Simplified cryopreservation of sperm in Grey mullet Mugil cephalus: A robust method for wide adoption

In this study, we developed a simple and robust method of cryopreservation of sperm in Mugil cephalus. A series of experiments were conducted to develop a simplified method by changing the extender, cryoprotectant and freezing height above the liquid nitrogen (LN2) surface. Fresh sperm were diluted 1:1 in a cryomedium (cryomedium = extender + cryoprotectant) containing cryoprotective agents (CPAs) namely, propylene glycol (PG), methanol (MeOH), glycerol (GLY), ethylene glycol (EG), dimethylsulfoxide (DMSO) and dimethylacetamide (DMA). The final concentration of CPAs in cryomedium was 5% and 10% (v/v). The cryovials (2.0 mL) were loaded with diluted samples, fixed in cryo canes, and frozen at 4, 6, 8, 10 and 12 cm above the LN2 surface and also by vitrification. After 7, 30 and 180 days of storage in LN2 (-196 °C), the sperm was thawed at 30 °C for 90-120 s and their quality was evaluated. Among the experimented factors, sperm diluted in cryomedium composed of 0.3 M glucose combined with 10% EG, frozen at 4 cm above the LN2 surface recorded significantly (P 0.05) differences were observed in sperm quality at different storage days. Thus, the results of the study revealed that using this simplified method of cryopreservation high-quality post-thaw sperm can be obtained. Besides, it is expected that this method can be successfully adoptable at hatchery, farm and on-site cryopreservation of sperm in grey mullet.