Cryopreservation of spermatozoa of critically endangered yellow catfish Horabagrus Nigricollaris (Pethiyagoda & Kottelat 1994)

Abstract

In order to develop gene banking techniques aimed at conserving the critically endangered black collared yellow catfish of the Western Ghats, Horabagrus nigricollaris and popularize this species in the aquaculture scenario a study on cryopreservation of spermatozoa was undertaken. Freshly collected milt was observed for its characteristics. Well water was used to activate the milt in various stages of the experiment. There was no significant difference in motility of spermatozoa or percentage hatching from fresh and milt cryopreserved using 10% dimethyl sulfoxide (DMSO). Screening of four extenders (A, B, C and D) containing NaCl, KCl, CaCl2, NaHCO3, KH2PO4, MgSO4.7H2O, Na2H PO4 and Glucose at various proportions clearly indicated that the extender composition had significant effect on the percentage of motility, fertilization and hatching. Selected milt samples were preserved under cryogenic condition and utilized for experimental spawn production. The quality of milt samples was analyzed for the spermatocrit value, sperm density, pH, motility score and time: values obtained ranged from 57.8 to 59.8%; 16.5 x 109 to 20.9 x 109 spermatozoa/ ml of milt; 7.2 to 7.4; 4+ to 5+ (i.e. 80-100%) and 30 to 70 seconds respectively. The most promising combination was extender - A with 10% DMSO as the cryoprotectant. The ratio of milt and diluent was 1:4. Samples were equilibrated at 0°C ± 4°C on ice and vapourised over liquid nitrogen fumes for 10 minutes and finally stored in liquid nitrogen for three months. Stored milt samples were thawed and utilized to fertilize the eggs. Quick thawing procedure (27°C ± 2°C for 7 seconds) was followed. A French straw containing 0.5ml milt was found to be sufficient to fertilize 200 eggs approximately. Average fertilization % obtained was 30.3 ± 2.8 for extender- A against 35.8± 0.4 for the control. Mean hatching % obtained was 35.0 ± 4.7 for extender - A and 41.6± 4.4 for the control. No significant difference in fertilization success was found between cryopreserved sperm and untreated sperm from the same milt samples. Viable hatchlings were produced from milt that had been cryopreserved for three months indicating the feasibility of establishing a cryopreservation protocol to bank the genes and popularize the aquaculture of this critically endangered species. With the rapid global expansion of aquaculture, there is a need for year-round availability of larvae that could, to some extent, be met through cryopreservation of gametes.