Freezing Protocol Optimization for Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm under Field Conditions

SIMPLE SUMMARY: The germplasm banks of wild species, such as Iberian red deer, are not widespread, mainly due to the difficulties of collecting and cryopreserving reproductive cells. Optimal freezing protocols under field conditions could be a breakthrough for these species. In this study, epididymal sperm was evaluated using two methods of sperm storage during refrigeration (tube and straw); four equilibration periods (0, 30, 60, and 120 min); and four methods of freezing (cryopreservation in liquid nitrogen vapors in a tank (control) or box, freezing in dry ice, or freezing over a metallic plate). The results showed that samples stored in straws during refrigeration produced less apoptotic spermatozoa and more viable spermatozoa withactive mitochondria. A long equilibration period (120 min) yielded a higher percentage of acrosomal integrity. Moreover, there was no difference in sperm quality between freezing in liquid nitrogen vapors in a tank or box. However, a worse quality was obtained when the samples were cryopreserved in dry ice or over a metallic plate compared to the control. ABSTRACT: Creating germplasm banks of wild species, such as the Iberian red Deer (Cervus elaphus hispanicus) can be challenging. One of the main difficulties is the obtention and cryopreservation of good-quality reproductive cells when the spermatozoa are obtained from epididymides after death. To avoid a loss of seminal quality during transport, developing alternative methods for cooling and freezing sperm samples under field conditions is necessary. The objective of this study was to evaluate the effects of different durations of equilibrium and different techniques of cooling and freezing on Iberian red deer epididymal sperm quality after thawing to optimize the processing conditions in this species. Three experiments were carried out: (I) evaluation of refrigeration in straws or tubes of 15 mL; (II) study of equilibration period (0, 30, 60, or 120 min); and (III) comparison of four freezing techniques (liquid nitrogen vapor in a tank (C), liquid nitrogen vapor in a polystyrene box (B), dry ice (DY), and placing straws on a solid metallic plate floating on the surface of liquid nitrogen (MP)). For all experiments, sperm motility and kinematic parameters, acrosomal integrity, sperm viability, mitochondrial membrane potential, and DNA integrity were evaluated after thawing. All statistical analyses were performed by GLM-ANOVA analysis. Samples refrigerated in straws showed higher values (p ≤ 0.05) for mitochondrial activity and lower values (p ≤ 0.05) for apoptotic cells. Moreover, the acrosome integrity showed significant differences (p ≤ 0.05) between 0 and 120 min, but not between 30 and 60 min, of equilibration. Finally, no significant differences were found between freezing in liquid nitrogen vapors in a tank or in a box, although there was a low quality after thawing when the samples were cryopreserved in dry ice or by placing straws on a solid metallic plate floating on the surface of liquid nitrogen. In conclusion, under field conditions, it would be possible to refrigerate the sperm samples by storing them in straws with a 120 min equilibration period and freezing them in liquid nitrogen vapors in a tank or box.