Increasing the proportion live

Fig. 1. The theoretical relationship between sperm number in the inseminate and fertility. Normally, for artificial insemination, the sperm dose should be sufficient to reach the asymptote. Both the slope and the maximum response are determined by semen characteristics. spermatozoa were required to achieve greater than 50 fertility, whereas it is recognised that cervical insemination requires 10 times that dose. If the insemination was made into Ž . the oviduct itself, less than 1 million spermatozoa were needed Maxwell et al., 1993 . Another observation relating to cryopreserved semen is manifested with boar semen. In the pig, ovulation can occur over an extended period of oestrus such that spermatozoa Ž . may be required to survive up to 40 h in the oviduct. Waberski et al 1994 have noted that fertility with cryopreserved semen may be high providing the insemination is made in the period 4 h before ovulation. Outside this period, fertility with cryopreserved spermatozoa declined dramatically, but fresh semen maintained its fertility for a much longer period. Cryopreserved spermatozoa do not survive in the female tract compared with fresh spermatozoa. In summary, the cryopreservation process results in reduced fertility compared with fresh semen. It has been shown that this arises from a combination of both loss of sperm viability and an impairment of function in the population of survivors. This situation needs to be borne in mind when strategies to improve the results are contemplated. We need to consider not only the cryopreservation protocol to optimise the number of survivors, but also the functional ability of the surviving population.

2. Increasing the proportion live

The cryopreservation protocol has a number of potentially damaging stresses: firstly, the change in temperature; secondly, the osmotic and toxic stresses presented by exposure to molar concentrations of cryoprotectants; and thirdly, the formation and dissolution of ice in the extracellular environment. We shall discuss these in turn. 2.1. Change in temperature It has long been known that cooling ungulate semen too rapidly between 30 8C and 8C induces a lethal stress in some of the cells proportional to the rate of cooling, the Ž . temperature interval and the temperature range Watson, 1981 . This phenomenon, known as cold shock, also variably affects a number of other species and, as a result, cooling in this range prior to cryopreservation is usually conducted carefully. In boar spermatozoa, the phenomenon is mostly manifested immediately after ejaculation but the cells become progressively less sensitive over the next few hours Ž . Pursel et al., 1972 . We have investigated this phenomenon, and found that boar sperm incubated at room temperature in their own seminal plasma become quite resistant to Ž . cold shock over 16 h after ejaculation Tamuli and Watson, 1994 . The membranes are altered such that they do not respond to the cold stress. This observation suggests that the ideal time to cryopreserve boar semen may not be as soon after ejaculation as possible, i.e. within 6 h, as is commonly practised, but after 18–24 h when the resistance is at a maximum. Modern diluents now permit the maintenance of a high proportion of spermatozoa in a viable state for at least 24 h. Even with slow cooling, however, temperature change induces stresses on mem- branes. It is probable that these are related to phase change in lipids and altered functional state of membranes. Cold shock is then seen merely as the extreme state of a continuum of stress, influenced by the rate of onset of the phenomenon. Such stresses on the membranes may be continued below 0 8C since phase changes are not complete at Ž . 8C, but our preliminary studies unpublished were unable to show cold shock injury in bull spermatozoa below 0 8C. However, it is well known that a major phase change Ž . occurs in the vicinity of 5–15 8C Drobnis et al., 1993 , and this may well be the prime temperature range for temperature dependent injury. The suggestion that membrane injury results from phase events in the lipid bilayer is well attested. Freeze fracture studies of membranes before, during and after cooling show clear evidence of phase separation events, which are only partially reversed after Ž . Ž . rewarming Holt and North, 1984; de Leeuw et al., 1990 . Pettitt and Buhr 1998 have shown the importance of modulation of the lipid environment of the plasma membranes during cooling, implicating the lipid component in mechanisms of injury. Nevertheless, one should not overlook the other membrane elements that may be altered by temperature stress. Integral membrane proteins are clustered by lipid phase separation, and this may be expected to alter function, especially of proteins which undergo a structural modulation to carry out their function, such as ion channel proteins. Ž Indeed, it is known that membrane permeability is increased after cooling Robertson . and Watson, 1986; Robertson et al., 1988 and this may be due to a generally increased membrane leakiness, but could be due to effects on specific protein channels. Calcium regulation is clearly affected by cooling and this undoubtedly has serious consequences Ž . in terms of cell function Bailey and Buhr, 1994 ; in severe cases, the change may be incompatible with continuing cell viability. The uptake of calcium during cooling Ž . contributes both to capacitative changes see below and fusion events between the plasma membrane and underlying outer acrosomal membrane. There are strong similari- ties between membrane damage during cooling and the acrosome reaction, the one being a disorganised version of the other. In addition, cytoskeletal elements are temperature-sensitive. The cooling of other Ž cells results in a premature depolymerisation of actin filaments Hall et al., 1993; . Ž . Saunders and Parks, 1999 . Spungin et al 1995 have postulated that the depolymeriza- tion of cytoskeletal F-actin is a necessary step permitting the approximation of plasma membrane and the underlying outer acrosomal membrane promoting acrosomal exocyto- sis. Perhaps, this also could contribute to a disorganised fusion of membranes following cooling or cryopreservation. 2.2. CryopreserÕatiÕe stresses The addition and removal of cryoprotectant in molar proportions applies a substantial but transient osmotic stress to the plasma membrane of spermatozoa, depending upon Ž . the relative permeability of the cryoprotectant Gao et al., 1993 . Generally, the Ž cryoprotectant of choice for spermatozoa is glycerol or occasionally, dimethyl sulphox- . Ž . ide , which induces osmotic stresses. Gao et al 1995 have shown that when human spermatozoa were exposed to 1 M glycerol in a single step, the volume excursion exceeded tolerable limits both on addition and removal. They showed that the stress could be reduced to tolerable limits by stepwise addition and removal and this substantially improved the proportion of spermatozoa surviving. Furthermore, spermatozoa are also sensitive to toxic effects of cryoprotectants, resulting in the unsuitability of some compounds commonly used for other cells being Ž . less useful for spermatozoa Storey et al., 1998 . Even with glycerol, care should be Ž . exercised in its use with spermatozoa Katkov et al., 1998 . 2.3. Ice crystal formation and dissolution The stresses induced by ice crystal formation are mainly associated with the Ž accompanying osmotic pressure changes in the unfrozen fraction Watson and Duncan, . 1988 . When a solution is cooled below the freezing point, ice crystals are nucleated and pure water crystallises out as ice. The solutes are dissolved in the remaining liquid water fraction and the osmotic strength of the solution rises. The proportion of the water crystallising out as ice, and hence the osmotic strength of the remaining solution, depends on the temperature — the lower the temperature, the smaller the unfrozen fraction and hence the higher the osmotic strength of the solution. It is generally recognised that the duration of exposure to such events should be minimised for optimal cell survival implying that the cooling rate should be rapid. However, the cooling rate must be slow enough to allow water to leave the cells by osmosis preventing intra- cellular ice formation which is lethal. Sperm cells are generally frozen at quite rapid rates in the range 15–60 8Crmin, which have been empirically determined as giving the best survival rates. Obviously, if the cell water permeability and its activation energy were known it should be possible to predict the maximal cooling rate compatible with osmotic equilibrium and so determine optimal cryopreservation protocols. Such consid- Ž . erations have been shown to be important for other cell types Mazur, 1984 . We and others have measured the water permeability of the membranes of spermato- Ž . zoa from a number of species. With the exception of the rabbit Curry et al., 1995a , most species seem to have a rather high water permeability in comparison to the Ž permeability of other cell types Watson et al., 1992; Noiles et al., 1993, 1997; Gilmore . et al., 1996 . We have also shown that modulating the glucose channels of the sperm Ž . membrane affects water permeability Curry et al., 1995b . Further studies of this phenomenon may offer novel ways to modify sperm cell responses to cryopreservation. However, when these figures for water permeability are used in equations to calculate the maximum cooling rate compatible with approximate osmotic equilbrium during Ž cooling, the rate works out much higher than is known empirically to be optimal Curry . et al., 1994 . We have, therefore, examined the assumptions on which the calculations are based. The first assumption is that the water permeability of the membrane remains unchanged in the presence of cryoprotectant, an assumption recently shown to be Ž . questionable Gilmore et al., 1998 although the magnitude of the change by no means accounted for all the discrepancy. Another possibility is that the permeability of the sperm plasma membrane is regionally variable. The assumption that a single value applies to the whole cell is unlikely to be true given the known regionalization of the sperm plasma membrane. Thus, a measured value may not represent, say, the sperm Ž head when the majority of the water transport may occur across the tail membrane Holt . and North, 1994 . Alternatively, the techniques for estimating water permeability simply overestimate the true value. More recent methodology showed this to be the case but Ž again, the magnitude did not rectify the discrepancy Gilmore et al., 1996; Curry and . Watson, unpublished . Fourthly, the activation energy measured above 0 8C may not reflect the activation energy below 0 8C, an hypothesis shown to be true for human spermatozoa but still inadequate to account for the discrepancy between calculated and Ž . empirical optimal cooling rates Noiles et al., 1993 . Perhaps, the discrepancy is explained by the sum of all these errors in the assumptions on which the theoretical calculations are based. A recent study with mouse spermatozoa has suggested that water permeability is markedly less at subzero temperatures in the presence of ice crystals and Ž cryopreservatives, and in this instance, the discrepancy was largely eliminated De- . vireddy et al., 1999 . Nevertheless, it is possible that there are other stresses that are additional to those Ž . included in these calculations. Holt and North 1994 have shown that the signs of distress were manifested during rewarming and that these were related to osmotic stress, although not necessarily implying that the damage occurred during rewarming phase. These considerations led us to hypothesise that there were indeed other factors determining the optimal cooling rate independent of the risk of intracellular ice formation. We and others have shown the extreme sensitivity of sperm membranes to osmotic stresses. We believe these may determine the optimum cooling rate, not by permeability to water but by the rate of displacement required of the plasma membrane to accommodate the volume change, and we suggested that this may stress the Ž . attachments of the cytoskeleton Watson, 1995 . In support of this hypothesis, when Ž mouse or Koala spermatozoa were exposed to cytochalasin D which disrupts f-actin . filaments they were more able to withstand extremes of osmotic stress without Ž . membrane rupture Noiles et al., 1997; Holt and Johnston, 1999 . It appears that we need a more elaborate theory of plasma membrane disruption during cryopreservation than is currently in vogue. The proportion of cells, which survive the cryopreservation protocol, is determined by the sensitivity to osmotic stress during cryopreservative addition and removal, and during cooling and rewarming. While there may be species differences in overall sperm sensitivity to cryopreservation, the ejaculate is heterogeneous with a variable resistance to osmotic stress amongst the cells. Those that succumb to lethal membrane damage, however, are not determined stochastically. The fact that indiÕiduals can often be classified as ‘‘good freezers’’ or ‘‘bad freezers’’ implies that certain characteristics of membrane structure, which may be genetically determined, predispose towards survival under cryopreservation stress. Attempts to modify lipid composition have not demon- strated any dramatic benefits, perhaps implying that other membrane elements may be more important, e.g. cytoskeleton. This is an area for further investigation. Nevertheless, even if we optimise the process and minimise the cell death, there will still be a proportion of cells which fail to survive. We need, therefore, to concentrate on the function of the surviving population.

3. Increasing the quality of survivors