Study on cryogenic preservation of grey mullet sperm

Abstract

Preliminary techniques of mass propagation of grey mullet were established in Taiwan some years ago. Among the many problems concerned, the cryogenic preservation of grey mullet sperm has been studied since 1971. The aim of this study is not only to ensure the availability of mullet semen anywhere and anytime it is needed, but also to contribute to the international cooperation of cross breeding of grey mullet in the future. Results of the study on cryogenic preservation of grey mullet sperm made during the experimental mass propagation of mullet in the Tungkang Marine Laboratory for the past 3 years are summarized in this paper.

The gonadosomatic index (GSI) of male grey mullet migrating near the coast of Tungkang ranged from 4 to 20 during the spawning season. The pH value of the semen was 7.4. Each spermatozoon was composed of a head part measuring 2.3 μ × 1.4 μ and a tail part four to five times as long as the head. There were about 5.3 × 1010 sperms in each ml of semen. Eosin-nigrosin staining was used for clearer identification. Sperm motility was preserved for up to 23 days in the case of raw semen at 5° C. Cryoprotective agents were needed at the ultra-low temperature (−196° C) of preservation in liquid nitrogen. Feasible procedures of freezing the grey mullet sperm were determined. Fresh semen diluted with cryoprotective agents was dispensed into 0.5 ml straws which were then sealed. These pretreatments prior to cryopreservation had to be done within the correct equilibration time of 1 h or less. Semen in straws was precooled in liquid nitrogen vapor until a temperature of −80° C was reached. Straws in the canister were then put into liquid nitrogen for long-term preservation. The optimum effect of cryoprotective agents was found with 5–10% glycerine or dimethylsulfoxide (DMSO) at 1:1, 1:5, and 1:10 dilutions. In this condition, both good motility and fertility before freezing and cryoprotection were obtained. So far the best result of frozen thawed mullet sperm was moderate motility and 2.7% fertility of the semen cryopreserved for 1 year and 4 days.