Effects of extender, storage and sperm-to-egg ratio on cryopreservation success of Atlantic cod (Gadus morhua L.) sperm

The study aimed at improving the protocol for cryopreservation of Atlantic cod sperm. Five diluents, four cryoprotectants, two time intervals between thawing and activation of spermatozoa, and two sperm collection times prior to freezing were tested for their effect on post-thaw motility and fertilization ability. Minimum sufficient sperm:egg ratio was determined. The study was expanded by quantitative assessment of cod sperm using computer–assisted sperm analysis (CASA). Fertilization success obtained with fresh sperm was generally higher than with the cryopreserved sperm. Hanks’ solution + 10% hen's egg yolk + 10% glycerol or 10% dimethyl actetamide (HBSS+EY+DMA and HBSS+EY+Gly, respectively) were the best among the tested variants and resulted in average hatching success of embryos produced with use of cryopreserved sperm (44 and 36%, respectively) not differing from control (36%). A 30 min delay between thawing and activation did not have a significant effect on post-thaw spermatozoa motility in 15 out of 20 tested variants. Hatching success of embryos produced with the use of cryopreserved sperm stored for 3 days prior to freezing, in two out of three individual sperm samples, did not differ significantly from control. . Percentage and straight-line velocity of motile spermatozoa were found to be good predictors for the fertilization potential of cryopreserved sperm, correlating highly and positively (r > 0.80) with fertilization and hatching success of embryos obtained with cryopreserved sperm, particularly when low sperm:egg ratios were applied. When using cryopreserved sperm, the minimum sperm:egg ratio required to maximize fertilization succes was 300 000 : 1.