The viability of cryopreserved tilapia spermatozoa

Abstract

Tilapia (Oreochromis spp.) spermatozoa protected with 12.5% methanol (MeOH) in fish Ringers, stored in 1.5-ml cryotubes and held in a vapour phase liquid nitrogen refrigerator remained viable for at least 13 months. The results, however, showed a high degree of variation; of the 16 samples tested only 66% produced viable cells on thawing. Mean fertilization rates of eggs were independent of storage time and ranged from 38.7 to 93.4%. To minimize variability a protocol using 0.5-ml plastic straws, which were held under liquid nitrogen, was developed and tested on the ability of post-thaw spermatozoa to fertilize eggs in comparison with an unfrozen control. Choice of cryoprotectant and its concentration, cooling and dilution rates and the fertilizing capacity of 0.5 ml of extended (ca. 25 μl milt) milt were investigated. The best pre-freezing activation of spermatozoa was obtained with MeOH and maximum cell protection was achieved using 10% MeOH. Cooling spermatozoa at rates of between 5 and 50°C/min and diluting milt at rates of between 1:2 and 1:20 had no significant (P greater than 0.05) influence on the rates of fertilization of eggs. Mean embryo yields ranged from 86.2 to 98.6%. Milt diluted at 1:20 could satisfactorily fertilize up to 500 eggs (140×103 spermatozoa/egg). The growth and survival of 30-day-old fry produced from milt stored for up to 12 months were not significantly (P greater than 0.05) different from normally produced fry.