Cryopreservation of Cynomolgus Macaque (Macaca fascicularis) Sperm by Using a Commercial Egg-Yolk-Free Freezing Medium

Conventional TRIS egg yolk (TEY) freezing medium for the cryopreservation of NHP sperm has the risk of contamination due to widespread zoonotic diseases. This study was aimed at determining the optimal glycerol concentration, freezing rate, and holding time in liquid N-2 vapor for the cryopreservation of cynomolgus macaque sperm by using a commercial egg-yolk-free freezing medium (SC medium) designed for human sperm cryopreservation. Sperm motility and acrosomal integrity after freezing were assessed. Sperm in SC medium (dilution ratio, 3:1) frozen at cooling rates of -67 degrees and -183 degrees C/min in liquid N2 vapor showed higher post-thaw motility than did samples frozen at -435 degrees C/min. At the cooling rate of 183 degrees C/min and dilution in SC medium at a 3:1 ratio, post-thaw motility was higher after a holding time of 10 min than after 30 min (recommended by the manufacturer). In addition, post-thaw motility of sperm frozen in SC medium was higher with dilution ratios of 3:1, 4.5:1, and 6:1 compared with 9:1, 10.5:1, and 12:1, and the sample diluted 12:1 showed the lowest percentage of thawed sperm with intact acrosomes. Sperm showed higher post-thaw motility after freezing in TEY than in SC medium; acrosomal integrity did not differ between the 2 media. Our results indicated that cynomolgus macaque sperm can be cryopreserved successfully by using a commercial egg-yolk-free freezing medium, which provides an option for genetic preservation with decreased zoonotic risk in this important NHP species.