Background and Aim: Dairy farm profitability is linked to milk yield and reproductive efficiency. High prolactin levels during lactation can negatively impact fertility. Timed AI protocols like PG7G are used to improve pregnancy rates. This study investigates the effects of extending the voluntary waiting period (VWP) from 63 to 73 days after the PG7G protocol on reproductive parameters, including progesterone and prolactin levels, pregnancy rates, and pregnancy losses, in lactating Holstein cows during summer and winter seasons. Materials and Methods: A total of 2100 lactating dairy cows were divided into groups and assigned to the PG7G protocol based on their parity. Two groups were formed based on the number of days in milk (DIM) after the first PGF2α injection. The first group (PG7G-63) received a PGF2α injection 30-day postpartum (pp) and followed a specific protocol. The second group, PG7G-73, followed the same protocol but received a PGF2α injection 40-day pp. Pregnancy was confirmed, and blood samples were collected for analysis. Temperature and humidity were also recorded throughout the study. Results: Extension of the VWP to 73-day (PG7G-73 group) significantly improved pregnancy rates on day 47 pp compared with the 63-day VWP (PG7G-63 group). However, the PG7G-63 group exhibited lower PLs. Seasonality markedly influenced reproductive outcomes, with higher P/AI in the moderate season for primiparous cows and in the hot season for multiparous cows. Conversely, PL in both groups increased during the hot season. Conclusion: Extending the VWP from 63 to 73 days pp in lactating Holstein cows significantly improved pregnancy rates, while seasonality affected reproductive outcomes, with higher pregnancy rates in moderate temperatures and increased pregnancy loss during hot seasons. Keywords: dairy cows, days in milk, pregnancy rate, prolactin, season, timed artificial insemination.
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