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Department of Animal Science, Michigan State University, East Lansing 48824
1 Corresponding author: pursleyr{at}msu.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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and GnRH before the first GnRH of Ovsynch. Lactating dairy cows (n = 137) were assigned to receive either no treatment before Ovsynch (control) or 25 mg of PGF2 (PreP) followed 2 d later by 100 µg of GnRH (PreG), administered 4 (G4G), 5 (G5G), or 6 (G6G) d before initiating the Ovsynch protocol. Transrectal ultrasonography was performed to assess follicular size and resulting ovulation, and blood samples were collected to measure circulating concentrations of progesterone and estradiol immediately before each hormonal injection. Cows were inseminated at a fixed time 16 h after final GnRH of Ovsynch. Pregnancy diagnosis was performed 35 d later by palpation per rectum of uterine contents. Proportion of cows that ovulated to first GnRH of Ovsynch was 56.0, 66.7, 84.6, and 53.8% for G4G, G5G, G6G, and controls, respectively, and was greater for G6G than for control cows. Luteolytic response to PGF2 of Ovsynch was greater in all treated than control cows (92.0, 91.7, 96.2, and 69.2% for G4G, G5G, G6G, and control, respectively). Synchronization rate to Ovsynch was greater (92 vs. 69%, respectively) in G6G than in control cows. In addition, cows that ovulated in response to first GnRH of Ovsynch had greater response to PGF2 of Ovsynch (92.7 vs. 77.1%, respectively) and greater synchronization rate to the overall protocol (87.9 vs. 62.9%, respectively) than those that did not ovulate. Concentrations of progesterone at PGF2 of Ovsynch, and estradiol and follicle size at final GnRH of Ovsynch, were identified as significant predictors of probability of pregnancy 35 d after artificial insemination. In summary, a PGF2-and-GnRH based pre-Ovsynch strategy consisting of a 6-d interval between PreG and first GnRH of Ovsynch resulted in a greater ovulatory and luteolytic response to first GnRH and PGF2 of Ovsynch, respectively, compared with control cows. This, in turn, optimized synchronization rate to Ovsynch.
Key Words: dairy cow • Ovsynch • gonadotropin-releasing hormone • preovulatory follicle maturation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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and GnRH (Ovsynch; Pursley et al., 1995) to control luteal regression and induce ovulation in lactating dairy cows.
Despite Ovsynch’s overall positive impact, 10 to 30% of Ovsynch-treated cows failed to synchronize ovulation in response to final GnRH (Vasconcelos et al., 1999; Navanukraw et al., 2004). Ovulatory response to first GnRH of Ovsynch was the key determinant for a successful synchronization outcome (Vasconcelos et al., 1999). Ovulation to first GnRH of Ovsynch synchronized follicular wave emergence within 1.6 to 2.5 d after injection (Pursley et al., 1995; Ryan et al., 1998). This event was key to coordination of a functional dominant follicle at the time of PGF2 and subsequent final GnRH of Ovsynch. Several features were proposed as potential indicators of follicular maturational status before ovulation; namely, follicle size (Vasconcelos et al., 1999; 2001; Perry et al., 2005), follicle life span and duration of dominance (Bleach et al., 2004; Burns et al., 2005), and circulating concentrations of reproductive hormones, such as progesterone (P4) and estradiol (E2; Sartori et al., 2004; Souza et al., 2005). Yet, the concepts of maturational status of the preovulatory follicle and its impact on fertility have not been elucidated.
Our overall long-term objective is to restructure Ovsynch to induce the ovulation of follicles with greater fertility potential. The objective of this study, the first in a series, was to develop a pre-Ovsynch hormonal strategy that increased the percentage of cows that ovulated in response to the first GnRH injection of Ovsynch. We hypothesized that if cows ovulated in response to first GnRH injection, the subsequent ovulatory follicle yielded by Ovsynch would also be synchronized in terms of maturational status and overall synchronization rate to Ovsynch would be improved. In addition, potential associations between indicators of follicular maturational status and fertility were evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Treatments
Lactating Holstein dairy cows (n = 137) were assigned to treatments on a weekly basis before first service between 62 and 70 DIM. Cows were blocked by parity (primiparous vs. multiparous) and assigned randomly to each of the following groups (Figure 1
). Cows assigned to treatments received 100 µg of GnRH (PreG; Ovacyst, IVX Animal Health, Inc., St. Joseph, MO) either 4 d (G4G, n = 33), 5 d (G5G, n = 31), or 6 d (G6G, n = 32) before the first GnRH injection of Ovsynch, then completed the Ovsynch program. Cows in each of these groups received 25 mg of PGF2 (PreP; Prostamate, IVX Animal Health, Inc.) 2 d before PreG. We refer to these 2 injections (PreP and PreG) as pre-Ovsynch treatments. Controls (n = 34) were treated with the Ovsynch protocol only (Pursley et al., 1995).
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Analysis of Luteal and Follicular Function
Cycling status before treatment was assessed in all cows based on 1) serum concentrations of P4 measured in 2 blood samples collected 10 or 11 d before and on the day of PreP in treated cows, and 2) detection of a corpus luteum (CL) by transrectal ultrasonography on day of PreP. Cows were considered to be cycling when concentrations of P4 were > 0.5 ng/mL in at least one sample. All cows with concentrations of P4 > 0.5 ng/mL also had a CL, as detected by transrectal ultrasonography. Two cows with concentrations of P4 < 0.5 ng/mL in both samples were considered to be cycling based on detection of a growing CL by transrectal ultrasonography throughout the 4 examinations performed between PreP and first GnRH of Ovsynch.
To assess luteal function at each hormonal injection of Ovsynch, additional blood samples were collected from an unbiased selected subset of cows (n = 101) immediately before the first GnRH injection, before PGF2, and before the final GnRH injection of Ovsynch. Cows were considered to have a functional CL at PGF2 when concentrations of P4 were > 1 ng/mL and no luteolysis before PGF2 of Ovsynch was detected. Luteolysis was defined as a decrease in P4 from > 1 ng/mL on the day of PGF2 of Ovsynch to < 1 ng/mL 2 d later. Three cows with concentrations of P4 < 1.5 ng/mL 2 d after PGF2 of Ovsynch were also considered to have undergone luteolysis in response to the injection based on a 
70% decline in P4 and a continuous decrease in size or disappearance of the CL during the subsequent 4 d, as measured by transrectal ultrasonography. Cows undergoing luteolysis before PGF2 of Ovsynch were defined as having < 1 ng/mL P4 on day of PGF2 of Ovsynch after having either concentrations of P4 > 1 ng/ mL at, or detection of a new CL in response to, first GnRH of Ovsynch. Ultrasonographic information also was used to verify presence and absence of CL. Two cows with P4 concentrations < 1.7 ng/mL immediately before PGF2 of Ovsynch also were considered to have had luteolysis before PGF2 of Ovsynch based on a significant decrease in the concentration of P4 and in CL size from first GnRH to PGF2 of Ovsynch. Circulating concentrations of E2 were measured in blood samples collected at the time of final GnRH of Ovsynch in cows that synchronized to Ovsynch.
All blood samples were collected by coccygeal venipuncture using tubes containing no anticoagulant additive (BD Vacutainer, Preanalytical Solutions, Franklin Lakes, NJ). Samples were refrigerated overnight and centrifuged at 3,000 x g for 20 min. Serum was collected and stored frozen at –20°C for later analyses of P4 and E2.
Analysis of Follicular Development
Ovarian structures of an unbiased selected subset of cows (n = 101) were recorded by transrectal ultrasonography using a 7.5-MHz linear array probe (Aloka SSD-900V, Aloka Co. Ltd., Wallingford, CT). Transrectal ultrasonography was performed immediately before each hormonal injection to identify and measure ovarian structures, and 2 d after each GnRH injection to observe the subsequent fate of each dominant follicle. Perpendicular cross-sections of the antrum of each follicle > 4 mm in diameter and all detectable luteal structures were measured using a built-in caliper. Follicular and luteal measurements were recorded within an ovarian map for each cow. Mean follicular diameter was obtained by averaging both cross-section measurements for each follicle. Ovulation was defined as disappearance of a follicle(s) 48 h after GnRH injection, followed by detection of newly formed CL. If new CL were not detected concurrently with disappearance of dominant follicles, cows were reevaluated 4 d later to assess luteal development. Dominant follicles were defined retrospectively as follicles that went on to ovulate in response to GnRH. Follicles that did not ovulate in response to GnRH were assumed to be not functional and already undergoing atresia. Preovulatory follicles were defined as dominant follicles measured and mapped at the time of final GnRH of Ovsynch that were not present 2 d later. Synchronization rate was defined as the percentage of cows that responded to both PGF2 (by regressing a functional CL) and final GnRH of Ovsynch (by ovulating; Pursley et al., 1995).
Hormonal Assays
Concentrations of P4 (ng/mL) in serum were determined by using a commercially available solid-phase radioimmunoassay (Coat-A-Count Progesterone, Diagnostic Products Corporation, Los Angeles, CA). Sensitivity of the assay was 0.1 ng/mL; intra- and interassay coefficients of variation were 3.4 and 4.3%, respectively.
Serum concentrations of E2 (pg/mL) at the time of final GnRH of Ovsynch were determined only in synchronized cows. Duplicate 400-µL serum samples were extracted with ether. Concentrations of E2 were measured using a modified version (Prendiville et al., 1995) of a commercially available radioimmunoassay MAIA kit (Polymedco Inc., Cortlandt Manor, NY). Intra- and interassay coefficients of variation were 2.5 and 13.9%, respectively, for control serum samples that averaged 2.6 pg/mL; and 4.1 and 19.8%, respectively, for control serum samples that averaged 7.3 pg/mL; sensitivity of the assay was 0.19 pg/mL.
Statistical Analyses
Of the 137 cows initially assigned to the study, only 130 were considered for analyses. The 7 excluded cows were culled from the herd before all data were collected or were inseminated prematurely after detected estrus following PreP. Statistical analyses on hormonal concentrations during Ovsynch, follicular data, ovulation to first GnRH, luteolytic response to PGF2 of Ovsynch, luteolysis before PGF2 of Ovsynch, and synchronization rate to Ovsynch were based on a subset of 101 cows. Cyclicity status and pregnancy outcome were analyzed for a total of 130 cows.
Binomial variables were analyzed using a generalized linear mixed model fitted with the GLIMMIX procedure of SAS (Version 9.1, SAS Inst. Inc., Cary, NC). The model considered treatment, parity, and their interaction as fixed effects and weekly groups as a random effect. For analysis of pregnancy data, milk production was included as a covariate, and service sire and AI technician were considered for inclusion as random effects. Predicted probabilities of pregnancy and odds ratios were computed using the LOGISTIC procedure of SAS.
Continuous variables were analyzed using a linear mixed model implemented with the MIXED procedure of SAS. Fixed effects considered in the model were treatment, parity, and their interaction; weekly group was included as a random effect. In the initial analysis, we observed a potential difference in variation of concentrations of P4 at first GnRH and preovulatory follicle size among treatments. Therefore, models of several covariance structures were compared for these variables. The null model had homogeneous residual variance, whereas the alternative models included heterogeneity of residual variance because of treatment or because of ovulation to first GnRH. The model of best fit was selected based on the Bayesian information criteria, Akaike’s information criteria and likelihood ratio test against the null model, as reported by the MIXED procedure of SAS. The Bayesian and Akaike’s information criteria values are used as statistical criteria to select the simplest model that best represents the data. Residual analysis was performed to check model assumptions and to detect potential outliers. The model selected based on this procedure was used in the comparison of the fixed effects mentioned above.
Preplanned pairwise comparisons were performed between each treatment and the control using Dunnett’s test from the MIXED procedure of SAS. Dunnett’s test was selected for statistical analyses because it allows the comparison of each treatment with a reference (control). In this study, the control was used as the reference group for all comparisons. Analysis of circulating concentrations of P4 at PGF2 of Ovsynch across groups required adjustments by P4 concentrations at first GnRH prior to treatment comparisons because 1) P4 at PGF2 was dependent (P < 0.001) on P4 at first GnRH of Ovsynch, and 2) the variability of P4 at first GnRH of Ovsynch was different (P < 0.001) across treatments (see Results). The adjustment was performed by including the covariate "concentration of P4 at first GnRH" in the statistical model used to analyze circulating concentrations of P4 at PGF2 of Ovsynch. This model also included the effects of treatment, parity, and cycling status.
Verification of the Randomization of Cows into Treatments
Percentage of cycling cows 7 to 8 d before the first GnRH injection of Ovsynch was not different for treated and control cows (P = 0.49) and averaged 89% across groups. Also, treated cows did not differ from controls in milk production within 7 d of AI (45 ± 1 kg/d, P = 0.90) DIM at AI (83 ± 2 DIM, P = 0.36), or BCS at calving (2.6 ± 0.3, P = 0.80), BCS during early lactation (2.4 ± 0.3, P = 0.28), and BCS at AI (2.4 ± 0.3, P = 0.64). Thus, treatment effects were likely not confounded by these factors.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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. Percentage of cows that ovulated in response to the first GnRH injection of Ovsynch was greater (P < 0.03) in G6G compared with control, whereas neither G4G nor G5G differed from controls. Percentage of cows that responded to PGF2 of Ovsynch was greater in G4G, G5G, and G6G compared with controls (P < 0.05 for each comparison). Synchronization rate in response to Ovsynch was increased (P = 0.05) in G6G compared with controls. Synchronization rate was not different between controls and G4G or G5G. Proportion of cows pregnant 35 d after AI tended (P = 0.08) to be greater in G6G compared with controls.
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) circulating concentrations of P4 than controls at the time of first GnRH of Ovsynch. Concentrations of P4 before the first GnRH injection of Ovsynch were less (P < 0.001) variable in treated vs. controls (estimated variances of 2.11 vs. 7.78, respectively).
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). As expected, follicle size at first GnRH of Ovsynch increased (P = 0.006) linearly from G4G to G6G (Table 1). Seventy-one percent of cows assigned to pre-Ovsynch treatments responded to both injections, PreP (luteal regression) and PreG (ovulation). Cows that responded to both PreP and PreG were considered to initiate a new estrous cycle.
Effect of Ovulatory Response to First GnRH Injection of Ovsynch
Cows that ovulated in response to first GnRH of Ovsynch were more (P < 0.03) likely to respond to PGF2 of Ovsynch than those that did not ovulate to the injection (92.7 vs. 77.1%, respectively). In addition, synchronization response to Ovsynch was improved (P < 0.005) in cows that ovulated in response to first GnRH of Ovsynch compared with those that did not (87.9 vs. 62.9%, respectively;).
Treated and control cows identified as noncycling before initiating treatments had a greater (P < 0.02) incidence of luteolysis between first GnRH and PGF2 of Ovsynch than cows identified as cycling before treatment (28.6 vs. 6.9%, respectively).
Concentrations of P4 at PGF2 of Ovsynch
Among cows that had a functional CL at PGF2 of Ovsynch, circulating concentrations of P4 were greater (P = 0.007) in G6G than in controls (Table 1), but were not different between either G4G or G5G and controls (Table 1). Number of functional CL present at PGF2 of Ovsynch was greater (P < 0.04) in G6G than in control (Table 1) but did not differ between G4G or G5G and controls (Table 1).
Among cows that had synchronized ovulation in response to Ovsynch, circulating concentration of P4 at PGF2 of Ovsynch was identified as a predictor of pregnancy 35 d after AI with an odds ratio of 1.7. The predicted probability of pregnancy increased (P < 0.007) with greater concentrations of P4 at PGF2 of Ovsynch (Figure 3).
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). Nevertheless, variability in follicle size at final GnRH of Ovsynch was decreased (P = 0.02) when cows had ovulated in response to first GnRH of Ovsynch than if they had not ovulated (Figure 4). Estimated variance of follicle size at final GnRH of Ovsynch was 2.5 and 6.0 for cows that did or did not ovulate in response to first GnRH of Ovsynch, respectively. Furthermore, a quadratic (P < 0.05) relationship between follicle size at final GnRH of Ovsynch and probability of pregnancy was identified (Figure 5).
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). Moreover, a positive linear (P < 0.001) relationship was identified between preovulatory follicle size and circulating concentrations of E2 at final GnRH of Ovsynch. The relationship between preovulatory follicle size and circulating concentrations of E2 at final GnRH of Ovsynch differed (P < 0.03) for cows diagnosed pregnant compared with those diagnosed not pregnant 35 d after AI. The linear slope between follicle size and E2 concentrations at final GnRH of Ovsynch was greater (Interaction P < 0.03) in cows diagnosed pregnant 35 d after AI than for those diagnosed not pregnant (Figure 7). Thus, for any given follicle 16 mm in diameter, pregnant cows had greater (P < 0.01) circulating concentrations of E2 than nonpregnant cows. In contrast, if preovulatory follicle was approximately 14 mm in diameter, circulating concentrations of E2 did not differ with pregnancy status.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Data from this study indicated that G6G increased the percentage of cows that ovulated in response to first GnRH of Ovsynch and yielded a greater percentage of cows that synchronized to Ovsynch. These data indicated that ovulation to the first GnRH of Ovsynch positively affected luteal and follicular responses to subsequent PGF2 and final GnRH of Ovsynch. Pregnancy information indicated that concentrations of P4 at PGF2 of Ovsynch, and concentrations of E2 and follicle size at final GnRH, were key indicators of fertility.
Effects of Pre-Ovsynch Treatments with PGF2 and GnRH
A new estrous cycle was initiated in 81% of cows treated with PreP and PreG. This included cycling cows that either responded to both injections, cows that were in latter stages of the estrous cycle in which CL regression already had occurred, or noncycling cows that responded only to PreG. In contrast, controls were assumed randomly distributed throughout the estrous cycle at the time of first GnRH of Ovsynch. Randomness of controls was evident by the greater variation in concentrations of P4 at the time of the first GnRH of Ovsynch compared with treated cows. Surprisingly, only 54% of controls ovulated in response to first GnRH of Ovsynch administered at random stages of the cycle. Previous reports indicate a range of 64 to 90 % in ovulatory response to first GnRH of Ovsynch administered at random stages of the cycle (Pursley et al., 1995; Vasconcelos et al., 1999). Ovulatory response to GnRH at random stages of the estrous cycle was variable between studies (Pursley et al., 1995; Ryan et al., 1998). Variation between studies may be explained by differences in stage of the estrous cycle and stage of follicle wave development at the time of the injection (Vasconcelos et al., 1999) or physiological differences of cows. Physiological differences could include differences in length of interwave intervals (time from onset of one follicle wave emergence to another) because of bST treatment (Lucy et al., 1994) or random differences in concentrations of GnRH, gonadotropins, E2, inhibin, or P4 (Ireland et al., 2000; Thatcher et al., 2002). If interwave intervals were longer, the chance of inducing an ovulation to a random injection of GnRH would increase as a result of having a lesser proportion of cows in early stages of follicular wave development at any given time. On the contrary, if shorter, the chance of a GnRH-induced ovulation would decrease as a result of having a greater percentage of cows in early stages of follicle development before acquisition of LH receptors, at any given time.
Treated cows that initiated a new estrous cycle following PreG received first GnRH of Ovsynch 4, 5, or 6 d later; thus, on d 4, 5, or 6 of the cycle. Previous studies showed that initiation of Ovsynch on d 5 to 9 of the estrous cycle was key to successful synchronization of ovulation (Vasconcelos et al., 1999; Moreira et al., 2000). A likely reason for this synchrony was the presence of a functional dominant follicle capable of ovulating in response to the LH surge induced by the first GnRH injection of Ovsynch (Vasconcelos et al., 1999; Moreira et al., 2000). In the current study, a greater percentage of cows ovulated in response to first GnRH of Ovsynch following a 6-d interval after PreG compared with controls. In contrast, the proportion of cows that ovulated to first GnRH of Ovsynch following a 4- or 5-d interval after PreG did not differ from controls. Given the high percentage of cows that ovulated to PreG, a 4- or 5-d interval to first GnRH of Ovsynch did not allow sufficient time for deviation and acquisition of LH receptors by the growing follicle. Indeed, as follicular size at first GnRH increased linearly from G4G to G6G, so did the proportion of cows that ovulated to first GnRH (56, 67, and 85%, for G4G, G5G, and G6G, respectively). Therefore, in this study, acquisition of ovulatory capacity progressed with time. This progression likely resulted in differences among cows in time to deviation of the dominant follicle from largest subordinate follicles. Previous studies indicated that expression of LH receptors in granulosa cells after deviation was required for ovulation (Xu et al., 1995). After deviation, development of ovulatory capability was associated with follicular size in an LH dose-dependent manner (Sartori et al., 2001).
Proportion of cows having a luteolytic response to PGF2 of Ovsynch was greater in all treatments compared with controls. Also, response to PGF2 of Ovsynch was more likely to occur in cows that ovulated in response to first GnRH. In a previous study, luteolytic response to PGF2 of Ovsynch was also an important contributor to successful synchronization of ovulation in response to Ovsynch (Moreira et al., 2000). In our study, 2 possible scenarios were observed in those cows in which synchronized luteolysis failed in response to PGF2 of Ovsynch. The CL in these cows either underwent luteolysis before PGF2 of Ovsynch or failed to respond to PGF2. Cows in which the CL was regressed before PGF2 of Ovsynch were more likely to show estrus and have a spontaneous preovulatory LH surge before induction of ovulation by the final GnRH injection of Ovsynch (Vasconcelos et al., 1999; Moreira et al., 2000). As a consequence, ovulation was not synchronized with timed AI. A previous study indicated that spontaneous luteolysis before PGF2 of Ovsynch was less likely to occur if heifers were started on Ovsynch before d 9 of the cycle (Moreira et al., 2000), as was the case for most treated cows in our study. PreP was intended to induce regression of all mid and late-cycle CL. As a result, probability of spontaneous luteolysis during the following 15 d (when Ovsynch was implemented) was minimized in treated cows. Therefore, the new CL induced by PreG, the first GnRH, or both, were more likely to remain functional until injection of PGF2 of Ovsynch.
Treatment with G6G outperformed other treatments by increasing the percentage of cows ovulating to first GnRH and the percentage of cows that had a luteolytic response to PGF2 of Ovsynch. As a result, significantly more G6G-treated cows synchronized to Ovsynch compared with controls. Based on treatment effects, it is clear that ovulatory response to first GnRH and luteolytic response to PGF2 are critical for successful synchronization of ovulation in response to Ovsynch. It is not clear if G6G alone can improve conception rates in Ovsynch-treated cows. Further testing with greater numbers of cows and farms would be required for validation of the accuracy of conception rates. It should be noted, though, that this study was not originally designed to test pregnancy outcome in response to pre-Ovsynch treatments. This study was designed to test ways to improve ovulation rate to first GnRH of Ovsynch, which is considered to be a critical factor for overall improved success to Ovsynch (Vasconcelos et al., 1999). Future studies will further evaluate control of follicle growth during Ovsynch and its effect on fertility.
Effect of Ovulation in Response to First GnRH of Ovsynch
Ovulatory response to the first GnRH injection of Ovsynch is a critical determinant of the overall synchronization outcome to Ovsynch (Vasconcelos et al., 1999; Moreira et al., 2000). The CL in cows that ovulated after the first GnRH of Ovsynch, regardless of treatment, were more likely to undergo luteolysis in response to PGF2 of Ovsynch and have a greater synchronization rate compared with cows that did not respond to first GnRH. Moreover, if cows ovulated after first GnRH, they were more likely to have a functional dominant follicle capable of ovulation at the final GnRH injection of Ovsynch (Vasconcelos et al., 1999). On average, a future dominant follicle takes 7 to 10 d to go through emergence, deviation, and dominance, before ovulating or becoming atretic (Ginther et al., 1989a). Induction of ovulation is possible before atresia of the dominant follicle (Silcox et al., 1993; Moreira et al., 2000). A dominant follicle that emerges after ovulation to first GnRH of Ovsynch is likely to still be functional 9 d later, at the final GnRH of Ovsynch. In contrast, a follicle that emerges within 3 d before the first GnRH of Ovsynch will probably not ovulate to first GnRH and may already be atretic before the final GnRH. Thus, it is not surprising that synchronization of ovulation to Ovsynch increased if cows ovulated to first GnRH.
Variability in follicle size at final GnRH was decreased when cows had ovulated to first GnRH of Ovsynch. Consequently, the ovulatory follicle of Ovsynch was more homogeneous in size. A growing body of evidence strongly supports an association between preovulatory follicle size and fertility in cattle (Cooperative Regional Research Project, 1996; Peters and Pursley, 2003; Perry et al., 2005). Taken together, the evidence indicates that preovulatory follicle size less than or greater than a corresponding minimal or maximal threshold is related to impaired fertility. Differences in type (beef vs. dairy) and age or physiological status (heifers vs. cows) of cattle used in studies make it difficult to provide absolute threshold values applicable to all scenarios. In spite of this limitation, a general appreciation of these studies indicates that for each case, there may be a defined window of preovulatory follicle size in which fertility may be optimized. Controlling ovulation in response to first GnRH of Ovsynch may provide an opportunity to control more precisely the size of the preovulatory follicle of Ovsynch and evaluate the direct effect of such precision on fertility.
Potential Indicators of Fertility
Our study identified 3 significant predictors of fertility. Concentrations of P4 at PGF2 of Ovsynch, concentrations of E2, and follicle size at final GnRH of Ovsynch were associated with an increased probability of pregnancy 35 d after AI.
Predicted percentage of cows pregnant increased with greater concentrations of P4 at PGF2 of Ovsynch. Fonseca et al. (1983) showed a positive relationship between concentrations of P4 at induction of luteolysis and conception rates. Conversely, lesser concentrations of P4 at induction of luteolysis were associated with a decline in CR (Xu et al., 1997). This evidence indicates that fertility might depend, at least partially, on the concentrations of circulating concentrations of P4 before luteolysis.
Competency of the dominant follicle, quality of its oocyte, or both, may be affected by the P4 environment in which they develop and mature, respectively (Mihm et al., 1994; Revah and Butler, 1996). Knowledge gained from the persistent-follicle model indicates that this may be the case. Greater LH pulsatility, as induced by maintenance of subluteal concentrations of P4, yielded a persistent preovulatory follicle of larger size and extended duration of dominance (Mihm et al., 1994; Bridges and Fortune, 2003). Greater LH pulsatility also induced oocytes to undergo premature resumption of meiosis (Revah and Butler, 1996). Fertility of cows that ovulated persistent follicles was compromised (Cooperative Regional Research Project, 1996). Thus, the P4 environment in which a follicle is induced to grow may affect its subsequent fertility potential. In our study, pre-Ovsynch treatment influenced P4 environment: circulating concentrations of P4 at PGF2 of Ovsynch in cows with a functional CL were greater in G6G vs. controls. This may be partially explained by more CL present at PGF2 of Ovsynch in the ovaries of G6G cows compared with controls. Nevertheless, the mechanisms underlying the association between P4 environment during follicular development and subsequent fertility are not completely understood.
Fertility also was associated with the size and function of the preovulatory follicle at final GnRH of Ovsynch. Follicle size at final GnRH of Ovsynch was a significant predictor of pregnancy 35 d after AI. A quadratic effect indicated that fertility was greater after ovulation of a follicle approximately 16 mm in diameter. A similar quadratic relationship was recently reported (Perry et al., 2005). These results reinforce the concept proposed previously in this discussion, regarding the possibility of a defined window of preovulatory follicle size in which fertility might be optimized.
Follicle size at final GnRH of Ovsynch was positively associated with the functionality of the preovulatory follicle in terms of production of E2. A similar relation-ship was previously indicated (Ireland and Roche, 1982). The linear slope of the association between follicular size and concentrations of E2 at final GnRH of Ovsynch differed with pregnancy status diagnosed 35 d after AI. Thus, for a given preovulatory follicle greater than 16 mm in diameter, concentrations of E2 were greater in cows subsequently diagnosed pregnant compared with cows diagnosed not pregnant. Moreover, probability of pregnancy increased with greater concentrations of E2 at the final GnRH of Ovsynch. Estradiol produced by the preovulatory follicle plays a key role in physiological events critical for reproductive success. Estradiol is required for development of the dominant follicle, triggering the LH surge essential to cause ovulation, and resumption of meiosis in the oocyte (Greenwald and Roy, 1994). Our data indicate that follicular size and function could be jointly used to define maturational status of the preovulatory follicle and to possibly predict subsequent fertility in dairy cows. The way in which these indicators interplay to predict a pregnancy, however, is not clear. Relative weight of each of these indicators on predicting fertility may be variable and interdependent. For example, in this case, the relative importance of circulating E2 concentrations for predicting fertility varies with preovulatory follicular size. We believe this is a novel concept regarding the relationship between E2, follicle size, and potential for a subsequent pregnancy.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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- and-GnRH–based strategy preceding Ovsynch. This strategy consisted of a PreP injection followed 2 d later by PreG, which in turn, was administered 6 d before the first GnRH of Ovsynch. This strategy also enhanced the response to PGF2 of Ovsynch and increased synchronization rate to Ovsynch compared with controls. Concentrations of P4 at PGF2 of Ovsynch, as well as concentrations of E2 and follicle size at final GnRH of Ovsynch, were identified as significant predictors of probability of pregnancy 35 d after AI. Our ultimate goal is to effectively and consistently control follicular and luteal development and function through a new synchronization program, and test its effect on fertility of lactating dairy cows. The present study constitutes the first of a series of experiments that will evaluate ways to accomplish our goal. From this study, it was clear that a 6-d interval between PreG and first GnRH of Ovsynch increased ovulatory response to the latter. Future studies will further evaluate the effectiveness of strategies to maximize ovulation to first GnRH of Ovsynch and ways to pharmaceutically control ovulatory follicle development to enhance fertility of lactating dairy cows.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication November 30, 2005. Accepted for publication March 10, 2006.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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indicates a tendency for a difference from controls.
) or was not (•) synchronized* in response to Ovsynch are indicated. *Cows having luteolytic response to PGF2
