Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilizationin African catfish (Clarias gariepinus)

A new integrated approach including computer-assisted sperm analysis (CASA) , viability staining and fertilization was used to study the quality of cry odiluents used in fish sperm cryopreservation. As an example the sperm qual ity of an African catfish, Clarias gariepinus (Burchell, 1822), was assesse d by its fertilizing ability, motility and viability at day 0 (fresh), afte r 2 days' storage at 4 degreesC and after 2 days, 5 months and 10 months fr ozen at -196 degreesC using solutions containing dimethyl sulphoxide (DMSO) or glycerol as permeating cryoprotectants. Four of the best freezing solut ions were used, namely, Steyn's extender (S1, S4) and Mounib's extender (M3 , M4) associating 10% hen's egg yolk. Progressive sperm movement measured b y CASA and expressed by the straight line velocity (VSL), the average path velocity (VAP) and the curvilinear velocity (VCL) was highly correlated wit h hatching rates obtained from fertilization using minimal sperm:egg ratios . After 2 days, the motility of spermatozoa frozen with DMSO and 10% egg yo lk had deteriorated less than that of spermatozoa stored at 4 degreesC. Pos t-thaw hatching rates reflected the post-thaw sperm viability, which was cr yodiluent dependent: 14.9+/-2.0% (S4), 17.0+/-4.2% (S1), 25.9+/-3.7% (M4) a nd 52.1+/-3.4% (M3) after 5 months of cryopreservation. The percent motilit y of IO-months-frozen spermatozoa was high in M3 (70.7+/-11.4%) and M4 (64. 0+/-2.0%) cryoprotected sperm when measured between 5 and 20 sec after acti vation, but decreased rapidly to 24.3+/-8.3% (M3) and 23.0+/-9.0% (M4) betw een 21 and 35 sec after activation. Mounib's extender (M3, M4) provided the best cryoprotection to the spermatozoa for all post-thaw sperm quality mea surements and at all freezing durations. Sperm motility was positively rela ted to fertility. Our method will make it possible to develop even better e xtenders and cryoprotectants. (C) 2001 by Elsevier Science Inc.