The objective was to develop a freezing protocol using a directional freezing (DF) technique for cryopreservation of rhesus macaque sperm and achieve a survival rate comparable to that achieved with a conventional freezing (CF) technique. Rhesus macaque sperm frozen with a DF technique, with cooling rates of 12 or 16 °C/min, had higher post-thaw motility (P < 0.05) than those cooled at 7 °C/min (59.3, 61.1, and 50.3%, respectively). Furthermore, sperm cryopreserved with 5% glycerol and a DF technique had similar frozen-thawed sperm motility to those cryopreserved by a CF technique (63.7 vs. 53.9%, P > 0.05). The function of sperm cryopreserved at the optimized cooling rate using a DF technique was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated rhesus macaques. Of the 38 mature oocytes collected, 78.9% were fertilized and 71.1, 47.4, and 42.1% of the oocytes developed to the 2-cell, morulae, and blastocyst stages, respectively. In conclusion, rhesus macaque sperm was effectively cryopreserved using a DF technique, providing a new and effective method for genetic preservation in this important species.
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