Short- and long-term cryopreservation of sperm and sperm suspension of the grouper, Epinephelus malabaricus (Bloch and Schneider)

Abstract

Essential factors which affect the feasibility of cryopreserving sperm of the grouper, Epinephelus malabaricus (Bloch and Schneider), were determined by comparing percentage of motile spermatozoa, duration of storage life and fertilization rate of eggs when available. Fertility of sperm after thawing was maintained by using 10% dimethyl sulfoxide (DMSO) as a cryoprotectant with less than 10 minutes equilibration. An optimal ratio of 85% for sperm suspension from sliced testis or fresh milt diluted 20 times in Ringer solution for marine fish: 10% DMSO: 5% glucose is suggested. The addition of 15% Menezo Medium B2 INRA to the milt mixture resulted in the retention of higher progressive motility of cryopreserved sperm. The use of isopropanol with dry ice at -75°C or liquid nitrogen vapor at -100°C as freezing medium simplified the freezing procedure. Analysis of the motility of post-thawing sperm using the Hamilton-Thorn motility analyzer showed that the performance of one-and three-month old cryopreserved grouper spermatozoa was slightly poorer in terms of path velocity, progressive velocity, track speed and straightness. The use of microwaves in thawing frozen milt in polyethylene straws was found feasible and convenient with an optimum degree of power at 40% and conditional duration of 30-70 seconds. The post-thawing fertility of the spermatozoa was better when frozen immediately upon collection or right after transporting the material of raw milt or sperm suspension at 4°C for 1-2 hours. Fertility records of 78±18%, 83±3%, 95±0%, 75±32%, 13±1%, 95±0% and 66±12% were obtained for 1-, 17-, 41-, 54-, 128-, 278- and 291-day cryopreserved sperm (65.58-91.92% in controls) during the spawning seasons of 1990 and 1991. Preliminary extension to a private hatchery in 1991 resulted in additional production of about 50 million fertilized eggs by using cryopreserved sperm of E. malabaricus eight times when fresh milt was not available in the field.