Accurate assessment of ram sperm quality is crucial to optimizing assisted reproductive technologies in sheep. However, semen preservation can induce sperm due to osmotic, biochemical, and thermal stress. Stabilizing sperm with a suitable cooling rate and adaptation period to the extender could mitigate these effects for a more reliable evaluation. This study aimed to determine: (1) the best time to assess ram sperm quality, and (2) the factor responsible for the altered state of ram sperm during the first hours of liquid storage. In Experiment 1, ejaculated sperm were diluted and assessed for sperm motility and functionality at four preservation times: 0, 3, 6, and 24 h as sperm damage control. Both sperm motility and functionality improved after 6 h. Experiment 2 investigated the factor responsible for sperm quality change by testing the interactions of seminal plasma and extender with sperm from epididymides independently and in combination. The evaluation of sperm was performed as in Experiment 1. Sperm in groups containing the extender showed altered motility at 0 and 24 h, and lower functionality at 0 h. Thus, we could assume that extender addition initially alters ram sperm, causing sublethal damage that is reversible after 3 to 6 h of semen preservation. In conclusion, ram sperm require an adaptation time of 3 to 6 h to the extender before an accurate quality assessment can be conducted. This has practical implications for reproduction centers, enabling better workflow organization and optimal expression of ram sperm attributes when cervical artificial insemination is routinely performed.
Keywords: cooled sperm; membrane stability; ovine; semen preservation; stabilization.