G.J.Morris1,3, E.Acton1, and S.Avery2

3To whom correspondence should be addressed Human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been achieved by the standard approaches of cryobiology. Conventional approaches to cryopreservation impose a linear change of temperature with time; however, the stresses that cells encounter during cryopreservation are all non-linear with time. In this paper it is shown that improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes instead of imposing a linear temperature reduction. Several treatments were compared: control of solidification to achieve constant ice formation with time was more damaging than the standard linear reduction in temperature. However, treatments which fol-lowed a chosen non-linear concentration profile, referred to as ‘controlled concentration ’ allowed recovery of almost all the cells which were motile before freezing. The bio-physical basis of these different responses was examined using the cryostage of a scanning electron microscope and freeze substitution and it was found that, surprisingly, all samples of spermatozoa in the frozen state were neither osmotically dehydrated nor had any visible intracellular ice. Viability on thawing did not appear to correlate with conventional theories of cellular freezing injury, which suggests that for human spermatozoa other factors deter-mine viability following freezing and thawing. Key words: cryopreservation/electron microscopy/freeze frac-ture/freeze substitution/spermatozo