Sperm cryopreservation of Indian major carp, Labeo rohita: cryodiluents, sperm: cryodiluent dilution ratio and cryoprotectan t concentration

Cryogenic preservation trials of spermatozoa of Labeo rohita were carried out. Twenty four cryodiluents (extender + cryoprotectant), with the combination of six extenders such as egg-yolk citrate, urea-egg-yolk, 0.9% NaCl, Kurokura-2, Ma and Mb and four cryoprotectants viz. DMSO, glycerol, methanol and ethanol, were used to screen out the suitable cryodiluents. Sperm was preserved in 0.25ml plastic straw in programmable freezer. Two step freezing method was followed. Sperm preserved with egg-yolk citrate and urea-egg-yolk containing 10% DMSO showed best post-thaw motility (80%) followed by 0.9% NaCl (60%) and Kurokura-2(30%) solutions. Sperm with the extenders M" and Mb clotted at the time of equilibration and also after few days of preservation. Egg-yolk citrate mixed with ethanol and methanol also showed good percentage of motility (80%) but egg-yolk citrate with glycerol showed less sperm motility (>60%). To determine suitable dilution ratio of milt and cryodiluent two best extender eggyolk citrate and urea-egg-yolk with four cryoprotectants such as DMSO, glycerol, methanol and ethanol at different ratio viz 1:2,1:4,1:7,1:10,1:15 and 1:20 were used. Highest post-thaw motility (>80%) was observed when milt was preserved with egg-yolk citrate containing 10% DMSO at 1:2, 1:4, 1:7 and 1:10 dilutions. Meanwhile using glycerol as cryoprotectants provided less post thaw motility at lower dilution ratio but with the increase of its dilution showed good sperm motility compared with other cryoprotectants. Finally, evaluation on the effect of cryoprotectant concentration on post-thaw sperm motility was conducted. Egg-yolk citrate and four cryoprotectant i.e. DMSO, glycerol, methanol and ethanol with six different concentrations namely 5%,7%, 10%, 15%, 20% and 30%.were evaluated. Among the cryoprotectants DMSO, methanol and ethanol showed highest post-thaw motility (about 80%) at 7% and 10% concentrations. Although glycerol was not suitable at low concentration but its 20% and 30% concentration levels provided best post-thaw motility. No post-thaw motility was obtained with DMSO at 30% concentration. The overall analysis on cryoprotectant concentration indicated that below 5% and above 20% cryoprotectant concentrations could not be suitable for effective cryopreservation of spermatozoa.