Estrus expression after removal of
Estrus ribosomal s6 kinase after removal of P4 insert and before TAI is a clear predictor of greater P/AI in protocols that use GnRH [6,8], EB, or ECP [10,18] as ovulation inducers. Therefore, our third hypothesis was that heifers showing estrus behavior would have greater P/AI, which was supported in both treatment groups (Table 3). A very few heifers in the ECP group did not show estrus (6.1%; 13 of 214) and none became pregnant; whereas 56.2% (113 of 201) of the ECP-treated heifers showing estrus became pregnant. Of the GnRH-treated heifers, approximately one third did not show estrus (32.3%; 65 of 201) and 33.8% (22 of 65) of these heifers became pregnant. In contrast, 71.3% (97 of 136) of the GnRH-treated heifers showing estrus became pregnant, a proportion much greater than GnRH-treated heifers that did not show estrus and even greater than ECP-treated heifers showing estrus (Table 3). In heifers that expressed estrus, the greater P/AI following GnRH treatment could be due to the tendency for a larger preovulatory follicle in the heifers treated with GnRH compared to ECP (P = 0.0776; Table 3). Clearly, ECP treatment increased manifestation of estrus in heifers with smaller preovulatory follicles and, thus, the reduction in fertility in the ECP group that expressed estrus may be a result of the smaller follicle size in this group (Table 3; Fig. 2A). Finally, our fourth hypothesis was that heifers ovulating smaller follicles would have greater fertility following GnRH treatment compared to ECP treatment. This hypothesis was definitively not supported, as heifers with small follicles at TAI had low fertility to either treatment (15.5%; 11 of 71) and heifers with medium-sized follicles (8.5–10.9 mm) tended to have better fertility following ECP (62.9%; 39 of 62) compared to GnRH (46.7%; 28 of 60; P = 0.07). Thus, GnRH treatment did not improve fertility in heifers ovulating smaller follicles. Instead, ECP may have benefited these heifers if a follicle larger than 8.5 mm was present. Supplementation with ECP during proestrus in TAI protocols improved P/AI in cows with preovulatory follicles smaller than 12.2 mm in either Bos taurus  or Bos indicus  beef cattle. To our knowledge, this is the first study that provides evidence of a beneficial effect of ECP upon fertility in Bos taurus heifers with medium-sized (8.5–10.9 mm) preovulatory follicles (Fig. 2B). In another recent study, Madsen et al.  observed that preovulatory E2 is essential for pregnancy establishment and maintenance in ovariectomized cows supplemented with P4. Based on these previous data and our current results, it seems likely that circulating E2 near breeding has an important role in pregnancy establishment, although the precise E2 concentration that is required has not been established. Thus, it appears that an exogenous source of E2 can increase P/AI, especially in cows and heifers with medium-sized preovulatory follicles. Some of the most intriguing observations in our study were the findings with heifers having larger preovulatory follicles (≥11 mm). Nearly 100% (98.1%; 105 of 107) of these heifers showed estrus, independent of GnRH or ECP treatment. Unfortunately, circulating E2 was not measured in this study, however, based on expression of estrus, these heifers had sufficient endogenous E2 to induce behavioral estrus without the need for an exogenous source of E2 (Fig. 2A). Interestingly, GnRH-treated heifers had greater P/AI than ECP-treated ones. This could be due to positive effects of GnRH or alternatively, to negative effects of ECP treatment in heifers with larger preovulatory follicles. Jinks et al.  and Martins et al.  did not observe beneficial effects of ECP on P/AI in follicles larger than 12.2 mm in beef cows. One possible explanation is that treatment with GnRH at TAI, following the expected LH surge associated with estrus, may have exposed the follicle to increased LH concentrations, which could have either altered the time of ovulation or perhaps improved luteal function thereafter . We believe, however, that the most likely explanation for this result is that ECP treatment acts to reduce fertility in heifers with larger preovulatory follicles. One possible mechanism could be an earlier time of estrus and ovulation in ECP-treated heifers. In addition, perhaps timing of AI and ovulation was less ideal compared to heifers that ovulate large follicles without ECP treatment, and this could have decreased fertility in these heifers . In a previous study, non-suckled beef cows treated with ECP had earlier time to estrus and ovulation when comparing with EB . Conversely, heifers treated with GnRH may have expressed estrus later than ECP-treated heifers and therefore may have ovulated later [6,34]. Unfortunately, we do not have information in this study on the timing of ovulation or on the percentage of heifers that ovulated or did not ovulate to a given protocol. It is possible that GnRH-treated heifers had an increase in percentage of heifers that ovulated to the protocol compared to ECP-treated heifers. In this study, the increase in fertility in GnRH-treated heifers that had large (∼80%) compared to medium-sized follicles (<50%) may be related to the fertility-enhancing effects of increased circulating E2 [8,29,30] or simply to an earlier LH surge and more optimal timing of AI and ovulation . It is intriguing that there was no increase in P/AI in ECP-treated heifers with large rather than medium-sized follicles at TAI. Future research is needed to determine whether GnRH treatment enhances fertility in heifers with large follicles or if ECP hinders fertility in these females. Acquiring information on ovulation in future experiments will be particularly useful to resolve these important questions that have arisen during the analysis of outcomes in our study.