Effect of Insemination Timing Within a <scp>TAI</scp> Program on Fertility Using Sex‐Sorted Semen in Lactating Dairy Cows

The objective of this study was to evaluate the effect of insemination timing with sex-sorted semen on fertility in dairy cows subjected to a timed artificial insemination (TAI) protocol. A total of 611 Holstein cows (46 ± 3 DIM) were enrolled and subjected to a presynchronized Ovsynch protocol (G7G; PGF₂α-2d-GnRH-7d-GnRH-7d-PGF₂α-56 h-GnRH), and randomly allocated to four treatment groups. The control group (CONV-14, n = 154) was inseminated with conventional semen 14 h after the final GnRH, while cows in the sex-sorted semen groups were inseminated at 14 (SS-14, n = 152), 18 (SS-18, n = 153), or 22 h (SS-22, n = 152) after the same treatment. The same bull was used for all inseminations. All cows were examined by ultrasonography to individually evaluate ovarian responses to the protocol and pregnancy status. No significant differences were observed among groups in body condition score, milk yield, cyclicity at the beginning of the protocol, response to the protocol, or follicle size at TAI. Pregnancies per artificial insemination were similar with 50.0% (77/154) in the CONV-14 group, 42.8% (65/152), 48.4% (74/153), and 43.4% (66/152) in the SS-14, SS-18, and SS-22 groups, respectively. No significant difference was observed in embryonic loss rates among groups: 5.2% in CONV-14, 9.2% in SS-14, 4.1% in SS-18, and 13.6% in SS-22, while SS-22 was numerically higher (~7%) than the average of the other SS groups. Overall, conception rates were higher in cows responding to the first GnRH than in non-responders (49.7% vs. 32.3%, p < 0.0005), with a significant difference observed only in the CONV-14 and SS-18 groups (p < 0.005). Estrous expression during TAI was associated with higher conception rates in the CONV-14 group (75.0% vs. 45.4%, p = 0.008), while no such difference was detected in the combined SS groups (51.8% vs. 43.3%). However, the conception rate in the SS-22 group (36.7%) was distinctly lower (p < 0.02) than in other SS groups (53.6% in SS-14, 68.0% in SS-18). In conclusion, contrary to the expectation that advancing insemination closer to ovulation with sex-sorted semen would be advantageous, fixed time insemination at 22 h within the TAI program showed poorer outcomes compared to 18 h, which achieved a relative conception rate of 97% compared with conventional semen. It was also concluded that TAI at 22 h should not be recommended in cows exhibiting estrus on the day of insemination.