Optimize Ethylene Glycol Concentrations, Freezing Rates and Holding Times in Liquid Nitrogen Vapor for Sperm Cryopreservation of Rhesus Macaque (Macaca mulatta)

概要

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Ethylene glycol (EG) has been speculated as the most appropriate penetrating cryoprotectant for sperm cryopreservation of rhesus macaque due to the higher permeability coefficient. The present study aimed to determine the optimal EG concentration, freezing rate and holding time in liquid nitrogen (LN2) vapor for rhesus sperm cryopreservation. Among six tested EG concentrations (0, 0.18, 0.35, 0.7, 1.4 and 2.1 M), 0.7 M EG showed the most effective cryoprotection (P<0.05). Sperm frozen with 0.7 M EG at －183℃/min showed higher post-thaw motility than sperm frozen at －10, －67 and －435℃/min (P<0.05). Sperm frozen in LN2 vapor at －183℃/min with 0.7 M EG by holding for 10 min showed the highest post-thaw motility than those for 5 and 15 min (P<0.05). The function of sperm cryopreserved at the optimized EG concentration, freezing rate and holding time was further evaluated by in vitro fertilization. Of the 36 oocytes collected from gonadotropin-stimulated rhesus macaques, 61.1% were fertilized and 61.1, 44.4, and 36.1% of the oocytes developed into 2-cell, morulae, and blastocysts, respectively. Our findings provided an alternative penetrating cryoprotectant and optimal protocol for genetics preservation purposes in this important species.