Comparison of long-term controlled internal drug release-based protocols to synchronize estrus and ovulation in postpartum beef cows 1

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1 Published November 25, 2014 Comparison of long-term controlled internal drug release-based protocols to synchronize estrus and ovulation in postpartum beef cows 1 J. M. Nash,* D. A. Mallory,* M. R. Ellersieck, S. E. Poock, M. F. Smith,* and D. J. Patterson* 2 *Division of Animal Science, S132 ASRC; Agricultural Experiment Station; and Department of Medicine and Surgery, University of Missouri, Columbia ABSTRACT: Two experiments were conducted to examine the necessity of adding a GnRH injection to a 14-d controlled internal drug release ()-based protocol for synchronization of estrus and ovulation in postpartum beef cows. The experiments were designed to characterize long-term -based protocols in cyclic and noncyclic postpartum beef cows on the basis of estrous response, follicular dynamics, and serum steroid hormone concentrations. In Exp. 1 and 2, crossbred lactating beef cows (n = 40 and 38, respectively) were randomly assigned to 1 of 2 treatments by age, days postpartum (DPP), BCS, and estrous cyclicity status: 1) cows received a from d 0 to 14 followed by GnRH 9 d after removal (d 23) and PGF 2α on d 30 ( ) or 2) administration from d 0 to 14 followed by PGF 2α 16 d later (d 30; Show- Me-). Estrus detection was performed using HeatWatch transmitters applied from removal to AI. Cows in Exp. 1 were artificially inseminated based on detected estrus whereas cows in Exp. 2 were inseminated at a fixed time. In both experiments, follicle turnover on d 25 of treatment was greater among -treated cows (P < 0.001) compared with Show- Me--treated cows. In Exp. 1, -treated cows tended to have a reduced (P = 0.06) variance for the interval to estrus after PGF 2α than treated cows. Also, cows assigned to the protocol had greater concentrations of progesterone (P < 0.05) on the day before PGF 2α administration as well as greater concentrations of estradiol-17β (P < 0.01) 48 h after PGF 2α administration. In Exp. 2, mean dominant follicle diameter on d 23 and at fixed-time AI (FTAI) did not differ between treatments (P > 0.10), but Show- Me--treated cows had larger follicles at d 28 (P < 0.001) and tended to have larger follicles at PGF 2α (d 30; P = 0.06) compared with cows assigned to. In summary, the administration of GnRH on d 23 of a long-term -based estrus synchronization protocol increased follicle turnover; however, both long-term -based protocols yielded similar physiological outcomes among estrous-cycling and anestrous postpartum beef cows. Key words: controlled internal drug release insert, estrus detection, estrus synchronization, fixed-time artificial insemination, postpartum beef cow 2013 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas Contribution from the Missouri Agricultural Experiment Station (Columbia, MO). This project was supported by National Research Initiative Competitive Grant No from the USDA National Institute of Food and Agriculture (Washington DC). The authors gratefully acknowledge Pfizer Animal Health (New York, NY) for providing the Lutalyse sterile solution and EAZI-Breed cattle inserts, Merial (Athens, GA) for providing the Cystorelin, Sires Inc. (Plain City, OH) for providing the semen, and the personnel of the University of Missouri Beef Research and Teaching Farm (Columbia, MO) for their support of this project. 2 Corresponding author: pattersond@missouri.edu Received August 22, Accepted March 11, INTRODUCTION Estrus synchronization protocols that facilitate use of fixed-time AI (FTAI) have yielded acceptable pregnancy rates in beef heifers (Mallory et al., 2011) and postpartum beef cows (Larson et al., 2006; Wilson et al., 2010). Short-term protocols have been the preferred method to synchronize estrus and ovulation in postpartum beef cows; however, long-term progestinbased protocols (presynchronization with a progestin before GnRH PGF 2α or PGF 2α only) produce more consistent results after FTAI in beef heifers (Busch et al., 2007; Leitman et al., 2009a,b; Mallory et al., 2011).

2 ronizing estrus in postpartum beef cows 3169 Several published studies questioned the utility of GnRH in estrus synchronization protocols for beef heifers (Wood-Follis et al., 2004; Lamb et al., 2006; Leitman et al., 2009a,b), and experiments conducted by Leitman et al. (2009a,b) and Mallory et al. (2011) failed to confirm the hypothesis that the addition of GnRH on d 23 of the controlled internal drug release () protocol results in a more highly synchronized estrus when compared with a 14-d PG protocol without GnRH between removal and before PGF 2α. Similar comparisons are not available in postpartum cows. Nash et al. (2012), however, recently reported that pregnancy rates were similar after FTAI in postpartum beef cows regardless of whether cows were assigned to a long- () or short-term (7d CO- + ) -based protocol. The experiments reported here were designed to characterize differences between 2 long-term -based estrus synchronization protocols ( and Show- Me-) in postpartum beef cows to test the hypothesis that GnRH is required to improve estrous response and synchrony of estrus after treatment administration. The objectives of these experiments were to characterize follicular dynamics, ovulatory response, estrous response and synchrony of estrus, and serum steroid hormone concentration patterns between estrous-cycling and anestrous postpartum beef cows during treatment. MATERIALS AND METHODS All experimental procedures were approved by the University of Missouri Animal Care and Use Committee. Experiment 1 Experimental Design. This experiment was conducted at the University of Missouri Beef Research and Teaching Farm (Columbia, MO). Crossbred, lactating beef cows (n = 40) were randomly assigned to 1 of 2 treatments by age, days postpartum (DPP; 24 to 69 d), BCS (1 to 9 scale, in which 1 = emaciated and 9 = obese; Richards et al., 1986), and pretreatment estrous cyclicity status. In October, blood samples were collected 10 and 1 d before initiating treatment to determine pretreatment estrous cyclicity status (progesterone 0.5 ng/ml at 1 or both sampling times; estrous cycling). Cows assigned to the treatment (n = 20; Fig. 1A) received an EAZI-Breed insert (1.38 g of progesterone; Pfizer Animal Health, New York, NY) from d 0 to 14 followed by an injection of GnRH [100 μg intramuscularly (i.m.); Cystorelin; Merial, Athens, GA] on d 23 and PGF 2α (25 mg i.m.; Lutalyse Sterile Solution; Pfizer Animal Health) on d 30. Cows assigned to the 14-d PG (; n = 20; Fig. 1A) protocol received Figure 1. (A) Cows assigned to the controlled internal drug release () protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows assigned to treatment were observed for estrus during a 144 h synchronized period after PGF 2α and were inseminated approximately 12 h after detection of estrus. (B) Cows assigned to the controlled internal drug release () protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows allotted to treatment were fixed-time inseminated 72 h after PGF 2α administration with GnRH (100 μg i.m.) administered at fixed-time AI. a insert from d 0 to 14 followed by an injection of PGF 2α on d 30. All cows were managed in the same pasture for the duration of the trial. Estrus Detection and AI. All cows were fitted with a HeatWatch estrus-detection transmitter (DDx Inc., Denver, CO) at the time of removal for continuous detection of estrus. Estrus was defined as having occurred when a cow received 3 mounts of 2 s in duration within a 4-h period (Busch et al., 2007). Transmitters were maintained on all cows until AI, and the synchronized period was designated as 0 to 144 h

3 3170 Nash et al. after PGF 2α. Artificial insemination was performed approximately 12 h after the onset of estrus by 1 of 2 experienced technicians, and semen from 1 of 3 sires was used. Cows were exposed to fertile bulls 10 d after AI for the remainder of a 60 d breeding season. Ultrasonography. Ovaries of all cows were scanned by transrectal ultrasonography (Aloka 500V equipped with a 7.5 MHz linear array transducer; Aloka, Wallingford, CT) on d 23 of treatment to characterize follicular dynamics. Follicles 5 mm in diameter and the presence of corpora lutea (CL) were recorded. Follicle diameter was determined by measuring diameter of the follicle at the widest point and then at a right angle to the first measurement. Ovaries were scanned on d 25 to determine ovulatory response to GnRH for -treated cows or follicle turnover for -treated cows coincident with timing of GnRH for ; this was defined as disappearance of the dominant follicle (DF) that was recorded from d 23. Ovaries were scanned on d 30 to record diameter of the DF at PGF 2α and again at AI to determine the diameter of the preovulatory follicle. Blood Collection and RIA. Blood samples were collected via jugular venipuncture into 10-mL vacuumevacuated tubes (Fisher Scientific, Pittsburgh, PA) 10 and 1 d before treatment initiation to determine the presence or absence of luteal activity. Cows were considered to be estrous cycling if progesterone (P 4 ) concentrations were 0.5 ng/ml at 1 or both of the pretreatment blood sampling times. Additionally, blood samples (10 ml) were collected daily from removal (d 14) until the time of AI. Blood samples were allowed to clot and stored at 4 C for 24 h. Serum was isolated by centrifugation (2,800 g for 25 min at 4 C). Serum samples were stored at 20 C until hormone analyses were performed. Serum concentrations of estradiol-17β (E 2 ) and P 4 were determined via RIA. Samples were analyzed for P 4 from removal to AI whereas samples were analyzed for E 2 at PGF 2α and 24 h (d 31) and 48 h (d 32) after PGF 2α. Serum E 2 concentrations were determined by validated extraction RIA (Kirby et al., 1997). Intra- and interassay CV were 7.61 and 7.6%, respectively, with an assay sensitivity of 0.25 pg/ml. Serum P 4 concentrations were determined with a Coat-A-Count kit (Siemens Medical Solutions Diagnostics, Los Angeles, CA) with intra- and interassay CV of 2.1 and 6.7%, respectively, and an assay sensitivity of 0.1 ng/ml (Kirby et al., 1997). Statistical Analyses. Differences between treatments in age, DPP, BCS, and DF diameter at d 23, PGF 2α, and AI were analyzed by PROC TTEST (SAS Inst. Inc., Cary, NC). Differences in interval to estrus after removal and PGF 2α were analyzed by ANOVA using the linear statistical model of treatment, estrous cyclicity status, and the interaction of treatment cyclicity (PROC GLM of SAS). Variances associated with interval to estrus after removal and PGF 2α were compared by performing an F-test (greater variance divided by the smaller variance; Snedecor and Cochran, 1989). Pretreatment estrous cyclicity status and follicular disappearance on d 25 were analyzed using a continuity adjusted χ 2 analysis (PROC FREQ of SAS). Estrous response after removal and PGF 2α was analyzed using a generalized linear mixed model (PROC GLIMMIX of SAS), using a binomial distribution and the link function of logit. The model for estrous response included the main effects of treatment, estrous cyclicity status, and the interaction of treatment cyclicity. Daily serum P 4 concentrations from the day of removal to AI and daily E 2 concentrations at the time of PGF 2α injection and 24 h and 48 h after PGF 2α were analyzed using repeated measures over time using the mixed model procedures (PROC MIXED of SAS) as outlined by Littell et al. (1998). A compound symmetry was used to model the covariance structure for the repeated measures. Experiment 2 Experimental Design. Experiment 2 was conducted at the University of Missouri Beef Research and Teaching Farm (Columbia, MO). Crossbred, lactating beef cows (n = 38) were assigned to treatments as in Exp. 1. In April, blood samples were collected 8 d before and immediately before initiation of treatment (d 0) to determine pretreatment estrous cyclicity status (progesterone 0.5 ng/ml at 1 or both sampling times; estrous cycling). Cows were assigned to either the protocol (n = 19; Fig. 1B) or to the Show- Me- protocol (n = 19; Fig. 1B). All cows were managed in the same pasture for the duration of the trial. Estrus Detection and AI. All cows were fitted with a HeatWatch estrus-detection transmitter (DDx Inc.) 1 d before removal to characterize estrous response. Transmitters were maintained on all cows until FTAI for continuous estrus detection. Fixed-time AI was performed 72 h after PGF 2α administration for cows in both treatments, and an injection of GnRH (100 μg i.m.) was administered at FTAI. Artificial insemination was performed by 1 of 2 experienced technicians, and semen from 1 of 3 sires was used. Cows were exposed to fertile bulls 11 d after FTAI for the remainder of a 65 d breeding season. Ultrasonography. Ovaries of all cows were scanned by transrectal ultrasonography (Aloka 500V equipped with a 7.5 MHz linear array transducer) on d 23 to characterize follicular dynamics, and follicles 5 mm in diameter and the presence of CL were recorded. Follicle diameter was determined as in Exp. 1. Ovaries were scanned on d 25 to determine ovulatory response to GnRH for -treated cows or follicle turnover for -treated cows coincident with

4 ronizing estrus in postpartum beef cows 3171 timing of GnRH for ; this was defined as disappearance of the DF that was recorded from d 23. On d 28, ovaries were scanned to determine diameter of the DF to elucidate the follicular dynamics at that time and again at d 30 to determine DF diameter at PGF 2α. The ovaries of all cows were then scanned at FTAI to determine diameter of the preovulatory follicle. Blood Collection and RIA. Blood samples were collected via jugular venipuncture into 10-mL vacuumevacuated tubes (Fisher Scientific, Pittsburgh, PA) 8 d before and immediately before treatment initiation (d 0) to determine estrous cyclicity status; cows were considered to be estrous cycling if P 4 concentrations were 0.5 ng/ml at either of the pretreatment blood sampling times. Additionally, blood samples (10 ml) were collected daily beginning the day after removal (d 15) until FTAI (d 33). Blood samples were allowed to clot and stored at 4 C for 24 h. Serum was collected by centrifugation (2,800 g for 25 min at 4 C) and stored at 20 C until hormone analyses were performed. Serum concentrations of E 2 and P 4 were determined via RIA. Samples were analyzed for P 4 daily from the day after removal to FTAI whereas samples were analyzed for E 2 daily from PGF 2α through FTAI. Serum E 2 concentrations were determined by validated extraction RIA (Kirby et al., 1997). Intra- and interassay CV were 7.61 and 7.6%, respectively, with an assay sensitivity of 0.25 pg/ml. Serum P 4 concentrations were determined with a Coat-A-Count kit with intra- and interassay CV of 1.99 and 8.3%, respectively, and an assay sensitivity of 0.1 ng/ml (Kirby et al., 1997). Statistical Analyses. Differences between treatments in age, DPP, BCS, and DF diameter at d 23, d 28, PGF 2α, and FTAI were analyzed by PROC TTEST of SAS. Differences in interval to estrus after removal and PGF 2α were analyzed by ANOVA using the linear statistical model of treatment, estrous cyclicity status, and the interaction of treatment cyclicity (PROC GLM of SAS). Variances associated with interval to estrus after removal and PGF 2α were compared by performing an F-test (greater variance divided by the smaller variance; Snedecor and Cochran, 1989). Pretreatment estrous cyclicity status and follicular disappearance on d 25 were analyzed using a continuity adjusted χ 2 analysis (PROC FREQ of SAS). Estrous response after removal and PGF 2α was analyzed using a generalized linear mixed model (PROC GLIMMIX of SAS), using a binomial distribution and the link function of logit. The model for estrous response included the main effects of treatment, estrous cyclicity status, and the interaction of treatment cyclicity. Daily serum P 4 concentrations from the day after removal to FTAI and daily E 2 concentrations from PGF 2α to FTAI were analyzed using repeated measures over time using the mixed model procedures of Table 1. Number of cows, estrous cyclicity status, age, days postpartum, and BCS (mean ± SE) of cows before initiation of the controlled internal drug release () and estrus synchronization protocols in Exp. 1 1 and Exp. 2 2 Item Exp. 1 Exp. 2 SAS (PROC MIXED) as outlined by Littell et al. (1998). A compound symmetry was used to model the covariance structure for the repeated measures RESULTS No. of cows Cows with increased progesterone 3,4 Proportion 17/20 16/20 3/19 4/19 Percent Age, 5 yr 4.4 ± ± ± ± 0.8 DPP, 6 d 54.4 ± ± ± ± 0.8 BCS ± ± ± ± Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows assigned to treatment were observed for estrus during a 144 h synchronized period after PGF 2α and were inseminated approximately 12 h after detection of estrus. 2 Cows in this experiment received the same protocol treatment as cows in Exp. 1; however, cows in this experiment were not inseminated based on detected estrus. All cows allotted to treatment were fixed-time inseminated 72 h after PGF 2α administration with GnRH (100 μg i.m.) administered at fixed-time AI. 3 Estrous cyclicity is equal to the number of cows with increased ( 0.5 ng/ml) concentrations of progesterone in blood serum before treatment initiation. Cows were considered to be estrous cycling if progesterone was increased in either 1 or both blood samples collected 10 and 1 d before treatment initiation. 4 Estrous cyclicity was determined similarly for cows in Exp. 2, with the exception that blood samples were collected 8 d before and on the day (d 0) of treatment initiation. 5 Age (years) of cows at initiation of treatments. 6 Number of days postpartum (DPP) at d 0. 7 Body condition scores of cows on d 0 of protocol initiation (1 to 9 scale: 1 = emaciated and 9 = obese). Experiment 1 The number, estrous cyclicity status, age, DPP, and BCS of cows before the initiation of treatments are shown in Table 1. There were no differences between treatments on the basis of estrous cyclicity status (P = 1.0), age (P = 1.0), DPP (P = 0.27), or BCS (P = 0.44). rony of Estrus after Controlled Internal Drug Release Removal. Estrous response after removal was similar between treatments (P = 1.0) and did not differ between treatments based on estrous cyclicity status. The mean interval to estrus and variance for interval to estrus did not differ between treatments

5 3172 Nash et al. Table 2. Estrous response rate (% in parentheses), interval to estrus (mean ± SE), and the variance for the interval to estrus of cows after controlled internal drug release () removal for cows assigned to the and estrus synchronization protocols in Exp. 1 1 and Exp. 2 2 Exp. 1 Exp. 2 Item 3,4 Estrous response 17/20 (85) 17/20 (85) 14/19 (74) 13/19 (68) Estrous cycling 15/17 (88) 14/16 (87.5) 3/3 (100) 4/4 (100) Anestrus 2/3 (66.7) 3/4 (75) 11/16 (69) 9/15 (60) Interval to estrus, h 47.9 ± ± ± ± 7.0 Estrous cycling 49.8 ± ± ± ± 13.0 Anestrus 33.2 ± ± ± ± 8.6 Variance for interval to estrus Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows assigned to treatment were observed for estrus during a 144 h synchronized period after PGF 2α and were inseminated approximately 12 h after detection of estrus. 2 Cows in this experiment received the same protocol treatment as cows in Exp. 1; however, cows in this experiment were not inseminated based on detected estrus. All cows allotted to treatment were fixed-time inseminated 72 h after PGF 2α administration with GnRH (100 μg i.m.) administered at fixed-time AI. 3 Estrous cyclicity is equal to the number of cows with elevated ( 0.5 ng/ml) concentrations of progesterone in blood serum before treatment initiation. Cows were considered to be estrous cycling if progesterone was elevated in either 1 or both blood samples collected 10 and 1 d before treatment initiation. 4 Estrous cyclicity was determined similarly for cows in Exp. 2, with the exception that blood samples were collected 8 d before and on the day (d 0) of treatment initiation. (P > 0.10; Table 2). During the peak response interval, 65 and 70% of cows in and groups exhibited estrus, respectively. Follicular Dynamics and Response to GnRH. Cows in both treatments had similar size follicles at d 23, at the time of PGF 2α, and at AI (P > 0.10; Table 3). However, cows assigned to the protocol had a greater incidence of follicular disappearance on d 25 than cows assigned to the protocol (P < 0.001;, 85%;, 5%; Table 3). rony of Estrus after PGF 2α Administration. The distribution of estrus after PGF 2α is illustrated in Fig. 2. There was no difference (P = 1.0) in the proportion of cows assigned to and that displayed estrus after PGF 2α, and estrous response was similar between treatments based on estrous cyclicity status. The mean interval to estrus was similar between treatments (P = 0.91; Table 4). Cows assigned to the protocol tended to have a reduced variance associated with the interval to estrus compared with cows assigned to the protocol (P = 0.06; Table 3. Diameter of the dominant follicle (mean ± SE) and dominant follicle disappearance or turnover rate (% in parentheses) of cows assigned to the controlled internal drug release () and estrus synchronization protocols in Exp. 1 1 Item Dominant follicle diameter at d 23 of treatment, 2 mm 10.8 ± ± 0.5 Dominant follicle disappearance or turnover at d /20 (85 a ) 1/20 (5 b ) Dominant follicle diameter at d 30 of treatment, 4 mm 11.1 ± ± 0.6 Dominant follicle diameter at AI, mm 14.1 ± ± 0.3 a,b Within a row, means without a common superscript differ (P < 0.001). 1 Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows assigned to treatment were observed for estrus during a 144 h synchronized period after PGF 2α and were inseminated approximately 12 h after detection of estrus. 2 Day 23 of treatment was coincident with the timing of GnRH (100 μg i.m.; Cystorelin; Merial, Athens, GA) administration for cows in whereas cows in did not receive GnRH. 3 Indicates disappearance of the dominant follicle that was measured on d Day 30 of treatment was coincident with the timing of PGF 2α (25 mg i.m.; Lutalyse; Pfizer Animal Health, New York, NY) administration. Table 4). During the peak response interval (60 to 84 h after PGF 2α ), 75 and 50% of cows assigned to and exhibited estrus, respectively. Serum Steroid Hormone Concentrations. Mean concentrations of P 4 and E 2 are shown in Table 5. There was no difference (P > 0.10) in mean concentrations of P 4 and E 2 at PGF 2α between cows in and Show- Me- treatment groups. Cows in both treatments had similar (P > 0.10) mean P 4 concentrations for all of the days sampled, with the exception that cows assigned to had elevated concentrations of P 4 compared with cows assigned to on the day before PGF 2α (d 29 of treatment; P < 0.05). Mean concentrations of E 2 were similar between treatments from PGF 2α until 24 h after PGF 2α (P > 0.10); however, -treated cows had greater E 2 concentrations 48 h after PGF 2α than -treated cows (P < 0.01; Table 5). Experiment 2 The number, estrous cyclicity status, age, DPP, and BCS of cows before the initiation of treatments are shown in Table 1. There was no difference between treatments for estrous cyclicity status (P = 1.0), age (P = 0.46), DPP (P = 0.83), or BCS (P = 0.66). rony of Estrus after Controlled Internal Drug Release Removal. There was no effect of treatment (P = 1.0) or estrous cyclicity status (P = 0.96) on estrous response after removal for cows assigned

6 ronizing estrus in postpartum beef cows 3173 Table 4. Estrous response rate (% in parentheses), interval to estrus (mean ± SE), and the variance for the interval to estrus of cows after PGF 2α administration and before fixed-time AI (FTAI) for cows assigned to the controlled internal drug release () and estrus synchronization protocols in Exp. 1 1 and Exp. 2 2, respectively Figure 2. Percentages of estrous cycling and anestrous cows that exhibited estrus after PGF 2α administration for cows assigned to the controlled internal drug release () and estrus synchronization protocols in Exp. 1; (black bar) and Show- Me- (gray bar); No response = no estrous response. Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows assigned to treatment were observed for estrus (via HeatWatch detection transmitters) during a 144 h synchronized period after PGF 2α and were inseminated approximately 12 h after detection of estrus. to the and treatments (Table 2). The mean interval from removal to estrus was similar (P = 0.99) between treatments and the variance for interval to estrus did not differ (P > 0.05) between treatments. During the peak response period, approximately 58 and 42% of cows in and exhibited estrus, respectively. Follicular Dynamics and Response to GnRH. Mean DF diameter on d 23 did not differ (P = 0.95) between the 2 treatments (Table 6). However, -treated cows had a greater incidence of follicular disappearance on d 25 than cows assigned to (P < 0.001;, 95%;, 0%). Cows assigned to had larger follicles on d 28 (P < 0.001) and tended to have larger follicles on the day of PGF 2α administration (d 30; P = 0.06) compared with cows assigned to (Table 6). However, cows in both treatment groups had similar sized follicles at the time of FTAI (P = 0.61). rony of Estrus after PGF 2α Administration. The distribution of estrus after PGF 2α before FTAI is shown in Fig. 3. Estrous response after PGF 2α before FTAI was similar between treatments (P = 0.17) and did not differ (P = 0.97) between cows in and groups based on estrous cyclicity status (Table 4). There was no effect of treatment (P = 0.90) on the mean interval to estrus after PGF 2α before FTAI. The variance associated with the interval to estrus did Exp. 1 Exp. 2 Item 3,4 Estrous response 19/20 (95) 19/20 (95) 15/19 (79) 10/19 (53) Estrous cycling 16/17 (94) 16/16 (100) 3/3 (100) 3/4 (75) Anestrus 3/3 (100) 3/4 (75) 12/16 (75) 7/15 (47) Interval to estrus, h 75.2 ± ± ± ± 1.9 Estrous cycling 76.1 ± ± ± ± 3.7 Anestrus 70.6 ± ± ± ± 2.4 Variance for interval to estrus a b a,b Within a row, means without a common superscript tended to differ (P = 0.06). 1 Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows assigned to treatment were observed for estrus during a 144 h synchronized period after PGF 2α and were inseminated approximately 12 h after detection of estrus. 2 Cows in this experiment received the same protocol treatment as cows in Exp. 1; however, cows in this experiment were not inseminated based on detected estrus. All cows allotted to treatment were fixed-time inseminated 72 h after PGF 2α administration with GnRH (100 μg i.m.) administered at FTAI. 3 Estrous cyclicity is equal to the number of cows with increased ( 0.5 ng/ml) concentrations of progesterone in blood serum before treatment initiation. Cows were considered to be estrous cycling if progesterone was elevated in either 1 or both blood samples collected 10 and 1 d before treatment initiation. 4 Estrous cyclicity was determined similarly for cows in Exp. 2, with the exception that blood samples were collected 8 d before and on the day (d 0) of treatment initiation. not differ (P > 0.05) between the 2 treatments. During the peak response period (48 to 72 h after PGF 2α ), 79 and 53% of the cows in the and -treated groups exhibited estrus, respectively. Serum Steroid Hormone Concentrations. Mean concentrations of P 4 and E 2 are shown in Table 7. Mean concentrations of P 4 and E 2 did not differ (P = 0.63 and P = 0.41, respectively) at PGF 2α. Cows in both treatments had similar mean P 4 concentrations for all days sampled from the day after removal to FTAI (P > 0.05). Also, cows in both treatments had similar mean E 2 concentrations for all days sampled from PGF 2α administration to FTAI (P > 0.05). DISCUSSION Short-term -based protocols have been the preferred method to synchronize estrus and ovulation in postpartum beef cows (Lamb et al., 2001; Bridges et al.,

7 3174 Nash et al. Table 5. Serum steroid hormone concentrations (mean ± SE) for cows assigned to the controlled internal drug release () and estrus synchronization protocols in Exp. 1 1 Item Serum concentrations of progesterone 4.2 ± ± 0.4 at PGF 2 2α injection, ng/ml Serum concentrations of estradiol-17β 7.0 ± ± 0.4 at PGF 2α injection, pg/ml Serum concentrations of estradiol-17β 9.7 ± ± h after PGF 2α injection, pg/ml Serum concentrations of estradiol-17β 48 h after PGF 2α injection, pg/ml 13.3 ± 1.0 a 10.4 ± 1.0 b a,b Within a row, means without a common superscript differ (P < 0.01). 1 Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows assigned to treatment were observed for estrus during a 144 h synchronized period after PGF 2α and were inseminated approximately 12 h after detection of estrus. 2 PGF 2α (25 mg i.m.; Lutalyse; Pfizer Animal Health) on d 30 of treatment. 2008; Wilson et al., 2010); however, pregnancy rates were greater among heifers assigned to long-term based protocols compared with short-term protocols (Busch et al., 2007; Leitman et al., 2009a,b; Mallory et al., 2011). Long-term -based protocols also provide the opportunity to administer any recommended prebreeding vaccines at insertion, which is more than 30 d before breeding. This ultimately decreases the number of times that cattle are handled, leading to decreased stress and less labor required to effectively implement the protocol. Numerous questions remain regarding the use of GnRH in estrus synchronization protocols designed for beef heifers (Leitman et al., 2009a,b; Mallory et al., 2011), based on the fact that a number of studies demonstrated that response to GnRH in heifers is inconsistent when compared with cows (Macmillan and Thatcher, 1991; Pursley et al., 1995). Leitman et al. (2009b) and Mallory et al. (2011) reported that GnRH was not required to synchronize estrus in beef heifers when administered sequentially after removal and before PGF 2A after administration of a 14-d -based protocol. In fact, results from those studies concluded that GnRH failed to facilitate an improvement in synchrony of estrus in beef heifers (Leitman et al., 2009b) and led to reduced pregnancy rates resulting from FTAI (Mallory et al., 2011). The literature is devoid of studies evaluating administration of long-term -based estrus synchronization protocols in postpartum beef cows. However, Nash et al. (2012) recently reported that pregnancy rates resulting from FTAI were similar Table 6. Diameter of the dominant follicle (mean ± SE) and dominant follicle disappearance or turnover rate (% in parentheses) of cows assigned to the controlled internal drug release () and estrus synchronization protocols in Exp. 2 1 Item Dominant follicle diameter 11.7 ± ± 0.5 at d 23 of treatment, 2 mm Dominant follicle disappearance 18/19 (95 a ) 0/19 (0 b ) or turnover on d 25 3 Dominant follicle diameter 10.3 ± 0.3 a 13.7 ± 0.5 b at d 28 of treatment, mm Dominant follicle diameter 11.7 ± 0.3 c 12.9 ± 0.5 d at d 30 of treatment, 4 mm Dominant follicle diameter at d 33 of treatment, 5 mm 13.7 ± ± 0.4 a,b Within a row, means without a common superscript differ (P < 0.001). c,d Within a row, means without a common superscript tended to differ (P = 0.06). 1 Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows allotted to treatment were fixed-time inseminated 72 h after PGF 2α administration with GnRH (100 μg i.m.) administered at fixed-time AI. 2 Day 23 of treatment was coincident with the timing of GnRH (100 μg i.m.; Cystorelin; Merial, Athens, GA) administration for cows in whereas cows in did not receive GnRH. 3 Indicates disappearance of the dominant follicle that was measured on d Day 30 of treatment was coincident with the timing of PGF 2α (25 mg i.m.; Lutalyse; Pfizer Animal Health) administration. 5 Day 33 of treatment was coincident with fixed-time AI. for cows assigned to either a long-term -based protocol () or the short-term 7-d CO- + protocol. Interestingly, similarities in pregnancy rates resulting from FTAI between longand short-term treatments occurred despite the fact that estrous response before FTAI was reduced among cows assigned to the long-term protocol. Similarities between treatments in pregnancy rates resulting from FTAI, despite differences in estrous response, suggest that long-term treated cows had follicles of adequate physiological maturity to ovulate and establish pregnancy on fertilization (Nash et al., 2012). Although 14-d -based protocols have been compared with and without the addition of GnRH after removal and before PGF 2α in beef heifers, similar data in cows is not available. Experiments 1 and 2 were conducted to compare response among postpartum beef cows to long-term -based estrus synchronization protocols ( and ) and to determine based on these comparisons the need for GnRH as part of a sequential treatment schedule. Comparing results from the 2 experiments, it was interesting to note that diameters of DF of cows were similar between treatments at AI even though cows in Exp.

8 ronizing estrus in postpartum beef cows 3175 Table 7. Serum steroid hormone concentrations (mean ± SE) for cows assigned to the controlled internal drug release () and estrus synchronization protocols in Exp. 2 1 Figure 3. Percentages of estrous cycling and anestrous cows that exhibited estrus after PGF 2α administration for cows assigned to the controlled internal drug release () and estrus synchronization protocols in Exp. 2; (black bar) and (gray bar); No response = no estrous response. Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows allotted to treatment were fixed-time inseminated 72 h after PGF 2α administration with GnRH (100 μg i.m.) administered at fixed-time AI (FTAI). 1 (inseminated after observed estrus;, 92.6 h;, 88.6 h) were inseminated later after PGF 2α than cows in Exp. 2 (inseminated at predetermined fixed times;, 72.7 h;, 72.5 h). However, DF diameters were greater at PGF 2α among cows assigned to the treatment in Exp. 2 compared with cows assigned to. These differences are difficult to explain and dissimilar to results from Exp. 1. The major difference however between cows in each of these experiments was that a greater proportion of cows in Exp. 2 were anestrus at the time treatments began compared with a greater proportion of cows in Exp. 1 being estrous cycling. Differences in season (i.e., fall vs. spring) could have contributed to the observed differences between these 2 experiments. Also, daily mapping of ovaries was not performed in either of these experiments and would provide a more descriptive characterization of ovarian follicular dynamics before PGF 2α for cows assigned to each of these protocols. Progesterone concentrations at PGF 2α did not differ between treatments in either experiment although concentrations of progesterone in serum were numerically greater in each experiment among cows assigned to the treatment. This numeric increase may have resulted from accessory CL that formed in response to GnRH among -treated cows. Mean concentrations of E 2 at 48 h after PGF 2α were greater among cows assigned to the vs. protocol, and may be a function of the synchronized development of Item Serum concentrations of progesterone 3.7 ± ± 0.5 at PGF 2 2α injection, ng/ml Serum concentrations of estradiol-17β 3.6 ± ± 0.6 at PGF 2α injection, pg/ml Serum concentrations of estradiol-17β 11.1 ± ± h after PGF 2α injection, pg/ml Serum concentrations of estradiol-17β 14.8 ± ± h after PGF 2α injection, pg/ml Serum concentrations of estradiol-17β at the time of FTAI, 3 pg/ml 9.5 ± ± Cows assigned to the protocol received a (Pfizer Animal Health, New York, NY; 1.38 g of progesterone) insert from d 0 to 14, GnRH [Cystorelin; Merial, Athens, GA; 100 μg intramuscularly (i.m.)] on d 23, and PGF 2α (Lutalyse; Pfizer Animal Health; 25 mg i.m.) on d 30. Cows assigned to the protocol received a from d 0 to 14 and PGF 2α on d 30. All cows allotted to treatment were fixed-time inseminated 72 h after PGF 2α administration with GnRH (100 μg i.m.) administered at fixed-time AI. 2 PGF 2α (25 mg i.m.; Lutalyse; Pfizer Animal Health) on d 30 of treatment. 3 Fixed-time AI (FTAI) on d 33 of treatment. follicular waves resulting from GnRH administered 7 d before PGF 2α. Differences in concentrations of E 2 between treatments in Exp. 2 for the respective sampling times were not observed. As expected, synchrony of estrus was similar between treatments after removal; however, -treated cows tended to have an improved synchrony of estrus after PGF 2α compared with cows assigned to the protocol in Exp. 1. This difference was again in all likelihood a result of an improvement in synchronization of follicular waves that resulted among cows that received GnRH on d 23. The relatively small number of cows involved in each experiment precludes any meaningful assessment of how these treatments may perform on the basis of fertility after treatment; therefore, all data pertaining to pregnancy rates has been omitted. Although long-term protocols have been used extensively in beef heifers, experiments have not yielded significant differences in pregnancy rates in beef heifers (Mallory et al., 2011) or postpartum beef cows (Nash et al., 2012) after insemination based on FTAI. It is important to again note that although the majority of cows were estrous cycling at treatment initiation in Exp. 1, the majority of cows in Exp. 2 were anestrus at the time treatments were initiated based on pretreatment blood samples. The authors acknowledge the potential for misclassification of cows on the basis of estrous cyclicity status determined from 2 blood samples taken before treatment initiation and the use of P 4 values of 0.5 ng/ml to confirm cyclicity. However, the potential for committing

9 3176 Nash et al. a Type II error is greatly reduced by classifying cows as anestrus when using a 0.5 ng/ml limit. The results reported here raise questions regarding the need for GnRH as part of a long-term -based protocol to synchronize estrus in postpartum beef cows and suggest that proper administration of these protocols requires careful consideration of the advantages and disadvantages that accompany each of them. Both protocols effectively synchronized estrus in estrouscycling and anestrous postpartum beef cows and suggest that each of these protocols may be especially useful in situations accompanied with high rates of anestrus. Producers should also understand that due to the length of these protocols, a major downfall to their use is a decrease in the number of cows that can be placed on treatment based on later calving dates. Regardless of which protocol a producer chooses to use, strict compliance with the assigned times and proper treatment administration is essential for successful application. In summary, these results in addition to those recently reported by Nash et al. (2012) indicate that long-term -based protocols successfully synchronize estrus and ovulation in postpartum beef cows and may provide an effective alternative to short-term estrus synchronization protocols classically used to facilitate FTAI in cows. These data suggest that similar to heifers, GnRH may not be required to increase the synchrony of estrus before FTAI as part of a long-term -based treatment schedule; however, studies involving larger numbers of cows will be required to fully answer this question. As previously mentioned long-term protocols provide the opportunity to combine animal health and reproduction in a single management step. This consideration is based on the fact that prebreeding vaccinations should be administered at least 30 d before the breeding season, a time coincident with the initiation of these long-term protocols. LITERATURE CITED Bridges, G. A., L. A. Helser, D. E. Grum, M. L. Mussard, C. L. Gasser, and M. L. Day Decreasing the interval between GnRH and PGF 2α from 7 to 5 days and lengthening proestrus increases timed- AI pregnancy rates in beef cows. Theriogenology 69: Busch, D. C., D. J. Wilson, D. J. Schafer, N. R. Leitman, J. K. Haden, M. R. Ellersieck, M. F. Smith, and D. J. Patterson Comparison of progestin-based estrus synchronization protocols before fixed-time artificial insemination on pregnancy rate in beef heifers. J. Anim. Sci. 85: Kirby, C. J., M. F. Smith, D. H. Keisler, and M. C. Lucy Follicular function in lactating dairy cows treated with sustainedrelease bovine somatotropin. J. Dairy Sci. 80: Lamb, G. C., J. E. Larson, T. W. Geary, J. S. Stevenson, S. K. Johnson, M. L. 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