Pregnancy Rates Per Artificial Insemination for Cows and Heifers Inseminated at a Synchronized Ovulation or Synchronized Estrus 1

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1 Pregnancy Rates Per Artificial Insemination for Cows and Heifers Inseminated at a Synchronized Ovulation or Synchronized Estrus 1 J. R. PURSLEY,*,2 M. C. WILTBANK,*,3 J. S. STEVENSON, J. S. OTTOBRE, H. A. GARVERICK, and L. L. ANDERSON *Department of Dairy Science, University of Wisconsin, Madison Department of Animal Sciences, Kansas State University, Manhattan Department of Animal Sciences, The Ohio State University, Columbus Department of Animal Science, University of Missouri, Columbia Department of Animal Science, Iowa State University, Ames ABSTRACT Received October 19, Accepted June 27, Partial funding for this project was provided by the Regional Project NC-113 of the Cooperative State Research Service, USDA. 2Current address: Department of Animal Science, Michigan State University, East Lansing Send reprint requests to M. C. Wiltbank, Department of Dairy Science, 1675 Observatory Drive, Madison Two synchronization protocols were tested for lactating dairy cows and heifers. Nulliparous dairy heifers (13 to 23 mo; n = 155) and primiparous and multiparous dairy cows (60 to 289 d postpartum; n = 310) were assigned randomly to two treatments. Controls received 25 mg of PGF 2a and were artificially inseminated according to the a.m. p.m. rule following detected estrus. All controls that were not detected in estrus were injected with 25 mg of PGF 2a at 14-d intervals until artificial insemination (AI) at a detected estrus or until timed AI at 72 to 80 h after a third sequential injection of PGF 2a. Treated cows and heifers received a protocol that used GnRH and PGF 2a to synchronize ovulation (Ovsynch). Cows and heifers that were treated with Ovsynch were injected i.m. with 100 mg of GnRH at a random stage of the estrous cycle. Seven days later, cows and heifers in this group received 25 mg of PGF 2a, followed by a second injection of 100 mg of GnRH 30 to 36 h later. Subsequently, the treated cows and heifers received AI 16 to 20 h after the second injection of GnRH. Pregnancy rates per AI were similar (38.9% vs. 37.8%) for control cows and cows treated with the Ovsynch protocol, respectively. However, pregnancy rate per AI was greater for control heifers (74.4%) than for heifers treated with Ovsynch (35.1%). Evaluation of serum progesterone concentrations at each hormonal injection indicated that the first injection of GnRH synchronized luteal function of lactating dairy cows but not of heifers. In summary, one fixedtime AI at a synchronized ovulation provided similar pregnancy rates per AI as did AI following the a.m. p.m. rule after estrus had been induced by PGF 2a in lactating cows, but the fixed-time AI was not effective for heifers because of the lack of synchronization. ( Key words: dairy cattle, synchronization of ovulation, gonadotropin-releasing hormone, prostaglandin F 2a ) Abbreviation key: CL = corpus luteum. INTRODUCTION Methods to synchronize estrus have been developed and used in many dairy herds to manage reproduction more efficiently. Management of AI is more efficient when synchronization schemes are used to reduce the variation to first and subsequent AI (5). Synchronization protocols using approved hormones for lactating dairy cows in the US have been limited to the use of PGF 2a (1, 4, 7, 9, 16, 17). Pregnancy rates per AI were similar when dairy cows were bred at a detected estrus after synchronization of estrus with PGF 2a or after a spontaneous estrus (16, 17). However, estrus is not synchronized precisely with PGF 2a because this treatment does not synchronize growth of follicles but only regulates the lifespan of the corpus luteum ( CL). Thus, detection of estrus is needed over a 7-d period after two doses of PGF 2a are administered 11 to 14 d apart (4, 6, 9, 14). Consequently, when cows received a fixed-time AI at 72 to 80 h after a second injection of PGF 2a, pregnancy rates per AI were considerably lower than those for cows receiving AI at a detected estrus (15, 16, 17). Recently, a novel protocol, Ovsynch, was developed to synchronize ovulation in lactating dairy cows using GnRH and PGF 2a (11). This protocol synchronized ovulation within an 8-h period from 24 to 32 h after the second injection of GnRH. This precise synchrony may allow for successful AI without the detection of estrus (11). The current study was designed to evalu J Dairy Sci 80:

2 296 PURSLEY ET AL. ate pregnancy rates obtained by timed AI after synchronization of follicular growth and ovulation following the Ovsynch protocol compared with those after a traditional synchronization program using PGF 2a and detection of estrus. In addition, a previous study (11) indicated that follicular changes during the Ovsynch protocol were different for lactating cows than for heifers. Thus, a second objective of this study was to compare pregnancy rates per AI for lactating cows and nulliparous heifers in response to these two synchronization strategies. MATERIALS AND METHODS This study was a collaborative project among stations of the NC-113 Project of the Cooperative States Research Service of the USDA entitled Methods for Improvement of Fertility in Cows Postpartum. Lactating dairy cows from three experiment stations (total of four dairy farms) and heifers from four experiment stations (total of five dairy farms) were used in this trial. In this experiment, 310 lactating dairy cows, 60 to 289 d postpartum, and 155 dairy heifers, 13 to 23 mo old, were assigned randomly by location to the two treatments described in Figure 1. Lactating cows were randomly assigned by lactation number (1, 2, and 3) and stage of lactation (60 to 99, 100 to 149, and 150 d postpartum). The mean number of days postpartum ( ±SEM) at the start of treatment was ± 4.3 and 96.0 ± 4.7 for the control and Ovsynch groups, respectively. Cows in the control group received up to three i.m. injections of 25 mg of PGF 2a 14 d apart. Only cows and heifers that were not detected in estrus were given subsequent injections. All control cows detected in estrus after each PGF 2a injection received AI according to the a.m. p.m. rule. All controls that did not exhibit estrus after the third injection of PGF 2a received one fixed-time AI at 72 to 80 h after PGF 2a. Detection of estrus was performed twice daily by herd personnel at each location for 7 d following the first two injections of PGF 2a and for 3 d following the third injection (Figure 1). Cows and heifers treated with the Ovsynch protocol were administered 100 mg of GnRH i.m. Seven days later, cows and heifers received 25 mg of PGF 2a. This group of cows and heifers then received a second injection of 100 mg of GnRH 30 to 36 h later and timed AI 16 to 20 h later (Figure 1). Cows and heifers in the Ovsynch group were not observed for estrus. The semen that was used for AI was chosen by the herd manager as part of routine management of the herd. All injections were performed by or under the supervision of NC-113 collaborators. All AI were performed by the farm personnel that were normally in charge of AI. Pregnancy diagnosis was determined by Figure 1. Description of the timing of injections for the two synchronization methods used in this experiment. Control cows received AI in relation to estrus unless they were not detected in estrus after any of the three PGF 2a injections. Ovsynch cows had timed AI. ultrasound at 25 to 30 d postpartum on approximately 80% of the cows and heifers and by rectal palpation between 35 to 49 d following AI on approximately 20% of the cows and heifers. The pregnancy rate per AI was defined as the percentage of cows or heifers that were confirmed pregnant at the single pregnancy diagnosis (ultrasound or palpation) after one AI. In a subset of cows and heifers, blood was collected to evaluate the progesterone status at the time of injections, to determine the effect of PGF 2a on CL regression, and to determine the estrus detection rate of controls. For control cows and heifers, blood was collected at the time of each PGF 2a injection and at 24 to 48 h after PGF 2a. Blood from cows and heifers in the Ovsynch group was collected at each hormone injection. Serum was evaluated for progesterone concentration using the validated ELISA or radioimmunoassay procedure at each station. Progesterone concentrations were determined to be high at 1 ng/ ml or low at <1 ng/ml. Effect of treatment on pregnancy rate per AI was evaluated by chi-square analysis. The interactions of treatment and stage of lactation (60 to 75 d, 76 to 100, and >100 d), treatment and parity (1, 2, and 3), and treatment and location were analyzed by logistic regression using the CATMOD procedure of SAS (12). No interactions were detected between treatment and parity or treatment and location. However, a trend ( P = 0.14) for an interaction between treatment and stage of lactation was observed. Therefore, stage of lactation (60 to 75 and >75 d) was reanalyzed using chi-square analysis. In addition, interactions of progesterone status and treatment and

3 PREGNANCY RATES USING SYNCHRONIZATION OF OVULATION 297 interactions of pregnancy rate, progesterone status, and treatment were analyzed by chi-square analysis. RESULTS The time from the start of treatment until AI was 13 ± 11 d ( x ± SD) for cows treated with PGF 2a and 9 ± 0 d for cows treated with Ovsynch. As shown in Table 1, lactating cows had a similar pregnancy rate per AI following the traditional PGF 2a protocol (control) or the Ovsynch protocol. In contrast, control heifers had a greater ( P < 0.01) pregnancy rate per AI than did heifers treated with Ovsynch. Also, pregnancy rate per AI was greater ( P < 0.01) for control heifers than for control cows. Cows in the Ovsynch group that were >76 d postpartum had a greater ( P < 0.05) pregnancy rate per AI than did cows that were 60 to 75 d postpartum (Table 2). Analysis of progesterone concentrations in control cows and heifers allowed calculation of an estrus detection rate [percentage of cows and heifers detected in estrus after high ( 1 ng/ml) followed by low (<1 ng/ml) progesterone concentrations] of 66% in cows (n = 160) and 60% in heifers (n = 77). Control cows with high concentrations of progesterone ( 1 ng/ ml) had a greater ( P < 0.05) pregnancy rate per AI (49.3%; n = 83) than did control cows with low concentrations of progesterone (21.2%; n = 19) on the day of PGF 2a injection; however, progesterone status had no effect on pregnancy rate per AI in heifers (high = 82.7%, n = 52; low = 71.4%, n = 7; P > 0.1). Most cows and heifers received AI after the first or second PGF 2a injection; only 18% (lactating cows) and 23% (heifers) received the third PGF 2a injection (Table 3). Of the lactating cows that received the third PGF 2a injection, almost all were bred by timed AI (92%), and the pregnancy rate from this AI was very low (Table 3). In contrast, more than half of the heifers that received the third PGF 2a injection were bred to a detected estrus (Table 3). Control cows that were inseminated at an observed estrus had a greater ( P < 0.01) pregnancy rate per AI than did control TABLE 1. Pregnancy rates in cows and heifers after treatment with PGF 2a (control) or synchronization of ovulation (Ovsynch). Pregnancy rate Control Ovsynch P (%) Cows 38.9 (n = 154) 37.8 (n = 156) >0.10 Heifers 74.4 ( n = 78) 35.1 ( n = 77) <0.01 P <0.01 >0.10 TABLE 2. Influence of stage of lactation on pregnancy rates in lactating cows after treatment with PGF 2a (control) or synchronization of ovulation (Ovsynch). 1 Stage of lactation Pregnancy rate Control Ovsynch Total (%) 60 to 75 d 39.4 (n = 33) 26.0 (n = 50) 31.3 (n = 83) 76 d 38.8 (n = 121) 43.4 (n = 106) 41.0 (n = 227) P No differences between treatments. cows that received timed AI (Table 3). However, pregnancy rate per AI was similar ( P > 0.10) for control cows inseminated only at estrus and for cows in the Ovsynch group. For cows and heifers with functional CL on the day of PGF 2a injection, CL regression occurred (high progesterone on day of injection followed by low progesterone 24 to 48 h later) in all control cows and heifers (100%) and in 109 of 116 cows (94%) and 74 of 77 heifers (96%) in the Ovsynch group. Analysis of progesterone concentrations for cows and heifers in the Ovsynch group demonstrated that a similar percentage of heifers and cows had high progesterone concentrations on the day of the first GnRH injection ( 60%; Table 4). At the PGF 2a injection for cows in the Ovsynch group, the percentage of lactating cows with high concentrations of progesterone increased to 86% ( P < 0.05) but remained at 60% in heifers. The pregnancy rate per AI was similar for cows, regardless of whether concentrations of progesterone were high or low at the time of PGF 2a, but heifers with low progesterone concentrations at the time of PGF 2a injection had a lower pregnancy rate per AI than did heifers with high progesterone concentrations at the time of PGF 2a injection (Table 4). DISCUSSION This study compared pregnancy rates per AI for lactating dairy cows and dairy heifers after two different synchronization strategies. Although both protocols allowed all dairy cows to be bred, the Ovsynch protocol provided a very predictable time of AI after the start of treatment. Ovsynch could benefit dairy operations because it allows for timed AI of lactating cows without detection of estrus. For lactating dairy cows, synchronization of ovulation with GnRH and PGF 2a resulted in a pregnancy rate per AI that was similar to the rate for cows receiving PGF 2a every other week. For those cows, 83% had AI after a detected estrus, and 17% had timed AI. In another study with 333 lactating dairy cows, we also found

4 298 PURSLEY ET AL. TABLE 3. Percentage of cows bred and pregnancy rates per AI after each treatment with PGF 2a. (% bred of total) Lactating cows (% pregnant of bred) (% bred of total) Heifers First PGF 2a Second PGF 2a Third PGF 2a AI to Estrus Timed AI (% pregnant of bred) 1Percentage of pregnant cows was lower ( P < 0.01) for cows bred after third PGF 2a injection than that after first or second PGF 2a injection. similar pregnancy rates for first, second, and third AI using the Ovsynch protocol compared with breeding at a detected estrus (10). In contrast to the 38% pregnancy rate per AI after Ovsynch, only 8.3% of cows conceived to the timed AI after the third PGF 2a injection in the control group. The control cows that received timed AI were probably from a less fertile population, because the other control cows had previously shown behavioral estrus, indicating estrous cyclicity. A similar number of this less fertile population was probably included in the Ovsynch group, which might have reduced pregnancy rate per AI for cows in the Ovsynch group. Thus, pregnancy rates per AI after Ovsynch were similar to breeding after a detected estrus in lactating dairy cows, but Ovsynch did not yield acceptable pregnancy rates per AI in heifers. Pregnancy rates per AI at an observed estrus were almost twice as great for heifers as for lactating dairy cows in the current study. This difference has been apparent in most dairy herds and has been reported previously (8). The reason for this large discrepancy in fertility between cows and heifers has not been well characterized. The obvious differences between cows and heifers include lactation, parity, age, and nutrition (2). These factors may produce differences in oocyte quality and uterine environment that reduce fertility. Second, the response to the Ovsynch protocol by heifers was dramatically different from the response by lactating cows. In contrast to the normal pregnancy rate per AI that was produced by Ovsynch in lactating cows, heifers had a lower pregnancy rate per AI after Ovsynch than after PGF 2a and detected estrus. Ovsynch may be the first synchronization protocol that has performed well in lactating cows but not in heifers. An understanding of the differences in follicular dynamics may explain differences in response to Ovsynch. Timed AI after PGF 2a is ineffective in lactating dairy cows because of anestrous cows (17) and because of the large variation in time from regression of the CL to expression of estrus (15, 16, 17). This variation in time to estrus is due to differences in the developmental stage of the preovulatory follicle at the time of PGF 2a injection ( 3 ) and is not related to the rate of progesterone decrease to basal concentrations (5). The developmental stage of the preovulatory follicle can be controlled by exogenous hormones, such as GnRH (19), which is the basis of the Ovsynch protocol. We previously reported that 85% of lactating dairy cows ovulate after an injection of GnRH at a TABLE 4. Pregnancy rates and progesterone (P 4 ) status in a subset of cows and heifers treated with the Ovsynch protocol. 1Percentage of cows with high P 4 was different ( P < 0.05) at first GnRH versus PGF 2a injection. 2Pregnancy rate was greater ( P < 0.05) for heifers with high ( 1 ng/ml) versus low (<1 ng/ml) P 4. First GnRH injection PGF 2a Injection >1 ng of P 4 /ml <1 ng of P 4 /ml >1 ng of P 4 /ml <1 ng of P 4 /ml (%) (no.) (%) (no.) (%) (no.) (%) (no.) Cows Pregnancy rate / / / /16 % of Cows / / / /116 Heifers Pregnancy rate / / / /30 % of Heifers / / / /74

5 PREGNANCY RATES USING SYNCHRONIZATION OF OVULATION 299 random stage of the estrous cycle, and a new follicular wave begins in all cows (11). One follicle from this new follicular wave is functionally dominant by d 7 (time of PGF 2a ) and can be induced to ovulate with a second injection of GnRH. Heifers are poorly synchronized by Ovsynch. As discussed previously, the success of the Ovsynch protocol is dependent on synchrony of both the ovulatory follicle and the CL. Greater synchrony of CL function was evident in lactating cows because high serum progesterone concentrations were detected at the time of PGF 2a injection in 86.2% of cows compared with only 59.5% of heifers (Table 4). This result agrees with that of our previous study in which we found that the first injection of GnRH stimulated ovulation of a follicle in only 54% of heifers compared with 85% of lactating dairy cows (11). Therefore, in contrast to heifers, cows appear to have a functionally dominant follicle that is responsive to LH for a greater proportion of the estrous cycle. The percentage of heifers with elevated serum progesterone concentrations was not different 7 d following the first injection of GnRH as was found at a random stage of the estrous cycle. Approximately 60% of heifers had high progesterone concentrations at the first injection of GnRH and at PGF 2a injection. Obviously, luteal development was not synchronized by the first injection of GnRH in heifers. The low ovulatory response in heifers, as well as other differences in follicular dynamics, might also decrease Ovsynch success because of a lack of follicular synchrony in heifers. To achieve successful synchronization with the Ovsynch protocol it is particularly critical to ovulate a follicle when the first injection of GnRH is given during the late luteal phase. If a follicle does not ovulate during this period, the cow or heifer may cycle normally and would most likely be in estrus before the second injection of GnRH. If ovulation occurs from the initial injection of GnRH during the late luteal phase, the newly induced CL appears to be unresponsive to the spontaneous pulses of PGF 2a, and, even though the spontaneous CL may regress, the new CL continues to grow until the day of PGF 2a injection (Pursley and Wiltbank, 1996, unpublished data). Lactating dairy cows primarily have two waves of follicular growth during an estrous cycle (18), which makes it likely that there would be a follicle that is responsive to LH during the late luteal phase. Thus, differences in numbers of follicular waves or length of follicular waves may be crucial for determining the success of synchronization with Ovsynch. Follicle growth seems to be more rapid for heifers than for lactating cows (11), and the length of a follicular wave seems to be longer (Pursley and Wiltbank, 1996, unpublished data). Thus, newly emerging follicles of heifers that are not ovulated by the first injection of GnRH may lose dominance status (turnover). Specifically, the heifers that do not respond to the first injection of GnRH would most likely be in the first 3 d of a new follicular wave. Seven days later, at the time of PGF 2a, some of these heifers would be at d 9 or 10 after the initiation of a follicular wave, a time when the old follicle loses dominance and a new follicular wave emerges. Thus, the newly emerging dominant follicle would not be sufficiently mature to respond to the second injection of GnRH. Unlike our previous study (11) in which heifers and cows received the second injection of GnRH at 24 and 48 h after PGF 2a, respectively, both heifers and lactating cows in this study were given the second injection of GnRH at 30 to 36 h after PGF 2a injection. The design of this study did not allow us to evaluate the degree of follicular synchrony in cows versus heifers because sequential ultrasound evaluations of the ovaries were not performed. Extrapolation from previous ultrasound evaluations (11), the present results, and results of others (13) indicate that Ovsynch synchronizes follicular and luteal development in lactating cows, but not in heifers. Practical Implications The ability to achieve acceptable fertility after a timed AI could have a major impact on reproductive management of lactating dairy cows. Breeding to a synchronized ovulation would allow more control in AI programs and would remove dependence on estrus detection before AI. This achievement opens the door to various ways of managing AI and the calving interval. For example, traditional selection of a voluntary waiting period might be replaced by selection of a day of first AI. Managers could eliminate first AI that occur too early or too late in lactation. The data from the present study indicate that this day of first AI should be after 75 d to achieve maximal pregnancy rates per AI (Table 2). Use of this protocol at the University of Wisconsin has allowed all first AI to occur at about 70 d of lactation, and all AI are performed on 1 d of the week. The timeliness of subsequent breedings can also be ensured because all open cows could receive an effective timed AI at 9 to 10 d after pregnancy diagnosis. Potentially, the need for detection of estrus could be eliminated from a reproductive management program. The effectiveness of such a program is improved if early pregnancy diagnosis is performed, for example with transrectal

6 300 PURSLEY ET AL. ultrasonography (10). Thus, synchronization of ovulation with GnRH and PGF 2a provides an effective way to manage reproduction in lactating dairy cows. However, this protocol was not an effective synchronization method for heifers. ACKNOWLEDGMENTS The authors thank Myron Brown of Sanofi Animal Health, Inc., Overland Park, Kansas, for providing GnRH (Cystorelin ) and John Chenault of The Upjohn Company, Kalamazoo, Michigan, for providing PGF 2a (Lutalyse ). Support was also provided by the Wisconsin, Kansas, Ohio, Missouri, and Iowa Cooperative Experiment Stations and Ohio Dairy Farmers Cooperative. We thank Crave Bros. Dairy, Waterloo, Wisconsin; Pfeifer Farms, Bucyrus, Ohio; and Stoll Farms, Marshallville, Ohio, for providing cows and heifers. In addition, we thank Jerry Guenther, University of Wisconsin, Madison; Ann Ottobre, Daniel Grooms, and David Smith, The Ohio State University; Yasuhiro Kobayashi and Betty Hensley, Kansas State University; and Brent Salfen, University of Missouri, for providing technical assistance. REFERENCES 1 Archbald, L. F., T. Tran, R. Massey, and E. Klapstein Conception rates in dairy cows after timed-insemination and simultaneous treatment with gonadotropin-releasing hormone and/or prostaglandin F 2a. Theriogenology 37: Butler, W. R., and R. D. Smith Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci. 72: Fortune, J. E., J. Sirois, A. M. Turzillo, and M. Lavoir Follicle selection in domestic ruminants. J. Reprod. Fertil. 43(Suppl. 1): Hafs, H. D., and J. G. Manns Onset of oestrus and fertility of dairy heifers and suckled beef cows treated with prostaglandin F 2a. Anim. Prod. 21:13. 5 King, M. E., G. H. Kirakofe, J. S. Stevenson, and R. R. Schalles Effect of stage of the estrous cycle on interval to estrus after PGF 2a in beef cattle. Theriogenology 18: Larson, L. L., and P.J.H. Ball Regulation of estrous cycles in dairy cattle: a review. Theriogenology 38: Lauderdale, J. W., B. E. Seguin, J. N. Stellflug, J. R. Chenault, W. W. Thatcher, C. K. Vincent, and A. F. Loyancano Fertility of cattle following PGF 2a injection. J. Anim. Sci. 38: Lee, C. N., E. Maurice, R. L. Ax, J. A. Pennington, W. F. Hoffman, and M. D. Brown Efficacy of gonadotropinreleasing hormone administered at the time of artificial insemination of heifers and postpartum and repeat breeder dairy cows. Am. J. Vet. Res. 44:11. 9 Lucy, M. C., J. S. Stevenson, and E. P. Call Controlling first service and calving interval by prostaglandin F 2a gonadotropin-releasing hormone and timed insemination. J. Dairy Sci. 69: Pursley, J. R., M. R. Kosorok, and M. C. Wiltbank Reproductive management of lactating dairy cows using synchronization of ovulation. J. Dairy Sci. 80: Pursley, J. R., M. O. Mee, and M. C. Wiltbank Synchronization of ovulation in dairy cows using PGF 2a and GnRH. Theriogenology 44: SAS/STAT User s Guide: Release SAS Inst., Inc., Cary, NC. 13 Schmitt, E. J.-P., T. Z. Diaz, M. Drost, and W. W. Thatcher Use of a GnRH-agonist for a timed insemination protocol in cattle. J. Anim. Sci. 72(Suppl. 1):1128.(Abstr.) 14 Momont, H. W., and B. E. Seguin Treatment of unobserved estrus in lactating dairy cows with prostaglandin F 2a products. Page 28 in The Compendium on Continuing Education for the Practicing Veterinarian on Reproductive Management in Food Animals. Univ. Minnesota, St. Paul. 15 Stevenson, J. S., M. O. Mee, and R. E. Stewart Conception rates and calving intervals after prostaglandin F 2a or prebreeding progesterone in dairy cows. J. Dairy Sci. 72: Stevenson, J. S., M. C. Lucy, and E. P. Call Failure of timed inseminations and associated luteal function in dairy cattle after two injections of prostaglandin F 2a. Theriogenology 28: Stevenson, J. S., and J. R. Pursley. Use of milk progesterone and prostaglandin F 2a in a scheduled artificial insemination program. J. Dairy Sci. 77: Taylor, C., and R. Rajamahendran Follicular dynamics, corpus luteum growth and regression in lactating dairy cattle. Can. J. Anim. Sci. 71: Thatcher, W. W., M. Drost, J. D. Savio, K. L. Macmillan, K. B. Entwistle, E. J. Schmitt, R. L. De la Sota, and G. R. Morris New clinical uses of GnRH and its analogues in cattle. Anim. Reprod. Sci. 33:27.