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.

Segregated water observed in a putative fish embryo cryopreservative.

Development of new cryopreservation strategies has major potential in medicine and agriculture and is critical to the conservation of endangered species that currently cannot be preserved. A critical property of any potential cryopreservative solution is its ability to prevent cell-damaging ice formation during cooling and subsequent heating. This study focuses on the freezing behaviour of promising model cryoprotective solutions. We perform neutron scattering analysis, combined with computer modelling, of the water structure after quench cooling these solutions. It is found that water in this solution forms nano-clusters encapsulated by the surrounding matrix of cryoprotectant solute molecules. We posit that these small volumes inhibit ice formation, because water does not have space for the structural relaxation required to crystallize on the timescale of the cooling process.

Sperm selection in the female mammalian reproductive tract. Focus on the oviduct: Hypotheses, mechanisms, and new opportunities.

Research over the past 3 decades has caused a major shift in the way that the oviduct, or fallopian tube, is perceived. Previously, it was regarded as little more than the anatomic site for fertilization, where spermatozoa and oocytes meet as they travel in opposite directions. However, this view has been radically altered by the realization that both spermatozoa and oocytes elicit changes in the biochemical composition of oviductal fluid through the induction of novel gene expression. Moreover, it has also been shown that only a privileged sperm population, selected on the basis of multiple criteria, is permitted to enter the oviduct, where they are subjected to even more selection processes that control their motility and capacitation status, thus either inhibiting or facilitating their progress toward the oocyte. Even more recently, it has become apparent that the oviduct has some ability to differentiate the genetic signatures of X- and Y-bearing spermatozoa. Although how exactly this is achieved is unknown, it prompts us to speculate that the oviduct may also be capable of distinguishing other genetically encoded properties of individual spermatozoa and that there must ultimately be a huge payoff in terms of selective animal breeding.

Heat shock protein A8 stabilizes the bull sperm plasma membrane during cryopreservation: Effects of breed, protein concentration, and mode of use.

Heat shock protein A8 (HSPA8) is a highly conserved member of the Hsp70 family, which is expressed in oviductal cells, translocated into oviductal fluid, and becomes attached to the sperm surface during sperm transport. Previous research has shown that HSPA8 supports mammalian sperm viability during in vitro incubation at both 5 °C and body temperature. The present series of experiments was designed to explore the possibility that bovine recombinant HSPA8 might therefore protect bull spermatozoa during cryopreservation through its beneficial effects on the sperm plasma membrane. Soy-based cryopreservation media were used in these experiments. The effects of HSPA8 addition before freezing were examined at concentrations ranging from 0.2 to 6.4 µg/mL, whereas the effects of postthaw HSPA8 addition were tested between 0.2 and 12.8 µg/mL. When bull spermatozoa (from beef and dairy breeds) were frozen in the presence of HSPA8, beneficial but complex effects on postthaw viability were observed. Low HSPA8 concentrations (0.2 and 0.4 µg/mL) resulted in significantly reduced postthaw sperm viability, but concentrations above 0.8 µg/mL improved plasma membrane integrity. If HSPA8 was added to spermatozoa after thawing, outcomes were also biphasic and beneficial effects on viability were only seen if the HSPA8 concentration exceeded 3.2 µg/mL. Beneficial effects were significantly more apparent with beef rather than dairy breeds. When HSPA8 was used in combination with cholesterol-loaded cyclodextrin, spermatozoa from the beef breeds showed significantly lower apoptotic effects. This was not observed with the dairy breeds.

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 alternate day collection on semen quality of Asian elephants (Elephas maximus) with poor initial fresh semen quality.

In captivity, male Asian elephants often yield poor quality semen after transrectal manually assisted semen collection; however, the reasons for the disappointing semen quality are not clear. Here we test the hypothesis that accumulation of senescent spermatozoa is a contributory factor, and that semen quality can therefore be improved by more frequent ejaculation. To this end we investigated the effect of collecting semen five times on alternate days, after a long period of sexual rest, on semen quality in Asian elephants known to deliver poor semen during infrequent single collections. All eight bulls initially displayed a high incidence of detached sperm heads and low percentages of motile (close to 0%) spermatozoa. After semen collection on alternate days, the percentages of detached sperm heads, and head and mid-piece abnormalities, were reduced significantly (p<0.05). In particular, one bull showed markedly improved sperm motility (increased from 0% to 60%) and membrane integrity (increased from 5% to 75%). In addition, advancing age significantly (p<0.01) correlated with lower percentages of sperm with intact membranes and a higher frequency of detached sperm heads. In contrast to sperm accumulation problems in other species, a small ampullary diameter correlated significantly (p<0.05) with reduced semen quality.

Dietary fatty acid enrichment increases egg size and quality of yellow seahorse Hippocampus kuda.

Seahorses populations in the wild have been declining and to restore them a better knowledge of seahorse reproduction is required. This study examines the effect of dietary quality on seahorse fecundity and egg quality. Two different diets were tested with Hippocampus kuda females: frozen mysis (control) and frozen mysis enriched with a liposome spray containing essential fatty acids. Diets were given to females (two groups of five) over a seven week period. After this period, males (fed the control diet) and females were paired and the eggs dropped by the females were collected. Fatty acid profile were analysed and eggs were counted and measured. Results showed that females fed on enriched mysis had larger eggs and that these had a higher content of total polyunsaturated fatty acids. The size of the egg was especially affected in the first spawn, where egg size for females fed the enriched diet was significantly higher than the egg size from control females. This effect was reduced in the following spawning where no significant differences were found. Egg size is an important quality descriptor as seahorse juveniles originating from smaller eggs and/or eggs of poor quality will have less chances of overcoming adverse conditions in the wild and consequently have lower survival and growth rates. This study shows that enriching frozen mysis with polyunsaturated fatty acids increases egg size and egg quality of H. kuda.

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.

The addition of heat shock protein HSPA8 to cryoprotective media improves the survival of brown bear (Ursus arctos) spermatozoa during chilling and after cryopreservation.

The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 µg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 µg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 µg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 µg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 µg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and "apoptotic-like" changes).

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 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.

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 dynamics of sperm DNA stability in Asian elephant (Elephas maximus) spermatozoa before and after cryopreservation.

The purpose of this study was to investigate the occurrence of sperm DNA fragmentation in Asian elephant (Elephas maximus) spermatozoa at various processing stages before and after cryopreservation. Five semen samples from four elephants were assessed at four different stages during processing; after (1) collection and reextension in TEST-egg yolk; (2) cooling to 5 °C; (3) equilibration for 1 h with glycerol; (4) thawing. An experimental approach was adopted that allowed comparisons of DNA fragmentation rates developed after the various processing stages. For this, spermatozoa were incubated in TEST-yolk media at 37 °C for 0, 4, 8, 24 and 48 h, and sperm DNA fragmentation rates were estimated using an elephant-specific Halosperm procedure. Incubation at 37 °C induced a rapid increase in DNA fragmentation, and significant differences between males were observed. The overall rate of increase over 4 h was estimated at about 5% per hour, and no significant changes to this rate were observed at the different processing stages, even, including the post-thaw samples. As semen quality of the five ejaculates was relatively poor, the basic semen parameter data were compared with nine different samples collected 11 mo earlier to see whether the tested samples were atypical or representative of the population, As there was no significant difference between the two sets of samples, it is believed that the samples tested for DNA stability were not unusually sensitive. These results suggest that Asian elephant spermatozoa are more susceptible to DNA fragmentation than spermatozoa of other mammals.

Soy lecithin interferes with mitochondrial function in frozen-thawed ram spermatozoa.

Egg yolk and milk are the 2 major membrane cryoprotectants commonly used in freezing media for the long-term preservation of semen (alone or in combination with others). However, in recent years, there have been increasing arguments against the use of egg yolk or milk because of the risk of introducing diseases through the use of cryopreserved semen. In this study, we analyzed the protective effect of lecithin as an alternative to egg yolk for the cryopreservation of ram semen, using a range of functional markers for sperm viability, motility, apoptosis, and mitochondrial functionality analyses (mitochondrial inner membrane surface [MIMS], mitochondrial inner membrane potential [MIMP], and cell membrane potential) as methods of assessment in samples diluted in 3 different media: Tris-citrate-glucose as control and 2 media supplemented with soy lecithin or egg yolk. The results showed that lecithin was able to effectively protect certain sperm quality characteristics against freezing-induced damage. However, lecithin induced loss of mitochondrial membrane potential or mitochondrial loss that was not reflected by modifications in sperm motility in fresh semen. MIMS and MIMP values decreased in thawed lecithin-treated samples, concomitant with a lower (P < .05) percentage of total and progressively motile cells, compared with those in egg yolk-containing samples. Further incubation of thawed samples revealed changes in motility and mitochondrial functionality that otherwise would not have been detected. These results indicated that lecithin may have affected the inner mitochondrial membrane in frozenthawed spermatozoa and confirmed that sublethal damages that seriously affect sperm functionality, not detected by classic sperm quality analyses, can be evidenced by changes in the inner mitochondrial membrane surface. These findings strengthen the relationship between mitochondrial membrane potential and motility and show that the mitochondrial alterations induced by the cryopreservation process could be specific targets for the improvement of semen cryopreservation protocols.

Antioxidant combinations are no more beneficial than individual components in combating ram sperm oxidative stress during storage at 5°C.

The unfrozen storage of ram semen for 2-4 days is an important goal for the acceptance of artificial insemination in sheep breeding programmes. The objective was to investigate the benefits of antioxidant supplementation on the production of hydrogen peroxide (H(2)O(2)) by ram spermatozoa stored at 5°C over 3 days. Ejaculates from 9 rams were split between two defined diluents, INRA-96 and RSD-1, and cooled slowly to 5°C for storage. Four different additives (vitamin E phosphate 6-100 µmol/L, catalase 500 IU+superoxide dismutase 9-150 µmol/L, and glutathione peroxidase, 20 IU) were investigated both separately and in combination. The amount of H(2)O(2) generated was assessed by use of a 1-step fluorometric micro-plate assay. Sperm viability, acrosome integrity and membrane fluidity were assessed by flow cytometry. H(2)O(2) production in INRA-96- compared with RSD-1-diluted spermatozoa increased approximately 2-3-fold after 24h in storage at 5°C and then declined up to 72 h, while that in RSD-1 showed no change over 72 h; this had no effect on the sperm characteristics. Addition of antioxidants singly reduced H(2)O(2) production in INRA-96, regardless of concentration. Optimal concentrations were vitamin E phosphate 12.5 µmol/L, catalase 500 IU, superoxide dismutase 37 µmol/L, and glutathione peroxidase, 20 IU. In any combination, none was more effective than others. Viability was reduced but acrosomal integrity protected by the antioxidants in INRA-96 but not in RSD-1; membrane fluidity was not affected. Based on this study, there were no combinations more efficient at combating oxidative stress than any one alone.

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.

Effects of porcine pre-ovulatory oviductal fluid on boar sperm function.

Sperm storage within the oviductal isthmus prior to ovulation typically involves binding to oviductal epithelial cells, which are thought to modulate sperm functions including internal calcium concentration, membrane fluidity, and motility. Around the time of ovulation the spermatozoa are gradually released so that they eventually encounter the oocytes within the oviductal ampulla. Previous studies have shown that the oviductal epithelial cells selectively sequester high quality spermatozoa, but the role of oviductal fluid as a selective modulator of sperm function has been investigated to a lesser extent. Here we address the hypothesis that oviductal fluid is also likely to modulate sperm function. Using samples of porcine oviductal fluid collected in the follicular phase of the estrus cycle, we show that short exposure (20 min to 50 microg/mL of oviductal fluid proteins) to either of two separate proteins fractions (> or < 100 kDa) promotes boar sperm viability and acrosomal integrity, decreases sperm plasma membrane fluidity (measured using merocyanine S540), and increases zona binding and polyspermy during in vitro fertilization. Exposure to the lower molecular fraction significantly inhibited, but did not abolish, the bicarbonate-induced stimulation of motility. The results show that subpopulations of spermatozoa respond differentially to oviductal fluid, and suggest that exposure to oviductal fluid in vivo could exert a further level of functional sperm selection.

Sperm storage in the vertebrate female reproductive tract: how does it work so well?

The capacity for sperm storage within the female reproductive tract occurs widely across all groups of vertebrate species and is exceptionally well developed in some reptiles (maximum duration, 7 yr) and fish (maximum duration, >1 yr). Amphibians (most salamanders and one species of frog; duration approximately 5 mo), all birds examined to date and some bats, have also evolved the ability to store spermatozoa in the female reproductive tract. Although there are many reports on both the occurrence of female sperm storage and its adaptive benefits, few studies have been directed toward explaining the mechanisms involved. Phylogenetic evidence suggests that the capacity for sperm storage has evolved independently within different taxonomic groups, and it is by no means clear whether these groups have established similar or different mechanisms or whether simple and common principles have been exploited during evolution. If the process has indeed developed by the invention of numerous different and species-specific mechanisms, it is surprising that none have yet been elucidated by technologists wishing to improve the long-term storage of fresh semen. On the other hand, if there is a simple and common solution to the problem, readily accessed by diverse groups of species, it is equally logical to suppose that the mechanism should be easily discovered in the laboratory. While recognizing that studies on wild species are usually neither practically or ethically easy to undertake, it is clear that there is a huge and largely unexplored field to be investigated.