Survival of bull sperm frozen at different rates in media varying in osmolarity

The effects of freezing procedures, osmolarity, trehalose, and sucrose on survival of bull sperm in whole milk (WM) and egg yolk-Tris (EYT), semen extenders used worldwide, were studied. Sperm were added to extenders at 25 degrees C, cooled slowly to 5 degrees C, glycerolated, packaged in 0.5-ml straws, and frozen. Different freezing rates were accomplished in two steps. Straws were transferred from +5 degrees C to nitrogen vapor at temperatures ranging from -10 to -100 degrees C in the first step and to liquid nitrogen in the second step. Straws were thawed in water at 35 degrees C. A substantial decrease in sperm motility occurred between -10 and -20 degrees C, as abrupt nucleation occurred following supercooling to -13 degrees C. To study the interactions between osmolarity x cooling rate, WM and EYT extenders were prepared to yield media measuring 220 to 420 mOsm/L. The optimal first-step range of cooling in the two-step procedure was -30 to -70 degrees C, and the highest proportions of motile sperm after freezing and thawing were 61 to 62 in 260 to 300 mOsm/L WM and 63 to 64% in 300 to 340 mOsm/L EYT, equivalent to the results with the control procedure used commercially. As the cooling rate increased (first step to -100 degrees C) sperm motility was much higher in hypertonic than in hypotonic extenders (P < 0.05), indicating the importance of partial dehydration before rapid cooling. Replacing part of EYT and WM with equivalent solutions (same mOsm/L) of sucrose or trehalose had no appreciable effect. These results provide a basis for further investigating simple freezing systems that might be more effective in preserving bull sperm than those currently available. (C) 1998 Academic Press.