MSU RETURNING MATERIALS: P1ace in book drop to remove this checkout from LIBRARIES .—3—_ your record. FINES_ W11] be charged if book is returned after the date stamped below. MAINTENANCE OF PROGESTERONE RELEASING INTRAVAGINAL DEVICES (PRIDE) AFTER pcrza INCREASES SYNCHRONIZATION OF LB SURGES AND ESTRUS IN HOLSTEIN HEIFERS BY Benon M. Kanyima AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1984 ABSTRACT MAINTENANCE OF PROGESTERONE RELEASING INTRAVAGINAL DEVICES (PRIDs) AFTER PGan INCREASES SYNCHRONIZATION OF LH‘SURGES AND ESTRUS IN HOLSTEIN HEIFERS BY Benon M. Kanyima Objective was to increase precision of LH surges and estrus with reduced progesterone and increased basal LH. Control and experimental heifers received PRIDs (2% progesterone) for 7 and 10 d. Controls received BLANKs (0% progesterone) for 3 d after PRIDs removal. All received PGFZQ on day 7. After PGFZa: blood from experimental heifers (Exp. I) was collected 4-hourly for 72 h. Then from all heifers, blood was collected 4-hourly for 12 h, 2-hourly for 72 h and 4-hourly for 36 h. Intervals from PRIDs removal to LH surges in experimentals (32.2 i_l.8 h; range 26-38 h) varied less (p < .05) than controls (59.0 i 6.1 h, range 34-78 h). Schedules for PRIDs and PGan in Exp. II were as for Exp. I. After PRIDs removal, observations for estrus were every 4 h for 96 h. Benon M. Kanyima Intervals from PRIDs removal to estrus in experi- mentals (40.0 i 1.2 h; range 24-56 h) varied less (p < .05) than controls (60.8 i 2.2 h; range 36-88 h). Maintaining PRIDs after PGan synchronizes LH surges and estrus in Holstein heifers. Dedicated to Mom, Dad, Brothers, Sisters, and Brenda ii ACKNOWLEDGMENTS My sincere thanks and appreciation are extended to: Dr. R. L. Fogwell, my major Academic Advisor for his faithful guidance, constructive criticisms and availabi- lity to assist me during production of this thesis, Dr. John B. Kaneene and Dr. David A. Morrow, for their dedication to my work while they served as members of my guidance committee; Faculty and Graduate Students parti- cularly Alex Villa-Godoy and William Enright for their collaborative effort and availability to assist me during my experiments; and to the rest of my friends for their moral support during the 2 years of my stay at MSU. Heartfelt thanks and love are extended to Brenda Mbabazi Kanyima for her patience and understanding during various stressful situations and to Susan Mwebesa, Penina Tukahiirwa, Metu Bagyenyi, Charles Atuheire and Nathan Twinamasiko for their continuous prayers and words of encouragement. Final gratitude is extended to my parents Rev. Canon K. and Mrs. M. Kanyima, my brothers Kenneth, David and Andrew and my sisters for their continuous prayers, encouragement and rememberance while I was on this mission. iii Last but not least, I don't forget the soul of Goretti Turyahabwe. I remember Fred T. Bujuuri for the encouragement he provided me while he lived. May he be in comfort wherever he is? God knows what happened! iv TABLE OF CONTENTS Page LIST OF TABLES O O O O O O O O O O O O O Vii LI ST OF FIGURES O O I O O O O O O O O 0 Vi ii INTRODUCTION . . . . . . . . . . . . . 1 REVIEW OF LITERATURE. . . . . . . . . . . 4 Introduction. . . . . . . . . . . . 4 General Survey of Bovine Estrous Cycles. . . 5 Artificial Control of Bovine Estrous Cycles . 8 Progestagens. . . . . . . . . . . 9 Long term progestagens . . . . . . 9 Short term progestagens . . . . . . ll Prostaglandins . . . . . . . . 13 Two injections of PGFZG spaced 10 to 12 days apart . . . . . . . . 14 Single injection of PGcmx . . . . 15 Combination of Short Term Progestagens with luteolytic substances. . . . . . . . 15 Short term progestagens combined with estrogens. . . . . . 16 Short term progestagens combined with single injection of PGan . . . . . 18 Causes of Variation in Intervals from PRID- PGF o.Synchronization Treatment to Estrus in Hei ers . . . . . . . . . . . . 20 Effect of Stage of an Estrous Cycle on Precision of Synchronized Estrus and Ovulation in Heifers . . . .. 21 Function of CL at removal of PRID and/or injection of PGFZa . . . 22 Effect of Rates of Growth and Deve10p- mental Status of Ovulatory Follicles. . 24 Effect of Exogenous Concentrations of Progesterone in Peripheral Circulation and Injections of PGF2a . . . . . . . . 26 Control of Preovulatory Follicular DevelOpment 29 Role of gonadotropic hormones . . . . . 30 Page Interaction of ovarian steroid hormones and pituitary gonadotroPic hormones during synchronized estrous cycles of heifers. . 31 Rationale of Increasing Precision of Estrus and Ovulation using PRID-PGFZG to Synchronize Estrus in Cattle . . . . . . . . . . 32 Outline of Review of Literature . . . . . 33 ”TERIES AND METHODS . O O O O O O O O O 3 5 Preliminary Trial. . . . . . . . . . 35 Objectives . . . 35 Animals, Treatments, Sampling of Blood Storage of Sera and Quantification of Progesterone . . . . . . . . . . 35 Experiment I . . . . . . . . . . . 36 Objectives . . . . . . . . . . . 36 Design . . . . . . . . . . . . 36 Sampling of Blood. . . . . . 37 Handling of Sera and Quantification of LH and Progesterone . . . . . . . . . 40 Statistical Analysis. . . . . . . . 40 Experiment II . . . . . . . . . . . 41 Objectives . . . . . . . . . . . 41 Design . . . . . . . . . . . . 42 Detection of Estrus . . . . . . . . 42 Statistical Analysis. . . . . . . . 45 RESULTS Preliminary Trial. . . . . . . . . . 46 Experiment I . . Experiment II . . o o o o o o o o o b 0 57 DISCUSSION . O 0 O O O O O O O O O O O 65 APPENDIX . . . . . . . . . . . . . . 77 BIBLIOGRAPHY . . . . . . . . . . . . . 81 vi TABLE LIST OF TABLES Page Occurrence of preovulatory surges of LH after removal of PRIDs from diestrous Holstein heifers presynchronized to days 6 to 8 of an estrous cycle. . . . . . 53 Occurrence of preovulatory surges of LH after removal of PRIDs from all diestrous Holstein heifers . . . . . . . . . 58 Detection and onset of estrus after removal of PRIDs from Holstein heifers. . 61 vii FIGURE LIST OF FIGURES Follicular and endocrine changes from luteal regression to resumption of luteal function in cows. . .. . . . . . . Design of Experiment‘I: Temporal rela- tionship of Treatments and Sampling of BlOOd O O O O O O O O O O O 0 Design of Experiment II: Temporal rela- tionship of Treatments and Observations for Estrus O O O O O O O O O O 0 Concentrations of Progesterone maintained by PRIDs after PGan in Diestrous Holstein heifers. . . . . . . . . Basal concentrations of LH and concentra- tions of Progesterone maintained during 3 days of PRIDs after PGF a in presyn- chronized Diestrous Experimental heifers. Basal concentrations of LH and concentra- tions of Progesterone, maintained during 3 days of PRIDs after PGF a in all Diestrous Experimental he fers . . . . Distribution of onset of Preovulatory surges of LH after removal of PRIDs from Diestrous Holstein heifers . . . . . Distribution of Onset of Estrus after removal of PRIDs from Holstein heifers . viii Page 39 44 48 52 56 60 INTRODUCTION Reproduction has been identified as most limiting to efficient production of farm animals (Cartwright, et a1, 1980). Consequently, it is justified to study control of reproduction in farm animals in order to achieve or increase their reproductive potentials. Since domestication, cattle have been an impor- tant source of food for man. Ability of cattle to uti- lize low quality nutrients in forages to synthesize high quality proteins (eg. milk and meat) makes cattle the least-cost and most efficient food resource for humans. Necessity to control reproduction of cattle arises from the need to increase reproductive efficiency through enhancement of superior genetic potentials for their productivity. This in turn ensures availability of food for humans. Improvements of reproductive efficiency of cattle is limited by biology of gestation. The average gestation period of 283 days means that, the maximal achievable reproductive performance in cattle is limited to annual calving. Research into breeding and genetics of dairy cattle has enabled the Holstein cattle to produce large quantities of milk at an early age (eg. 24 mo.). There 1 is great advantage in perpetuating this milk production potential both within and among cattle breeds and success- ful reproduction is the key to perpetuation of this poten- tial. Artificial insemination (AI) is presently, the common and most available practical method to perpetuate superior genetic material among cattle. For AI to succeed, cows or heifers ideally, should be inseminated at a stage of estrus which ensures maximal conception. One major obstacle to increased usage of AI is detection of estrus especially in Beef cattle and Dairy heifers. Methods to increase ability of cattlemen to detect estrus accurately or better eleinate the requirement to detect estrus would enhance use of AI in the cattle industry. Ferti- lity range of bovine spermatozoa is 24h; within which ovulation should occur. Thus fixed time AI or AI without detection of estrus is a group of cattle results into fertilization if precision of synchronized estrus or ovulation is within a range of 24h. In addition to AI, faster perpetuation of superior genetic material in cattle can be achieved through superovulation and embryo transfer (ET) technologies. However, for successful transfer of embryos to a large number of recipients, stage of an estrus cycle of the donor and recipients needs to be synchronous. It appears that for full realization of reproduc- tive potentials of cattle, through AI and ET, methods to artificially synchronize estrous cycles have to be employed. Thus increased precision of estrus and ovula- tion following estrous synchronization would reduce inaccurate timing of AI and ET. This would increase reproductive efficiency and enhance genetic progress of cattle. It is the intention of this research to investi- gate if maintaining l to 2 ng/ml of progesterone for 3 days after PGan will increase precision of preovula- tory surges of LH and estrus in Holstein heifers. REVIEW OF LITERATURE Introduction Cattle are polyestrous and capable of reproduc- tion independent of season. Duration of bovine estrous cycles varies within and among cattle but ranges from 18 to 24 days (average a 21 d) Asdell, 1964, Desjardins and Hafs, 1968; Morrow, 1969). Ovarian steroids (progesterone and estradiol 178) are two major hormones, controlling bovine estrous cycles. Concentrations of ovarian steroid hormones influence secretory patterns of gonadotropic hormones (luteinizing hormone (LH) and Follicle Stimulating hormone (FSH)). Concentrations of gonadotropic hormones secreted from anterior pituitary gland, are required for growth and development of ovarian follicles (Peters, 1979). Artificial methods have been develoPed to mimick and/or modify secretion of reproductive hormones for purposes of controlling reproduction. Progesterone, estradiol 178 and luteolytic prostaglandins can be used for synchronizing estrus and ovulation. Various routes of administration for synchronization treatments are employed for the different regimens used in synchroniza- tion of estrus and ovulation of cattle. Currently, 4 regimens for estrous synchronization, use either proges- tagens combined with either estradiol 178 (Roche, 1978; Roche, 1979a) or prostaglandin F20 (PGFZa) or its analo- gues. Use of combination regimens for estrous synchroni- zation increases percentages of cattle, responding to estrous synchronization treatments (Britt, 1979). In addition, PGF a can be administered alone for estrous 2 synchronization of cattle. Single injections or double injections of PGFZa, spaced 11 days apart can synchronize estrus in cattle (Thatcher, 1976). General Survey of Bovine Estrous Cycles A diagramatic description depicting hormonal and ovarian follicular changes during bovine estrous cycle is shown in Figure 1 obtained from a review article (Hansel and Convey, 1983). Follicular growth and func- tion occur continuously throughout the bovine estrous cycle. Ovulation takes place 10 to 12 h after surge of gonadotropic hormones (luteinizing hormone (LH) and Follicle Stimulating Hormone (FSH)) when concentrations of progesterone in serum are less than 1 ng/ml. Concentrations of progesterone in serum of cattle begin to decline on day 16 or 17 of an estrous cycle (Hansel et a1, 1973) following endogenous secretion of estrogens by ovarian follicles (Ireland, 1984). Secretion of estrogen by ovarian follicles occurs after middle of Figure l.--Follicular and endocrine changes from luteal regression to resumption of luteal function in cows (Hansel and Convey, 1983). Progesterone (no/ml) LH (no/mi) Fol liculor Growth // 6.- 4-é/ I'L—Ic—g“ (’9 2. l I PROGESTERONE g/ 05"- 4—H-—u———II-x/ 60 40—- ‘u’ (x 20 "’ ‘ x 4 “ [LH]2 NW1 [LH] 2 '0: fla f“. 0‘ HOURS {:2 . -' 20 E SILL-OAA-F-ASH T \ \‘ ," p._o.‘_.:‘ou_§s [IZA—IL '0 g 4 . Jr 2 l . ”2.x“ \\ I‘, I lessees, \ ’ ‘a'sfia‘r/ \\ , "' 6 U) I. A I yak \ I - 4 ‘1- LHW \ }/ K‘ \ I H ,. I,/"‘ \x . . J ' I 1m»)? \u—x—xJ—a—x 2 l 1 1 1 ' 1 L 1 1 l 1 l l _J ’3 '2 'I O l 2 3 4 5 6 7 8 Days Relative to the Gonodotropin Surge the bovine estrous cycle (Ford et a1, 1975). Estrogens stimulate the endometrium to synthesize and release prostaglandin F20 (PGan) (Horton and Poyser, 1976; Bartol et a1, 1980). PGan is locally transferred via utero-ovarian vasculature (Ginther, 1976) to the ovary bearing a corpus luteum (CL) to cause luteal regression. Concurrent with declining concentrations of progesterone, after PGF2a—induced luteal regression, basal concentra- tions of LH increase (Hobson and Hansel, 1972; Chenault et a1, 1975). Increased basal concentrations of LH after luteal regression probably promote follicular growth when concentrations of progesterone in serum, are low (Roche and Ireland, 1981b). Artificial Control of Bovine Estrousggycles Objective of artificial methods for controlling reproduction in cattle, is to synchronize estrous cycles in a group of cattle without impairing fertility so that maximum numbers of synchronized-cattle can be bred within a specified time interval. Artificial control of estrous cycles is achieved through regulation of endogenous secre- tion of reproductive hormones. Ideally, inhibition or induction of follicular growth and ovulation achieved by regulating lifespan of CL, will synchronize bovine estrous cycles (Hafez, 1969). Thus, basis of artificial methods for controlling bovine estrous cycles is to prevent ovulation by administering progestagens or to induce luteal regression by administering luteolytic substances. Following withdrawal of progestagens or luteal regres- sion, follicular growth and ovulation occur (Hafez, 1969; Roche, 1976b). For purposes of this review, progestagens and luteolytic substances (estrogens and prostaglandins) used for synchronizing estrus of cattle, will be dis- cussed. Progestagens Administration of sufficient dose of progesterone or synthetic progestagens to cows, will suppress estrus without affecting lifespan of the CL (Christian and Casida, 1948). To-control estrus progestagens can be administered for 18 to 21 days (Long term progestagens) or 7 to 12 days (Short term progestagens). Luteolytic substance can be administered at the beginning (Wiltbank and Kasson, 1968) or the end (Wishart, 1974a; Roche, 1976b) of a short-term progestagen treatment, to enhance estrous response. Long term progestagens Several progestagen compounds can be administered to control estrus and ovulation for a duration of 18 to 21 days. In cattle some progestagens can be administered 10 by oral route (eg. Fluorogestone Acetate (FGA), Chloro- acetoxy progesterone (CAP)) but others (eg. norproges- terone (SC 21009) and melangesterol acetate (MGA)) are administered via subcutaneous route (Mauleon, 1974). Intramuscular route for SC 21009 or MGA cannot maintain sufficient concentrations of progesterone in serum of cattle to block estrus (Mauleon, 1974), hence intra muscular route is not commonly used for administering progestagens. Intravaginal route for administering progestagens indicated that FGA could diffuse across vaginal mucosa easily. Thus long term progestagens can be administered intravaginally to control estrus in cattle. Progestagens are withdrawn after 18 to 21 days affla:corpora lutea in all treated cows have regressed spontaneously (Britt, 1979). Essentially all treated cows will be in follicular phase of an estrous cycle when progestagens are withdrawn (Roche, 1979a). Given that lifespan of CL is not affected by progestagens dura- tion of exposure to progestagens among follicular phase animals is variable. However, most of the treated cattle manifest behavioral signs of estrus 2 to 6 days after withdrawal of progestagens (Roche, 1976a). The degree of synchronization within a specific period of 24 h to 48 h after withdrawal of progestagens ranges from 68 to 98 percent (Mauleon, 1974). 11 Non-synchronization occurs in less than 10 percent of the animals (Mauleon, 1974). Theoretically precision of synchronized estrus after long term progestagen treatment would be sufficient for application of artificial insemi- nation (AI) at a fixed time. But fertility of cattle following either natural or artificial insemination was low (Jochle, 1972; Roche, 1974). There was a possibility that high concentrations of progesterone in serum due to administered progestagens, was detrimental to fertility. However, it appeared that detrimental effecr was associated with duration of progesterone and variable onset of follicular phase among treated cattle. Residual effect of exposing ovarian follicles t0 long term progestagens further reduced fertility of treated cattle in the ensuing estrous cycle by 7 to 10 percent (Mauleon, 1974). Long term progestagens have been withdrawn from being used for synchronizing estrous Cycles of cattle but MGA can still be administered via subcutaneous implants orfeed to prevent occurrence of estrus and ovulation in feedlot heifers. Short term progestagens Short term progestagens for synchronizing estrus in cattle have been described (Wiltbank and Kasson, 1968; Mauleon, 1974; Roche, 1974; Sreenan and Mulvehill, 1975b). Subcutaneous route for administering short term 12 progestagens is commonly used. However, Roche (1975) described a method which used Progesterone Releasing Intravaginal Devices (PRID) to administer short term progestagens. Description of the method for installing PRID'sinvagina of cattle was discussed (Roche, 1979a). Fertility of cattle after synchronized estrus using short-term progestagens for 12 days is not impaired (Mauleon, 1974; Roche, 1974; Sreenan, 1975a; Wiltbank and Gonzalez-Padilla, 1975; Roche, 1976c) in contrast to using short term progestagens for 14 days. Thus impair- ment of fertility of cattle synchronized with progestagens is alleviated by minimizing duration of treatment with progestagens. However, precision of synchronizing estrus within 24 to 48 h is reduced unlike when long term progestagens are used. Percentage of synchronized cattle, manifesting estrus within a specified period of 48 h after short term progestagens ranged from 45 to 67 per- cent in contrast to 68 to 98 percent after long term progestagens (Mauleon, 1974). It is evident that despite ability of short term progestagens to maintain fertility of treated cattle, possibility for using short term progestagens for AI at fixed time, is limited because of assynchrony of intervals from withdrawal of progestagens to estrus or ovulation among cattle. Consequently there is need 13 to increase precision of synchronized estrus and ovulation. Prostaglandins Local or systemic injections of PGan or its analogues effectively cause luteolysis between day 5 and 16 or 17 of bovine estrous cycle (Rowson et a1, 1972; Lauderdale, 1972; Louis et a1, 1972; Cooper, 1974a). PFGza or its analogues do not cause regression of CL in cattle during day 0 to 4 or 17 to 21 of an estrous-cycle (Rowson However 0 to 4) come if istered et a1, 1972; Louis et a1, 1973; Hafs et a1, 1974). non responsiveness of CL during metestrus (days or follicular phase (days 17 to 21) can be over- injections of PGFZa or its analogues are admin- using either of the following options: 1. two injections of PGF 1 spaced 10 to 12 2a days apart (Cooper and Furr, 1974b; Heersche et a1, 1974; King and Robertson, 1974; Graves et a1, 1974; Hafs et a1, 1975; CooPer, 1974a). 2. One injection of PGan with modifications in the management as follows: 1PGonL will refer to both PGFZa and its analogues. 14 (a) prior ascertainment of CL by palpation of ovaries per rectum before injection of PGFZa, or (b) breed all animals detected in estrus 4 to 6 days prior to injection of PGan into the remaining animals (Lauderdale et a1, 1980). Two injections of PGan spaced 10 to 12 days apart: This protocol causes luteal regression in all randomly cycling cows or heifers (Cooper, 1974a; Hafs et a1, 1975). Intervals from second injection of PGde to estrus range from 48 h to 120 h (Cooper, 1974a) up to 168 h (Tanabe and Hann, 1984; Smith et a1, 1984). Pre- cision of synchronized estrus varies with parity of cattle (Roche, 1979a) and stages of an estrous cycle (Macmillan et a1, 1978; Jackson et a1, 1979, Refsal and Sequin, 1980; King et a1, 1982; Tanabe and Hann, 1984). Assynchrony of intervals from treatment to estrus does no facilitate AI at fixed time (Britt, 1979). Thus, conception rates after AI performed at fixed time eg 72 h (Cooper, 1976; Roche et a1, 1977; Hansel et a1, 1978) or 80 h (Thimonier et al, 1975; Smith et a1, 1984) are lower than when AI is scheduled by detection of estrus. However, conception rates following two artificial 15 inseminations at fixed times eg-72 h and 90 h (Lauderdale, 1975) or 72 h and 96 h (Roche, 1976b), are comparable to conception rates after AI scheduled by detection of estrus; or spontaneous estrus (Lauderdale et a1, 1974; Louis et a1, 1975; Hafs et a1, 1975). Single injection of PGFZo This treatment protocol requires availability of labor as an essential component for it to work. Assynch- rony of intervals from single injection of PGan without employing modifications in management as described, only qualifies this technique as aid to detection of estrus rather than a method to use for fixed time AI. It appears that unless two injections of PGan are to be used to synchronize estrus of cattle for fixed time AI, new methods should be developed to overcome the inability of single injection of PGan to synchronize estrus in randomly cycling cattle. Combination of Short Term Progestagens with luteolytic substances: Additional to methods using progestagens or prostaglandins independently, combination of the two synchronization agents may result into synchronization of estrus (Lauderdale, 1975; Thimonier et al, 1975; Nancarrow and Cox, 1976a; Roche, 1976a; DeBenedetti et a1, 1977, Chupin and Pelot, 1977). 16 The major advantage from using combination proto- cols is that synchronized estrus occurs in all randomly cycling cattle (Wishart, 1974a; Roche, 1976a) and dura- tion of progestagen treatments is reduced (Roche, 1976a; O'Farrell, 1977; Roche, 1978) without impairment of fer— tility (Thimonier et a1, 1975; Roche, 1978). Both estrogens (Wiltbank et a1, 1975) and PGan (Thimonier et al, 1975) have been used to reduce duration of short term progestagens to 9 days (eg. with synchromate B) or to 7 days (eg. with PRID-PGan). Short term progestagens combined with estrogens Administration of estrogens to cattle after middle of spontaneous estrous cycle, causes luteal regression (Wiltbank et a1, 1961; Niswender et a1, 1965; Brunner, 1969). However, injections of 5 mg of estradiol benzoate at beginning of short term progestagen treat- ments in cattle,reduce estrous response to synchroniza- tion (Roche, 1974). Piper and Foote (1965) reported that estrogens administered early during an estrous cycle of ewe, cause lengthening of the cycle due to increased hflfibtflxfic support. However, if 5 mg of estra- diol benzoate are administered together with 50 mg of progestagen at beginning of short-term progestagen treatment, estrous response to synchronization is 17 increased (Roche, 1974; Wiltbank and Gonzalez-Padilla', 1975; Sreenan, 1975a; Webel, 1976). Controversy exists about dosage of estradiol benzoate to use in synchroniza- tion regimens. Injection of 5 mg instead of 7.5 mg of estradiol benzoate synchronizes estrus without impairing fertility (Whitman et a1, 1972; Burrell et a1, 1975). Thus high precision is required for determining dosage of estradiol benzoate which may curtail applicability of short term progestagen-estrogen protocol. However, the protocol is recommended for synchronizing estrus in beef cattle (Wiltbank et a1, 1971; Whitman et a1, 1972; Burrell et a1, 1972; Roche, 1976a; Wiltbank and Gonzalez- Padilla', 1975). Intervals from removal of short term progestagen (subcutaneous implant) to estrus, range from 1 to 4 days (Wiltbank et a1, 1971, D'addammio et a1, 1972). Approxi- mately 65 percent of synchronized cattle manifest signs of estrus within 48 h to 72 h of withdrawing progestagens (Wishart and Drew, 1977). Consequently, this synchroni- zation method is recommended for fixed time AI, 48 h to 54 h after withdrawal of progestagen. In spite of the method of synchronization being recommended for fixed time AI in beef cattle, require- ment of high precision in determining dosage of estradiol benzoate probably curtails fertility cattle since 18 conception rates after fixed time AI are lower than would be expected from estrous response. Short term progestagens combined with single injection of PGan Concentrations of progesterone released from progestagens block occurrence of estrus and ovulation without affecting lifespan of CL. Injections of 25 mg of PGan into cycling cattle after receiving progestagens for 6 to 7 days, causes luteal regression in all CL- bearing cattle. Cattle originally between days 0 to 5 of an estrous cycle at installment of progestagens would be between day 6 and 12 of an estrous cycle. Con- sequently protocols using short term progestagens for 6 to 7 days before PGan, synchronize estrus in all randomly cycling cattle (Thimonier et a1, 1975; Britt, 1979). Methods for administering short term progestagens have been reviewed. The common method for administering short term progestagens is via PRID (Roche, 1979a; Smith et a1, 1980; Smith et a1, 1984) especially if synchroni- zation protocol involves PGFZa. Reports indicate that more cows are bred to synchronized estrus with PRID-PGFZa combinations than by two injections of PGan spaced 11 days apart (Thimonier et a1, 1975; Chupin, 1977; Roche, 1979a; Smith et a1, 1980; Smith et a1, 1984). l9 Intervals from removal of progestagens to estrus range from 24 h to more than 120 h (Smith et a1, 1984; Tanabe and Hann, 1984). Percentage of synchronized cattle 48 h to 72 h after removal of progestagen (40 to 60 percent) is low (Stauffer et a1, 1976; Roche, 1979a). Overall, lack of precisely synchronized estrus and ovula- tion reduces fertility to fixed time AI in contrast to AI, scheduled by detection of estrus (Macmillan et a1, 1980). Variation of intervals from treatment to estrus and ovulation has been discussed (Britt, 1979; Macmillan et a1, 1983/84) as a major limitation to successful A1 at fixed time (Nancarrow and Cox, 1976a). There are con- troversial reports regarding conception rates or fertility of cattle inseminated artificially after detection of estrus following synchronization with PRID-PGFZa. Reports agree that conception rates or fertility of cattle after synchronization with PRID-PGan combinations, are similar to those of controls (Wishart, 1974a; Thimonier et a1, 1975; Delatang, 1975; Heersche et a1, 1979). However, Chupin and Pelot (1976) reported that there is tendency for conception rates to be lower when AI is scheduled by estrus after PRID-PGan (35.6%) than after two injections of PGan (45.9%) or Norgestomet-estradiol benzoate (48.4%). On the contrary, conception rates following AI at a fixed time are not different between treatments 20 (Chupin and Pelot, 1976). The cause of reduced fertility reported above is not known given that results in a recent report (Smith et a1, 1984) favor the opposite view. To make AI by appointment (AI at fixed time) feasible possible causes of variation in fertility must be identified and understood. Then methods must be developed to control the identified causes, so as to ultimately increase precision of ovulation after treat- ments used for estrous synchronization. Causes of Variation in Intervals From 'PRID-PGan'Synchronization 'Treatment To Estrus in Heifers In a previous discussion, intervals from removal of PRID to estrus were reported to range from 24 h to more than 120 h, however, Smith et a1 (1984) has reported that injection of PGan, one day before removal of PRID increases precision of synchronized estrus. Similar results were reported by Thimonier et a1 (1975). Smith et a1 (1984) reported that conception rates of heifers synchronized with PRID-PGan and bred to AI at fixed hour of 84 h (66%) were comparable to conception rates of non-synchronized controls, (73%) bred to AI after detection of estrus. It is possible to increase precision of estrus by modifications of PRID-PGFZa 21 regimen. However, modifications of currently used pro- cedure of PRID-PGan is limited. PRID is required to prevent occurrence of estrus for a minimum of 6 days in order for injections of PGan to induce luteal regression in all randomly cycling cattle (Wishart and Young, 1974b;Roche, 1979a). Duration of PRID in vagina of the cow should not exceed 12 days (Roche, 1979a) otherwise fertility after removal of PRID will be reduced. To understand variation of intervals from treat- ment with PRID-PGFZa to estrus and ovulation, possible causes and variation have been identified: 1. stage of an estrous cycle at injection of PGan and (or) removal of PRID (Roche, 1979a; Tanabe et a1, 1984). 2. concentrations of progesterone in peripheral circulation at removal of PRID. Effect of Stage of an Estrous Cycle on Precision of Synchronized Estrus and Ovulation in Heifers Possible influence of stage of an estrous cycle on precision of synchronized estrus has been reported (Macmillan et a1, 1978; Jackson et al, 1979; Refsal and Sequin, 1980; King et al, 1982; Tanabe and Hann, 1984; Stevenson et al, 1984). Stage of an estrous cycle at removal of PRID and (or) injection of PGFZo may be influenced by: 22 a) function of CL at removal of treatments for estrous synchronization. b) rate of growth and development (Scaramuzzi et a1, 1980) or developmental status of ovulatory follicles (Nancarrow and Cox, 1976 Ireland and Roche, 1982b). Function of CL at removal of PRID and/or injection of PGF a 2 Variation of onset of estrus after injections of PGan on any day of diestrus (King et a1, 1982) has been attributed to variable response of CL to luteolytic doses of PGF2a (Momont and Sequin, 1984). The CL becomes fully functional by day 10 of an estrous cycle. However, estrous response of heifers to luteolytic doses of PGFZa administered on day 11 of an estrous cycle (13.3%) is less than when PGan is administered on either day 7 (88.4%) or day 15 (73.5%) (Tanabe and Hann, 1984). Similar to the report above, Stevenson et al (1984) observed 97 percent of heifers in estrus if PGFZa administered on days 5 to 8 in contrast to 83 percent of heifers observed in estrus if PGan is administered on days 14 to 16 of an estrous cycle. Detection of few heifers in estrus after PGFZG on day 11 of an estrous cycle is probably related wave patterns of bovine follicular growth. Two periods of 23 follicular growth and atresia occur in heifers before their midcycle (Ireland and Roche, 1982b). From days 3 to 7, of an estrous cycle a single ovarian follicle develops in heifers but does not ovulate because of inhibitory effect of high (> 1 ng/ml) concentrations of progesterone in peripheral circulation. Atresia of non- ovulatory follicles occurs concurrently with onset of a second wave of non ovulatory follicular growth between day 7 and 13 of an estrous cycle (Ireland and Roche 1982b). Given that around midcycle of heifers, func- tional transition for estradiol 178 occurs simultaneously with attainment of full functional status of CL; these ovarian changes may affect estrous response to injections of PGFZa around day 11 of an estrous cycle. On the contrary, insufficient control of luteal function in heifers (Roche, 1978) by injections of PGFZa (Nancarrow et a1, 1974) may be associated with concentra- tions of progesterone secreted by CL (Watts et a1, 1984). Rates of estrous response after injections of PGFZa into diestrous heifers were positively correlated to concen- trations of progesterone at the time of injections (Watts et a1, 1984). Further evidence is required to define relation- ship between follicular growth and CL function at mid— cycle of cattle. In general however, data by Momont 24 and Sequin (1984), Tanabe and Hann (1984) and Stevenson et a1 (1984) suggest that maximal precision of synchro- nized estrus within a 24 h period, can be attained if regimens for synchronization are designed to maximize numbers of cattle receiving PGFZG on day 8 or 15 of an estrous cycle. Effect of Rates of Growth and Developmental Status of Ovulatorprollicles Wave patterns of bovine follicular growth were described (Rajakoski, 1960; Mariana and Nguyen Huy, 1973; Staigmiller et a1, 1982). Determination of concentra- tions of follicular fluid steroid hormones and receptors for gonadotrOpins in qranulosa cells of bovine follicles on different days of bovine estrous cycle, gave further evidence for existence of wave patterns of bovine folli- cular growth (Ireland and Roche, 1983). Preovulatory follicles develop from small antral follicles into large ovulatory follicles (Richards, 1980). During bovine estrous cycle follicular growth and atresia occur con- currently and continuously independent of stages of an estrous cycle (Choudary et a1, 1968). Luteal regression enhances ability of bovine ovaries to develop ovulatory follicles (Matton et a1, 1981) and facilitates the largest antral follicle, present on the ovary, 3 days before estrus, to ovulate (Dufour et a1, 1972). Developmental 25 status of ovarian follicles affects precision of synchro- nized estrus (Nancarrow and Cox, 1976a; Ireland and Roche, 1982b; Macmillan et a1 1983/84). On the contrary rate of growth and development of ovarian follicles at the end of treatments for synchronizing estrus, could affect preci- sion of estrus and ovulation (Scaramuzzi et a1, 1980). However, Ireland and Roche (1982b) hypothesized that pre- sence or absence of ovulatory follicle on ovary at removal of PRID and (or) injection of PGan may be more critical with respect to precision of estrus and ovulation than just rates of growth and development of ovarian follicles. In principle, PRID-PGan regimen mimicks hormonal events preceeding occurrence of spontaneous estrus but probably does not ensure homogeneous stages of ovulatory follicles among cattle. Variation in stages of ovarian follicles among synchronized cattle is likely to affect precision of estrus and ovulation. Therefore, failure to precisely synchronize estrus or ovulation in a group of cattle may be inherent in the complexity of bovine ovarian follicular system. Modifications of synchronization pro- cedure so as to homogenize follicular growth among cattle should probably increase precision of ovulation. However, through understanding of involvement of steroid and gonado- tropic hormones in control of bovine ovarian follicles is required (Nancarrow and Cox, 1976a). 26 Effect of Exogenous Concentrations of Progesterone in Peripheral Circulation and Injections ofPGF2_ In ewes, long term progestagens impair sperm transport and consequently reduce fertility (Quinilivian and Robinson, 1972; Jennings and Crowley, 1972). Short term progestagens are recommended for synchronizing estrus of cattle (Manuleon, 1974) on the basis that ferti- lity is not impaired (Roche, 1979a) but do not induce sufficient estrous response unless combined with either estrogens (Wiltbank and Kasson, 1968) or PGfo (Roche, 1976b). Pathological effects of progesterone from short term progestagens have not been established in cattle (Gordon, 1976). Manifestation of estrus occurs in pre- sence of less than 1 ng/ml of serum progesterone (Webel et al, 1974; Roche, 1977b). Variation in intervals from injection of PGFj: to estrus is related to decline in serum progesterone. However, other changes associated with decline in serum progesterone and injection of PGan are probably important. Injections of PGFZa, 2 days before removal of a 9-day subcutaneous implant (SC 80996) increased precision of synchronized estrus (Thimonier et al, 1975; Chupin and Pelot, 1977) because concentrations of progesterone in serum of synchronized cattle at removal ofimplant were .5 ng/ml (Thimonier et a1, 1975). Smith et a1 (1984) however, observed increased precision of 27 synchronized estrus, if PGFéa was injected 1 day before removal of a 7-day PRID.2 Increased synchrony of estrus was attributed to PRID being able to maintain 3.5 to 4.0 ng/ml of progesterone in serum of heifers, 24 h after PGFZa as previously Suggested (Mauer et a1, 1975; Hansel and Beal, 1979). However, mechanism by which presence of progesterone in serum of heifers, after PGan increases precision of estrus, is not known. Increased precision of estrus and consequent high conception rates following AI at fixed time (Smith et a1, 1984) was perhaps due to both decreased variability of rates of decline in postluteolytic progesterone and prim- ing effect of PGF a (Hansel and Beal, 1979). However, 2 intervals from removal of PRID after PGan to occurrence of LH surge, vary directly with percentage of progesterone contained in PRID (Roche and Ireland, 1981b) suggesting possible effect of postluteolytic concentrations of pro- gesterone on LH. Given that 75 percent decline of proges- terone from CL occurs within 12 h after PGan (Spicer et a1, 1981) and luteolysis is possibly complete within 24 h of PGan; feasibility of PRID reducing rate of decline in serum progesterone is difficult. Perhaps if differ- ence in progesterone between PRID and serum after PGFZa 2PRID is 6.75% progesterone by weight of silastic. 28 is small, retaining PRID for more than 24 h after luteo- lysis may maintain progesterone in serum, within ranges that are steadily declining. PGFZG induced priming would possibly manifest via release of GnRH to influence secre— tion of endogenous gonadotropic hormones from pituitary gland. In turn, gonadotropic hormones, would influence develOpment of ovarian follicles. In contrast to this view, progesterone inhibits secretion of gonadotropic hormones at hypothalamic level (Shoenmann et a1, 1983). Given that half-life of PGFZG is very short (Glew, 1982) and concentrations of both endogenous (CL) and exogenous progesterone (PRID) in serum are likely to still be high enough to inhibit secretion of gonadotropins, influence of PGan-induced priming on observed precision of synchro- nized estrus and ovulation is still to be determined. Nancarrow and Redford (1976b) hypothesized that induction of follicular diapause for a specific period, in synchronized cattle which normally would manifest estrus and ovulation early after synchronization, could facilitate development of ovulatory follicles in the late responders, such that developmental stages of ovarian follicles will be homogeneous among synchronized cattle. HOwever this hypothesis depends on possibility of increas- ing basal concentrations of gonadotropic hormones, to prevent normal occurrence of atresia (McNatty et a1, 1982) during follicular diapause. 29 Further understanding of relationship or interaction between progesterone and gonadotropic hormones after luteal regression, is required, before better methods to increase precision of synchronized estrus. Control of Preovulatory Follicular Development Follicular development depends on interplay of two major control mechanisms, located in central nervous system and ovary respectively (Peters, 1979). Follicular growth begins with emergence of ovarian follicles from non-growing pre-antral state, via transitory stages of antral follicle and graafian follicle until ovulation or atresia. Endogenous or exogenous gonadotropic hormones or estradiol 178 do not initiate follicular growth (Ashkol et a1, 1970; Peters et a1, 1973). However, in mice and rats, gonadotropic hormones are required to support orderly development of growing follicles (Richards and Midgley, 1976). Follicular growth, initiated and established before puberty (Peters et a1, 1969) continues throughout all estrous cycles in the animals' lifetime (Govan, 1970; Pedersen and Peters, 1971). 30 Role of gonadotropic hormones Influence of FSH and LH during preovulatory phase affects ovulation (Richards and Midgley, 1976; Richards, 1980). In mice, (Peters et a1, 1975) and ewe (McNatty et a1, 1982), injections of pregnant mare serum gonado- tropin (PMSG) prevent atresia of large follicles. In contrast, hypophysectomy in rats, reduces atresia of medium and large follicles (Ingram, 1953). In ewe, injec- tions of PMSG, 10 h after PGan-induced heteolysis, pre- vents occurrence of normal atresia in small antral follicles yet enhances secretion of estradiol 173 by large antral follicles (McNatty et a1, 1982). Estradiol 178, in rats, is required for FSH to increase its receptors on granulosa cells before FSH promotes follicular growth (Tonetta and Ireland, 1983). In prepubertal rats, basal concentrations of LH, sustained by physiological concen- trations (50 to 70 ng/ml) of exogenous progesterone in serum, support growth of small antral follicles to pre- ovulatory stage (Richards et a1, 1982). In prepubertal heifers, estrus and ovulation are induced following with- drawal of l to 2 ng/ml of serum progesterone (Gonzalez- Padilla' et a1, 1975; Sheffield et a1, 1982). It appears that steroid and gonadotrOpic hormones interact, to control preovulatory follicular development. 31 Interaction of ovarian steroid hormones and pituitary onadotropic hormones during synchronized estrous cycles 0 heifers Development of better techniques to control estrous cycles of cattle require understanding of inter- actions between ovarian steroid and pituitary gonadotropic hormones (Roche and Ireland, 1981a). Role of gonado- tropic hormones during follicular development has been discussed. However, regulatory role of ovarian steroid hormones on secretion of gonadotropic hormones requires further understanding. Progesterone, secreted by CL, is key steroid hormone regulating secretion of LH in sheep (Hauger et a1, 1977) and cattle (Convey et a1, 1977; Roche and Ireland, 1981b). Using a 7 day PRID + PGFZa to synchronize estrus in heifers, Ireland and Roche (1981a) observed a 2 to 3- fold increase in basal concentrations of LH, as concen- trations of progesterone in serum declined after PGFZa. Basal concentrations of LH increased in heifers receiving progesterone via 2% PRID (Roche and Ireland, 1981a) sug- gesting that low but not high (Rahe et a1, 1980) concen- trations of progesterone do not block increase in basal concentrations of LH in heifers. Tonic LH stimulates follicular growth in metestrus rat (Peluso et a1, 1983) but pulses of LH, in ewe (Baird et a1, 1976) stimulate 32 preovulatory follicles to secrete estradiol 178. Role of estradiol 178 during follicular growth as been dis- cussed. However, increased concentrations of estradiol 178, also: 1. modulates response of pituitary gland to endogenous or exogenous gonadotropin releas- ing hormone (GnRH) (Convey, 1973). GnRH stimulates preovulatory gonadotropin surge of LH in-cattle (Beck and Convey, 1977; Kesner et a1, 1982) ewe (Howland et a1, 1977) and gilt (Elsaesser et a1, 1979). In turn increased concentrations of LH (pre- ovulatory LH surge) induce final maturation of ovulatory follicles (Richards, 1980). induce behavioral signs of estrus in cattle, sheet and swine (Asdell et a1, 1945; Glencross et a1, 1980). Peak of behavioral signs of estrus occurs, when concentrations of proges- terone in serum are less than 1 nq/ml (Katongole et a1, 1971; Lemon et a1, 1975; Esslemont et a1, 1981). Rationale for'Increasing Precision of Estrus and Ovulation Using PRID-PGFZE to Synchronize Estrus in Cattle Data reported (McNatty et a1, 1982) suggest that LH and FSH interact synergistically to prevent follicular 33 atresia after PGan-induced luteolysis. Changes in secre- tion of gonadotropic hormones after luteolysis are associated with promotion of 1 to 2 ovarian follicles to ovulation (Brand and deJong, 1973; McNatty et a1, 1981). Ovarian steroid hormones may regulate promotory effects of ovarian follicles through modulated secretion of gonadotropic hormones. It appears possible to increase precision of synchronized estrus and ovulation in cattle. However, artificial methods for synchronizing estrus have to be modified such that concentrations of proges- terone maintain increased basal concentrations of LH after PGFZa, for a period long enough to facilitate homo— geneous stages of follicular development among synchro- nized cattle. Thus, following removal of exogenous progesterone (PRID) synchronized cattle would manifest preovulatory surges of LH, estrus and possibly will ovulate, with increased precision. Outline of Review of Literature . Introduction to Review of Literature. General survey of bovine estrous cycles. . Artificial control of bovine estrous cycles. :5 u N H O . Causes of variation in intervals from PRID- PGFZa synchronization treatment to estrus in heifers. 34 5. Control of preovulatory follicular develOp- ment. 6. Rationale for increasing precision of estrus and ovulation using PRID-PGFZa to synchroni- zation of estrus in cattle. The objective of this thesis was to study if precision of estrus and ovulationis increased by maintain- ing 1 to 2 ng/ml of progesterone in serum of heifers for 3 days with PRIDs, after injection of 25 mg of PGFZG. Increased precision of estrus or ovulation will be moni- tored by magnitude of variances for intervals from removal of PRIDs to peaks of preovulatory LH surge and onset of estrus. MATERIALS AND METHODS PreliminarygTrial Objectives 1. To identify approximate time required for PRID to remain in the vagina of Holstein heifers before concentrations of progesterone in serum are between 1 and 2 ng/ml. 2. To evaluate whether PRIDs will maintain l to 2 ng/ml of progesterone in serum, for at least 3 days after interval defined above (1). Animals, Treatments,'S§mpling of Blood, Storage of Sera and Quantification of ProgeSterone Reproductive organs of five pubertal Holstein heifers were palpated via rectum for evidence of corpus luteum. Heifers were then injected intramuscularly with 25 mg of prostaglandin an (PGan).3 Immediately after injection of PGFZa we installed Progesterone Releasing Intravaginal Devices (PRIDs, containing 2% progesterone by weight of silastic, Appendix 1), into the vagina of heifers. Coccygeal arterial (or) venous blood was sampled once, daily, over an interval of 12 days. Blood was 3Lutalyse,o Upjohn Co., Kalamazoo, MI USA. 35 36 alloWed to coagulate at room temperature of approximately 20°C for 8 h before centrifugation at 30009 to separate serum. Sera were stored at -18°C until concentrations of progesterone were determined by radioimmunoassay (RIA) (Louis et a1, 1973). Experiment I Objectives 1. To determine if basal secretion of LH would change during 3 days when progesterone was 1 to 2 ng/ml. 2. To determine if retention of PRIDs for 3 d after PGF2a affects precision of preovulatory surges of LH which occur after removing PRIDs. Desiqg Seventeen pubertal Holstein heifers were observed for signs of estrus for 30 d and were then synchronized by 3 injections of PGFZa spaced 11 days apart. At the start of the experiment, heifers ranged between 6 to 15 days of an estrous cycle (mode = day 7). All heifers were in diestrus but at injection of PGan (7 days after installment of PRIDs), PRIDs were assumed to be maintain- ing at least 2 ng/ml of progesterone in serum of heifers. Therefore all heifers (n = 12) having more than 3‘: 2 ng/ml of progesterone at injection of PGF2a were presumed 37 in diestrus and heifers (n = 5) having less than 3 i 2 ng/ml of progesterone were in follicular phase of an estrous cycle. PRIDs were installed into vagina of heifers. Seven (7) days after installing PRIDs (approximately day 14 of an estrous cycle for 17 heifers), 25 mg of PGan were injected intramuscularly to cause luteal regression. In experimental heifers (n = 9) PRIDs remained in the vagina for 3 d after injection of PGFZa, thus in experimental heifers, PRIDs stayed for a total of 10 d. In contrast, PRIDs remained in vagina of control heifers (n a 8) for a total of 7 d and was replaced by BLANKs (0% progesterone, Appendix 1) which remained in vagina for 3 d after injection of PGFZa (Figure 2). Sampling of Blood Jugular venous canulae (Ico-Rally Corp., Palo Alto, Calif.) were installed into all heifers 6 days after installing PRIDs (one day before injection of PGFZG) . For experimental and control heifers, blood was sampled immediately before injection of PGFZa (oh). After PGan, blood was sampled from experimental heifers every 4 h for 72 h (Figure 2). After removal of PRIDs, from both experimental and control heifers, blood was sampled every 4 h for 12 h, then every 2 h for 72 h and finally, every 4 h for 36 h (Figure 2). 38 Figure 2.--Design of Experiment I: Temporal rela- tionship of Treatments and Sampling of Blood. 39 Z. x2<4m .50 Ema 330d 000.5 “.0 mam—.524"... p 54 . 3N 3W— zuuzfimm 34218.2. . . . .50 xz<.._m 2. 9mm e I. EQQNQH u (405.200 hwaEwaxu n. v. m. N. I O. m m e. m n v m N _ 0 L0 m><0 q a 1 a . . . s a . . q a 1 . 14 w o ”I44h2m25mexw .50 Sad summon z. Ema 000.5 “.0 mu4e2mwhz. 1 . . . .3 gm .5. “8.5.2sz . mu: JOthOU mu: 4¢mmmO .11 So xzesm I 2. 9%. s e Ashes. E .. 30528 m_¢_m_N___0_mm~.0 menu _ 0 m>mmmmo q an 1: 1.05.200 amt: 45.2msimdxw So: mememz 26.6.5... 44.5%.: "34.2.24 HH hzméEmaxw 45 a heifer that mounted others was observed to discharge clear mucus at the vulva. Artificial insemination (AI) of heifers was 8 to 12 h after detection of estrus. Statistical Analysis Intervals from removal of PRID to onset of estrus were determined from data collected during observations for estrus. Variances of intervals from removal of PRIDs to onset of estrus were contrasted between experimental and control heifers using ratio of variances (Gill, 1978). Chi-square test was used to contrast numbers and percent- ages of experimental versus control heifers detected in estrus or diagnosed pregnant.. RESULTS Preliminary Trial Data from one heifer was eliminated from this study because concentrations of progesterone during the trial indicated that it did not respond to PGan, so data presented.is from 4 heifers. Concentrations of progesterone (Figure 4) during 12 days with PRIDs present, declined from an average of 3.63 i .52 ng/ml at injection of 25 mg of PGF a to .90 i 2 .03 ng/ml at end of trial. Concentrations of progesterone declined (p < .05) from 3.63 i .52 ng/ml to 2.60 i .35 ng/ml within 24 h of installation of PRIDs (Figure 4). Concentrations of progesterone were maintained between 1 and 2 ng/ml (Figure 4) by PRIDs, 3 to 11 days after injection of PGFZa. Given that injections of PGFZa do not induce luteolysis between days 0 and 5 of an estrous cycle, installations of PRIDs for a minimum of 6 to 7 days before injection of PGFZa is necessary to ensure estrous response in all injected cattle regardless of day of an estrous cycle. Thus, for purposes of experiment I and II, installing PRIDs for 7 days prior to PGij was chosen to 46 47 Figure 4.--Concentrations of Progesterone maintained by PRIDs after PGFZa in Diestrous Holstein heifers. 48 xmeoa 5,52 m> .05) from those in 50 control heifers (8.63 i to .63 ng/ml). However, concen- trations of progestrone in serum of experimental heifers (2.64 i .57 ng/ml) 24 h after PGFZd were greater (p < .05) than in controls (1.18 i .11 ng/ml). Concentrations of progesterone were maintained between 1 and 2 ng/ml in experimental heifers (Figure 5), 24 h after PGan until removal of PRIDs. In contrast, concentrations of proges— terone in control heifers, declined below 1 ng/ml when BLANKs were present. At removal of BLANK from control heifers, concentrations of progesterone (.50 i .04 ng/ml) were lower (p < .05) than in experimental heifers (1.07 i .02 ng/ml) at removal of PRIDs. Basal concentrations of LH in experimental and control heifers, increased (p < .02) with time after PGF Following injection of PGan, and before removal 2°" of PRIDs or BLANKs, increase in basal LH among experi- mental and control groups did no differ (p‘) .05) until 60 h after PGFZa; when preovulatory surges of LH occurred in 4 of 5 control heifers with BLANKs present. Range of intervals from removal of PRIDs to pre- ovulatory peak of LH in experimental heifers (12 h) was not different from that in controls (14 h). However, preovulatory surges of LH occurred earlier after PRIDs in experimental heifers (26 to 38 h) than in controls (64 to 51 Figure 5.--Basa1 concentrations of LH and concentra- tions of Progesterone maintained during 3 days of PRIDs after PGon: in presynchro- nized Diestrous Experimental heifer 52 (- _.) Iw/fiu ENOHBISBSOHd 0. N. «Need more was m N 1 a _ _ _ ,I-"il‘l", - a 101/ So QE 1 LLLLI guise N0 g0 0.0 0.0 0.. N._ v.- (o—o) IUJ/GU H‘I 53 78 h). Mean of intervals from PRIDs to preovulatory peak of ' LH (Table 1) in experimental heifers (32.9 i 1.8 h) was shorter (p < .05) than in controls (70.8 i 2.3 h). Vari- ation in intervals from PRIDs to preovulatory peaks of LH (Table l) in experimental heifers was not different (p > .05) from that in controls. TABLE l.--Occurrence of preovulatory surges of LH after removal of PRIDs from.heifers presynchronized to day 6 to 8 of an estrous cycle Groups of heifers Sources of variation Experimental ICSntrol Number of heifers 7 5 Intervals from removal of PRIDs to peak LH: Mean (n) 32.9 + 1.8a 70.8 + ‘ 2.3a Standard deviation (h) 4.7a 5.2a Variance (hz) 22.5a 27.2a Coefficient of variation (%) 14.4a 7.4a Range (h) 26 - 38 64 - 78 aData in a row without common superscripts differ (p < .05). 2. Data from all diestrous heifers was used to determine overall effect of l to 2 ng/ml of progesterone maintained by PRIDs for 3 days after PGFZoL, on basal concentrations LH and precision of preovulatory surges of LH. 54 After 7 days of PRIDs, injections of 25 mg of PGan caused a precipitous decline (9 < .05) of proges- terone in serum of experimental heifers (Figure 6) from 9.40 i 1.32 ng/ml to 2.36 i .36 ng/ml, within 24 h. In contrast, concentrations of progesterone in control heifers, declined from 4.63 i 1.15 ng/ml to .95 i .15 ng/ml within 24 h. Concentrations of progesterone in serum of experimental heifers (Figure 6) were maintained between 1 to 2 ng/ml, 24 h after PGFZa until removal of PRIDs whilst those in controls were less than 1 ng/ml, 24 h after PGFZa when BLANKs were present. Concentrations of progesterone in experimental heifers were greater (p < .05) than in controls before and after injection of PGan. However, decline in concentrations of progesterone was 5-fold in control heifers in contrast to 4-fold in experimental heifers. There was tendency (p < .10) for basal concentrations of LH to increase above concentra- tions at pretreatment time (0b) in experimental heifers (Figure 6) after injection of PGFZa. However, in 2 of 9 experimental heifers, basal concentrations of LH did not increase after PGFZa but declined below baseline for approximately 24 h and then tended to increase, 12 h before removal of PRIDs. In contrast basal concentrations of LH at pretreatment time (Oh) were not different (p > .05) among experimental vs controls. However there was 55 Figure 6.--Basal concentrations of LH and concentra- tions of Progesterone, maintained during 3 days of PRIDs after PGFZOL in all Diestrous Experimental heifers. 56 (.._.) Iw/bu BNOHBISBOOHd Anon. mus: 93 n N _ 1 . _ _ _ ‘ Tllll‘l'l'l‘lllll‘l / N0 v.0 0.0 (0—0) nun/bu H') 57 tendency for basal concentrations of LH in experimental heifers to be lower (p < .10) than controls, 24 h after PGan because preovulatory surges of LH were due to occur in 2 of 8 controls. Range of intervals from removal of PRIDs to pre- ovulatory peak of LH (Table 2) was shorter in experimental heifers than controls. Mean of intervals from removal of PRIDs to preovulatory peak of LH (Table 2).in experi- mental heifers was shorter (p < .05) than in controls. Variation in intervals from removal of PRIDs (Table 2) in experimental heifers, was less (p < .05) than in controls. Percentage of preovulatory peaks of LH occurring within 1 12 h of mean interval (Figure 7) from removal of PRID to preovulatory surges of LH was greater (p < .01) in experimental heifers than in controls. Experiment 11 Overall, 5 of 100 heifers lost PRIDs (experimental) n = 2, control, n = 3). Data on estrus from these heifers were excluded from analysis. Estrus was detected in 81 of 95 heifers (85.3%). Therefore all data analyzed and reported in experiment II are derived from these 81 heifers. Based on ovarian norphology assessed rectally and wide range in concentrations of progesterone (0.2 to 11.3 ng/ml) when PRIDs were installed it appeared that 58 TABLE 2.--Occurrence of preovulatory surges of LH after removal of PRIDs from all diestrous Holstein heifers. Sources of variation Groups of heifers. Experimental Control Number of heifers Intervals from removal of PRIDs to peak of LH: Mean (h) Standard deviation (h) Variance (hz) Coefficient of variation (%) Range (h) 32.2 + 1.8a 59.0 + ” 6.1b ‘ 5.2a 17.2b 27.4a 297.1b 16.0a 29.0a 24 - 38 34 - 73 ab .05) 0 Data in a row without common superscripts differ (p < 59 Figure 7.--Distribution of onset of Preovulatory surges of LH after removal of PRIDs from Diestrous Holstein heifers. PERCENT OF HEIFERS WITH SURGES OF LH 80 —- MEAN: |2h INTERVAL TO PEAK LH (h) 60 - I<———9I =37.5°/o 59.0i6J 4O - f CONTROL . . (n:8) 20 "' 7 / / / / // O l I V l T I MEANiIZh INTERVAL TO PEAK LH (h) 80 - I¢——vl =lOO°/o 32.2i|.8 60 - 2 / EXPERIMENTAL / (n=9) 40 b / ¢ 20 I" / O /1‘ I I H I I I I 0 IO 203040506070 80 90 TIME (h) 61 most, if not all, stages of an estrous cycle were repre- sented. Detection of estrus (Table 3) in experimental heifers was not different (p > .05) from that in controls. However percentage of heifers detected in estrus (Table 3) in experimental group, was greater (p‘< .01) than in controls. TABLE 3.--Detection and onset of estrus after removal of PRIDs from Holstein heifers. Groups of heifers Sources of variation Experimental Control Number of heifers in estrus 44 37 Percent of heifers detected c d in estrus 92 79 Intervals from removal of PRIDs to onset of estrus: Mean (h) 40.0 + 1.2a 60.8 + ‘ 2.2b ‘ Standard deviation (h) 8.0a 13.1b Variance (hz) _ 64.0a 171.7b Coefficient of variation (%) 20.0a 22.0a Range (h) 24 - 56 35 - 88 abData in a row without similar superscript differ (p < .05) o CdData in a row without similar superscript differ (p < .05). 62 Range of intervals from removal of PRIDs to estrus (Table 3) was short in experimental heifers in contrast to controls. Mean of intervals from removal of PRIDs to onset of estrus (Table 3) in experimental heifers was lower (p < .05) than in controls. Variation in intervals from removal of PRIDs to onset of estrus (Table 3) in experimental heifers was less (p < .05) than in controls. Distribution of intervals from removal of PRIDs to estrus (Figure 8) indicated that percentage of experimental heifers detected in estrus within 1 12 h of mean interval from removal of PRIDs to estrus was greater (p < .05) than for control heifers detected in estrus within same range of corresponding mean interval. There was tendency for conception rate (Table 3) in experimen- tal heifers to differ (p < .10) from that in control heifers. 63 Figure 8.--Distribution of Onset of Estrus after removal of PRIDs from Holstein heifers. PERCENT OF HEIFERS DETECTED IN ESTRUS 64 ‘40 " MEAN :I2h Io————oI 365%: 30 T HEIFERS DETECTED INTERVAL TO IN ESTRUS (°/a) ESTRUSHT) 20 .. CONTROL 37(79) 60.8:22 (n84?) IO " / ' T I —r I r If I T r j 50 " IMEAN i-I2h '95 °/o 4o I- a .. a % . EXPERIMENTAL 44(92) 400: I2 (n=48) 20 - /4 '0 / 0L4 ,/<::T7U ' I T I I T T Tfi 243 I 40 48 56 64 72 8O 88 96 TIME (h) DISCUSSION The goal for using PRID to maintain l to 2 ng/ml of progesterone is serum of heifers (Preliminary Trail) so that basal concentration of LH could increase was attained (see Exp. 1(1)). The main objective for increas- ing basal concentrations of LH when PRIDs were maintain- ing 1 to 2 ng/ml of progesterone in serum of heifers was to provide a hormonal milieu facilitatory to follicular development. Thus removal of PRIDs after decreased con- centrations of progesterone in serum of heifers for 3 days after PGan, could permit occurrence of preovulatory surges of LH and estrus with increased precision. This objective was achieved (see Exp. I and II). In Experiment I, concentrations of progesterone declined to less than 1 ng/ml in serum of control heifers within 24 h of injecting PGF 0. Changes in concentration 2 of progesterone after PGFZa in controls were similar to those reported in other studies (Rowson et a1, 1972; Louis et a1, 1973; Lauderdale, 1975; Renegar, 1978). Thus presence of higher concentrations of progesterone in experimental heifers than in controls, 24 h after PGon, was due to presence of PRIDs. 65 66 Increased basal concentrations of LH after PGan occurred in 7 of 9 experimental heifers which originally were at days 6 to 8 of an estrous cycle before PRIDs were installed (Exp. I(l)). Increase in mean LH, 1 to 3 h after PGFZa was in agreement with earlier studies (Louis et a1, 1974, Stellflug et al, 1977, Milvae and Hansel, 1983). After PGFZa, decrease in serum progesterone and increase in concentrations of LH are associated with regression of CL (Ireland and Roche, 1982a). Similar results were observed in the present study (Experi- ment I). Basal concentrations of LH in serum of 7 experimental heifers were maintained for 3 d after PGFZa, when l to 2 ng/ml of progesterone from PRID maintained in serum of heifers. In contrast, increased basal concentrations of LH in 5 control heifers were maintained despite presence of BLANKs, until preovulatory surges of LH. These results suggest that presence of l to 2 ng/ml of progesterone did not prevent post-PGFZa increase of basal LH. Increased basal concentrations of LH after PGFZa in heifers is not maintained 36 h after injection of PGFZa (Schallenberger et a1, 1984). However, the results suggest that in presence of 1 to 2 ng/ml of progesterone, increased basal concentrations of LH were maintained in 7 of 9 experimental heifers up to time of removal of 67 PRID (72 h after PGde) similar to observations during proestrous of cattle (Rahe et a1, 1980). However increased basal concentrations of LH, after spontaneous or PGan-induced luteal regression (Ireland and Roche, 1982a) normally OOmnBIduring decline of progesterone from CL and often preceeds occurrence of preovulatory surges of LH. Thus increased endogenous basal concentrations of LH for 3 days in spite of 1 to 2 ng/ml of progesterone in serum of heifers, suggests that low concentrations of progesterone do not block basal concentrations of LH from increasing after PGFZa but may block preovulatory surges of LH in diestrous heifers (Short et a1, 1979). Increased basal concentrations of LH do not occur if PGan is administered in absence of CL (Hafs et a1, 1975; Furr et al, 1981). Similar results were observed in 2 of 9 experimental heifers in which basal con- centrations of LH did not increase after injection of PGFZa. Concentrations of progesterone at injection of PGan indicated that the two heifers were in follicular phase of an estrous cycle. Thus failure to observe increased basal concentrations of LH in the two heifers after PGFZa was probably due to stage of an estrous cycle. The largest antral follicles on the ovary regress after injection of PGFZd on day 8 or 9 of an estrous cycle 68 of the cow (Ireland and Roche, 1983 ). Atretic follicles become non-responsive to stimulation by LH due to lack of LH receptors in qranulosa and theca layers of the follicles (Ireland and Roche, 1982b). Results from the study suggest that injection of PGan probably caused ovarian follicles to become atretic in the two heifers and presence of 1 to 2 ng/ml of progesterone in serum of heifers caused further decline in basal concentrations of LH observed 36 to 40 h after PGFZa. Progesterone inhi- bits secretion of basal LH by pituitary cells in response to LHRH challenge in vitro (Padmanabhan et a1, 1982; Keech et a1, 1984). Concentrations of progesterone, greater than 1 ng/ml in serum of postpubertal heifers (Roche and Ireland, 1981b), suppress pulsatile secretion of LH after mid luteal phase (Rahe et a1, 1980). Similar observations have been reported in ewe (Karsch, 1977) and rats (Richards et a1, 1982). There is evidence that progesterone treatments result in atresia of follicles in cattle (Maracek et al, 1977) probably because of inhibitory effect of progesterone on FSH-induced increase of estradiol in granulosa cells reported in rats (Schreiber et a1, 1981). Consequently, presence of l to 2 ng/ml of progesterone in sera of 2 experimental heifers having follicular phase, may have caused atresia of large antral follicles. However, 1 to 2 ng/ml in sera 69 of the 2 heifers, did not prevent increased basal LH 12 h before removal of PRIDs. Thus, the two heifers manifested preovulatory surges of LH after removal of PRIDs, which suggests a possibility of development of new ovulatory follicles 12 h before removal of PRIDs. Therefore 1 to 2 ng/ml of progesterone maintained in serum of heifers for 3 d after PGF 0 may facilitate follicular growth. 2 Given that follicular growth and artesia occur continuously throughout an estrous cycle (Rajakoski, 1960; Schams et a1, 1977; Matton et a1, 1981) results in Experiment I(l) and II suggest that possible follicular growth preceeding preovulatory surges of LH coincided with facilitatory effects of 1 to 2 ng/ml of progesterone and increased basal secretion of LH in serum of experi- mental heifers. Sustained secretion of basal LH in ewes (Baird et a1, 1981; England et a1, 1981) and rats (Richards et a1, 1982) stimulates preovulatory follicular growth (McNatty et a1, 1982). Thus increase of basal LH observed in diestrous heifers (Exp. 1(1)) possibly stimu- lated ovulatory follicles, to grow in 7 to 9 experimental heifers which were in diestrus at injection of PGFZa. But occurrence of preovulatory surges of LH in all experi- mental heifers (Exp. I(2)) after removal of PRIDs obviates, stage of diestrus as part of synchronization regimen. 70 Preovulatory surges of LH (Exp. I) and estrus (Exp. II) occurred with increased precision in experi— mental heifers than in controls after 3 days of l to 2 ng/ml of progesterone. These results suggest that 1 to 2 ng/ml of progesterone and its associated changes in LH (and) or probably over reproductive hormones, were neces- sary to increase precision of preovulatory surges of LH and estrus regardless of stage of an estrous cycle at time of injecting PGFZa. However, the mechanisms involved for increasing precision of preovulatory surges of LH and estrus using PRID for 3 days after PGFZa remain to be resolved. In control heifers, range of intervals from removal of PRIDs and injection of PGFZa, to occurrence of preovulatory surges of LH, were similar to intervals, previously reported (Roche and Ireland, 1981b). Inter- vals from removal of PRIDs to estrus were similar to intervals reported after 2 injections of PGFZa, spaced 11 days apart (Kinkie et a1, 1976; Smith, 1976; Macmillan et a1, 1980; Refsal et a1, 1980; Tanabe and Hann, 1984; Stevenson et a1, 1984). Similar results were obtained when PRIDs were installed for 6 days with-PGan on removal of PRIDs (Smith et a1, 1984) or PRIDs for 7 days with PGFZa 1 day before removal of PRIDs (Graves et al,'1975; Stauffer et a1, 1976; Smith et a1, 1984). Regimens for 71' synchronizing estrus with PRID and PGde combination or two injections of PGFZa, do not increase precision of preovulatory surges of LH or estrus. Thus, maintaining 1 to 2 ng/ml of progesterone for 3 days after PGFZa as in experimental heifers was probably a better method to increase precision of preovulatory surges of LH and estrus in cattle. Preovulatory surges of LH occurred shortly after removal of PRIDs, in experimental heifers. There results suggest that physiological changes associated with l to 2 ng/ml of progesterone and increased basal LH probably simulates events of proestrus. Overall, experimental heifers were less variable with respect to occurrence of estrus and preovulatory surges of LH. However, PRIDs maintained for 10 days in contrast to 7 days, (Exp. 1(1)) did not decrease variation of intervals from removal of PRIDs to LH surges in diestrous heifers. Thus, increased precision of estrus and surges of LH in experimental heifers was due to possible presence of homogeneous proestrous state rather than disparity in duration of PRIDs. Synchronization of estrus increases frequency of displaying behavioral signs of estrus because more than one cow are in estrus at the same time (Hurnik et a1, 1975; Esslemont et a1, 1981; Glencross et a1, 1980). 72 Maintaining 1 to 2 ng/ml of progesterone in serum of heifers for 3 days after PGde, increases detection of estrus (Exp. II) in experimental heifers (Table 3). Results indicate that the number of experimental heifers detected in estrus within 1 12 h of mean interval to estrus after PRIDs was greater than in controls. Thus, better detection of estrus in experimental group versus control group, was due to increased synchronization of estrus. There was tendency for conception rate in experi- mental heifers (Table 3) to be lower than in controls. The results suggest that maintaining 1 to 2 ng/ml of progesterone or other experimental conditions could affect fertility of cattle. However, conception rates of experimental and control heifers (Table 3) were compar- able to conception rates reported in other studies (Lauderdale, 1975; Roche, 1976a). The fact that two- thirds of this study was carried out during winter could have affected fertility Normally, cattle in temperate zone have lowest fertility during winter and summer in contrast to spring or fall (Mercier and Salisbury, 1947; Spalding, Everett, and Foote, 1975). In extreme-cases of short day light and low temperatures (Sweetman, 1950). lowest fertility occurred during winter. If season was detrimental to 73 fertility, both experimental and control heifers would have been affected unless subfertility in experimental group was due to interaction between 1 to 2 ng/ml of progesterone or changes associated with it, and environ- ment. However winter season affects fertility of cattle regardless of previous concentrations of progesterone in serum if fixed time AI is performed at 65 h versus 80 h (Jester et a1, 1982). Effects of climatic conditions on plasma progesterone of cattle are conflicting (Wolff Vaught et a1, 1977; Rosenberg et a1, 1977; Roman-Ponce et a1, 1981; Rosenberg et a1, 1982). High basal concen- trations of LH are associated with high concentrations of progesterone in serum of cattle, subjected to stress of high ambient temperature but occurrence of pre- ovulatory surges of LH is not affected (Roman-Ponce et a1, 1981). Given that AI in this study was scheduled by detection of estrus, subfertility observed in experi— mental heifers was probably due to factors other than season. Subfertility due to long term progestagen (Gordon, 1976) can occur because of: 1. altered sperm transport 2. too rapid sperm capacitation 3. defective fertilization 4. abnormalities in ova 74 5. reduced embryo survival 6. change in cervical mucus 7. release of LH out of phase with estrus 8. high estrogen during progestagen treatment However, use of short term progestagen (5 12 days) over- comes subfertility previously due to long term proges- terone (Roche, 1979a). Results from the study (Exp. II) suggest that sub- fertility in experimental heifers was not due to occur- rence of preovulatory surges of LH out of phase with estrus (Table 3) since surges of LH occurred within ranges of intervals from PRIDs to estrus. However,syndhronized cattle may express varying shades of estrous activity and behavioral signs not necessarily indicative of physiological status (Wishart and Young, 1974b). Thus, subfertility in experimental group could be due to AI of some heifers which probably were manifesting false posi- tive signs of estrus due to increased synchronization. There were changes observed in vaginal mucus which were associated with nylon string used to remove PRIDs. Vagi- nal mucus in both experimental heifers and controls, became cloudy whenever the nylon string was in contact with vaginal mucosa. However, more heifers in experi- mental group were affected than in control group. Thus vaginitis due to nylon string could have interferred with 75 insemination and fertility of heifers. Care to minimize contact between nylon string and vaginal mucosa may reduce vaginitis along with other inflammatory changes in vagi- nal mucus and probably to increase fertility of heifers. Major goal of underlying this kind of study is to develOp a method to increase precision of estrus and ovulation so that fertility at AI is increased. Informa- tion learned from the study along with that reviewed sug- gests that maintaining 1 to 2 ng/ml of progesterone in serum of Holstein heifers for 3 days after PGFZa increases precision of preovulatory surges in LH and estrus. Given that 95 percent of heifers or greater, manifested surges of LH or estrus within an interval of 24 h (Figures 4 and 5), maintenance of 1 to 2 ng/ml of progesterone in serum of Holstein heifers, after PGFZa enhances feasibility of AI at fixed time. However further understanding of physiological status of entire reproductive system during presence of l to 2 ng/ml of progesterone in serum of heifers after PGan through to after removal of PRID, is required. Then field application to test this method should follow. In conclusion, 2% PRIDs, installed in vagina of Holstein heifers for 7 days prior to PGan can be main- tained to deliver 1 to 2 ng/ml of progesterone in serum for 3 days after PGF 0, 2 76 Concentrations of progesterone (l to 2 ng/ml) maintained by PRIDs in serum of heifers, for 3 days after PGde, allowincreased basal concentrations of LH in serum of diestrous heifers which probably facilitate ovarian follicular growth to increase precision of LH surges and estrus. Increased precision of estrus and LH surges occur in all heifers regardless of stage of an estrous cycle and fertility of heifers is not affected. 77 APPENDI X APPENDIX Procedure for Making 2% PRIDs or BLANKS The materials for making 9 PRIDs are: 450 g Silastic 382® Medical Grade Elastomer (Dow Corning Corp., Midland, MI). 59.4 g Silastic® RTV Thinner (Dow Corning Corp., Midland, MI). 10.46 g 13-0130® 4 Pregnene-3, 20-dione (Sigma Chemical Company, St. Louis, MO). 90 drops Catalyst M® (Dow Corning Corp., Midland, MI). 9 strips of stainless steel (27.5 cm long, 3 cm wide, and .02 mm thick) supplied by Dept. of Agric. Engineering, Michigan State Univ., East Lansing, MI). Food color (Spartan Stores Inc., Grand Rapids, MI) Silastic 382® Medical Grade Elastomer (450 g.), ® RTV Thinner (59.4 q) and P-0130® Pregnene-3, Silastic ZO-dione (10.46 g) were mixed together vigorously in a plastic jar (1 litre) for 5 to 10 minutes. Then Catalyst M® (90 dr0ps) was added to the mixture Simultaneously with food color (approximately 5 mls). Mixing of the 78 79 five ingredients continued for approximately 1 minute. Food color was used to color-code the product regarding percent progesterone, and to indicate homogenity of mixing. Steel strips were centered in the enclosed mold such that a space of 2 mm was left between perimeters of mold and steel strips. Soon after mixing the thick past was injected under manual pressure, with 50 c.c. Catheter tip-syringes (Plastipak® Disposable syringe Becton-Dickson, Co., Rutherford, NJ) into molds of plexi-glass; so that a thin layer of Silastic (1.14 mm thick) coated the flat sides of the stainless steel strips. A layer of Silastic (2.0 mm thick) coated the perimeter of the stainless steel. Total thickness of PRID was about 2.3 mm. The final products (PRIDs) contained approximately 2% progesterone by weight of Silastic. ® to mixture, further CAUTION: After adding catalyst M mixing lasts approximately 1 minute so as to avoid vulcanization of Silastic mixture before injection into plexiglass molds. Thus working time for injecting Silastic mixture was approximately 10 minutes. After injection of Silastic mixture into molds, curing of PRIDs occurred after 4 to 8 h, depending on ambient temperature. Ambient temperatures between 21 to 24.5°C were ideal. 80 Procedure for making BLANK'S Procedure for making BLANKS was same as for PRIDs except no progesterone (P-0130® Pregnene-B, 20- dione), was involved. BLANKS contained 0% progesterone. After curing and recovery from molds, PRID'S or BLANK'S were each coiled manually to a final coiled diameter of approximately 5.5 cm and length approxi- mately 10 cm. A piece of nylon cord (100 cm long, folded in half) was fixed at one of the ends of PRID or BLANK. Cleaning of molds Following recovery of PRIDs or BLANKS from molds, a blunt instrument (eg. screw-driver) was used to clear out thick remnants of cured Silastic from sides and corners of the molds. Then gauze Sponges (4 in. by 4 in.) (Parke-Davis and Co., Detroit, MI) impregnated with Xylene (AR® Mallinckrodt Inc., Paris, Kentucky) were used to wipe out remnants of cured Silastic until molds were thoroughly clean. Then molds were left to dry in air for at least 2 h before assembling them for new batches of PRIDs or BLANKS. 81 BIBLIOGRAPHY BIBLIOGRAPHY Asdell, S. A., J. DeAlba and J. S. Roberts. 1945. The level of ovarian Hormones required to induce heat and reactiOns in ovariectomized cow. J. Anim. Sci. 4:277. Asdell, S. A. 1964. In 'Patterns of Mammalian Reproduc- tion' (2nd Ed.), Cornell Univ. Press. Ashkol, A., B. Lunefeld and H. Peters. 1970. Ovarian Development in Infant Mice. Dependence on gonadotropic hormones. In 'Gonadotrgpius and Ovarian Development, W. R. Butt, A. C. 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