3. a c. _ ._. _ _ ._ . 0/ owv/hu 0 «NV .0 «UV 0 o v 2 v v «0% 9%....03 me ....o a. m. at Tootoa; T||o~_coEoc>mlll_ «:mEamek >8 ... 35 .m meLe :04 :mwmwc Faucmsecwaxm .a mesmwd mhm 0mm or mNP 0mm mw mNF ODN Vv ODN Om“ mw men one E a mam 8% 032:5 .N 2925. 2228. an a mam 3% 22980 .2 35:2 amexéoauvmam mammmoo : mucmczmmck >mo VN wwhwmwmw Nr—F o m. VF. _ _ _ _ LW _ AW mv _ W W & x 09/ .0OVVJ/00v OOVS NUJmWO/vxl J/nosV Ac WV 8 vVhU Luv Amw.ao a? Avuu no Au Au 0 9 I v o/ 90% Theorem; T|lm~_coEoc>wllll_ Aw «cochmck >8 m 36 tubes and left at room temperature for 1 to 2 hr. Tubes were then stored in a refrigerator for 24 to 48 hr before being centrifuged (3000 g) for 15 min. Serum was poured into 12 x 75 mm disposable culture tubes. The tubes were capped and stored at -20°C until ready for hormone assays. A sample of jugular venous blood was taken at the time of the second PGan injection during synchronization of estrus. Blood samples were taken every 12 hr for a 132 hr period starting at the time of the first gonadotropin injection. An additional blood sample was taken at the time of slaughter. Figure 5 shows the schedule for sampling of blood. SDay Treatment 1 - L - 1 I o , 1 1_Perlod_1 6". g} 0' 9 0‘ a, \ ‘6‘ «'1. Q” 6° ('1' 86° 0° 0 Q Q? {3 «9%? 9 l l l l I l 0 12 15161718 2 Q 1 4 Day Every 12 hrs. A nows Indlcate time of blood sampling. Figure 5. Blood sampling schedule for Trials 1 and 2. 37 Necropsy Procedures On day 7 postestrus cattle were hauled 52 kilometers to Milligan Pack at Parma, Michigan. Slaughter began, under Federal inspection, at 9:00 a.m. Reproductive tracts were removed, identified, placed on ice and returned to campus for data collection. Processing of tracts began at 1:00 p.m. Ovarian and Embryonic Data Collection All ovarian and embryonic data were collected and recorded separately for the right and left side. Ovaries were removed, weighed individually and the number of corpora lutea counted. The diameter of unovulated follicles at the surface of the ovary was measured and recorded. Follicular fluid was drained and, following blotting, the weight of stromal and luteal tissue was taken. Follicular fluid weight was obtained by subtracting stromal and luteal tissue weight from total ovarian weight. Flushing procedures for Trial 1 were as follows. Each oviduct was dissected free from the mesovarium. Each uterine horn was then cut 15 cm posterior to the utero-tubular junction (UTJ). A two—way Foley catheter (Bard, l6 Fr.) was placed into a uterine horn and the cuff inflated. Using a blunt needle attached to a 50 ml syringe, 20 ml of phosphate buffered saline (PBS) was flushed through the oviduct, uterine horn and Foley catheter into a 200 x 38 mm test tube. The media had previously been sterilized by a 0.20 micron membrane filter (Nalge Sybron Corporation, Rochester, New York). The oviduct was then removed at the UTJ and 50 m1 of PBS were flushed 38 through the uterine horn and collected. The uterine horn was allowed to balloon with fluid several times by clamping the Foley catheter. Of 70 m1 used per horn, 67 to 68 ml were recovered. Following a settling period of not less than 15 min, two 10 m1 aliquots of fluid were pipetted from the bottom of each collection tube and placed in 100 x 15 m1 petri dishes. Using a binocular dissecting microscope each aliquot was examined for embryos. The number of embryos recovered was recorded. Procedures for Trials 2 and 3 were the same as those described for Trial 1 with the following exceptions. Each uterine horn was dis— sected 10 cm posterior to the UTJ. This was necessary due to small size of the reproductive tracts from the virgin heifers. Forty m1 of media were used on the second step of flushing the uterine horn. The flushing media used was Dulbecco's PBS (Grand Island Biological Company, Grand Island, New York) to which 100 ml heat inactivated fetal calf serum plus 100,000 units penicillin, 100,000 mcg streptomycin and 250 mcg FungizoneCD had been added per liter. Flushing fluid was col- lected directly into a 90 x 50 mm dish (Fisher Scientific Company) for observation. This eliminated the pipetting step used in Trial 1. A microscope with 100X magnification was used for determining condition of embryos. 39 Quantification of Progesterone Serum progesterone concentration was determined by radioimmunoassay similar to that of Louis et_gl. (1973). Depending upon expected concentration, duplicate aliquots (50—100 01) of each unknown were placed in 16 x 100 mm disposable culture tubes. About 2000 cpm of 3H-1,2,6,7-progesterone (104 Ci/m mole, repurified by column chromatography) was added to six randomly selected samples to estimate procedural losses. These were mixed 10 sec and allowed to equilibrate for 30 min before extracting. For comparison among assays, duplicate aliquots of standard sera (3/assay) with high and low progesterone concentrations and blank extraction tubes (4/assay) were assayed with each set of unknown samples. Each tube was mixed with 2 m1 benzene-hexane (1:2) for 30 sec, then stored at -20°C for at least 1 hr to freeze the aqueous phase. The organic solvent from tubes with 3H-progesterone was decanted into a scintillation vial for quantification of the recovered radioactivity. The solvent in the extraction tubes of unknowns was decanted into 12 x 75 mm disposable culture tubes for radioimmunoassay as follows. Three sets of standard tubes containing 0, 1, 2, 5, 10, 25, 50, 75, 100, 150 and 200 pl of stock progesterone (10 ng/ml in methanol, Sigma Chemical Company) were included in each assay and treated similarly to the unknowns. Standard progesterone and serum extracts were dried in a vacuum (-29 lbs) oven (50°C) with a dry ice 40 trap between the oven and vacuum pump. Antibody (MSU #74 produced in rabbits against progesterone-20-oxim-human serum albumin, diluted 1:2000) diluted to 200 pl phosphate-buffered saline (0.1 M), pH 7.4 containing 0.1% gelatin (PBS-G) was added. Crossreaction of rabbit antiprogesterone (MSU #74) has previously been reported by Convey ._p._1. (1977). After addition of antibody, each tube was mixed 2 sec. Then about 24,000 cpm 3H-l,2,6,7-progesterone (104 Ci/m mole, repurified by column chromatography) diluted in 200 pl PBS-G was added to each tube. The tubes were mixed 2 sec and incubated at 5°C for 12 to 18 hr. To separate free from antibody-bound progesterone, 0.5 ml of dextran-coated charcoal (0.5 9 Carbon Decolorizing Neutral Norit, Fisher Scientific Company, and 1 g Dextran T-70, Pharmacia Inc., Uppsala, Sweden, in 100 ml distilled water) was added at 5°C. Each tube was mixed for 2 sec and centrifuged (3000 9) immediately for 15 min at 5°C. Antibody bound 3H-progesterone in 0.5 ml of the supernatant fluid was measured in a liquid scintillation spectro- meter and recorded on a cassette tape. Using the CDC 6500 computer, the unknown progesterone concentrations were calculated. Procedural losses resulted in 88.5: 2.0% extraction efficiency (n= 9) for Trial 1 samples and 86.9: 2.1% extraction efficiency (n= 7) for Trial 2 samples. Values for all blank extraction tubes were negligible. Intra- and inter-assay coefficients of variation are presented in Table 2. 41 TABLE 2. PROGESTERONE INTRA— AND INTER-ASSAY COEFFICIENTS OF VARIATION Intra-Assay Inter—Assay Standard Sera (3/assay) (%) (%) -------------- Trial 1 (n= 9) - - - - - - — - - - - - - - Low (no. 4009—-estrus cow) 25.54 34.72 High (no. 4010--pregnant cow) 8.38 14.21 -------------- Trial 2 (n= 7) - — - - - - - - - - - — - - Low (no. 4016--estrus cow) 11.54 21.86 High (no. 4008--diestrus cow) 6.19 7.65 Quantification of Estradiol—178 Serum estradiol-178 concentration was determined by radioimmunoassay similar to that of Butcher _£._1. (1974). a. Extraction. One millileter of each unknown was placed in a 16x 100 mm disposable culture tube. To account for procedural losses, 2000 cpm of 3H-2,4,6,7,16,17-estradiol (100 Ci/m mole repurified by column chromatography) was added to two randomly selected samples for each assay. These were mixed 10 sec and allowed to equilibrate for 30 min before extracting. Duplicate aliquots of distilled water, ovariectomized cow serum (ovex) and ovex to which 5, 40 and 100 pg of stock estradiol (100 pg/ml in benzene, Sigma Chemical Company) had been added, were included in each assay. Estradiol was extracted by mixing with 3 m1 of freshly opened anesthesia grade ether (Mallinckrodt, Inc., St. Louis, Missouri) for 2 min. Following centrifugation for 10 min at 3000 g, the aqueous 42 phase was frozen on dry ice and the ether decanted into 12x 75 mm disposable culture tubes. Extracts were then dried in a vacuum (—29 lbs) oven (no heat) with a dry ice trap between the oven and vacuum pump. b. Chromatography. Glass 5 m1 disposable pipettes (Kimble #72120, Toledo, Ohio) were used for columns. A 4 mm glass bead was placed in the column and a slurry of 0.8 g of Sephadex LH-20 (Pharmacia, Inc., Piscataway, New Jersey) soaked overnight in methylene chloride: methanol (90:10, Budrick and Jackson Laboratories, Inc., Muskegon, Michigan) was added. A disc of glass filter paper (2.1 cm GF/A, Whatman Ltd., England) was placed on top of the column. Columns were rinsed with 10 m1 of the 90:10 eluting solution which had been allowed to equilibrate for 30 min. Samples were added to the columns in 0.2 m1 of the eluting solution and allowed to enter. Then 3.7 m1 of the solution was added and the elute discarded. A final 4.0 m1 of eluting solution was placed on the column and collected into 12x 75 mm disposable culture tubes. Samples for procedural losses were collected into scintillation vials. c. Radioimmunoassay. Three sets of standard tubes containing 0, 1, 2, 4, 6, 10, 20, 40, 60, 100 and 200 pg of stock estradiol were included in each assay and treated similarly to the unknowns. Standard estradiol and serum extracts were dried in the vacuum (-29 lbs) oven (50°C). Assay tube walls were rinsed down with 250 pl of methanol, and the rinse methanol was evaporated. PBS-G was used to dilute the estradiol antiserum to l:40,000. MSU anti-estradiol #74 was prepared in rabbits against estradiol-6-oxim-human serum albumin. 43 Crossreactivity has previously been reported by Oxender et_g1, (1977). Antibody (200 pl) was added and tubes were mixed 2 sec. PBS-G (200 pl) with 5000 cpm of 3H-2,4,6,7,16,17-estradiol (100 Ci/m mole, repurified by column chromatography) wasadded to each tube and mixed 2 sec. All tubes were incubated 12 to 18 hr at 5°C. To separate free from antibody-bound estradiol, 0.5 m1 of dextran coated charcoal was added at 5°C. Each tube was mixed for 2 sec and centrifuged (3000 9) immediately for 15 min at 5°C. Antibody- bound 3H—estradiol in 0.5 ml of the supernatant fluid was quantified in a liquid scintillation spectrometer and recorded on a cassette tape. Using the CDC 6500 computer, unknown estradiol concentrations were calculated. Procedural losses resulted in 79.3: 5.5% extraction efficiency (n= 4). Blank tubes containing distilled water (2/assay) averaged 1.84: 2.77 pg/ml. Intra— and inter-assay coefficients of variation are presented in Table 3. TABLE 3. ESTRADIOL 17B INTRA- AND INTER-ASSAY COEFFICIENTS OF VARIATION n= 4 Intra-Assay Inter-Assay Standard Sera (2/assay) (%) (% Ovex (no. 4015——ovariectomized cow) 9.91 22.79 Ovex + 5 pg E2 178 7.74 11.00 Ovex + 40 pg E2 178 13.29 30.31 Ovex + 100 pg 02 178 14.16 33.52 44 Data Calculations and Statistical Analysis Analysis of variance was used to examine main effects and interactions of ovarian and embryonic data within each trial (Snedecor and Cochran, 1967). Hormone data were analyzed by split plot analysis of variance (Gill and Hafs, 1971). Significant differences between hormone profiles were determined using Bonferroni's tftest (Miller, 1966), modified for serial correlation according to Albers (1978). Comparison of slopes was used to detect differences between hormone regression lines (Gill, 1978). RESULTS Estrus Synchronization To facilitate collection of ovarian and embryonic data, estrous cycles of all animals within each trial were synchronized. Results are presented in Table 4. Progesterone concentrations > 1 ng/ml were assumed to represent presence of a functional corpus luteum (CL). In Trial 1, 24 of 27 cows had progesterone levels in excess of 1 ng/ml. Even though the remaining 3 animals had less than 1 ng/ml of progesterone, standing estrus was observed within 56 hours following PGF20c injection. At the time of the second PGFZQ injection in Trial 2, all heifers had progesterone concentrations in excess of 1 ng/ml, indicating a CL was present. Eighty-one percent of cows in Trial 1 were observed in standing estrus with the remainder showing definite estrual behavior. Similarly, in Trial 2, 25 of 26 heifers displayed standing estrus while the remaining heifer exhibited estrual behavior. In Trial 3, 5 of 8 heifers manifested standing estrus with the remaining 3 demonstrating estrual behavior. The length of time from the second PGFZQ injection to estrus was nearly equal across all 3 trials and averaged close to 60 hr. However the variation was quite high with a range from 28 to 120 hr. 45 46 TABLE 4. ESTROUS SYNCHRONIZATION OF EXPERIMENTAL ANIMALS Progesterone c Concentration b Body Weight Trial (ng/ml) Hours to Estrus (kg) 1 mean 2.34: 1.22 58.2: 19.7 518.5: 74.2 range 6.02 to 0.38 120 to 28 658 to 376 2 mean 4.91: 1.34 58.9: 17.2 405.7: 24.5 range 7.49 to 1.87 96 to 34 447 to 356 3 mean __d 61.4: 7.0 467.3: 46.0 72 to 52 526 to 386 aSerum progesterone concentration at time of 2nd PGFZd injection. bHours to standing estrus or estrus behavior following 2nd PGFZu injection. CWeight taken at time of 2nd PGFZQ injection. dNo blood samples were taken from cattle in Trial 3. 47 Trial 1 The objective of Trial 1 was to test two doses of EPE with and without the addition of HCG for effectiveness to induce multiple ovulations (Table 5). EPE treated animals had a greater number of anovulatory follicles 2 10 mm relative to controls (P< .05) demonstrating that EPE is a very potent stimulus of follicular growth. As a result of large variation, number of corpora lutea observed (0 to 96) and number of embryos recovered (0 to 21) in cows treated with EPE did not differ from controls. Compared to EPE treatments without HCG, EPE treatments with HCG resulted in fewer corpora lutea and a greater number of anovulatory follicles 210 mm (P< .05). HCG was effective at suppressing ovulation at both low and high dose of EPE as no interaction between dose of EPE and HCG was present. Total ovary weight, stromal and luteal tissue weight and follicular fluid weight increased as dose of EPE increased. The addition of HCG reduced the weight of stromal and luteal tissue and increased follicular fluid weight which is consistent with the decreased number of corpora lutea and increased number of anovulatory follicles 210 mm observed in HCG treated cows. Trial 2 The objective of Trial 2 was to determine the effect of once or twice daily injections of 2 different doses of EPE. A comparison between the same dose of EPE injected once daily for 2 or 3 days was made. In addition, a five day, twice daily treatment of FSH—LH used 48 50523.3 Emcee”; 23 .3 32058 in; .8 2983 cam... 93 mm 3393.8 9:. 33m? £33223 BEES.» 2: .3 33028 $33.58 3.7.36 52: :8 Leg .395... :8... 9:. we 33¢..me PB $33.. £35383; .m— E... m— .3 :2 .3 :2. :o .2; 8m .8... .02: .02: .o u 82o .3338..me .3 use .2 .3 .2 .3 want :o 75.2.: as. 3827303.: com 58 .83 .83 .83 u Eu 3.8 53:9 .3 E; m— .3 .m. .m. 9:6 co A325 as. 33.....3035 omm 6mm 68 .08 63 u may 38 33m £23.58an m3. mmo. mmo. 723.62 983.3 N.Nwflm.8_ 93.“va ".416 mm T»: 3:: .33228 2o. 35. moo. 9: “a.mm m.$ No.3 M23 “9mm 9; 3...: md .40.: mm ..u: 2.33 :33 EB 3523 N2. owe. Bo. mézuwdpw “.mmflwdt o.¢mwp.e: fiwvflwdg mam—6.. um 703 350 Fave.— emm. 02. «aw. ”.mwed NKAmK o.—mw.o Tweed w.oflm.o “53.389. 3.295 .oz vmw. m8. v3. odwwwd— mécemdv Ndpwefi 72:15 oduo; wwwfi: .2038 .02 mom. So. So. m.mflo.m~ 92.1.72 wdawfiw oéwmg 90.36 nee. 328—328 “.3339... .02 NS. ..8. am. :2; 2.198 3.3.? 2.2.3 9:35 H...... 33.328 “.8229... .2. .5509.qu 8: ”Eu 30o 08: 3 an: obs ecu: 3 8: 3: 3.5.5”. 33.. moanitcmmm $3 38 :3: muau $8 :3 — 4551132231 zo .m .39: 49 by Colorado State University (Elsden et_al,, l976) was included to provide a positive control. The data for cows treated with EPE for 3 days is included in Table 6. None of the parameters measured were affected by dose of EPE or number of injections per day and no interaction was detected. A single daily injection of EPE for 3 days resulted in more corpora lutea and embryos recovered than the same dose of EPE given once daily for 2 days (Table 7). The number of anovulatory follicles le mm was not affected by treatment. Cows treated with FSH-LH had more corpora lutea and embryos than cows treated with EPE for 3 days (Table 8). Further reduction in the number of anovulatory follicles 210 mm was observed with the FSH-LH treatment relative to EPE treatments. Trial 3 An objective of Trial 3 was to ovulate anovulatory follicles le mm incurred on EPE treatments. The EPE treatment period was extended from 3 to 5 days and estrous cycles were synchronized with progestogen (Syncro-Mate 8CD). Results of comparing an equal total dose of EPE given by 2 regimens are presented in Table 9. Number of corpora lutea and number of embryos recovered were greater for constant dose EPE than for variable dose EPE. Number of unovulated follicles le mm were significantly higher for variable dose EPE than constant dose EPE (P< .05). The large dose of EPE given on day 5 of the variable dose treatment appeared to be detrimental to the number of ovulations. Many follicles were stimulated on this treatment but a low percentage ovulated. 50 :53:ng P895? 93 .3 35:8 “3:358 .5323... 32.53 93 .3 33028 .350 .3 332. 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N.wuflw.m m.m Ho.¢ cm ..pz Uwzrm Lm—zorFFOL mam. w.~ H_.o_ F.0F “N.m~ no ..u; mammvp mep3F vcm _mEogpm omm. m.m Ho.m_ o.o_ Hm.n— um ..pz xgm>o _mpOH mmF. m.o Hm.m F.m Hm.w omew>oqu moxgnEw .oz m¢_. m.w Hm.¢ o.m Hm.__ ummy:_ mgoagou .02 five. m.qm Ho.NN m.m Ho.m 055 OF Nmmruw__0$ umpm_:>oc: .02 mo_. a.mp Hm.mm m.m Ho.m_ uEE m wmm_uT_—om vaa_:>o:: .oz mocmmwmwfimwm swam wmoo anmwgm> mmmm wmoo pcmpmcou m 4o .m m4m .l). In Trial 2, heifers with a poor ovulatory response had significantly lower progesterone levels 55 8 0 TRIAL 1 Poor response 2 _ l \ G d 0 se (25 6.0- A 00 resp n u: _ 2: 8 4.0- f u: 5 _ Control 3 o 2.0- I: n. )- c I l I l l J Hr: 800 2000 800 2000 800 2000 1 72 3 DAY OF GONADOTROPHIN INJECTION 8-0 TRIAL 2 3 L 5 Good response \ (g 5.0 - E m . z 2 4,0- Poor response ., m *- F u: 3 o 2.0 - c: a. _ o A 1 n I 1 4 4 Hr: 800 2000 800 2000 800 2000 800 1 2 3 4 DAY OF GONADOTROPHIN INJECTION Figure 6. Progesterone profiles during the period from initiation of gonadotropin injection to PGFZd injection. PROGESTERONE (NG/ML) PROGESTERONE (NG/ML) 3.0- TRIAL 1 2.5r 20' 1'5" rPoorresponse Goodresponse 1.0- _5- bControl c J 1 l 1 l l 1 1 l J 4 I l 1;] I) 12 18 24 30 36 42 48 HOURSAFTERPGFZG 3.0 [ TRIAL 2 2.5- 2.0 - 1.5- 1.0- l‘ r/Poor response '5‘ \ Good response 0 l l l l l J l l l l l l l J (J 12 18 24 30 36 42 48 HOURSAFTERPGF2a Figure 7. Progesterone profiles during the period from 12 to 48 hrs after PGan injection. 57 throughout the period of gonadotropin treatment prior to injection of PGan (P< .0l). Following injection of PGan (Figure 7), both cows with good and poor ovulatory response in Trial 1 maintained elevated progesterone concentrations relative to controls (P< .05). However, the progesterone profiles did not differ between good and poor responders. Similarly, in Trial 2 animals having good or poor ovulatory response did not differ in progesterone level following PGFZa injection. Concentration of estradiol l78 in jugular serum is plotted in Figure 8 with regression lines drawn. In Trial l the rate of increase in estradiol l7B for cows with good and poor ovulatory response was significantly higher than that for control cows (P< .Ol). Cows with good ovulatory response reached peak estradiol l78 vluaes on day 5 while cows with poor ovulatory response did not reach peak values until day 6. Comparison of animals with good and poor ovulatory response revealed no difference in the slope of regression lines for both Trials 1 and 2. As seen in Figure 9, a significant positive correlation existed between the number of corpora lutea and serum progesterone concentration on day 7 postestrus in both Trials 1 and 2 (P< .0l). The correlation in Trial l was 0.52 while a correlation of 0.77 was observed in Trial 2. Embryo Recoveryiand Condition of Embryos Cows within each trial were grouped according to the ovulation rate and the mean within each group was determined (Table ll). The number of embryos recovered was then expressed as a percentage of the ESTRADIOL (PG/M L) ESTRADIOL (PG/M L) 58 TRIAL 1 Poor response 80.0 I A 60.0 — Goodresponse 40.0 - 20.0 - Control 0 1 2 3 4 5 6 DAY OF GONADOTROPHIN INJECTION L 2 80.0 TRIA 60.0 40.0 Goodresponse 20.0 A i Poorresponse o l J l J 1 2 3 4 5 6 DAY OF GONADOTROPHIN INJECTION Figure 8. Estradiol I78 concentrations during period of gonadotropin injection with regression lines. 59 TRIAL 1 40 [ R ==O.52 A O -‘ P < .01 :5 - A (\5 Y = 2.28 + 0.18X E w 20- E: (3 fi 0 ° *— (0 Lu (5 \C) E o o L 160 .L, 9 <10 1 1 J 1 0’ J 0 20 40 60 80 100 NO. OF CORPORA LUTEA TRIAL 2 4o 3 ’- 2 r- \ (D E w 20 E a: R = 0.77 E P < .001 U) A w v = 2.07 + max (5 C) L: Q 0 1 l l 1 J 1 l J 0 20 40 60 80 100 NO. OF CORPORA LUTEA Figure 9. Relationship between number of corpora lutea and serum progesterone concentration On day 7 postestrus. 6O o o q_ N m.¢m 00A 0 o «F m o.m¢ own—m mo — o.¢N mm N m.oN RN m m.NN omi—N mm _ o.u_ mm N m.w_ oN m N.NF ON-©_ oo— _ o.__ FR 9 m.m— o _ o.N_ m_-__ COP _ 0.0 mm m o.o o o_-m ON N m.N me w ©.N mN o N._ m-— ANV z :mmz ANV 2 com: Axv z cam: mcovpm_:>o umgw>oumm uwcw>oowm uwcm>oowm mo cwnszz mozgnsm mozgnEm moxgasm m Fave» N _wwLH P _mwgh ommm>oumm mo>mmzm 4~az~v m4m mzothm o.< momo BAN: uo uqu on 95an 0 A NA AA 5: a.m.A n.$ m 0 0 3 _mA A.3 0.NA A.3 A N gouge: 0N: 0 A m a 0.0N 0.2 0.2.. m 0 0 0A 5 mén 0.3 0.0m A n vuououox anon o a 0 N 0.5 «.3 0.: AA 0 NA 0 o A.oA n.NA 0.NN A n ouxcfio on: A 0 0A 0 0.02 n.mN A.moA AA 0 A AN 2 0.0NA mNN a.m.: A n A5895: mm: o 0 2 e A.m.~ N.0A a.m.o A com 0 0 NA AA a.mm 06A 0.0m A m A5895: on: + .A 0N 0N 0 «.00 fimm A.n0A m mam 0 0 0 0 0.3 n.0N 900 A o anemone: we: 0.8 30A 0 AN 0 «A A.mm 0.: A.nAA A N on N nA ANA 0.: mNm A N 033.20 .3: 0 2 m N m.wN n.0m A.3 m .A 2 N 0A m.A.A 0.2.. 0.2 A n vuououom no: 0 Nn n MN n.Am Con 0.00 A 0 3 n 2 0.2 A.mN 5.: A .A 9:55: :2 m NA < 2 TN oéN 93 AA m AA A 0 N.AN 0.NN 5.3 A 0. 0.53.3: Nuns 0 A 0 MA 0.¢N mJA 0.0m AA 0 A A NA A.0N A.mA NJ} A N Aux—.25: AnNN n NN A n 06 0.3” m8.» AA mmw n 3 o .A A6 «.mN n.mm A 0 vnouuuum <0: anon 30A 0 0 0 N n. «.0 A.m m 0 A 0 0 n. n.“ 0.0 A N cucuouom an: A A 0 N m. 0.0 0.» AA 0 0 A q n.N in 5m A n Amaze: fine“ 0 0 0 mA n.A 0.m 0.0 AA 0 A A NA 0A 0.0 N.AA A .A 2330: nNnN A A 0 N n.N N; 0.2 AA 0 0 0 o «N nfi 0.2 A N B893: 0n: Aouuaoo a 5.803 nousA an. 0AM as aw w ..u3 w ..u3 w ..u3 augm om< 000.5 .02 J: nahunam can mumu «...—canoe noAUAAAom noAUAAAom 33h 033:. Anon—3A .396 .EAE .02 A38. .02 .02 .oz “333:8 0cm Aweouum A38. A Amm2m oz< zo AA02A m.< mAmo A<20A>A02A m.< H.305. LITERATURE CITED LITERATURE CITED A1bers, w. 1978. Testing the mean of a norma1 popu1ation under dependence. Anna1s. Statist. 6:1337. A11en, D. M., and G. E. Lamming. 1961. Nutrition and reproduction in the ewe. J. Agr. Sci. 56:69. Avery, T. L., M. L. Fanning and E. F. Graham. 1962. Investigations associated with the transp1antation of bovine ova. II. Superovu1ation. J. Reprod. Ferti1. 3:212. Be110ws, R. A., D. C. Anderson and R. E. Short. 1969. Dose-response re1ationships in synchronized beef heifers treated with fo11ic1e stimu1ating hormone. J. Anim. Sci. 28:638. Betteridge, K. J. 1977. Embryo transfer in farm anima1s. A review of techniques and app1ications. Canada Dept. of Agricu1ture, Monograph 16. Booth, W. D., R. Newcomb, H. Strange, L. E. A. Rowson and H. B. Sacher. 1975. P1asma oestrogen and progesterone in re1ation to super- ovu1ation and egg recovery in the cow. Vet. Rec. 97:366. Brand, A., M. H. Aarts, D. Zaayer and N. D. Oxender. 1979. Recovery and transfer of embryos by non-surgica1 procedures in 1actating dairy catt1e. (In press.) Burfening, P. J., D. C. Anderson, R. A. Kinkie, J. Wi11iams and R. L. Friedrich. 1978. Synchronization of estrus with PGF2 in beef catt1e. J. Anim. Sci. 47:999. a Butcher, R. L., w. E. Co1ins and N. w. Fugo. 1974. P1asma concen- tration of LH, FSH, pro1actin, progesterone and estradi01-17B throughout the 4—day estrous cyc1e of the rat. EndocrinoI. 94:1704. Cahi11, L. P., J. C. Mariana and P. Mau1eon. 1979. Tota1 fo11icu1ar popu1ations in ewes of high and 10w ovu1ation rates. J. Reprod. Ferti1. 55:27. Convey, E. M., T. w. Beck, R. R. Neitze1, E. F. Bostwick and H. D. Hafs. 1977. Negative feedback contro1 of bovine serum LH concentration from comp1etion of the preovu1atory LH surge unti1 resumption of 1utea1 function. J. Anim. Sci. 45:792. 80 8T Danner, M. L., w. D. Oxender, R. L. Fogwe11, and R. H. Doug1as. 1979. Use of an equine pituitary extract with and without HCG to superovu1ate cows. Theriogeno1ogy 11:96. Abstr. Dickman, Z. 1969. Shedding of the zona pe11ucida. In Advances in Reproductive Physio1ogy. V01. 4. Edit., A. McLaven. Dow1ing, D. F. 1949. Prob1ems of the transp1antation of ferti1ized ova. J. Agric. Sci. Camb. 39:374. Dunn, T. G., J. E. Inga11s, D. R. Zimmerman and J. N. Ni1tbank. 1969. Reproductive performance of 2-year—o1d Hereford and Angus heifers as inf1uenced by pre— and post—ca1ving energy intake. J. Anim. Sci. 29:719. E1sden, R. P., S. Lewis, I. A. Cumming and R. A. S. Lawson. 1974. Superovu1ation in the cow fo11owing treatment with PMSG and prostag1andin an. J. Reprod. Ferti1. 36:455. Abstr. E1sden, R. P., J. F. Has1er and G. E. Seide1 (Jr.). 1976. Non— surgica1 recovery of bovine eggs. Theriogeno1ogy 6:523. Eisden, R. P., L. D. Ne1$on and G. E. Seide1 (Jr.). 1978. Superovu1ating cows with fo11ic1e stimu1ating hormone and pregnant mare's serum gonadotrophin. Theriogeno1ogy 9:17. Ensminger, M. E. 1970. Breeding sheep. In Sheep and W001 Science. The Interstate Printers and Pub1ishers, Danvi11e, I11inois. Fevo1d, H. L. 1939. Extraction and standardization of pituitary fo11ic1e-stimu1ating and 1uteinizing hormones. EndocrinoT. 24:435. Fevo1d, H. L., and S. L. Hisaw. 1934. Interactions of gonad stimu1ating hormones in ovarian deve1opment. Am. J. Physio1. 109:655. Ford, S. P., and F. Stormshak. 1978. Bovine ovarian and pituitary responses to PMSG and GnRH administered during metestrus. J. Anim. Sci. 46 1701. Gengenbach, D. R., N. Butendieck, P. M. Riek, R. L. Scipioni, E. B. 01tenacu and R. H. Foote. 1978. Contro11ed superovu1ation in dairy heifers using prostag1andin F2a and pregnant mare serum gonadotropin. J. Anim. Sci. 46:1293. Gi11, J. L. 1978. Design and Ana1ysis of Experiments in the Anima1 and Medica1 Sciences. 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Anim. Sci. 29:602. Zimmerman, D. R., H. G. Spies, E. M. Rigor, H. L. Se1f and L. E. Casida. 1960. Effect of restricted feeding, crossbreeding, and season of birth on age at puberty in swine. J. Anim. Sci. 19:687. 'MOLGQ padweas 019p aqi 401;e pauannaa s; xooq JL pafiueuo aq [HM 33]ng 'pqooaq .mox u wou; inoxoaqo $1111 aAomaJ SEIHVHGI‘I 01 doap xooq u; aoeld 381V1831VW SNINMDLEIH “SW 6891.1) 1.861 1.2 ["0 9136 .Iossapxd 101']: an aouapg LPUJLUV u; wasp—Tw— .10} smoulexinbex 0111 go wowugmg spmmoi poidaooe uaeq seq JBUUQG alpu L'] UOJKN Aq pewasexd SMOCJ 31V10A0d3dfl$ 0i iDVdiXE AdVlInlId 3NIT103 NV :10 ADV/31333 P91111119 $159111 9111 1qu 6311190 01815qu 1111111111111111111|1111 IIIIIIIIIIIIIIIIIIIIIIIIII E N “”11111111111111111 1 999999999999