Impact of cold storage on semen quality in two divergent rabbit lines selected for resilience under heat stress condition

Artificial insemination in rabbit farms is commonly performed using either fresh semen or doses stored at 14-18 °C for up to 3 days. However, exposure to elevated temperatures can impair sperm motility, concentration, and overall semen quality. This study aimed to evaluate the effect of cold storage on semen from two rabbit genetic lines divergently selected for resilience to thermal stress. A total of 254 ejaculates from 46 males belonging to the HE line (selected for reduced litter size variability) and the HO line (selected for increased litter size variability) were analyzed. Sperm motility and kinematic parameters, viability, acrosome reaction, reactive oxygen species (mROS), and mitochondrial membrane potential (MMP) were assessed. Bayesian methods were applied for statistical analysis. In fresh semen, both lines showed similar proportions of motile and progressive spermatozoa under both comfort and heat stress conditions. In contrast, after refrigeration, the HO line maintained higher motility than the HE line under comfort (75.3% vs. 66.0%; <i>P</i> = 94%) and heat stress conditions (85.3% vs. 75.7%; <i>P</i> = 97%). The HO line also exhibited a higher proportion of fast progressive spermatozoa in fresh semen under comfort (22.2% vs. 17.5%; <i>P</i> = 96%) and heat stress (22.4% vs. 18.1%; <i>P</i> = 93%) conditions. Conversely, mitochondrial activity was reduced in the HO line compared to the HE line under heat stress (56.9% vs. 67.1%; <i>P</i> = 95%). These findings suggest that genetic selection for resilience in rabbits could improve the preservation and functional quality of refrigerated semen, particularly under thermal stress.