The first hypothesis better fertility
The first hypothesis, better fertility in heifers that had CL at day 0, was clearly supported by our results. Heifers with a CL had over 60% P/AI compared to ∼50% in heifers without CL. These results are consistent with several other studies reporting that cycling heifers have greater fertility than non-cycling heifers [3,4,16,21]. For example, Gutierrez et al.  evaluated 2260 heifers using reproductive tract score (RTS; scale - 1 immature to 5 - mature) at beginning of TAI protocol and reported that heifers with CL (RTS 5) had greater P/AI (64.6%; 788 of 1220) compared with heifers without CL (RTS 1, 2, 3 and 4 combined; 52.4%; 756 of 1440), very similar to our current results (Table 2). Thus, improvements in TAI protocols for beef heifers should clearly be focused on increasing the percentage of heifers that are cycling at beginning of protocol . The increase in fertility is probably related to a combination of improvements in circulating hormones, follicle development, oviduct/uterine environment, and maturation of the hypothalamus-pituitary-gonadal axis . It should be noted that stage of CL would be expected to be highly variable because heifers were not presynchronized and therefore would be at a random stage of the estrous AICAR at the beginning of the protocol. In addition, heifers without CL on day 0 could still have been cycling and just at a stage of the cycle that did not have a detectable CL. The preovulatory follicle was larger in heifers with CL present at the beginning of protocol (10.4 mm vs 9.6 mm; P = 0.0058). It has been reported that post pubertal heifers have larger dominant follicles than pre pubertal heifers . In addition, beef heifers with high serum P4 concentration at PGF injection tend to have increased preovulatory follicle size at the end of a TAI protocol , so ovulation of a larger follicle may underlie some of the benefit observed in P/AI. Nevertheless, excessive P4 concentrations during the TAI protocol have generally been viewed as negatively affecting fertility in beef cattle, possibly due to inhibition of GnRH/LH pulses by the increased circulating P4 potentially causing a reduction in follicle growth [22,23]. Although we only evaluated the presence of CL using ultrasonography and did not measure circulating P4, this does not seem to be the case in our study. It is intriguing that GnRH but not ECP led to greater P/AI in heifers with CL but not in heifers without CL. The decrease in circulating P4 after PGF treatment requires 24 h to reach below 1 ng/mL and approximately 36 h to reach nadir concentrations . It seems possible that ECP is less likely to induce ovulation in heifers that have CL at the end of the protocol due to an inhibition of E2 action by the delayed decrease in circulating P4 during CL regression. Obviously, this concept still needs to be tested in future experiments by evaluating ovulation to the protocol and determining circulating P4 and presence of CL at both, the beginning and the end of a TAI protocol. Our second hypothesis, that heifers treated with ECP would be more likely to manifest estrus, was somewhat predictable, based on previous studies and cattle physiology [12,16,, , ]. In our study, treatment with ECP increased expression of estrus compared to GnRH treatment, regardless of the presence of CL at the beginning of protocol (Table 1, Table 2). Occurrence of estrus after ECP treatment is well documented for lactating dairy and beef cows [13,18] and in heifers [19,25,26]. A high proportion of ECP-treated heifers with medium-sized or large follicles were observed in estrus (98.2%; 108 of 110) and the majority of heifers with small follicles (<8.5 mm) were also in estrus after ECP treatment (77.1%; 27 of 35; Fig. 2A). A previous study with lactating dairy cows demonstrated that treatment with 1 mg of ECP increased circulating E2 for 50.7 h with a peak concentration of 3.4 pg/mL at 30.7 h after treatment . Thus, our treatment with 0.5 mg of ECP is likely to induce a prolonged but lower increase in circulating E2, but it appeared to be enough to induce estrus in nearly all heifers. In contrast, endogenous E2, secreted by the dominant follicle was the single source of circulating E2 in GnRH-treated heifers and therefore, there was a clear relationship between follicle size and estrus manifestation in GnRH-treated heifers but not in heifers treated with exogenous ECP [8,27,28]. In our study, a low proportion of GnRH-treated heifers with small follicles expressed estrus (5.6%; 2 of 36), whereas estrus was observed in over 60% of heifers with medium-sized follicles (61.7%; 37 of 60), and nearly 100% of GnRH-treated heifers with large follicles showed estrus (98.3%; 58 of 59; Fig. 2A).