Genome resource banking for wildlife conservation: promises and caveats.

The value of cryopreserved germplasm in agriculture, aquaculture and medicine was recognized in the mid-twentieth century following the discovery in the late 1940s of a method for recovering viable spermatozoa after freeze-thawing. Sir Alan Parkes (a founder of cryobiology as a discipline) remarked that "time and space has been abolished for cattle breeding", a phrase that continues to summarise the potential value of the Genetic Resource Bank (GRB) concept for all species. The underlying principle behind these remarks was based on the recognition that spermatozoa could remain viable for many years, and still achieve pregnancies even long after the semen donor had died. Nowadays, live mammalian embryos, amphibian spermatozoa and cultured somatic cells can also be stored for future use in conservation breeding programmes, where the overarching aim is to mitigate the deleterious impacts of inbreeding on the fitness and survival of populations. Revolutionary advances in the cryobiology of coral spermatozoa, embryos and larvae are also helping to counter the damaging effects of climate change and toxic chemicals on coral reefs. In this article we review the ways in which GRBs can contribute to global conservation activities, noting that species-specific biological differences can limit the success of standard animal breeding technologies such as artificial insemination and embryo transfer. These limitations mean that there is still a need for the development of novel, and possibly species-specific, GRB technologies.

Protamine composition of koala and wombat spermatozoa provides new insights into DNA stability following cryopreservation.

To investigate differences in the post-thaw DNA stability of koala and wombat spermatozoa, protamine amino acid sequences were compared and it was found that there were three more arginine residues for the wombat. Koala and wombat spermatozoa, cryopreserved using identical protocols, were examined for changes in sperm DNA fragmentation (SDF) dynamics over 24h of post-thaw incubation. Following validation of a wombat sperm chromatin dispersion test, wombat DNA showed a rate of SDF that was 6-fold higher than for koala spermatozoa (P=0.038). Finally, we examined whether expected differences in chromatin compactness, associated with protamine sequence, had an effect on restriction site accessibility of sperm DNA. Thawed spermatozoa were exposed to Alu I and EcoR1 endonuclease restriction enzymes and the SDF dynamics were observed. Koala spermatozoa exposed to Alu I showed a greater rate of SDF (P=0.01), whereas wombat spermatozoa exposed to EcoR1 showed a greater rate of SDF (P=0.032). We conclude that restriction sites in these species are differentially present or exposed and potentially account for differences in SDF dynamics. Although differences in the arginine composition of protamine may explain relative differences in SDF following cryopreservation, they do not support the hypothesis that increased arginine composition increases DNA stability; rather, increased arginine composition in the wombat may reduce post-thaw chromatin swelling.

Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa.

There is growing concern over the effect of sperm cryopreservation on DNA integrity and the subsequent development of offspring generated from this cryopreserved material. In the present study, membrane integrity and DNA stability of Xenopus laevis and Xenopus tropicalis spermatozoa were evaluated in response to cryopreservation with or without activation, a process that happens upon exposure to water to spermatozoa of some aquatic species. A dye exclusion assay revealed that sperm plasma membrane integrity in both species decreased after freezing, more so for X. laevis than X. tropicalis spermatozoa. The sperm chromatin dispersion (SCD) test showed that for both X. tropicalis and X. laevis, activated frozen spermatozoa produced the highest levels of DNA fragmentation compared with all fresh samples and frozen non-activated samples (P<0.05). Understanding the nature of DNA and membrane damage that occurs in cryopreserved spermatozoa from Xenopus species represents the first step in exploiting these powerful model organisms to understand the developmental consequences of fertilising with cryopreservation-damaged spermatozoa.

An introduction to epigenetics as the link between genotype and environment: a personal view.

Lamarck was one of the first scientists who attempted to explain evolution, and he is especially well known for formulating the concept that acquired characteristics can be transmitted to future generations and may therefore steer evolution. Although Lamarckism fell out of favour soon after the publication of Darwin's work on natural selection and evolution, the concept of transmission of acquired characteristics has recently gained renewed attention and has led to some rethinking of the standard evolutionary model. Epigenetics, or the study of heritable (mitotically and/or meiotically) changes in gene activity that are not brought about by changes in the DNA sequence, can explain some types of ill health in offspring, which have been exposed to stressors during early development, when DNA is most susceptible to such epigenetic influences. In this review, we explain briefly the history of epigenetics and we propose some examples of epigenetic and transgenerational effects demonstrated in humans and animals. Growing evidence is available that the health and phenotype of a given individual is already shaped shortly before and after the time of conception. Some evidence suggests that epigenetic markings, which have been established around conception, can also be transmitted to future generations. This knowledge can possibly be used to revolutionize animal breeding and to increase human and animal health worldwide.

Effect of pre-freeze semen quality, extender and cryoprotectant on the post-thaw quality of Asian elephant (Elephas maximus indicus) semen.

Semen cryopreservation and artificial insemination (AI) are potentially valuable methods for supporting the breeding management of endangered species like the Asian elephant. Cryopreservation of Asian elephant semen has however proven problematic with respect to maintenance of both adequate semen quality and fertility post-thaw. In this study, nine ejaculates from three adult bulls were used to compare the influence of extender (TEST versus INRA96®) and penetrating cryoprotectants (3% glycerol, 5% glycerol and 4% methylformamide) on post-thaw semen quality. We demonstrate that not only the freezing process, but also the quality of the semen before freezing, significantly influences the freezability of Asian elephant semen. Pre-freeze motility, viability, semen volume, semen pH, sperm concentration and the incidence of sperm mid-piece and tail abnormalities all significantly (p<0.05) affected post-thaw semen quality. While extender and cryoprotectant did not significantly affect any of the above semen quality parameters post-thaw, the skim-milk based extender (INRA96®) preserved DNA integrity better (p<0.05) than the egg yolk extender (TEST). Considerable between-ejaculate variation in all post-thaw semen quality parameters was also noted. It is concluded that strict criteria for semen quality is essential for the selection of Asian elephant bull ejaculates suitable for cryopreservation; stricter initial selection should improve the mean post-thaw quality.

Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic injury model appears to explain post-thaw changes in koala SDF, the mechanisms resulting in relaxed chromatin require further study. A lack of correlation between the percentage of sperm with relaxed chromatin and SDF suggests that the timing of these pathologies are asynchronous. We propose an integrative model of cryo-induced osmotic injury that involves a combination of structural damage (rupture of membrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.

The oviducal protein, heat-shock 70-kDa protein 8, improves the long-term survival of ram spermatozoa during storage at 17°C in a commercial extender.

Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical artificial insemination (AI). Heat-shock 70-kDa protein 8 (HSPA8), found within the oviduct, prolongs boar, ram and bull sperm survival at body temperatures in vitro. Here, we aimed to determine whether supplementing extenders (INRA-96 and RSD-1) with HSPA8 (4 µg mL⁻¹) would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 17°C. Sperm function was assessed at 1, 6, 24 and 48h and this experiment was repeated using 25 × 106 and 800 × 106 spermatozoa mL⁻¹. INRA96 supplemented with HSPA8 maintained sperm viability significantly better than INRA96 alone at both sperm concentrations. However, sperm nuclear DNA fragmentation (DF) increased significantly during storage using the higher sperm concentration, irrespective of the extender and the protein treatment used. Increasing levels of sperm nuclear DF over time could explain why poor fertility rates are often observed following cervical AI using stored ram semen. However, further research is required to ascertain whether supplementing the commercially available INRA96 extender with HSPA8 will improve fertility rates following cervical AI using stored ram semen.

Effect of cooled storage on quality and DNA integrity of Asian elephant (Elephas maximus) spermatozoa.

Artificial insemination (AI) is a potentially useful tool for breeding captive elephants because it facilitates efforts to minimise inbreeding. However, cooled storage of elephant semen markedly reduces fertility. This study compared the effects on semen-quality parameters, including sperm DNA fragmentation, of storing elephant semen at 4°C or 15°C in a commonly-used diluent (TEST) or a diluent developed to protect against sperm DNA damage (BullMax). Storing elephant semen for >24 h in either extender at either temperature resulted in decreases in sperm motility, viability, acrosome integrity and DNA integrity (P < 0.05); the decrease in motility was especially rapid. A subjective impression of circular sperm movement in TEST was confirmed by a higher curvilinear velocity and amplitude of lateral head displacement, but lower straight-line velocity and linearity than in BullMax. Initial percentages of spermatozoa with fragmented DNA (%SDF) did not differ between extenders or temperatures, but the rate of increase in %SDF during a 48-h incubation at 37°C was higher in TEST than in BullMax (P < 0.05). In conclusion, BullMax allows more linear movement and better preserves DNA stability of stored elephant spermatozoa than TEST. Sperm DNA stability during incubation at 37°C is a promising, discriminative parameter for selecting semen storage conditions of bulls for elephant AI.

The long and the short of sperm selection in vitro and in vivo: swim-up techniques select for the longer and faster swimming mammalian sperm.

Sperm competition and sexual selection outcomes are sometimes reported as depending on sperm velocity and flagellar length, suggesting that sperm shape may be optimized for maximum efficiency. This is a largely unexamined assumption regarding sperm performance. Here, we examine this idea using a 'swim-up' selection technique as a proxy for sperm transport within the female tract, testing the hypothesis that variation in sperm tail length should be reduced by this procedure. We detected small but significant (P < 0.001) increases in mean flagellar length in brown hare, pig and bull spermatozoa without reduction in variance. Applying the swim-up technique to boar ejaculates confirmed that the selected populations were enriched for fast motile spermatozoa. These effects were also reflected in vivo where boar spermatozoa with both short and long flagellae were able to reach and colonize the oviductal sperm reservoir. The benefits of possessing a longer flagellum thus appear to be marginal, suggesting that sperm selection in vivo is based on more complex criteria.

Directional mapping of DNA nicking in ejaculated and cauda epididymidal spermatozoa of the short-beaked echidna (Tachyglossus aculeatus: Monotremata).

Prototherian spermatozoa are unique amongst the Mammalia in terms of their filiform morphology, tandem arrangement of chromosomes and formation of sperm bundles. In the present study, we provide observations of echidna spermatozoa and note that the superstructure of the bundle is engineered around the shape of the individual sperm head and that this in turn may be a consequence of the unusual circumferential and helicoidal condensation of the DNA during spermiogenesis. Frozen-thawed ejaculated echidna spermatozoa were incubated and examined for the presence of non-typical DNA conformation by means of in situ labelling of DNA breaks using Klenow polymerase and via alkaline single-cell comet assays for detection of fragmented DNA. Both techniques successfully revealed the presence of what appeared to be directional DNA nicking, co-localised with the presence of highly sensitive alkali sites along the length of the sperm nucleus. It was not possible to define whether these alternative DNA configurations were associated with a failure of the sperm nucleus to condense appropriately during spermiogenesis or were evidence of DNA fragmentation following post-thaw incubation or a sequential structural chromatin rearrangement necessary for fertilisation.

Effects of oviductal proteins, including heat shock 70 kDa protein 8, on survival of ram spermatozoa over 48 h in vitro.

Following insemination, ram spermatozoa are transported to the isthmus region of the oviduct where they bind to the oviductal epithelial cells (OEC), remaining viable for several hours. The aim of the present study was to begin to decipher which component(s) of the ewe oviduct actively participates in maintaining the viability of ram spermatozoa. A series of experiments was conducted to investigate whether: (1) soluble OEC apical plasma membrane proteins (sAPM) isolated from ewes prolong survival of ram spermatozoa over an extended (48 h) coincubation period at 39 degrees C; (2) a recombinant form of one of these oviductal proteins, namely heat shock 70 kDa protein 8 (HSPA8), prolongs survival of ram spermatozoa; and (3) pretreatment with HSPA8 antibody compromises the ability of sAPM to prolong the survival of ram spermatozoa. Both sAPM and recombinant HSPA8 had a beneficial effect on the viability of ram spermatozoa during coincubation, although both these effects were dose dependent. In contrast, pretreatment with HSPA8 antibody significantly negated the ability of sAPM to maintain the viability of ram spermatozoa. These findings suggest that HSPA8 is an active component of the ewe oviduct that participates in maintaining the viability of ram spermatozoa. This is a potentially valuable observation given that there is a great deal of room for improving existing diluents for storing fresh ram semen.

Artificial insemination for the propagation of CANDES: the reality!

Conservation is about protecting and nurturing species so that they can survive, not only now, but also into the future. Ideally this means protecting genetically diverse populations and not simply breeding a few individuals. Unfortunately, this point is often overlooked by reproductive technologists, especially if they are more accustomed to working with humans, companion animals or agricultural species, where the goals are more usually directed towards obtaining offspring from particular individuals. This approach has tended to antagonise the conservation community, who are quick to develop an unreasonable suspicion of technological solutions, partly because they are unfamiliar with the scientific principles that underpin the reproductive technology. Unfortunately, this mutual failure to recognise that all parties are actually well meaning, has led to separate cultures that barely communicate with each other and thus fail to capitalise on the potential benefits that would come from a good working relationship. Notable successes with reproductive technology have only emerged where such relationships have been forged. In this review, we highlight, mainly for the benefit of the technologist community, the need to foster good working relationships with conservation managers and to recognise that the latest hi-tech approach to animal breeding is more likely to engender suspicion than enthusiasm.

Controlled freezing studies on boar sperm cryopreservation.

Boar spermatozoa from different males were frozen at a number of cooling rates using a controlled-rate freezing machine designed to minimise thermal variables involved in the cooling process, to see whether inter-boar sperm cryosurvival may be improved by changing cooling rate. Four cooling rates in the range 3 degrees C min(-1) to 24 degrees C min(-1) from +5 degrees C to -5 degrees C and five cooling rates in the range 5 degrees C min(-1) to 80 degrees C min(-1) from -5 degrees C to -80 degrees C were tested. Motile spermatozoa were assessed by CASA, plasma membrane integrity by fluorescent probes (SYBR14/propidium iodide) and flow cytometry, and acrosome membrane integrity by lectins (PSA-rhodamine) and fluorescent microscopy. Cooling rate affected sperm cryosurvival from different boars in different ways; that is, spermatozoa from some individuals were less susceptible than those from others. For some individuals, sperm cryosurvival was poor regardless of cooling rate, but for others it was better with faster rates. This confirms cooling rate effects on sperm cryosurvival depend on inter-individual boar differences more than on the cooling process itself.

Is semen analysis useful to predict the odds that the sperm will meet the egg?

Any mammalian spermatozoon that achieves successful in vivo fertilization has to perform almost perfectly in many disparate functions and overcome a series of obstacles imposed by the female reproductive tract. This implies that during formation in the testis and epididymis, the spermatozoa did not incur any morphological, metabolic, immunological or genetic abnormalities. Given that the spermatozoa are such highly differentiated cells, this means that every cellular compartment must not only be intact but must also respond appropriately to intracellular and extracellular signals. Assuming that a spermatozoon possesses this level of perfection, and is able to reach and penetrate an oocyte, it can only be regarded as 'fertile' if the DNA it carries is intact and able to sustain embryonic development. Although the proportion of inseminated spermatozoa that meet these criteria is vanishingly small, the female reproductive tract applies stringent selection criteria during sperm transport and, as a result, the probability of conception around the time of ovulation is very high. If laboratory tests of semen quality could approach the efficacy of the female reproductive tract, it would be possible to predict the odds of spermatozoa meeting the egg; however, this is not possible at present. In this article, I suggest a simple model to illustrate how a battery of laboratory tests could eventually be used to make these predictions.

Temporal dynamics of ram sperm binding and survival during 48-h coculture with oviducal epithelial cells.

Following insemination, ram spermatozoa bind to oviducal epithelial cells (OEC) in vivo and remain viable for several hours before fertilisation. In the present study, we investigated whether OEC monolayers reproduce this effect in vitro, performing an analysis of ram sperm binding and survival over an extended (48 h) period at 39 degrees C. We wanted to determine whether the reproductive cycle phase and/or oviducal region would influence ram sperm binding and survival in coculture with OEC and whether reproductive and non-reproductive epithelial cells bound and maintained the viability of ram spermatozoa equivalently. Oviducts were separated into groups based on their ovarian state (follicular or luteal) and then divided into two parts (isthmus and ampulla) for OEC isolation. Sheep kidney epithelial cells (Madin-Darby ovine kidney; MDOK) were purchased commercially. Reproductive cycle phase, but not oviducal region, affected sperm binding to OEC. Although more spermatozoa bound to luteal OEC than to follicular OEC at 1 h, at 24 h follicular OEC had bound more spermatozoa than luteal OEC. Generally, spermatozoa that were bound to OEC and MDOK had enhanced viability at each of the time points investigated (1, 6, 24 and 48 h), but the viability of the OEC-bound spermatozoa was greater than that of the MDOK-bound spermatozoa at 48 h. In conclusion, ram sperm-epithelial cell interactions are temporal, dynamic and depend on the origin of the epithelial cells.

Variable sperm size and motility activation in the pipefish, Syngnathus abaster; adaptations to paternal care or environmental plasticity?

Like seahorses, some of the closely-related pipefish species (Family Syngnathidae) incubate their eggs within a male brood pouch. This has contributed to considerable confusion about sperm transfer mechanisms to the eggs; some authors have reported that ejaculates are released directly into water before they reach the eggs, while others have suggested that eggs are fertilised using spermatozoa deposited directly into the brood pouch via an internal sperm duct. Here we present anatomical evidence from the freshwater pipefish, Syngnathus abaster, showing not only that direct sperm deposition into the pouch is impossible, but that spermatozoa must somehow travel a significant distance (>4 mm) outside the body of the male, to reach and fertilise eggs in the pouch. We have also used several putative sperm-activating solutions to identify the type of environment most conducive to sperm activation. Spermatozoa released from the testis were active for a brief period (<5 min) in water or 150 mm saline, but showed prolonged (>25 min) motility in ovarian fluid. This suggests that spermatozoa are released into a mixture of ovarian fluid and eggs while the male and female are in close contact. Our data also suggest that the fertilisation mechanism is highly efficient (sperm : egg ratio <200 : 1) even though this pipefish species produces dimorphic spermatozoa (with long and short flagellae). The shorter (<40 microm) morphotypes were not capable of motility activation, and are therefore probably incapable of fertilisation. If so, the sperm : egg ratio reported here would represent an overestimate.

Dynamics of sperm DNA fragmentation in domestic animals III. Ram.

From a biological viewpoint spermatozoa are ejaculated by the male and received into the female while maintaining roughly constant temperature, which in most mammals is below the temperature of the soma. When ejaculated spermatozoa are used for artificial reproductive purposes a temperature excursion episode is produced, because the spermatozoa are often stored as frozen or chilled samples and the biological temperature is only recovered after insemination. In this study we have analyzed the effects of cooling (to 15 degrees C) and freezing ram spermatozoa on the subsequent sperm DNA fragmentation index (sDFI) during a varying period of storage at 37 degrees C. The aim was to emulate in vivo processes that cooled or frozen-thawed spermatozoa experience after insemination. The study was performed using commercial semen samples derived from rams regularly used for reproductive purposes. Semen samples were studied after a cooling or cryopreservation episode followed by biological temperature recovery and incubation up to 48 h. The results indicated that when spermatozoa experience a severe (frozen) or mild (cooled) temperature excursion episode, major effects on sperm viability and DNA fragmentation are induced and cause the subsequent rapid decline of ram sperm quality. This effect could be detected just at the onset of the biological temperature recovery. Sperm DNA damage in cooled samples was observed after 5 h of incubation at 37 degrees C, while this time was reduced to less than 60 min in frozen-thaw samples. The dynamics of sDFI in different animals, analyzed under the same experimental conditions, was different from one sample to another, regardless of the method used for storage. Sperm viability was better preserved in cooled rather than in frozen samples. While for the frozen-thawed samples sperm viability was almost abolished after 5 h of incubation, a stable proportion of viable spermatozoa (ranging from 20% to 60%) was observed in the cooled samples at the corresponding time points. Finally, with respect to the prevalence of sDFI in ram, the level commonly found was lower than 5% at the onset of the experiment. However, sDFI was higher than 5% in 25% of the samples and in 15% of rams this index exceeded 10%.

One-sided ejaculation of echidna sperm bundles.

We report for the first time an unusual ejaculatory mechanism in the short-beaked echidna in which each side of the bilaterally symmetrical, rosettelike glans penis is used alternately, with the other being shut down. This is unparalleled in mammals but is reminiscent of the use of hemipenes in squamate reptiles, providing further reproductive evidence of a sauropsidian lineage in the Monotremata. Further, we describe the occurrence of motile sperm bundles in ejaculated echidna semen and provide scanning electron micrographs of their morphology. Sperm bundling appears to confer increased sperm motility, which may provide the potential for sperm competition between males.

Dilution of spermatozoa results in improved viability following a 24 h storage period but decreased acrosome integrity following cryopreservation.

This study was designed to investigate the physiological factors affecting the reduced viability of cryopreserved spermatozoa following dilution. Ninety-six ejaculates were collected from 13 bulls and diluted to 10 x 10(6) and 60 x 10(6) sperm/ml in a commercial long term extender (Eqcellsire; IMV) and in an egg yolk extender. Samples diluted in the egg yolk extender were frozen in 0.25 ml straws. Samples diluted in the Eqcellsire were stored at room temperature for 24 h and assessed for sperm cell viability using SYBR14 and PI (Molecular Probes) and osmotic resistance. Frozen samples were thawed and assessed for viability, osmotic resistance and acrosome intergrity. Acrosome integrity was measured using Mitotracker, PI and PNA-FITC (Molecular Probes). Spermatozoa diluted to 10 x 10(6) sperm/ml and stored at ambient temperature had a higher proportion of viable spermatozoa (P < 0.01) and were less susceptible to osmotic stress (P < 0.01) than sperm diluted to 60 x 10(6) sperm/ml. Following cryopreservation there was no concentration-related difference in the proportion of viable spermatozoa and their relative susceptibility to osmotic stress. Spermatozoa diluted to lower cell concentrations had a higher proportion of viable cells that were acrosome reacted (P < 0.001). It is suggested that the higher proportion of acrosome reacted cells may result from an increased proportion of cells in a capacitated-like state in the spermatozoa diluted to lower concentrations. A Spearmans ranked correlation demonstrates a relationship between individual bull spermatozoa following dilution or cryopreservation for viability (r2 = 0.98; P < 0.001) or osmotic resistance (r2 = 0.87; P < 0.001) suggesting a variation in these characteristics between bulls.

Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw.

There are no reports on the use of antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP) for the use of bull sperm cryopreservation despite studies in the ram, mouse and chimpanzee. The effect of freezing and thawing on bull sperm viability, osmotic resistance and acrosome integrity were observed following the addition of AFP1, AFPIII and AFGP at four concentrations (0.1, 1, 10 and 100 microg/ml). In a second part of the experiment, fluorescein was conjugated to the AFPs and AFGP and observations were made using fluorescence microscopy to determine whether binding occurred between the sperm cell membranes and the proteins. In the final part of the study the cryopreservation media were cooled in the presence of the AFPs and AFGPs at the four concentrations on a cryomicroscope to mimic similar cooling curves as those used in the presence of sperm. Following freeze-thaw, AFPI resulted in increased osmotic resistant cells at 0.1-10 microg/ml compared to the control (P<0.01). AFPI and AFPIII did bind to the sperm cells. There was no visual difference in ice structure between the control, AFPIII and AFGP but AFPI resulted in parallel crystals at 0.1, 1 and 10 microg/ml. We suggest that the increased osmotic resistance in the spermatozoa cryopreserved in AFPI is due to the cells orientating between the ice crystals, reducing mechanical stress to the cell membrane. Previous research has shown that osmotic resistance correlates with bull fertility, suggesting that bull spermatozoa cryopreserved in the presence of AFPI may have increased fertility in vivo.