Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold-shocked ram spermatozoa.

Capacitation of spermatozoa, a complex process occurring after sperm ejaculation, is required to produce fertilization of the oocyte in vivo and in vitro. Although this process results from a poorly understood series of morphological and molecular events, protein tyrosine phosphorylation has been associated with sperm capacitation in several mammalian species, but it still remains to be demonstrated in ram spermatozoa. Studies of capacitation in ram spermatozoa are of great interest, since several reports have suggested that the reduced fertility of cryopreserved spermatozoa is due to their premature capacitation. In this work, we report for the first time, to our knowledge, that tyrosine phosphorylation of ram sperm membrane proteins is related to the capacitation state of these cells. Capacitation induced tyrosine phosphorylation of some plasma membrane proteins of ram spermatozoa freed from seminal plasma by a dextran/swim-up procedure. It has also been proved that cold-shock induces protein tyrosine phosphorylation as well as a decrease in plasma membrane integrity. Addition of seminal plasma proteins prior to cold-shock not only improved sperm survival but also promoted a decrease in protein tyrosine phosphorylation.     

Effect of sex sorting on CTC staining, actin cytoskeleton and tyrosine phosphorylation in bull and boar spermatozoa

Sperm sorting is a useful technology that permits sex preselection. It presents some troubles because of low fertility after the process. The main aim of this work was to analyze the putative existence of capacitation-like changes in both boar and bull sperm subjected to sex sorting that could lead to a detriment of semen quality. The parameters used were CTC staining patterns, actin cytoskeleton organization and tyrosine phosphorylation patterns; the last two were determined by both western blotting and immunofluorescence. Sex sorted spermatozoa were compared with fresh, in vitro capacitated and in vitro acrosome reacted sperm. In both species, sex sorted sperm showed a CTC staining pattern similar to that observed after in vitro capacitation. The actin pattern distribution after sperm sorting also tended to be similar to that observed after in vitro capacitation, but this effect was more pronounced in bull than in boar spermatozoa. However, actin expression analysis through western blot did not show any change in either species. The tyrosine phosphorylation pattern in boar sperm was practically unaltered after the sex sorting process, but in bulls about 40% of spermatozoa had a staining pattern indicative of capacitation. Additionally, western blotting analysis evidenced some differences in the expression of protein tyrosine phosphorylation among fresh, capacitated, acrosome reacted and sex sorted sperm cells in both species. Our results indicate that not all the sex-sorted-related modifications of the studied parameters were similar to those occurring after “in vitro” capacitation, thus suggesting that sex sorting-induced alterations of sperm function and structure do not necessarily indicate the achievement of the capacitated status of sorted sperm.     

Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro

Both fertilization promoting peptide (FPP) and adenosine stimulate capacitation and inhibit spontaneous acrosome loss in epididymal mouse spermatozoa; these responses involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway. However, it was unclear whether these responses were restricted to the mouse or possibly common to many mammalian species. To address this question, the response of boar spermatozoa to FPP and/or adenosine was evaluated. FPP is found in nanomolar concentrations in seminal plasma of several mammals, but not the pig. When cultured in caffeine-containing Medium 199 for 2 hr, chlortetracycline fluorescence evaluation indicated that neither FPP nor adenosine stimulated boar sperm capacitation per se but did inhibit spontaneous acrosome loss. However, in caffeine-free medium, FPP and adenosine both stimulated capacitation and inhibited spontaneous acrosome loss, suggesting that boar spermatozoa have receptors for both FPP and adenosine. Gln-FPP, a competitive inhibitor of FPP in mouse spermatozoa, has recently been shown to inhibit mouse sperm responses to adenosine as well, suggesting that FPP receptors and adenosine receptors interact in some way. Used with boar spermatozoa, Gln-FPP also significantly inhibited responses to both FPP and adenosine. These responses suggest that mechanisms whereby FPP and adenosine can regulate sperm function, via AC/cAMP, are of considerable physiological significance. Mouse, human, and now boar spermatozoa have been shown to respond to FPP, suggesting that these mechanisms may be common to many mammalian species. We also suggest that the effects of FPP and adenosine could also be exploited to maximize monospermic fertilization in porcine in vitro fertilization.     

Improving the fertilizing ability of sex sorted boar spermatozoa

The sex sorting of spermatozoa by flow cytometry induces damage, since sperm cells are highly diluted, affecting their functionality and fertilizing ability. In this work it was investigated whether the concentration of sex sorted spermatozoa by the sedimentation method, rather than centrifugation, in combination with the presence of the seminal plasma protein PSP-I/PSP-II heterodimer may improve their fertilizing ability. Spermatozoa were sorted by flow cytometry and collected in BTS with 10% of seminal plasma (group C: control) or with 1.5mg/mL of PSP-I/PSP-II heterodimer (group H). Collected spermatozoa from each medium were split into two aliquots. One aliquot of each group was centrifuged (800 x g/5 min) just after sorting and stored 16-18 h at 17 degrees C (groups Cc and Hc) at 6 x 10(6)sperm/mL. The second aliquot was directly stored at 17 degrees C for 16-18 degrees C (group Cs and Hs). After storage the supernatant was discarded and the sedimented pellet adjusted to 6 x 10(6)sperm/mL. Membrane integrity, acrosome status and motility characteristics of spermatozoa from all groups were assessed. Post-weaning pre-ovulatory sows were inseminated by laparoscopy into the oviduct with 0.3 x 10(6) sex sorted spermatozoa to assess their ability to penetrate oocytes in vivo. Putative zygotes were collected 18 h after insemination by washing the oviduct. Penetration and monospermic rates were evaluated. After 16-18 h of storage, centrifuged spermatozoa collected with 10% seminal plasma or 1.5 mg/mL PSP-I/PSP-II heterodimer after sex sorting showed lower (p<0.05) percentages of membrane integrity, motility and fertilization than sedimented spermatozoa. Overall, the presence of 10% seminal plasma or PSP-I/PSP-II heterodimer did not affect the results. However, a positive effect of PSP-I/PSP-II heterodimer (p<0.05) was observed in sedimented spermatozoa. Hence, our results indicate that the sedimentation method in the presence of PSP-I/PSP-II heterodimer improves the in vivo fertilizing ability of sex sorted boar spermatozoa.     

Stimulating effect of pyroglutamylglutamylprolineamide,a prostatic,TRH-related tripeptide,on mouse sperm capacitation and fertilizing ability in vitro

Pyroglutamylglutamylprolineamide, a prostatic tripeptide with structural similarities to thyrotrophin-releasing hormone (TRH), has been found in the seminal plasma of several mammalian species, suggestive of a biological function relating to spermatozoa. Using chlortetracycline (CTC) fluorescence analysis and in vitro fertilization, we have obtained evidence that the tripeptide stimulates mouse sperm capacitation and fertilizing ability in vitro. The tripeptide at concentrations from 5–500 nM was added to sperm suspensions and cells were assessed with CTC after 40 min, insufficient time for complete capacitation by a majority of spermatozoa under standard conditions of incubation. Concentrations of 25 nM and higher significantly promoted capacitation, as evidenced by a decrease in the proportion of acrosome-intact F pattern spermatozoa, characteristic of uncapacitated cells, and an increase in the proportion of acrosome-intact B pattern spermatozoa, characteristic of capacitated cells. However, there was no significant stimulation of acrosomal exocytosis. These results suggested that peptide-treated cells would be more fertile than their untreated counterparts. This was confirmed using in vitro fertilization, where the presence of 100 nM peptide during sperm preincubation and gamete coincubation significantly stimulated fertilizing ability (peptide, 56.5% of oocytes fertilized; controls, 26.5%). Comparison of the prostatic tripeptide and TRH effects on capacitation revealed that TRH at a concentration of 250 nM was as effective as the prostatic tripeptide in promoting the F & B transition but was less effective or ineffective at lower concentrations. In vitro fertilization assessment of the two peptides, at 100 nM, revealed that only the prostatic tripeptide significantly stimulated fertility. Again, this was consistent with the CTC analyses. Because the prostatic tripeptide can stimulate sperm function in vitro, it is possible that it plays a similar role in vivo and promotes fertilizing ability of ejaculated spermatozoa. We therefore propose that this tripeptide be referred to as fertilization promoting peptide (FPP). © 1994 Wiley-Liss, Inc.     

Evidence for the autocrine induction of capacitation of mammalian spermatozoa

Mammalian spermatozoa require a maturational event after ejaculation that allows them to acquire the capacity for fertilization. This process, known as capacitation, occurs spontaneously in simple defined medium implicating a potential role of autocrine induction. This study shows that the ether phospholipid 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF) meets the criteria for an autocrine mediator of capacitation. Sperm released PAF after their dilution into capacitation medium and expressed a receptor for PAF on their membranes. PAF stimulated changes in the motility of sperm and enhanced fertilization in vitro. These actions were inhibited by a PAF receptor antagonist (UR-12519) and by extracellular recombinant PAF:acetylhydrolase (an enzyme that degrades PAF to a biologically inert form). Seminal plasma contained an acid-labile PAF:acetylhydrolase, whereas capacitation was inhibited by an acid-labile factor within seminal plasma, implicating this factor as a potential decapacitation factor within seminal plasma. Sperm from a PAF receptor knock-out mouse strain failed to express the receptor and displayed a significantly (p < 0.01) reduced rate of capacitation, as assessed by the spontaneous onset of the acrosome reaction in vitro. When used for in vitro fertilization, sperm from PAF receptor knock-out mice gave a significantly lower rate of fertilization (21.5%) than did wild-type sperm (66.7%). The study shows for the first time the operation of an autocrine loop that induces capacitation in sperm in vitro and shows that this loop acts in concert with other mediators of capacitation to promote efficient fertilization.     

Boar spermatozoa in the oviduct

In the pig, a functional tubal sperm reservoir (SR) is established before ovulation to ensure availability of suitable numbers of viable spermatozoa for fertilization. The boar's large ejaculate is split: most spermatozoa are delivered in a sperm-rich fraction (SRF) followed by a post-SRF fraction containing increasing amounts of the spermadhesin PSP-I/PSP-II-rich seminal vesicle secretion. This heterodimer acts as leukocyte chemoattractant both in vitro and in vivo, contributing to the phagocytosis of those spermatozoa not reaching the SR. Sequential ejaculate deposition of marked spermatozoa and SR screening showed that most spermatozoa in the SR arose from the fortuitous PSP-poor, first portion of the SRF fraction, escaping phagocytosis and replenishing the SR within 2-3 h. The SR-sperm numbers diminish gradually in relation to ovulation, spermatozoa being continuously redistributed toward the upper isthmus. In vitro, only uncapacitated spermatozoa bind to epithelial explants, suggesting that the SR influences sperm capacitation. In vivo, most viable spermatozoa--usually harbored in the deep furrows in the pre- or peri-ovulatory SR during spontaneous standing estrus--are uncapacitated, but capacitation significantly increases after ovulation. Pre-/peri-ovulatory SR spermatozoa promptly capacitate in vitro when exposed to the effector bicarbonate, an influence that can be reversed by co-incubation with SR fluid or its component hyaluronan. Fluid collected from the ampullar segment (rich in bicarbonate) induces capacitation in vitro. In conclusion, the lack of massive sperm capacitation in the SR and the diverse individual response to capacitation shown by tubal spermatozoa would relate both to the insurance of full sperm viability before ovulation and the presence of spermatozoa at different stages of capacitation in the upper oviduct, thus maximizing the chances of normal fertilization.     

Effect of cooling and seminal plasma on the capacitation status of fresh boar sperm as determined using chlortetracycline assay

Insemination of sows with frozen-thawed spermatozoa results in lower fertility, in part due to spermatozoa having undergone a capacitation-like reaction. The present study employed chlortetracycline (CTC) staining analysis to investigate the effect of adding 20% (v/v) boar seminal plasma (SP) to boar spermatozoa on the temporal progress of capacitation and the acrosome reaction in spermatozoa cooled to 5 degrees C or incubated at 39 degrees C. Based on CTC staining patterns, seminal plasma appeared to reverse capacitation in spermatozoa that had undergone capacitation while incubated at 39 degrees C in a capacitation-supporting medium from 59.7 to 36.6% capacitated (P<0.001). Similarly, the addition of SP to boar spermatozoa cooled to 5 degrees C resulted in both the prevention of the capacitation-like reaction, and the reversal of an established capacitation-like reaction from 63.3 to 34.2% capacitated (P<0.001). These observations indicated that some constituent(s) of boar SP both prevent spermatozoa from undergoing capacitation as well as reverse capacitation in spermatozoa that have already undergone the process.     

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21-39 for SMI and 50-61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2-10 and 23-40%, respectively). The incidences of sperm penetration were high (61-89% of inseminated oocytes) when the sperm were preincubated for 0-60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10-72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.     

Practical application of seminal plasma

Natural mating deposits a large number of sperm in a high volume of seminal plasma. Semen processed for AI has the seminal plasma greatly diluted, and processing of boar sperm for cryopreservation removes all seminal plasma. Boar sperm were evaluated for the impact of seminal plasma on the functional status of boar sperm. Seminal plasma prevented or reversed capacitation in frozen-thawed boar sperm. However, supplementing frozen-thawed semen doses with 10% seminal plasma did not significantly affect the number of sperm found in the utero-tubal sperm reservoir, nor did it affect farrowing rates.     

Arylsulfatases are present in seminal plasma of several domestic mammals.

Mammalian spermatozoa and seminal plasma both contain high levels of arylsulfatases (AS), enzymes that remove sulfate from sulfated glycoconjugates. In ejaculated semen of boars, 85% of AS was found in seminal plasma whereas only 13% was found in spermatozoa. A comparable distribution of AS between spermatozoa and seminal plasma was observed in other domestic mammals. The presence of AS in seminal plasma was not due to leakage from spermatozoa because sperm cells had intact acrosomes and plasma membranes after their separation from seminal plasma, and because 84% of the acrosomal marker enzyme hyaluronidase was retained in washed spermatozoa. Spermatozoa in boar semen diluted with Beltsville Thawing Solution (BTS) deteriorated faster during storage at 17 degrees C than spermatozoa stored in BTS without seminal plasma. This suggests that seminal plasma has a deleterious effect on mammalian spermatozoa. We propose that (1) sulfated glycoconjugates stabilize sperm plasma membranes; (2) AS present in seminal plasma contribute to the deterioration of spermatozoa by desulfating these glycoconjugates; and (3) AS present in seminal plasma could well play a role in sperm capacitation.     

Characterization of low Mr zona pellucida binding proteins from boar spermatozoa and seminal plasma.

A group of low Mr (16 kDa-23 kDa) glycoproteins on ejaculated boar spermatozoa have been shown to have high affinity for homologous zona pellucida glycoproteins (ZPGPs). These ZPGP binding proteins are derived from seminal plasma as shown by their absence from epididymal spermatozoa and their presence in seminal plasma as identified by N-terminal amino acid sequence analysis. They bind to ZPGPs by a polysulphate recognition mechanism similar to that found for proacrosin-ZPGP interactions. The haemagglutination activity of boar seminal plasma is also associated with these low Mr glycoproteins. It is suggested that they play a role in regulating the rate of sperm capacitation and survival in the female reproductive tract.     

Ganglioside GM1 mediates decapacitation effects of SVS2 on murine spermatozoa

Prior to fertilization, mammalian spermatozoa need to acquire fertilizing ability (capacitation) in the female reproductive tract. On the other hand, capacitated spermatozoa reversibly lose their capacitated state when treated with seminal plasma (decapacitation). Previously, we demonstrated that a mouse seminal plasma protein, SVS2, is a decapacitation factor and regulates sperm fertilizing ability in vivo. Here, we examined the mechanisms of regulation of fertilizing ability by SVS2. Capacitation appears to be mediated by dynamic changes in lipid rafts since release of the cholesterol components of lipid rafts in the sperm plasma membrane is indispensable for capacitation. When the ejaculated spermatozoa were stained with a cholera toxin subunit B (CTB) that preferably interacts with ganglioside GM1, another member of the lipid rafts, the staining pattern of the sperm was the same as the binding pattern of SVS2. Interestingly, SVS2 and CTB competitively bound to the sperm surface with each other, suggesting that the binding targets of both molecules are the same, that is, GM1. Molecular interaction studies by the overlay assay and the quartz crystal microbalance analysis revealed that SVS2 selectively interacts with GM1 rather than with other gangliosides. Furthermore, external addition of GM1 nullified SVS2-induced sperm decapacitation. Thus, ganglioside GM1 is a receptor of SVS2 and plays a crucial role in capacitation in vivo.     

Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family

A family of proteins designated BSP-A1, BSP-A2, BSP-A3, and BSP-30-kDa, collectively called BSP (bovine seminal plasma) proteins, constitute the major protein fraction of bull seminal plasma. BSP proteins can stimulate sperm capacitation by inducing cholesterol and phospholipid efflux from sperm. Boar seminal plasma contains one homologous protein of the BSP family, named pB1; however, its physiological role is still unknown. In the current study, we report a novel method to purify pB1 from boar seminal plasma by chondroitin sulfate B-affinity chromatography and reverse-phase-high performance liquid chromatography. We also studied the effect of pB1, BSP-A1/-A2, and whole boar seminal plasma on boar sperm capacitation. Boar epididymal sperm were washed, preincubated in noncapacitating medium containing pB1 (0, 2.5, 5, 10 or 20 microg/ml), BSP-A1/-A2 (0 or 20 microg/ml) proteins, or whole seminal plasma (0, 250, 500, or 1000 microg/ml), then washed and incubated in capacitating medium. Acrosomal integrity was assessed by chlortetracycline staining. The status of sperm capacitation was evaluated by the capacity of sperm to undergo the acrosome reaction initiated by the addition of the calcium ionophore, A23187. The pB1 and BSP-A1/-A2 proteins increased epididymal sperm capacitation as compared with control (sperm preincubated without proteins). This effect reached a maximum level at 10 microg/ml pB1 and at 20 microg/ml BSP-A1/-A2 (2.3- and 2.2-fold higher than control, respectively). Whole boar seminal plasma did not induce sperm capacitation. In addition, pB1 bound to boar epididymal sperm and was lost during capacitation. These results indicate that BSP proteins and their homologs in other species induce sperm capacitation in a similar way.     

Effects of exposure of epididymal boar spermatozoa to seminal plasma on the binding of zona pellucida proteins during in vitro capacitation

The purpose of the investigation was to determine whether seminal plasma plays a role in the increase during in vitro capacitation of the number of boar spermatozoa with enhanced binding of zona pellucida proteins. Ejaculated spermatozoa and spermatozoa collected from the caudae epididymides of boars were incubated at 39 degrees C in a Tyrode's IVF medium. During incubation, the zona binding ability of individual spermatozoa was assessed with fluorescein-conjugated solubilized zona pellucida proteins (FITC-sZP), using a flow cytometer. Propidium iodide (PI) was included to simultaneously monitor cell viability. During incubation of ejaculated spermatozoa, a percentage of the spermatozoa expressed enhanced binding of FITC-sZP. The percentage of viable spermatozoa with enhanced binding reached a maximum of 37% (S.D.=8, averaged over five boars) after 2-3 h. In epididymal sperm, a similar maximum was observed after incubation in vitro, but a longer time of incubation was needed (6 h). Also, the rate of cell death of epididymal sperm was much lower than that of ejaculated sperm. When epididymal spermatozoa was exposed to seminal plasma in vitro, the time needed to reach a maximal percentage of viable spermatozoa with enhanced FITC-sZP binding was similar to that in ejaculated semen. However, the rate of cell death was still much lower than in ejaculated sperm. We concluded that the binding sites on the sperm surface that are involved in the increased binding of zona proteins during incubation under IVF conditions were not derived from the seminal plasma. The cellular processes leading to the increased binding capacity were accelerated by exposure of the sperm to seminal plasma.     

Actin polymerization in boar spermatozoa: Fertilization is reduced with use of cytochalasin D

The aggregational state of actin in boar spermatozoa after capacitation and the acrosome reaction has been examined by several methods. In vitro fertilization (IVF) experiments were conducted in the presence and absence of cytochalasin D (CD) to evaluate the role of actin polymerization in the events of fertilization. The fertilizing capacity was very high in controls, but, when CD (an inhibitor of the polymerization of actin) was added to the capacitation medium, there was a marked decrease in the fertilizing capacity of the boar spermatozoa. There was a further decrease when CD was present during both capacitation and fertilization processes. In addition to the IVF tests, biochemical and immunoelectron microscopic methods were used to analyze the state of aggregation of actin in boar spermatozoa after capacitation, and the acrosome reaction. By immunoelectron microscopy with a phalloidin probe, there were no gold particles, indicating the presence of F-actin on boar sperm heads capacitated and acrosome-reacted in media containing CD. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis there were differences in NP-40 solubility, reflecting actin polymerization, between CD-treated and untreated sperm. These results suggest that actin polymerizes during capacitation and the acrosome reaction and that this polymerization is essential to the fertilization process. © 1993 Wiley-Liss, Inc.     

Influence of storage time on functional capacity of flow cytometrically sex-sorted boar spermatozoa

Sex-sorting of boar spermatozoa is an emerging biotechnology, still considered suboptimal owing to the slowness of the process, which requires long sorting periods to obtain an adequate number of spermatozoa to perform a non-surgical insemination. This period involves storage of sorted cells that could impair their functional capacity. Here, we have studied how the storage of sex-sorted boar spermatozoa affects their functional capacity. Sorted spermatozoa were assessed at various times (0, 2, 5h or 10h) during storage after sorting and compared with diluted and unsorted spermatozoa for sperm motility patterns, plasma membrane and acrosomal integrity and their ability to penetrate homologous IVM oocytes. Sex-sorted sperm motility and membrane integrity only decreased significantly (p<0.05) by the end of the storage period (10h) compared to unsorted spermatozoa. Sperm velocity, ALH and Dance increased significantly (p<0.05), immediately post-sorting, returning to unsorted sperm values during storage. Acrosome integrity was not seriously affected by the sorting process, but decreased (p<0.05) during storage after sorting. Sorted spermatozoa stored 2h after sorting did not differ from unsorted in penetration rates and numbers of spermatozoa per oocyte, reaching the highest (p<0.05) penetration rates and sperm numbers per oocyte, when co-cultured for 6 or more hours. Non-storage or storage for 5h or 10h negatively (p<0.05) affected sperm penetration ability. In conclusion, although flow cytometrically sex-sorted spermatozoa are able to maintain motility, viability and acrosomal integrity at optimal levels until 10h of storage after sorting, fertilizing ability is maintained only over shorter storage times (<5h).     

In vitro Capacitation of Ejaculated Rabbit Spermatozoa

Prior to fertilization mammalian spermatozoa undergo physiological changes in the female reproductive tract. These changes are collectively known as capacitation. In essence capacitation is a further differentiation that the sperm cell must acquire beyond the maturational changes that it undergoes in the epididymis. Although capacitation in vitro has been easily achieved in rodents, its accomplishment in the rabbit is inconsistent and difficult. We report here successful in vitro capacitation of ejaculated rabbit sperm, assessed by in vitro and in vivo fertilization of rabbit ova. Sperm were used from pooled ejaculates collected from bucks of proven fertility since sperm collected from individual bucks resulted in significant differences in fertilization levels. Conditions favoring in vitro capacitation were: (1) extended incubation time of 12 h, (2) addition of 20% heated rabbit serum to incubation medium, and (3) an atmosphere of 5% CO2, 8% O2, and 87% N2 during incubation. In vitro capacitation of sperm under these conditions resulted in 67% fertilization compared to 89% for control sperm capacitated for 13 h in the uterus.     

Importance of sperm-binding assays for fertility prognosis of porcine spermatozoa

There has been a considerable effort to establish correlations between the outcome of in vitro sperm-binding assays and the fertility achieved by individual males under conditions of commercial AI. During passage through the oviduct, a fertilizing spermatozoon has to bind to and interact with several targets. Generally, it is assumed that these interactions can be mimicked by in vitro binding assays. However, there is little evidence that assays based on zona binding, zona penetration, or IVF: (a) have been adequately validated; (b) provide data with a high degree of correlation to a boar of average fertility; (c) provide accurate predictions as to pregnancy rate and litter size from a given boar when used for commercial AI. This is due partly to the variability in measurements of pregnancy rate and litter size in a commercial setting and partly to the fact that sperm fertility is multifactorial. A recently developed in vitro test is based on the fact that spermatozoa bind in vivo to oviduct epithelium, creating a functional sperm reservoir, and that fertilization-competent spermatozoa are released in a time-dependent manner from these cells. Mating or insemination occurs usually hours before ovulation thus rendering such temporary sperm binding to the epithelial cells, a prerequisite for successful sperm-oocyte interaction. In vitro binding of porcine spermatozoa to explants derived from fresh oviduct epithelium may provide a useful test system to predict fertility, although detailed validation has not been published. The sperm-oviduct-binding assay tests for multifunctional characteristics of the plasma membrane and may be a valuable in vitro test to identify subfertile boars. We believe that boar subfertility might be indicated in vitro by reduced capacity of his spermatozoa to bind to oviductal cells and that this may provide information as to whether an adequate sperm reservoir will presumably be established in vivo from the sperm population that successfully has passed the barriers of the utero-tubal junction.