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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
* Department of Animal Science,
and
Agricultural Experiment Station, University of Missouri, Columbia 65211
Abstract
Two progestin-based protocols for the synchronization of estrus in beef cows were compared. Cyclic, nonlactating, crossbred, beef cows were assigned by age and body condition score to one of two treatments. Cows assigned to the MGA Select protocol were fed melengestrol acetate (MGA; 0.5mg•cow-1•-1) for 14 d, GnRH was administered (100 µg i.m. of Cystorelin) 12 d after MGA withdrawal, and PGF2
(25 mg of i.m. Lutalyse) was administered 7 d after GnRH. Cows assigned to the 7-11 Synch protocol were fed MGA for 7 d and were injected with PG on d 7 of MGA, GnRH on d 11, and PG on d 18. Transrectal ultrasonography was performed daily to monitor follicular dynamics from the beginning of MGA feeding through ovulation after the synchronized estrus. All cows exhibited estrus in response to PG. Mean interval to estrus was shorter (P < 0.01) for 7-11 Synch-treated cows (56 ± 1.5 h) than for cows assigned to the MGA Select protocol (73 ± 4.7 h). Mean interval from estrus to ovulation did not differ between treatments (P > 0.10). Variances for interval to estrus differed (P < 0.01) between treatments. Mean follicular diameter at GnRH injection, PG injection, and estrus did not differ (P > 0.10) between treatments. Relative to MGA Select, serum estradiol-17ß concentrations were higher (P < 0.01) for 7-11 Synch 2 d and 1 d before, on the day of GnRH injection, in addition to 4 d after GnRH, and 24 h after PG. Mean progesterone concentrations were greater (P < 0.01) for MGA Select cows from 4 d before to 7 d after GnRH. Forty-four percent of the variation in interval to estrus between treatments was explained by differences in estradiol-17ß concentrations 24 h after PG. This study suggests that follicular competence is likely related to steroidogenic capacity of the follicle and the endocrine environment under which growth and subsequent ovulation of the dominant follicle occurs.
Key Words: Beef Cow • Synchronization of Estrus • GnRH • Progestin • Steroids
Introduction
The development of methods that effectively synchronize a fertile estrus in beef cattle is needed to increase the use of AI and enhance the genetic merit of beef herds. Current surveys indicate that fewer than 5% of the beef cows in the United States are artificially inseminated and only half of the cattle producers that practice AI use any of synchronization of estrus protocol (NAHMS, 1994
, 1998). The inability to predict the time of estrus for individual females in a group often makes it impractical to use AI because of the labor required for the detection of estrus. The development of an economical method of fixed-time AI with high fertility would theoretically result in a dramatic increase in the adoption of AI in beef herds (Patterson et al., 2003).
We propose the general hypothesis that progestin treatment before a GnRH-PG protocol will successfully do the following: 1) induce ovulation in anestrous postpartum beef cows; 2) decrease the incidence of a short-luteal phase among anestrous cows induced to ovulate; 3) increase estrous response, synchronized conception, and pregnancy rates; and 4) increase the likelihood of successful fixed-time AI. Two protocols were recently developed to synchronize estrus in postpartum beef cows. These include the MGA Select and 7-11 Synch protocols (Wood et al., 2001; Kojima et al., 2000). Previous studies showed differences between these protocols in length of interval to estrus and the resulting synchrony of estrus following treatment (Kojima et al., 2000; Patterson et al., 2001; Wood et al., 2001). The objectives for this study were to compare the MGA Select and 7-11 Synch protocols for the synchronization of estrus in beef cows by 1) characterizing follicular dynamics during and after MGA feeding; 2) comparing timing of estrus and ovulation after PG among cows assigned to these two treatments; and 3) characterizing steroid hormone concentrations during and after treatment.
Materials and Methods
Animals.
Cyclic, crossbred, non-lactating, beef cows were assigned by age and BCS (1-to-9 scale, 1 = emaciated and 9 = obese) to one of two treatments. Cows assigned to the MGA Select protocol (n = 9) were fed melengestrol acetate (MGA; 0.5 mg•cow-1•d-1) for 14 d followed by an injection of GnRH (100 µg i.m. of Cystorelin; Merial, Athens, GA) on d 26 and PGF2 (PG; 25 mg i.m. of Lutalyse Sterile Suspension; Pfizer Animal Health, New York, NY) on d 33 (Figure 1). Cows assigned to 7-11 Synch (n = 8) were fed MGA for 7 d followed by injection of PG on d 7 of MGA, GnRH on d 11, and PG on d 18 (Figure 1). The day of GnRH administration was designated d 0 for this experiment. Cows were observed for signs of behavioral estrus beginning 1 d before and continuing for 7 d following the final PG injection.
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Ultrasonography, Estrus, and Ovulation.
Transrectal ultrasonography was performed daily to characterize ovarian follicular dynamics during and after treatment. An Aloka 500V equipped with a 7.5-MHz linear-array transducer (Aloka, Wallingford, CT) was used to monitor follicular dynamics. Ultrasonography began on the day on which MGA feeding was begun and continued daily until 48 h after PG. Subsequent ultrasounds were performed every 12 h until ovulation was confirmed.
Blood Collection and RIA.
Blood sampling was done via jugular venipuncture. Blood samples were collected 10 and 1 d before treatment to confirm cyclicity status, and were collected daily beginning the first day of treatment and continuing through ovulation. Blood samples were stored at 4°C for 24 h. Serum was collected by centrifugation and stored at -20°C until hormone analyses.
Serum concentrations of estradiol-17ß (E2) and progesterone (P4) were determined by RIA. Serum E2 concentrations were determined by validated extraction assay (Kirby et al., 1997). The assay sensitivity was 0.25 pg/mL. The intra- and interassay coefficients of variation were 6 and 20%, respectively. Serum P4 concentrations were determined by a Coat-A-Count Kit (Diagnostic Products Corporation, Los Angeles, CA; Kirby et al., 1997). The assay sensitivity was 0.5 ng/mL. The intra- and interassay coefficients of variation were 5 and 14%, respectively.
Statistical Analysis.
Effects of treatment on follicle size at the time of GnRH injection, PG injection, estrus, and 24 to 72 h after PG injection were tested by Fisher’s least significant difference test and by using repeated-measures analyses over time using mixed model procedures of SAS (SAS Inst. Inc., Cary, NC). Variances associated with intervals to estrus and ovulation were compared by dividing the greater variance by the lesser variance and performing an F-test (Snedecor and Cochran, 1989). The effects of treatment on hormone concentrations were tested by repeated measures analyses over time using mixed model procedures of SAS (SAS Inst. Inc.). Stepwise regression analyses were also performed to rank variables contributing to variance in interval to estrus after treatment (SAS Inst. Inc.).
Results
The subsequent results are for cows that responded to GnRH and initiated a new follicular wave following GnRH administration (MGA Select, n = 8; 7-11 Synch, n = 7). Two cows were considered unresponsive to treatments. One cow assigned to the 7-11 Synch protocol ovulated during the MGA feeding period, responded to GnRH, but failed to respond to PG. One cow assigned to the MGA Select protocol did not ovulate in response to GnRH but exhibited estrus after PG.
Follicular Dynamics.
Development of dominant follicles for individual cows during and after treatment with the MGA Select (Figure 2) and 7-11 Synch (Figure 3) protocols are presented. Follicular development during MGA feeding was poorly synchronized. Most cows ovulated after MGA withdrawal. Administration of GnRH after MGA withdrawal caused ovulation followed by a new follicular wave in both protocols.
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; Table 1). The range in estrous response was 48 to 96 h and 48 to 58 h following PG for cows assigned to the MGA Select and 7-11 Synch protocols, respectively (Figure 4; Table 1). Variance in interval to estrus was less for 7-11 Synch compared to MGA Select-treated cows (P < 0.01). Mean interval from estrus to ovulation did not differ between treatments (P > 0.10; Table 1).
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; Figure 5). It is important to note that despite a lack of difference in follicle diameter between treatments, there were distinct differences in steroid hormone profiles between the two treatments as indicated by the significant (P < 0.01) treatment x day interaction. Serum concentrations of E2 were greater for 7-11 Synch compared to MGA Select 2 d and 1 d before GnRH administration, the day GnRH was administered, 4 d after GnRH administration, and 24 h after PG (Figure 6). Conversely, mean concentrations of P4 were greater (P < 0.01) for MGA Select-treated cows than for 7-11 Synch-treated cows from 2 d before and 7 d after GnRH (Figure 7). Stepwise regression analyses ranked variables contributing to variance in interval to estrus after treatment. Forty-four percent of the variation in interval to estrus was explained by E2 concentrations 24 h after PG, and increased by 8% with the inclusion of P4 concentrations 4, 5, 6, or 7 d after GnRH.
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The MGA Select and 7-11 Synch protocols were effective for synchronizing estrus in mixed populations of cyclic and anestrous beef cows (Kojima et al., 2000; Patterson et al., 2002). Previous studies found that addition of GnRH to the MGA-PG protocol prevented short cycles and increased synchrony of estrus (Patterson et al., 2003). Although these protocols effectively synchronize estrus, there are distinct differences in interval to and synchrony of estrus following their administration (Kojima et al., 2000; Patterson et al., 2002). Our hypothesis was that differences in length of interval to estrus between cows treated with MGA Select and 7-11 Synch may be explained by differences in follicular development and steroid hormone concentrations of cows assigned to the two treatments.
The longer interval to estrus for MGA Select-treated cows may be caused by higher P4 concentrations and lower E2 concentrations during the growth phase of the dominant follicle (Figures 6 and 7). The aforementioned hormonal milieu is similar to the mid-luteal phase of the estrous cycle. We assume that high P4 concentrations in the MGA Select cows led to low-LH pulse frequency and high-LH pulse amplitude. This pattern of LH secretion creates an environment that results in follicles and/or oocytes that may be less physiologically mature compared with those that develop during the 7-11 Synch treatment. The 7-11 Synch-treated cows were in the early luteal phase of the estrous cycle at the time PG was administered. Follicles in 7-11 Synch-treated cows grow and develop under low-circulating P4 concentrations and create higher circulating concentrations of E2 (Figures 6 and 7). The low P4 concentrations may have caused high-LH pulse frequency and low-LH pulse amplitude. The greater frequency of LH pulses apparently stimulates E2 secretion by the follicle. These results are similar to previous reports in which high circulating concentrations of P4 resulted in reduced LH pulse frequency and high-LH pulse amplitude during the mid-luteal phase of the estrous cycle (Rahe et al., 1980; Walters and Schallenberger, 1984; Walters et al., 1984; Cupp et al., 1995).
The distinct differences in serum E2 despite similar follicular diameter suggest that the maturation rate of follicles depends on the hormonal milieu under which they develop, independent of measurable differences of follicle diameter. Perhaps follicles in the 7-11 Synch cows grow under greater influence from LH than cows assigned to the MGA Select protocol. Follicles in the 7-11 Synch cows, therefore, were more physiologically mature at the time PG was administered. A more intensive blood-sampling schedule will be needed in order to characterize LH patterns during and after treatment and to substantiate or refute this hypothesis.
Cows in this study had a decrease in P4 before PG injection (Figure 7). The decrease in P4 preceding PG may relate to the stage of cycle at which the PG was administered. The decrease in P4 was followed by a continued decline in P4 concentrations following PG-induced luteolysis. Three cows assigned to the MGA Select protocol that ovulated 2 to 3 d following MGA withdrawal would be on approximately d 15 or 16 of the estrous cycle the day PG was administered. This time is coincident with declining P4 concentrations that occur during the late estrous cycle.
Perry et al. (2002a) reported differences in embryonic mortality following fixed-time AI among cows assigned to a "CO-Synch" protocol that involves sequential administration of GnRH, PG, and GnRH, followed by AI (Geary et al., 2001). Higher rates of embryonic mortality occurred when cows were induced to ovulate follicles 
11 mm in diameter. Small follicles (11 mm) are, most likely, less physiologically mature at the time of GnRH-induced ovulation and may be associated with reduced oocyte and/or luteal competence. When cows were detected as being in standing estrus, follicle size did not affect pregnancy rates or late embryonic mortality (Perry, 2003). The authors suggested that oocyte and luteal competence were more dependent on steroidogenic capacity of the follicles than on follicle size at the time of GnRH-induced ovulation (Perry et al., 2002a; Perry, 2003). Our study demonstrated that steroidogenic capacity and follicular diameter may be independent measurements in dominant follicles because follicles of similar size in 7-11 Synch and MGA Select had vastly different steroid profiles. Consequently, follicular competence may be important not only for conception and resulting fertility but also for the maintenance of pregnancy (Perry, 2003). This theory is further supported by Mussard et al. (2003), who reported that premature ovulation of a dominant follicle results in decreased ovulatory size, reduced luteal function, and compromised conception rates compared to animals induced to ovulate a larger, more mature dominant follicle.
The MGA Select and 7-11 Synch protocols have been successfully used in conjunction with fixed-time artificial insemination (Kojima et al., 2002; Perry et al., 2002b; Kojima et al., 2003a,b; Stegner et al., 2004). In this case, follicles are induced to ovulate during the peak in estrous response that occurs after PG for the respective protocol (60 h for 7-11 Synch; 72 h for MGA Select). Mean follicle diameter at the time ovulation is induced when these protocols are used in conjunction with fixed-time AI suggest that both treatments potentially overcome the situation described by Perry et al. (2002a). Figure 5 helps to illustrate this point. Perry et al. (2002a) reported that high rates of embryonic mortality were associated with induced ovulation of follicles 11 mm in diameter among cows assigned to the CO-Synch protocol. Mean follicle diameter at the time ovulation is induced (Kojima et al., 2002; Perry et al., 2002b; Kojima et al., 2003a,b; Stegner et al., 2004) among cows assigned to the MGA Select or 7-11 Synch protocols exceeds the range described by Perry et al. (2002a).
It is important to note that these sequential approaches to the synchronization of estrus—MGA Select and 7-11 Synch (Perry et al., 2002b; Kojima et al., 2002; Kojima et al., 2003a,b; Stegner et al., 2004)—avoid the scenarios described by Perry et al. (2002a) and Mussard et al. (2003). Collectively, these studies with beef cows, and those reported in dairy (Thatcher et al., 2001), demonstrate the importance of presynchronization before the administration of GnRH and PG, and the resulting associated effects related to follicular development and subsequent fertility.
The data from this study represent a novel contribution to the literature related to estrous cycle control in beef cattle. Specifically, the physiological maturity of the dominant follicle is not solely related to its diameter. Follicular competence may be primarily related to steroidogenic capacity of the follicle and the endocrine environment under which the dominant follicle grows and develops.
Implications
Differences in circulating serum estradiol-17ß and progesterone concentrations in cows treated with the MGA Select and the 7-11 Synch protocols may explain differences in interval to estrus and resulting synchrony of estrus after treatment. A better understanding of these differences may lead to improved methods of synchronization of estrus and increase opportunities to inseminate beef cows at a fixed time without detecting estrus.
Footnotes
1 Contribution from the Missouri Agric. Exp. Stn. 
2 This research was supported by National Research Initiative Competitive Grant 00-35203-9175 from the USDA Cooperative State Research, Education, and Extension Service. The authors gratefully acknowledge Pfizer Animal Health, New York, NY, for providing the Lutalyse Sterile Suspension, and Merial, Athens, GA, for providing the Cystorelin for this research.
3 Correspondence—phone: 573-882-7519; fax: 573-882-4798;e-mail: pattersond{at}missouri.edu.
Received for publication July 30, 2003. Accepted for publication October 6, 2003.
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