CRYOPRESERVATION OF HUMAN SPERMATOZOA .4. THE EFFECTS OF COOLING RATEAND WARMING RATE ON THE MAINTENANCE OF MOTILITY, PLASMA-MEMBRANE INTEGRITY, AND MITOCHONDRIAL-FUNCTION

Objective: To test the hypotheses that there is a two-factor aspect of cellular damage during cryopreservation that occurs in human sperm (o smotic effects versus intracellular ice formation) and that there is a cooling rate by warming rate interaction related to this damage. Desi gn: Ejaculates from healthy men were cooled at 0.1, 1.0, 10, 175, or 8 00-degrees-C/min to -80-degrees-C in a solution of 0.85 M glycerol and plunged into liquid nitrogen. Samples were warmed at 400-degrees-C/mi n (experiment 1) or either 1-degrees-C or 400-degrees-C/min (experimen t 2). After warming, sperm were assessed for survival using motility a s the endpoint in experiment 1 and motility, plasma membrane integrity , and mitochondrial function in experiment 2. Results: In experiment 1 , over the various cooling rates with a standard 400-degrees-C/min war ming rate, a plot of motility versus cooling rate produced a classical inverted U-shaped curve (n = 6) with maximum motility at the 10-degre es-C/min cooling rate. In experiment 2, over the various cooling rates , both 1 and 400-degrees-C/min warming rates produced similar but shif ted plots of motility, plasma membrane integrity, and mitochondrial fu nction versus cooling rate, which also produced inverted-U-shaped patt erns (n = 11). Maximal survival for each of the three endpoints occurr ed at 10-degrees-C/min cooling rate for the rapidly warmed sperm and a t 1-degrees-C/min for the slowly warmed sperm. Conclusions: These data support the hypotheses that a two-factor hypothesis of cryodamage app lies to human spermatozoa and that an interaction exists between cooli ng rate and warming rate. These data also suggest that motility, plasm a membrane integrity, and mitochondrial function are not differently a ffected by cooling and warming during cryopreservation.