Improving long-term storage of liquid-preserved bovine semen: Effect of extender, cooling protocol and holding temperature on sperm quality and fertility.

Liquid preservation of bull semen provides a resource-efficient method for utilizing elite genetic material. However, the optimal preservation method including extender, cooling rate, and holding temperature is not fully defined. This study aimed to improve the long-term storage of liquid preserved bull semen (LS) for at least seven days. Semen from nine Holstein-Friesian bulls was diluted in a split-sample procedure with Caprogen or a fully synthetic extender (FSE) and subjected to three cooling protocols: direct cooling to 16 °C (A), direct cooling to 5 °C (B), and stepwise cooling with holding phase at 16 °C before further cooling to 5 °C (C). Sperm quality was assessed on day one (d1), four (d4), seven (d7), and ten (d10) of semen storage based on thermo-resistance test after 30 min and 120 min incubation at 38 °C (TRT30/120), morphologically intact spermatozoa (MIS), and plasma membrane and acrosome integrity (PMAI). Generalized linear mixed models revealed that Caprogen outperformed FSE across all parameters (P < 0.001) on d1, d4, and d7. On d7, storage at 5 °C (B/C) resulted in significantly (P ≤ 0.05) higher sperm quality compared to 16 °C (A). In a second trial, 99 Holstein-Friesian cows were inseminated with either LS stored for seven days at 5 °C (LS7, Protocol B, n = 53 cows) or frozen-thawed semen (FS, control, n = 46 cows). In vivo, the first-service conception rate was 49.05 % for LS7 and 52.17 % for FS (P = 0.38). Therefore, LS stored at 5 °C in Caprogen maintains sperm quality for seven days and represents a viable alternative for artificial insemination.