SUBLETHAL DAMAGE DURING CRYOPRESERVATION OF RAINBOW-TROUT SPERM

Although cellular damage during cryopreservation of freshwater fish sp ermatozoa has been reported in several studies, there is a lack of cor relation between this damage and the Fertility rates of eggs using pos tthawed milt. The apparent lack of such correlation may be due to othe r undetected sublethal cryodamage, which could affect the cell functio nality and viability. This may be extremely important for freshwater f ish spermatozoa whose ability to fertilize the egg requires dilution i n water or hypoosmotic solutions, an hazardous environment for the cel ls. This study tested the change in cell permeability during cryoprese rvation, using Hoechst 33258 to assess cell permeability. The permeabi lity of spermatozoa at different times after dilution in several hypoo smotic media were investigated. Ln the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied . Three CPT were tested (Me2SO, DMA, and methanol). In the second tria l, the addition of egg polk as a membrane stabilizer was investigated. Samples were frozen at -20 degrees C/min in a programmable cooler and thawed in a 25 degrees C water bath. Dilution in the CPTs slightly in creased the susceptibility of cells to damage but freezing/thawing cau sed a dramatic increase in the fragility of cells, which were killed i n a few seconds after their contact with the hypoosmotic solutions. Eg g yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization medi a. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated su blethal cryodamage in the function of the sperm membrane that was not detected by standard procedures, (C) 1998 Academic Press.