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Theriogenology 53:47-58, 2000© 1999 by Elsevier 5cience Inc.

Acknowledgments:Healthbred Ltd, PIC International and the UK Pig Reproduction Research Liaison Group for Iam grateful to the Ministry of Agriculture, Fisheries and Foods (United Kingdom), JSR financialand moral support.Correspondence and reprint requests to W. V. Holt, Institute of Zoology, Regent's Park,London NWl 4RY. E-mail;bill.holt@ioz.ac.uk

Semen storage technology was revolutionized approximately 50 years ago by the discoverythat glycerol could act as a cryoprotectant (36). This important observation enabled spermatozoa tobe frozen, stored for prolonged periods, and then used successfully for artificial insemination.Fortunately for the pioneers ofthis technology they chose to work with bull and fowl spermatozoa.Had they elected to begin with rodent, marsupial or porcine spermatozoa instead, they would haveencountered a series of species-specific problems that even today remain difficult to solve. Whilesorne species differences, such as sperm head shape, are obvious or measurable, other factors aremore complexo Sperm transport in the femaIe tract is one such species-specific difference; access tothe site of fertilization within the oviduct involves overcoming a series of obstacles that may haveevolved specifically to impede poor quality spermatozoa. Since even the best cryopreservationtechniques cause extensive lethal and sublethal cryoinjury, it would not be surprising that theefficiency of sperm transport may be severely reduced after cryopreservation.

INTRODUCTION

While semen cryopreservation is successfully used for a few species, application to otherspecies can be problematic. Here, 1 argue that species differences in female tract anatomy, subtledifferences in sperm transport mechanisms, ability to time inseminations and deliver spermatozoaeffectively are powerful determinants of fertility with cryopreserved spermatozoa. Poor spermsurvival represents one major aspect of the problem and determining biophysical characteristics ofthe sperm plasma membrane is an established approach to solving it. However, this approach isunable to account for the consistent differences in post-cryopreservatíon sperm quality betweenindividual males, an effect that is recognized in many species although only documented in a few.Searching for genetic differences between these individuals might offer a genomically-baseddirection in semen cryopreservation research. Cryopreservation of spermatozoa and spermatogeniccells for intracytoplasmic sperm injection has been developed primarily to deliver an intact genomeand presents a very different set oftechnical problems.© 1999 by Elsevier Science Inc.

Key words: semen cryopreservation, spermatozoa, sperm transport, artificial insemination.

ABSTRACT

Institute ofZoology, Zoological Society ofLondon, Regent's Park, London NWl 4RY, UK

W.V.Holt

FUNDAMENTAL ASPECTS OF SPERM CRYOBIOLOGY: THE IMPORT ANCE OFSPECIES AND INDIVIDUAL DIFFERENCES

As the magnitude of the water flux is highly dependent on cooling rate, optimal values forwater fluxes should be achievable by calculating optimal freezing rates. These argurnents havebeen applied to embryo and oocyte freezing (e.g., 29, 38), where theoretical predictions matchedobservations with a considerable degree of success, and their relevance to spermatozoa has beenstudied in some detail (e.g., 8, 13, 15). At present, with one exception which employed a noveldifferential scanning calorimetric technique to measure biophysical parameters of mousespermatozoa (10), the theoretically-derived optimal cooling rates for spermatozoa do not matchthe observations. Several likely reasons for this discrepancy can be suggested. Calculation ofwater flux depends upon the derivation of membrane-specific parameters, hydraulic conductivity

When cells are frozen, they are subjected to stresses resulting from the water-soluteinteractions that arise through ice crystallízation. Crystallízation induces the formation ofunfrozenpockets of hyperosmotic solution while cooling to approximately -50°C is in progress. Freeze­substitution electron microscopic studies (3) of spermatozoa within frozen diluent have shownthat sorne cells become excluded from the space occupied by the ice crystals and are, therefore,preferentially exposed to the anisosmotic environment. This results in withdrawal of intracellularwater, consequent cell shrinkage and possible influx ofions (4,30). Thawing involves a reversalof these effects, and the consequent inward water flux may cause cell membrane disruption. Sinceoverly rapid freezing causes lethal intracellular ice formation, the optimal cooling rate is thoughtto be slow enough to prevent this lethal effect, but fast enough to minimíze the harmful effects ofprolonged exposure to high salt concentrations ("solution effects"). High salt treatments arestandard textbook techniques used preparatively and analytically to release peripheral proteinsfrom cell surfaces. Exposing sperm to the hypertonic solutions formed by ice crystal developmentmeans that they will be injured by the removal of membrane proteins during this phase of thecryopreservation process if it is too prolonged.

Cellular Responses to Cryopreservation and Cryoprotectants

The field of semen cryopreservation research has, over the past few decades, beenweighted heavily towards empirical studies. Many papers have been published in which the effectsof varying a few technical parameters, such as the concentration of cryoprotectants and otheradditives, or cooling and warming rates, have been examined. While these may help improve post­thaw sperm quality under a particular set of circumstances, they fail to identiíy important cornmonthemes in sperm cryobiology, and are of limited use in predicting the way forward. Sorne researchgroups have recognized the limitation of this approach and have focused on modeling thehydraulic fluxes that spermatozoa experience during the freeze-thaw process. For this, they haveundertaken detailed studies of membrane permeability in spermatozoa and then used biophysicalapproaches to predict minimally damaging cooling protocols. Sorne of this work, especially thatrelating to the addítion and removal of cryoprotectant, has been very successful in allowingoptimal sperm recovery (14), although the accurate prediction, on theoretical grounds, of optimalcooling rates is still problematíc. In this review I surnmaríze sorne of this fundamental work.However, 1 will also highlight other issues that probably affect the success of spermcryopreservation techniques and that may be species- or taxon-specific. These issues presentmajor problerns for the development of sperm storage methods for threatened species wherebackground data are often sparse and opportunities for research may be limited.

Theriogenology48

The implications of species variability in both the beneficial and detrimental effects ofglycerol are worth considering in this respect. Bull spermatozoa are routinely cryopreserved using4-8% glycerol, and acceptable results have generally been obtained with wild ruminants when thisrange is used (red deer, scirnitar horned oryx and gaur; references 12, 16 and 24, respectively).Similarly, studies of primate sperm cryopreservation have focused upon this range as optimal.However, spermatozoa from other species do not tolerate exposure to these concentrations. Forexample, porcine spermatozoa suffer severe acrosornal damage if the glycerol concentrationsexceed 3%, and although little comparative work has been done on rodents, it is known thatmouse spermatozoa are damaged by glyceroI concentration exceeding 1.75%. Indeed, sornerecent techniques for mouse sperm cryopreservatíon avoid glycerol completely. That this is aspecies-specífíc effect, and not a taxon-specifíc effect, can be deduced from studies of chinchillaspermatozoa, where optimal re~lts were obtained with 6% glycerol (37). Further inconsistency isdetected when marsupial studies are considered. Taggart and colleagues (44) showed that 4-8%gIyceroI allowed very good post-thaw recovery of sperm motility (70-90%) in sorne marsupials(brush-tailed possum, northern brown bandicoot), while only 3% motility was recovered in fat­tailed dunnart. Surprisingly, in sorne species (e.g., koala, brush-tailed possum) there was

While this theoretically based approach is a logical way forward for developingcryopreservation methods for particular species, it might be argued that if cooling and warmingrates could be controlled appropriately, the problem would be solved. As the theory and practicedo not match, other factors must also be important. The major significance of the empirical workis the demonstration that the chemical and osmotic environment of the spermatozoa is of sorneconsequence in obtaining good sperm survival. Choice of buffer system, nature of cryoprotectantsand additives such as sugars, calcium chelators, antioxidants and milk or egg yolk proteins haveall been shown to influence sperm survival profoundly. It is unlikely that the fundamental role ofthese substances is to modify the water permeability of the sperm plasma membrane and,therefore, other explanations for their different and species-species effects must be sought.

Determinants of Species Differences in the Susceptibility of Sperm to Cryoinjury

As the estimates of sperm surface area needed in order to develop the theoreticalapproach have typically been derived from models which regard the spermatozoon as acombination of simple geometric shapes such as cylinders and cubes, considerable experimentalerror may also have been introduced. It should be possible to improve the accuracy of thesemeasurements by using novel techniques such as atomic force microscopy. This method, whichessentially involves the use of a tapping stylus to create a molecular scale 3-D representation ofthe cell surface, has the added advantage that the spermatozoa do not require pretreatment otherthan irnmobilization by mild fixation. This avoids the artifacts introduced when cells are processedfor electron microscopy.

and activation energy, but as the spermatozoon is such a complex cell, these parameters may varyover the various rnembrane domains of the sperm surface. Furthermore, changes in rnembranelipid packing may induce temperature-dependent variation in the derived parameters andcryoprotectants, especially glycerol, are known to interact with rnembrane lipids and maythemselves affect the derived parameters (for references, see 20).

49Theriogenology

Attempting to understand the source of differences in sperm cryosusceptibility amongspecies is complicated because there are so many potentially confounding factors to consideroOneway to simplify this analytical approach is to look instead at differences between individuals ofthesame species. Many practitioners of artificial insemination with frozen spermatozoa relate

ObviousIy, cryopreservation invoIves exposure to non-physiologically low temperatureseven before freezing occurs. This process is known to induce changes in two-dimensionalmembrane lipid organization or "packing" (lipid phase transitions) and, in tum, to modify thekinetic properties ofintramembranous enzymes (11, 22, 23). Thus membrane fusogenicityand theresponses of signaI transduction pathways couId also be affected by such changes, contributing tothe possibility that post-thaw sperm longevity is reduced through accelerated capacitation (49).Efforts to correlate susceptibility to cryoinjury with membrane lipid composition across specieshave suggested that cold-shock, a phenomenon by which cryoinjury is induced by sudden coolingbut without freezing, is more severe when sperm membrane sterol concentrations are low andpolyunsaturated fatty acid concentrations are high (9, 50). This classification distinguished fowland human spermatozoa as more cold-shock resistant than bull and ram spermatozoa. However,it cannot explain the major differences in post-freeze/thaw survival and fertility between, e.g.,porcine and bovine spermatozoa. Technical procedures such as slow cooling, and additives suchas egg yolk or milk proteins, are routinely incIuded in cryopreservation protocols to minimizecold-shock effects. The most significant cryoinjuries are therefore probably sustained during thefreezing and thawing process; this suggests that differences in membrane lipid composition are notas important as was once thought. Despite this concIusion, sorne attempts to manipulate the lipidcomposition ofspermatozoa through diet have been undertaken in the beliefthat this would alIowthe generation of more cryoresistant cells. So far, this research has shown that, as one mightexpect physiologically, sperm lipid composition' is unresponsive to manipulation (28); however,this work is being continued.

Such comparative data show that the effective range of glycerol concentrations inunfrozen diluent spans an order of rnagnitude «0.2 M to approxirnately 2 M). It seemsimplausible that this is exclusively attributable to species differences in membrane permeability,and more reasonable to consider all aspects of membrane structure and function. The molecularinteraction of gIyceroI with the sperm plasma membrane was reviewed in sorne detail byHammerstedt and Graham (20), who suggested that it might both alter membrane fluidity, bybecoming intercalated into the lipid bilayer, and change the intracellular cytoplasmic viscosity thusaffecting all metabolic reactions. Disruption of cytoskeIetal protein-membrane interactions inmouse spermatozoa by the action of glycerol has also been proposed (34). In this context, thepotential dísruptive effects were regarded as beneficial because tolerance to the osmotic stressesassociated with water fluxes may be enhanced. This hypothesis may help explain the need for suchhigh glycerol concentrations for sorne marsupial spermatozoa, as the group of species in whichcryopreservation is both probIematic and require the highest glycerol concentrations in thecryodiluents (kangaroos and wallabies), are also those with elaborate submembranous cytoskeletalstructures (43).

considerable benefit from increasing the glycerol concentrations to levels (15-20%) which wouldnormally be considered excessively high (25, 26, 31).

Theriogenology50

The observation that tolerance to osmotic stresses during the addition and removal ofcryoprotectant is highly predictive of sperm survival after cryopreservation can be viewed in twoways. On the one hand it shows that differences in membrane structure, permeability and elasticitydetermine the extent to which sperm will survive the cryopreservation process. However, at atechnical level this information suggests that sperm survival can be optimized by avoiding orminimizing osmotic shock as much as possible. Detailed biophysical studies of membranepermeability characteristics of human spermatozoa have allowed prediction of strategies thatminimize volume excursions during the addition and removal of cryoprotectants (14). The optimaltechnique, which involved the gradual addition and removal of glycerol in a series of

These results also formally test and support the hypothesis that inter-individual differencesin sperm "freezability" are genetically inherited rather than being random. Previous experimentson mouse sperm cryopreservation had suggested the existence of genetically determined straindifferences (42). Indeed, the difficulty of working with a transgenic mouse strain (C57BL/6J)prompted the use of partial zona dissection to improve the fertilization rate (33). These findingsappear to provide the basis for a molecular approach to sperm cryobiology. Within species, andeven within strains or breeds, identiíying groups of males that only differ in the response to spermcryopreservation protocols might be possible. Comparisons of pooled genomic DNA among thegroups should highlight gene differences, whose function may be correlated with spermcryopreservation. This is a potentially powerful approach, although novel and yet untested in thiscontexto

anecdotes about individual males whose spermatozoa either consistently fail to survive thefreeze/tbaw process or altematively, survive particularly well. Although such observations aregenerally anecdotal, they apply across a range of species, including dogs, bulls, boars, stallionsand humans. The clearest demonstrations of these inter-individual differences come from twoseries of experiments. Heterospermic insemination trials using mixed samples of bull spermatozoashowed tbat the cryopreservation process changed the proportions of conceptions attributable toparticular sires when the sperm were in competition with each other (2). This implies tbat thosespermatozoa tbat are normally advantaged in their ability to reach and fertilize oocytes are notnecessarily those most suited to withstand cryopreservation. This is an important observationbecause the experimental design eliminated many factors, especially those related to female tractanatomy and ovulation tirning, which ofien confound inter-specific comparisons. The secondseries of experiments to reveal the importance of inter-individual differences comes fromexperiments on mouse sperm cryopreservation. Working with three strains ofmice Songsasen andLeibo (40) showed tbat simple exposure of spermatozoa to cryoprotectíve medium, wíthoutfreezing, reduced fertilization rates in two strains while the third strain tested (B6D2Fl) remainedunaffected by this treatment. After freezing, spermatozoa from the B6D2Fl strain produced61.2% 2-cell embryos afier IVF, while fertility was severely depressed in the other two strains(17.2 and 3.0% 2-cell embryos for 129/J and C57BL/6J, respectively). Further experimentsshowed tbat these differences were mainly attributable to differential sensitivity to the osmoticshocks associated with the addition and removal of cryoprotectant. These observations not onlypoint once more to plasma membrane properties as highly influential determinants of post-tbawsperm survival and fertility, but are especially valuable because gross species difference effects areclearly not relevant.

51Theriogenology

Insemination tirning is critical when using frozen spermatozoa as the duration of spermsurvival is typically OOIfthat of fresh sperm. Success of Al programs is thus highly dependentupon the availability of good techniques for predicting, controlling or detecting ovulation. Ingeneral, insemination prior to ovulation is better than post-ovulation insemination. These technicalissues are compounded by anatomical variations and sperm transport mechanisrns. Intrauterineinsemination is difficult in species such as sheep, where the cervix efIectively blocks catheterinsertion, thereby reducing the number of spermatozoa reaching the uterus and oviduct. In otherspecies, such as cattle and pigs, insemination devices can easily pass through the cervix, increasingthe efficiency of sperm deposition. Entry of sperm into the oviduct is, however, restricted by theutero-tubal junction; sperm showing defective motility or advanced capacitation-like phenomenamay be selectively prevented from entering the oviductoDirect intrauterine deposition of sperm bylaparoscopy is often more successful than intra- or transcervical insemination, demonstrating thatdefective sperm transport is a major consequence of sublethal cryoinjury. Seminal volume andsperm numbers in the ejaculate reflect anatomical difIerences in the female tract. In pigs, semenvolume exceeds 300 ml and normally causes uterine distention and pressure; consequently, largevolumes of fluid and large numbers of cryopreserved spermatozoa are required for highconception rates. In cattle and many large ungulates semen volume is <10 ml and is naturallydeposited into the anterior vagina. Consequently, efficiency of sperm cryopreservation can belower in these species without significant loss of fertility, because smaller absolute numbers of

There are major differences among species in sperm morphology, especially in acrosomalshape and flagellar length, but their relevance to cryopreservation is unclear. Many rodents haveelaborate sperm heads, often being hook-shaped and possibly more susceptible to distortion anddamage. Bull, ram and boar sperm heads are very similar, i.e., "paddle-shaped", but the boarspermatozoa are more susceptible to cryoinjury. Sorne of the macropodid marsupials (kangaroosand wallabies) have elaborate submembranous cytoskeletal assemblies (43) that may render themparticularly susceptible to cryoinjury. Other marsupial spermatozoa pair by head-head attachmentduring sperm maturation in the epididyrnis (e.g., south American opossurns) and cannot swimprogressively if this mutual arrangement is disrupted (32); cryopreservation is likely to beparticularly difficult with such species.

Owing to space limitations, the aboye discussion has considered only a limited number ofcharacteristics which may be relevant to the relative success of cryopreservation protocols indifferent species. However, many different aspects ofreproduction undoubtedly contribute to theease of both cryopreserving spermatozoa and subsequently using them successfully forinseminations. The following paragraphs aim to provide a brief indication of these topics, withoutnecessarily going into much detail.

approximately eight incremental or decremental steps, permitted recovery of approximately 90%live spermatozoa. This considerably outperformed the results obtained when cryoprotectant wasadded or diluted all at once, which is the procedure routinely used in most laboratories. The morepainstaking and elaborate approach to cryoprotectant addition and removal is, of course, lesspractical in a busy laboratory. However, the availability ofthis technique for human spermatozoais worthy of note, especially for those dealing with samples from chemotherapy patients andwhich may be irreplaceable or of extremely poor quality.

Theriogenology52

When the aim of sperm preservation is for use with artificial insemination, few options areavailable except to freeze appropriately high numbers of sperrnatozoa and ensure tbat they retainfunctionality as much as possible. Ibis is true for insemination procedures applied to agricultura!

Recent Developments in the Preservation ofMale Gametes

As the source of semen and the collection method affects sperm concentration, motilityand probably also fertility, differences in ease of collection can bave major implications for thesuccess of cryopreservation. The use of artificial vaginae and trained semen donors provides thebest and most natural samples for cryopreservation. Electroejaculation under general anaesthesiais widely used with wild animals, but the quality of semen obtained is variable. Recovery of semenfrom the vagina after natural mating is sometimes useful where electroejaculation is inadvisable;however, vaginal secretions are detrimental to sperrnatozoa. Although seminal plasma containsmany components wbich are beneficial to sperrnatozoa and may complete their maturationprocess, it should be noted tbat during natural ejaculation the sperrnatozoa do not usually stay inprolonged contact with it. This is, however, an almost inescapable drawback of electroejaculationand spermotoxic effects of seminal plasma bave been noted.

Cryopreservation is directly letbal to a significant proportion (circa 50%) of spermatozoain a typical semen sample and consequently the number of sperrnatozoa required for successfulinseminations has to be increased aboye tbat needed for fresh spermatozoa. However, therequisite increase may be ten or a hundred fold, suggesting tbat many spermatozoa, although liveand motile, are unable to reach and fertilize the egg. The avoidance of sperm transport effects bylaparoscopic insemination shows tbat some of the inseminated spermatozoa can achievefertilization, even though they may bave sustained sublethal darnage impairing their ability topenetrate cervical mucus or pass through the utero-tubal junction. Whether these spermatozoapossess innate genetic advantages over other sperm in the ejaculate is unclear. There is also astrong possibility tbat sperrnatozoa selectively bind to oviductal epithelia, where they are storedand protected until ovulation occurs (for review, see 41).

Watson (49) linked observations tbat exposure of spermatozoa to low temperaturesshortens their capacitation time, with changes in membrane lipid architecture, membranepermeability and the reduced efficiency of homeostatic enzymes, especially those extrudingcalcium ions. Together, these changes resemble capacitation, and are likely to reduce long-termsperm viability and alter physiological functions such as motility. Capacitation-like changes mayalso influence the nature of interactions between sperrnatozoa and immune cells or epithelial cellsin the female reproductive tracto Within tbis context, species differences in sperm membranephysiology, structure and biochemistry would probably induce different responses to cooling andfreezing, thus influencing the subsequent fertility of spermatozoa.

fertile spennatozoa can stilI achieve high conception rates if deposited beyond the cervix, Whilethese remarks are especially relevant to large eutherian mammals, the principIes also apply toother groups of species. Marked differences in femaIe reproductive tract anatomy are especiallynotable in the marsupials, where two separate uteri are each connected to lateral vaginae by twincervices.

53Theriogenology

Intracytoplasmic sperm injection is being increasingly adopted for human clínical use inspecial cases where sperm quality is very poor or sperm numbers are very low due to testicularfailure or epididymal obstruction. In the last two years at least twenty-five publications bavedescribed the use of ICSI with immotile human testicular or epididymal spermatozoa recoveredfrom frozenltbawed tissue samples (e.g., 1,11). ICSI seems to offer an effective means ofusingthese recovered spermatozoa. To date, no deleterious effects bave been attributed to thecryopreservation technique itself. Despite the intention to use ICSI, sorne authors bave beenconcemed with optimizing the post-tbaw recovery of sperm motility in minced testicular tissue.Glycerol has therefore been included as a cryoprotectant for this purpose (11). Following similarlogic, Cohen et al. (6) developed an ingenious technique for the cryopreservation of singlespermatozoa. Their technique involved encapsulation of spermatozoa, individually or in smallgroups, within empty zonae pellucidae preloaded with cryoprotective media. Aftercryopreservation the spermatozoa were recovered with considerable success (14/15 spermatozoarecovered). Ten of these spermatozoa were used for ICSI and eight successfully fertilized theoocytes. Given tbat ICSI bypasses many physiological processes involved in fertilization, thejustification for using cryoprotectants for the preservation of motility and cellular integrity seemsquestionable. Nevertheless, recent studies bave indicated tbat human fertilization and pregnancyrates obtained using ICSI are considerably reduced if immotile, rather than motile, spermatozoaare used. Shibahara et al. (39) obtained high fertilization rates when epididymal spermatozoashowing normal motility were used (68.6% and 68.4%; fresh and frozen spenn, respectively).However, the fertilization rate was halved (31.6%) when immotile frozen spermatozoa were used.This result suggests tbat a correlation exists between motility and other important qualities of

This direct approach to mouse sperm preservation was tested (45, 46) and shown to befeasible. Spermatozoa were suspended in culture media, with and without the inclusion ofraffinose as a debydrating agent. Aliquots (50 IlL) ofsperm suspension were dispensed into 1mLcryotubes tbat were then placed in a freezer (-20°C, -50°C or directly into liquid nitrogen).Viability assays confirmed tbat these spermatozoa had damaged plasma membranes and weretherefore immotile. ICSI performed with tbese spermatozoa, baving first separated the heads andflagellae, resulted in 95-100% of injected oocytes being activated and fertilized normally wbateverthe final storage temperature. The authors noted sorne differences among the variousmodifications ofthe sperm freezing procedure, but qualified their remarks by saying tbat all oftbetreatments were successful, and tbat females produced litters of normal size with no apparentabnormalities. In view of these results and the limitation tbat cryopreserved mouse spermatozoamust, in any case, be used for in vitro fertilization, it seems likely tbat storage of mousespermatozoa may in the future be undertaken routinely by this simple, glycerol-free, method.

species on an industrial scale, or with wild species where the ability to recover oocytes andperfonn intensive procedures is limited by practical and ethical considerations, However, thedevelopment of techniques for helping the penetration of eggs by functionally defectivespermatozoa (e.g., zona drilling, subzonal insemination and intracytoplasmic sperm injection(ICSI)) has, for sorne purposes, eliminated the need to retain physiological integrity in storedspermatozoa. Initíal observations that isolated spenn nuclei can support embryonic developmentin cattle, human and hamster (19,21) prompted more recent investigations on mouse spermcryopreservation without cryoprotectant.

Theriogenology54

Since the literature on sperm eryopreservation is vast and has been reviewed extensively(21,47-49), the aim ofthis review was to explore sorne aspects ofthe topie which are cruciallyimportant but frequently overlooked. One of my intentions in writing this article was to emphasizethat while preserving sperm integrity during freezing and thawing is clearly important, the in vivofertilizing ability of cryopreserved spermatozoa also depends 00 the biologieal eontext in which itis used. Thus, anatornical features of the femaIe reproductive traet, and the ability to detect orcontrol ovulation aecurately, will ultimately govern the sueeess of artificial inserninations just aspowerfully as the percentage of acrosome-intact or motile spermatozoa. In fact, it can be arguedthat there will nearly always be enough spennatozoa which are capable of fertilizing the egg,provided they are able to reach it at the appropriate time. Another intention was to develop theidea that one potentially fruitful route to understanding the basis of cryoinjury may be through thestudy of differences between individuals of the same species. Studies in the mouse are beginningto reveal genetically-based trends in the success of sperm cryopreservation procedures, and rnight

CONCLUSIONS

An extension to the use of ICSI with cryopreserved testicular spermatozoa is the direetintracytopIasrnic injeetion of round spermatids and even spermatocytes into oocytes. Thisapproach was pioneered using mouse testicular cells (35), and has since been applied to humanpatients. A potential demand for round spermatid cryopreservation thus exists; however, as theyare only likely to be used with ICSI, the retention of nuclear integrity may be the only importantrequirement Nevertheless, successful cryopreservation of human spermatogenic cells has beenreported (1). In a parallel development the feasibility of cryopreserving rat sperrnatogonia andtranspIanting them into irradiated mouse testes was demonstrated experirnentally (5). A standardsomatic eell cryopreservation procedure was used in this case. The eryopreservation probablysucceeded because, being stem cells, sperrnatogonia can multiply to regenerate a viable populationeven if many sustain lethal damage. A1though the original aim of the sperrnatogonialtranspIantation was the study of spermatogenesis, this approach clearly offers sorne potential forthe reseue of the male germline in, for example, the preservation of genetic resources inagrieulture or wildlife conservation. At present, if spermatozoa are unavailable, there are no otheroptions for the storage of germpIasm from genetically valuable individuals. Considerable care willbe required to ensure that functional markers of DNA integrity are developed for monitoring theeffectiveness of this approach.

individual spermatozoa. Use of cryoprotectants to maintain optimal sperm motility seemstherefore highly advisable. However, there is an alternative way of viewing this data which isreminiscent of the arguments used in support of sperm competition theory (18). This suggests thatthe spermatozoa which are most effective in reaching and fertilizing oocytes also convey superiorgenetic qualities into the next generation. The ICSI studies cited above imply that cryoinjuriesaffecting motility may be more severe when nuclear structures are defective; alternatively theycould indicate that individual spermatozoa with the most eryosuseeptible motility-generatíngstruetures carry the poorest quality DNA. Given the complex meehanisms whieh have evolved inthe female reproductive traet to seleet the fertilizing spermatozoa from among the millions presentin the ejaculate, the absence of selectivity when performing ICSI should be a matter of concernand vigilanee, whether using fresh or frozen spermatozoa.

55Theriogenology

1. Aslam 1, Fishel S. Short-term in-vitro culture and cryopreservation of spennatogenic cellsused for human in-vitro conception. Hum Reprod 1998;13:634-638.

2. Beatty RA, Stewart DL, Spooner RL, Hancock JL. Evaluation by the heterospermicinsemination technique of the differential effect of freezing at -196°C on the fertility ofindividual bull semen. J Reprod FertilI976;47:377-379.

3. Bwanga CO, Ekwall H, Rodriguez-Martinez H. Cryopreservation of boar semen. III.Ultrastructure of boar spermatozoa frozen ultra-rapidly at various stages of conventionalfreezing and thawing. Acta Vet Scand 1991;32:463-471.

4. Castellini C, Bizzarri AA, Cannistraro S. Water volume oí rabbit spennatozoa measured byelectron paramagnetic resonance at different temperatures. 6th World Rabbit CongressToulouse 1996;55-58.

5. Clouthier DE, Avarbock MR, Maika SD, Hammer RE, Brinster RL. Rat spennatogenesis inmouse testis. Nature Lond 1996;381:418-421.

6. Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT.Cryopreservation of single human spermatozoa. Hum Reprod 1997;12:994-1001.

7. Crabbe E, Verheyen G, Tournaye H, Van Steirteghem A. Freezing oftesticular tissue as arninced suspension preserves sperm quality better than whole-biopsy freezing when glycerol isused as cryoprotectant. Int J Androl 1999;22:43-8.

8. Curry MR, Millar ID, Watson PF. Calculated optima1cooling rates for mm and human spermcryopreservation faíl to conformwith empiricalobservations.Biol Reprod 1994;51:1014-1021.

9. Darin-Bennett A, Poulos A, White IG. The effect of cold-shock and freeze-thawing on releaseofphospholipids by raro, bull and boar spermatozoa. Aust J Biol Sci 1973;26:1409-1420.

10. Devireddy RV, Swanlund DJ, Roberts KP, Bichof JC. Subzero water permeability parametersof mouse spermatozoa in the presence of extracellular ice and cryoprotective agents. BiolReprod 1999;61:764-775.

11. Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH. Cold shockdamage is due to lipid phase-transitions in cell-membranes - a demonstration using sperm as amodel. J Exp ZooI1993;265:432-437.

12. Fennessy PF, Mackintosh CG, Shackell GH. Artificial insemination of fanned red deer(Cervus elaphus). Anirn Production 1990;51:613-621.

13. Gao DY, Ashworth E, Watson PF, Kleinhans FW, Mazur P, Critser JK. Hyperosmotictolerance of human spennatozoa - separate effects of glycerol, sodium-chIoride, and sucroseon spermolysis. Biol Reprod. 1993;49:112-123.

14. Gao DY, Liu J, Liu C, McGann LE, Watson PF, Kleinhans FW, Mazur P, Critser ES, CritserJK. Prevention of osmotic injury to human spennatozoa during addition and removal ofglycerol. Hum Reprod 1995;10:1109-1122.

REFERENCES

ultimately provide clues which point to specific differences between strains. Ideally, one wouldhope to see that the differences corre late with the presence of particular gene products in thespennatozoa, such as membrane channels or structural proteins. The extraordinarily rapiddevelopments which are now taking place in functional genomics, especially of the mouse and thepig, mean that this is now a realistic avenue of study.

Theriogenology56

15. Gao DY, Mazur P, K.leinhansFW, Watson PF, Noiles EE, Critser JK. Glycerol permeabilityofhuman sperrnatozoa and its activation-energy. Cryobiology 1992;29:657-667.

16. Garland P. Artificial insernination of scimitar-homed oryx (Qm dammah). Bull ZooManagement 1989;27:29-30.

17. Garrels KL, Zini AS, Casper RF, Meriano JS, Jarvi KA. Fresh and frozen epididymal spermyield comparable pregnancy rates for intracytoplasmic sperm injection. Arch Androl1998;41:159-65.

18. Gomendio M, Harcourt AH, Roldan ERS. Sperm competition in mammals. In: Birkhead TR,Moller AP (eds), Sperm competition and sexual se1ection. San Diego, London, Boston:Academic Press, 1998;667-755.

19. Goto K. Kinoshita A, Takuma Y. Fertilization of bovine oocytes by the injection ofimmobiIized, killed, sperrnatozoa. Vet Rec 1990;127:517-520.

20. Harnmerstedt RH, Graham JK. Cryopreservation of poultry sperm: the enigma of glycerol.Cryobiology 1992;29:26-38.

21. Harnmerstedt RH, Graham JI<. Nolan JP. Cryopreservation of marnmalian sperm: what weask them to survive. J Androl 1990;11:73-88.

22. Ho1t WV, North RD. Partially irreversible cold-induced lipid phase transitions in marnmaliansperm plasma membrane dornains: freeze-fracture study. J Exp ZooI1984;230:473-483.

23. Holt WV, North RD. Thermotropic phase transitions in the plasma membrane of ramsperrnatozoa. J Reprod FertilI986;78:445-457.

24. Hopkins SM, Armstrong DL, Hummel S, Junior S. Successful cryopreservation of gaur (Bosgaurus) epididymal sperrnatozoa. J Zoo AnimMed 1988;19:195-201.

25. Johnston SD, McGowan MR, Carrick FN, Tribe A, Douglas R. Prelirninary investigationsinto the feasibility of freezing koala (Phascolarctos cinereus) semen. Australian Vet J1993;70:424-425.

26. Johnston SD, McGowan MR, Carrick FN, Tribe A. Douglas R. Prelirninary investigationsinto the feasibility of freezing koala (Phascolarctos cinereus) semen. Australian Vet J1993;(correction published in 72:473).

27. Katayose H, Matsuda J, Yanagirnachi R. The ability of dehydrated hamster and human spermnuclei to develop into pronuclei. Biol Reprod 1992;47:277-284.

28. Kelso KA, Cerolini S, Noble RC, Sparks NHC, Speake BK. The effects of dietarysupplementation with docosahexaenoic acid on the phospholipid fatty acid composition ofavian sperrnatozoa. Comp Biochem Physiol B 1997;118:65-69.

29. Leibo SP. Water permeability and its activation energy of fertilized and unfertiIized mouseova. J Memb Biol 1980;53: 179-188.

30. Mazur P. Freezing of living cells: mechanisrns and implications. Amer J Physiol1984;247:CI25-CI42. .

31. Molinia FC, Rodger JC. Pellet-freezing sperrnatozoa of 2 marsupials - the tammar wallaby,Macropus eugenii, and the brushtail possum, Trichosurus vuloecula, Reprod Fertil Dev1996;8:681-684.

32. Moore HDM, Taggart DA. Sperm pairing in the opossum increases the efficiency of spermmovement in a viscous environment. Bio1Reprod 1995;52:947-953.

33. Nakagata N, Okamoto M, Ueda 0, Suzuki H. Positive effect of partial zona-pellucidadissection on the in vitro fertilizing capacity of cryopreserved C51BL/6J transgenic mousesperrnatozoa oflow motility. Bio1Reprod 1997;57:1050-1055.

57Theriogenology

34. Noiles EE, Thompson KA, Storey BT. Water permeability, L-p, ofthe mouse sperm plasmamembrane and its activation energy are strongly dependent on interaction of the plasmamembrane with the sperm cytoskeleton. Cryobiology 1997;35:79-92.

35. Ogura A, Yanagirnachi R. Round sperrnatid nuclei injected into hamster oocytes formpronuclei and participate in syngarny. Biol Reprod 1993;48:219-225.

36. Polge C, Smith A, Parkes A. Reviva! of sperrnatozoa after vitrification and dehydration atlow temperatures. Nature Lond 1949;164:166.

37. Ponce AA, Aires VA, Carrascosa R, DeCuneo MF, Ruiz RD, Lacuara JL. Functional activityof epididyrnal Chinchilla laniger sperrnatozoa cryopreserved in different extenders. Res VetSci 1998;64:239-243.

38. Shabana M, McGrath J1. Cryomicroscope investigation and thermodynamic modeling of thefreezing ofunfertilized hamster ova. Cryobiology 1988;25:338-354.

39. Shibahara H, Harnada Y, Hasegawa A, Toji H, Shigeta M, Yoshimoto T, Shima H, KoyarnaK Correlation between the motility of frozen-thawed epididyrnal sperrnatozoa and theoutcome of intracytoplasmic sperm injection. Int JAndroI1999;22:324-328.

40. Songsasen N, Leibo SP. Cryopreservation of mouse sperrnatozoa. JI. Relationship betweensurvival after cryopreservation and osmotic tolerance of sperrnatozoa from three strains ofmice. Cryobiology 1997;35:255-269.

41. Suarez SS. The oviductal sperm reservoir in mamrnals; mechanisms of forrnation. BiolReprod 1998;58:1105-1107.

42. Tada N, Sato M, Yarnanoi J, Mizorogi T, Kasai K, Ogawa S. Cryopreservation ofmousesperrnatozoa in the presence ofraffinose and glycerol. J Reprod FertilI990;89:511-516.

43. Taggart DA, Leigh CM, Breed WG. Ultrastructure and motility of sperrnatozoa in the malereproductive tract ofperarneloid marsupials. Reprod Fertil Dev 1995;7:1141-1156.

44. Taggart DA, Leigh CM, Steele VR, Breed WG, Temple-Smith PD, Phelan J. Effect ofcooling and cryopreservation on sperm motility and morphology of several species ofmarsupial. Reprod Fertil Dev 1996;8:673-679.

45. Wakayarna T, Whittingham DG, Yanagimachi R. Production ofnormal offspring from mouseoocytes injected with sperrnatozoa cryopreserved with or without cryoprotection. J ReprodFertil 1998;112:11-17.

46. Wakayarna T, Yanagimachi R. Development of normal mice from oocytes injected withfreeze-dried sperrnatozoa. Nature Biotechnology 1998;16:639-641.

47. Watson PF. The preservation of semen in rnamrnaIs. In: Fino CA (ed), Oxford Reviews ofReproductive Biology. Oxford: Clarendon press, 1979;283-350.

48. Watson PF. Artificial insemination and the preservation of semen. In: Lamming G (ed),Marshall's Physiology of Reproduction. Edirturgb, Lon:lon: CbJrchiIl~ 1990;747-869.

49. Watson PF. Recent developments and concepts in the cryopreservation of sperrnatozoa andthe assessment oftheir post-thawing function. Reprod Fertil Dev 1995;7:871-891.

50. White IG, Darin-Bennett A. The lipids of sperm in relation to cold shock. 8th Congress AnimReprod ArtifInsem 1976; 951-954.

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