Basic principles of semen cryopreservation and cryoinjury

procedures necessarily involves more than the identification or application of novel cryoprotectants and additives. If large numbers of spermatozoa are required for concep- tion there will be less tolerance of poor sperm survival during cryopreservation. It could also be argued that species bearing large litters must generate a minimum number of fertilized eggs otherwise they fail to produce sufficient concentrations of pregnancy-re- cognition factors. Having mentioned that the quirks of individual species can have major influences upon the success of artificial insemination procedures with cryopreserved semen, it has been left to the authors of accompanying reviews to deal with species-specific require- ments. This review will mainly treat the spermatozoon as a generic cell type which undergoes various stresses during cryopreservation leading to survival, cell death or functional impairment. The reader should be aware that because of space limitations the literature has not been exhaustively cited. The principles and practice of semen Ž cryopreservation have been reviewed several times Bwanga, 1991; Hammerstedt and Graham, 1992; Hammerstedt et al., 1990; Hofmo and Almlid, 1991; Salamon and . Maxwell, 1995a,b; Watson, 1979, 1990, 1995; Wolf and Patton, 1989 , and these authors refer to most of the original literature up to the early 1990s. The reviews by Salamon and Maxwell are notable for their inclusion of research performed in the former Soviet Union.

2. Basic principles of semen cryopreservation and cryoinjury

When cells are frozen they are subjected to stresses resulting from the water-solute interactions that arise through ice crystallization. Exposure of cells to the hyperosmotic, yet unfrozen, solution causes withdrawal of intracellular water, consequent cell shrink- Ž . age and possible influx of ions Mazur, 1984 . Thawing involves a reversal of these effects, and the consequent inward water flux may cause cell membrane disruption. The detrimental effects of slow freezing, and therefore prolonged exposure of cells to Ž . hyperosmotic conditions ‘‘solution effects’’; Mazur et al., 1970 , have been viewed as balancing the deleterious consequences of rapid freezing which encourage intracellular ice crystallization. Cytoplasmic disruption through intracellular ice formation may be Ž . further compounded by the growth of ice crystals during thawing recrystallization . It has been suggested that a compromise freezing rate exists where the damaging effects of Ž . these two different sources of cryoinjury can be minimized Watson, 1990 . Ž . In reviewing cell freezing hypotheses, Mazur 1984 argued that the microscopic architecture of the freezing environment was an important determinant of cell survival. Early cryomicroscopic observations showed that slowly frozen erythrocytes became Ž . sequestered in narrow channels between plates of ice Nei, 1970 . Subsequent observa- tions with fluorescence cryomicroscopy have shown that as spermatozoa can also lie across the channels, single cells are exposed to high and low salt concentrations Ž . simultaneously Fig. 1 . The incorporation of micro-architectural considerations into hypotheses of cryoinjury may help to explain why some spermatozoa survive the cryopreservation process intact, while others suffer acrosomal disruption, plasma mem- Fig. 1. Ram spermatozoa surface-labelled with fluorescein isothiocyanate and cooled to y408C on the cryomicroscope stage at 308Crmin in the presence of 300 mgrml rhodamine in phosphate-buffered saline. Ž . The fluorescence in the background highlights regions where rhodamine and other salts have become concentrated; conversely, dark areas indicate regions of low salt concentration. Single spermatozoa can span areas of both high and low salt concentration. brane damage and loss of motility. If cell survival were dependent upon spatial orientation in relation to regions of heterogeneously distributed solute concentration, then different types of microstructure would alter the random likelihood of achieving optimal orientations. Hypotheses of sperm cryoinjury must account for the known thermodynamic and structural properties of the sperm plasma membrane. It is well known that the sperm Ž plasma membrane contains an unusual array of lipids Lin et al., 1993; Parks et al., . Ž 1987 and that the plasma membrane is organized into different domains Friend, 1984; . Holt, 1984 . The phospholipids typically adopt unusual configurations, with a high proportion of plasmalogens that contain ether-linked fatty acids instead of the more usual ester linkages. Phospholipids account for 65–70 of the total, and a large proportion of these contain a docosahexaenoic acid side chain, which may confer membrane fluidity and instability. Possibly to counteract these destabilizing effects, sperm plasma membranes contain variable amounts of sterols. The sperm plasma membrane lipids respond to temperature changes by alterations in their physical phase state. Although regions of fluid and gel phase lipids coexist at physiological tempera- tures, reductions of temperature favour fluid to gel transitions; the presence of sterols is thought to inhibit these phase changes. As spermatozoa are not adapted to undergo the temperature changes involved in cryopreservation, they cannot modify their lipid content to suit the environmental conditions. This useful strategy is widely used in nature to compensate for the changes Ž in ionic permeability and enzyme activity which result from phase transitions for . Ž review, see Hazel, 1989 . Spermatozoa undergo these lipid phase transitions Crowe et . al., 1989; Drobnis et al., 1993; Holt and North, 1984; 1986; Parks and Lynch, 1992 typically within the temperature range 17–368C. Their occurrence shows species depen- dence, which could go some way towards explaining the variations in cryopreservation sensitivity seen in spermatozoa from different species. It is also likely that during a typical freeze–thaw cycle, the sperm membranes must undergo phase transitions during both cooling and rewarming. Ž . Evidence that cold shock i.e. damage due to rapid cooling above 08C is caused by Ž . Ž . lipid phase transition effects was presented by Drobnis et al. 1993 . Holt et al. 1992 obtained some evidence that phase transitions might be involved in the manifestation of cryoinjury during the rewarming of cells after thawing. Ram spermatozoa were stained Ž . with fluorescein diacetate FDA , a fluorescent probe of cell membrane integrity, cooled Ž . to a series of minimum temperatures 58C, y108C and y208C and then rewarmed to 308C. Plasma membrane integrity was retained throughout cooling, but fluorescein leakage, indicating membrane disruption, occurred during the rewarming process. Conversely, performing similar experiments in the presence of external adenosine Ž . triphosphate ATP and a sperm reactivation medium, showed that spermatozoa rendered immotile by cooling could be restored to motility by an influx of ATP when the plasma membrane was breached. The threshold temperatures causing loss of membrane integrity were correlated with the minimum temperature reached during cooling. One interpreta- tion of this data is that as the post-thaw temperature increases, the plasma membrane is subjected to structural rearrangements involving lipids and proteins, the extent and nature of which are governed by interactions of temperature and solute effects during the freezing process. Besides causing physical disruption of the plasma membrane by the induction of lipid packing faults, lipid phase transition effects cause non-linear kinetic responses in some enzymes, including some of the membrane ATPases whose activity depends upon the Ž . physical state of annular lipids Kimelberg, 1977 . It is likely that such effects are partly responsible for the poor control of intracellular calcium concentration which is evident Ž . at temperatures below about 178C Bailey et al., 1994; Robertson and Watson, 1986 . Ž . This is probably the rationale for including ethylenediaminetetra-acetic acid EDTA and citrate in some semen diluents; these would chelate calcium and diminish the concentra- tion gradient across the sperm plasma membrane. Intracellular calcium concentrations Ž . ; 0.1 mM are four orders of magnitude lower than those in the external milieux. EDTA chelates other metallic ions, however, and might also act by inhibiting lipid peroxidation.

3. Cryoprotectants and additives