New research on the cooling and cryopreservation of mammalian spermatozoa is reviewed in the context of the older literature. Cryoinjury to a variety of cell organelles is regarded as being due to the two major stresses of cryopreservation, i.e., the change in temperature, and the formation and dissolution of ice and its consequences. Since the cryopreservation process involves departure of the cells from and return to body temperature, both cold shock and warm shock are included as potential stresses to be considered, as well as the stages involving cooling below the freezing point of the medium. The causes of cryoinjury are reconsidered and new concepts concerning the influence of osmotic stress are presented. Heterogeneity of the sperm population is discussed in the context of the success with which spermatozoa can be cryopreserved between and within ejaculates and individuals. The functional state of frozen and thawed spermatozoa is examined on the basis of published results of structural and functional tests of sperm competence. The hypothesis is advanced that cryopreserved mammalian spermatozoa are in a state resembling capacitation, which accounts for their relatively reduced longevity and their readiness to undergo egg penetration without incubation. The importance of this to the utilization of cryopreserved spermatozoa is examined, and proposals are made for new avenues of research to overcome these problems.

Acrosomal structures of ram spermatozoa were prominently stained when air dried smears of diluted semen were fixed for 15 minutes in buffered formal saline and stained for 90 minutes in a 6 per cent (v/v) buffered solution of Giemsa stain. Progressive disruption of the acrosomes was demonstrated during chilling and deep-freezing of the spermatozoa, and the degree of damage was systematically scored. A rapid and repeatable estimate of the state of the acrosomes in a sample could be made from the mean score of 20 spermatozoa examined per slide.

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Ram spermatozoa were stored at 5°C in diluents containing various fractions of egg yolk prepared by dialysis, ultrafiltration and ion-exchange chromatography. They survived storage best in the presence of components of egg yolk which were non-dialysable and were not filtered through membranes which retained substances of molecular weight greater than 100000. The substances isolated in peak B of the ion-exchange chromatogram of whole egg yolk described by Seideman et al. (1969) gave greater protection than those from other fractions from this chromatographic system. These data indicate that the low-density lipoprotein fraction of egg yolk is the most likely source of protection to ram spermatozoa against the effects of storage at 5°C.