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 ext enders at 25 degrees C, cooled slowly to 5 degrees C, glycerolated, pa ckaged in 0.5-ml straws, and frozen. Different freezing rates were acc omplished in two steps. Straws were transferred from +5 degrees C to n itrogen vapor at temperatures ranging from -10 to -100 degrees C in th e first step and to liquid nitrogen in the second step. Straws were th awed in water at 35 degrees C. A substantial decrease in sperm motilit y occurred between -10 and -20 degrees C, as abrupt nucleation occurre d following supercooling to -13 degrees C. To study the interactions b etween osmolarity x cooling rate, WM and EYT extenders were prepared t o yield media measuring 220 to 420 mOsm/L. The optimal first-step rang e of cooling in the two-step procedure was -30 to -70 degrees C, and t he 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 E YT, equivalent to the results with the control procedure used commerci ally. As the cooling rate increased (first step to -100 degrees C) spe rm motility was much higher in hypertonic than in hypotonic extenders (P < 0.05), indicating the importance of partial dehydration before ra pid cooling. Replacing part of EYT and WM with equivalent solutions (s ame mOsm/L) of sucrose or trehalose had no appreciable effect. These r esults provide a basis for further investigating simple freezing syste ms that might be more effective in preserving bull sperm than those cu rrently available. (C) 1998 Academic Press.