Effect of pre-freezing conditions on semen cryopreservation in rhesus monkeys

Although sperm cryopreservation has been studied in at least 17 non-human primate species, systematic factor optimization for any single species is lacking. Gene banking of non-human primate sperm is still in its infancy. The objective of the present study was to initiate a systematic approach to optimize the process of sperm cryopreservation for rhesus macaques, specifically, factors related to pre-freezing conditions (e.g., straw freezing position, sperm concentration, sperm washing, equilibration methods, and equilibration time periods). Straw position had no effect on post-thaw motility (P = 0.193). Sperm concentration was tested in a range from 5 × 106 mL−1 to 5 × 108 mL−1; post-thaw motility of sperm samples frozen at 5 × 107 cell mL−1 (51.0 ± 10.6%; mean ± S.D.) and 5 × 108 cell mL−1 (48.1 ± 7.3%) were higher than samples frozen at 5 × 106 cells mL−1 (33.0 ± 12.0%, P = 0.003). Comparison of motility immediately after thawing between samples with (51.2 ± 6.2%) and without washing (53.9 ± 6.8%) revealed no differences (P > 0.05). However, washing improved sperm forward progression within 1 h after thawing, whereas unwashed sperm retained higher post-thaw motility and progression during extended incubation (4 h) after thawing (P < 0.05). Equilibration methods (with or without pre-cooling) made no difference on post-thaw motility (P > 0.05), and the most effective equilibration time was the duration required for samples to acclimate to 4 °C prior to freezing. Evaluation and optimization of these pre-freezing conditions will help to minimize sources of injury, maximize survival, and contribute to the development of an optimized cryopreservation protocol for rhesus macaque sperm.