In vitro-cultured bovine granulosa and oviductal cells secrete sperm motility-maintaining factor(s)

Since bovine cumulus oophorous and oviductual cell cultures are known to support and maintain frozen-thawed bovine sperm viability and motility for extended time periods, we investigated whether granulosa cell (GC)- and oviductual cell (OC)-conditioned media have similar effects. GC and OC were cultured for 3 days in TCM-199 medium supplemented with 10% fetal calf serum. At that time, the supernatant was discarded from GC and the monolayers were covered with Sp-TALP medium containing 6 mg/ml bovine serum albumin, while the OC were recovered by centrifugation and transferred to culture bottles containing Sp-TALP. Two days later, GC-conditioned and OC-conditioned Sp-TALP were recovered and dialyzed, and their retentates were lyophilized. Bovine follicular fluid (BFF) was also dialyzed, and its retentate was lyophilized. When sperm were incubated in GC- or OC-conditioned media, motility remained above 62% and 42% at 6 hr and 30 hr, respectively, and motility was higher than that of the control both at 6 hr (39%; P < 0.001) and at 30 hr (9%; P < 0.0001). Similarly, when sperm were incubated in the lyophilized retentates of GC- and OC-conditioned media and in BFF at a dose of 0.1, 0.5, or 1.0 mg/ml, the motility rates were higher both at 6 hr (P < 0.05) and at 30 hr (P < 0.01) compared to the control. The increase in motility was dose dependent; a 1.0 mg/ml dose improved (P < 0.05) motility compared to a 0.1 mg mg/ml dose. Heat treatment of the retentates of GC, OC, and BFF at 55°C for 30 min did not destroy their ability to support and maintain motility. However, heating at 100°C for 5 min destroyed their ability to support motility. Molecular sieving of retentates on Sephancryl S-300 yielded fractions that were highly effective (P < 0.01) in enhancing and maintaining motility compared to the other fractions. In conclusion, GC and OC secrete nondialyzable, heat-labile factor(s), which support and maintain sperm viability and motility for up to 30 hr. © 1994 Wiley-Liss, Inc.     

Annexins are candidate oviductal receptors for bovine sperm surface proteins and thus may serve to hold bovine sperm in the oviductal reservoir

The sperm of eutherian mammals are held in a storage reservoir in the caudal segment of the oviduct by binding to the mucosal epithelium. The reservoir serves to maintain the fertility of sperm during storage and to reduce the incidence of polyspermic fertilization. Bovine sperm bind to the epithelium via seminal vesicle secretory proteins in the bovine seminal plasma protein (BSP) family, namely, PDC109 (BSPA1/A2), BSPA3, and BSP30K, which coat the sperm head. Our objective was to identify the receptors for bull sperm on the oviductal epithelium. Proteins extracted from apical plasma membrane preparations of bovine oviductal epithelium were subjected to affinity purification using purified BSPs bound to corresponding antibodies conjugated to Protein A agarose beads. Oviductal protein bands of approximately 34 and 36 kDa were eluted by EGTA from the beads and identified by tandem mass spectrometry as annexins (ANXAs) 1, 2, 4, and 5. Subsequently, antibodies to each of the ANXAs were found to inhibit sperm binding to explants of oviductal epithelium. Anti-ANXA antibodies labeled the apical surfaces and cilia of the mucosal epithelium in sections of bovine oviduct. Western blots confirmed the presence of ANXAs in apical plasma membranes. Because fucose had been determined to be a critical component of the oviductal receptor, the ANXAs were immunoprecipitated from solubilized apical plasma membranes and were probed with Lotus tetragonolobus lectin to verify the presence of fucose. Thus, these ANXAs are strong candidates for the sperm receptors on bovine oviductal epithelium.     

Fertilizing capacity of bovine sperm may be maintained by binding of oviductal epithelial cells

The ability of the bovine oviduct to maintain the motility and fertilizing capacity of bovine sperm was investigated by incubating frozen-thawed sperm with endosalpingeal epithelial cells cultured on either tissue culture plastic (nonpolarizing) or Matrigel-coated Millicell (polarizing) substrata. Sperm were also incubated in medium alone or with cultured bovine tracheal epithelial cells. Motility was determined at 6-h intervals over a 48-h period. The fertilizing capacity of sperm was evaluated after 0, 24, and 30 h of incubation by adding oocytes to the culture and determining the incidences of fertilization and polyspermy. Motility was maintained for 48 h in sperm that bound to endosalpingeal epithelial cells, but to a greater extent with polarized cells (38.4% motile) than with nonpolarized cells (0.8%). Fertilizing capacity was maintained for 30 h in sperm incubated with endosalpingeal epithelial cells on Matrigel/Millicell, but not in sperm incubated in medium alone or with tracheal cells. Only sperm incubated with oviductal cells developed hyperactivated motility. Scanning electron micrographs revealed that sperm were bound by the rostral portion of the intact acrosome to the apical surface of polarized endosalpingeal cells. These results suggest that the oviduct may not only store sperm but may also maintain sperm viability and fertilizing capacity during the preovulatory period.     

Sulfated glycoconjugates are powerful modulators of bovine sperm adhesion and release from the oviductal epithelium in vitro

The mechanisms of sperm adhesion and release within the mammalian oviduct are still poorly understood. In this in vitro study, a previously developed adhesion assay was used to analyze the effects of heparin, N-desulfated heparin, fucoidan, dextran sulfate, and dextran on bovine sperm-oviductal cell adhesion and release. Results showed that 1) all sulfated glycoconjugates were powerful inhibitors of sperm binding to oviductal monolayers in a dose-dependent manner, whereas N-desulfated heparin and dextran had no effect; 2) sperm pretreatment with heparin and fucoidan markedly inhibited adhesion; 3) treatment of oviductal monolayers with heparinase I, II, or sodium chlorate (an inhibitor of sulfation) had no effect on sperm adhesion; 4) sulfated glycoconjugates were also powerful and quick inducers of sperm release from oviductal monolayers; and 5) addition of sulfated glycoconjugates to the cocultures caused a sudden increase of bound-sperm flagellar beat frequencies, followed by a release of highly motile sperm. In conclusion, these data support the hypothesis that sulfated glycoconjugates may act as signals that induce sperm release and migration from the oviductal reservoir.     

Ability of sulfated glycoconjugates and disulfide-reductants to release bovine epididymal sperm bound to the oviductal epithelium in vitro

In Bos taurus, at ejaculation, epididymal sperm acquire a number of proteins secreted in the seminal plasma that increase their ability to interact with the female reproductive tract. Sperm-oviduct interaction comprises a transient sperm adhesion to the isthmus, the lower portion of the oviduct, followed by sperm release around ovulation. Oviductal fluid molecules, such as sulfated glycoconjugates and disulfide-reductants, are able to release bovine ejaculated sperm bound to the oviductal epithelium in vitro through the reduction of sperm surface protein disulfides to sulfhydryls. To understand whether the sperm molecules sensitive to releasing signals are already exposed on the surface of epididymal sperm, we studied the ability of cauda epididymal sperm to adhere to the oviductal epithelium and to be released by sulfated glycoconjugates and the disulfide-reductant penicillamine. Surface protein sulfhydryls in cauda epididymal sperm were analyzed in the initial suspension, in sperm bound to the in vitro-cultured oviductal epithelium, and in released sperm. Results showed that epididymal sperm are able to bind the oviductal epithelium in vitro, although at a lower extent than frozen-thawed ejaculated sperm; the interaction is mediated by oviductal cell microvilli that closely bind to the plasma membrane of the sperm head rostral region, as previously shown for ejaculated sperm. The sulfated glycoconjugates heparin, fucoidan, and dextran sulfate, as well as the disulfide-reductant penicillamine, are all powerful inducers of sperm release. The level of sulfhydryls in sperm surface proteins was (1) high in the initial sperm suspension; (2) low in bound sperm; (3) markedly increased in sperm released by heparin or by penicillamine. In conclusion, epididymal sperm are already able to bind the oviductal epithelium and to respond to the inducers of release through the reduction of sperm surface protein disulfides to sulfhydryls.     

Recombinant porcine zona pellucida glycoproteins expressed in Sf9 cells bind to bovine sperm but not to porcine sperm

The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal alpha-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.     

In situ detection and localization of lipid peroxidation in individual bovine sperm cells

Reactive oxygen species (ROS) have been implicated in many pathologies, including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries in order to perform artificial insemination. This freeze/thaw procedure is known to induce ROS in sperm samples. Lipid peroxidation in fresh and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous phospholipid class, phosphatidylcholine, and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in living sperm cells. Lipid peroxidation was particularly strong in the midpiece and tail of frozen/thawed spermatozoa and significantly less intense in the head. Induction of peroxidation in fresh sperm cells with the lipid soluble ROS tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze/thawing.     

CCHX zinc finger derivatives retain the ability to bind Zn(II) and mediate protein-DNA interactions

Classical (CCHH) zinc fingers are among the most common protein domains found in eukaryotes. They function as molecular recognition elements that mediate specific contact with DNA, RNA, or other proteins and are composed of a betabetaalpha fold surrounding a single zinc ion that is ligated by two cysteine and two histidine residues. In a number of variant zinc fingers, the final histidine is not conserved, and in other unrelated zinc binding domains, residues such as aspartate can function as zinc ligands. To test whether the final histidine is required for normal folding and the DNA-binding function of classical zinc fingers, we focused on finger 3 of basic Krüppel-like factor. The structure of this domain was determined using NMR spectroscopy and found to constitute a typical classical zinc finger. We generated a panel of substitution mutants at the final histidine in this finger and found that several of the mutants retained some ability to fold in the presence of zinc. Consistent with this result, we showed that mutation of the final histidine had only a modest effect on DNA binding in the context of the full three-finger DNA-binding domain of basic Krüppel-like factor. Further, the zinc binding ability of one of the point mutants was tested and found to be indistinguishable from the wild-type domain. These results suggest that the final zinc chelating histidine is not an essential feature of classical zinc fingers and have implications for zinc finger evolution, regulation, and the design of experiments testing the functional roles of these domains.     

Identification of ampullary and isthmic oviductal fluid proteins that associate with the bovine sperm membrane

The objective of this study was to define the complement of ampullary and isthmic oviductal fluid proteins that associate with the sperm membrane during the non-luteal and luteal stages of the oestrous cycle. Oviductal fluid was obtained from three dairy cows via indwelling cannulae in the ampulla and isthmus of the same oviduct. Daily samples of oviductal fluid were combined by region and stage of the cycle to create pools consisting of non-luteal isthmic, luteal isthmic, non-luteal ampullary, and luteal ampullary oviductal fluid. An aliquot from each oviductal fluid pool was biotinylated by incubation in biotin reagent. Spermatozoa from three fertile bulls were pooled, washed and incubated for 4 h in each of the four pools of biotinylated oviductal fluid. After incubation, sperm membrane proteins were solubilized, and proteins subjected to one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biotinylated oviductal fluid proteins that associated with the sperm membranes were detected by avidin–horseradish peroxidase and diaminobenzidine substrate. Eight oviductal fluid proteins with apparent molecular weights of 97, 75, 66, 55, 48, 34, 28 and 24 kDa were consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected.     

Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells

One-cell bovine embryos fertilized in vivo were cultured in TCM-199 and bovine oviductal epithelial cells, in TCM-199, or in explanted immature mouse oviducts supported by TCM-199 to compare development to the blastocyst stage. The morphological stage of development and cell number were determined following 144 hours of culture. Of the embryos that cleaved at least once, 52.6, 30.4 and 0.0% developed to the morula/blastocyst stage after culture in oviductal epithelial cells, in TCM-199 alone, or in explanted mouse oviducts, respectively. The mean total cell number for embryos cultured in oviductal epithelial cells (24.5) was higher than for embryos cultured in TCM-199 (12.8) or in explanted mouse oviducts (5.9; P<0.05). The mean cell number of embryos cultured in TCM-199 or in explanted mouse oviducts did not differ. The explanted immature mouse oviduct supported by TCM-199 did not provide an environment adequate for development of one-cell bovine embryos to the blastocyst stage. Development of one-cell bovine embryos was best supported by co-culture with oviductal epithelial cells in TCM-199 medium.     

In vitro methylation of bovine papillomavirus alters its ability to transform mouse cells.

Bovine papillomavirus (BPV) was methylated in vitro at either the 29 HpaII sites, the 27 HhaI sites, or both. Methylation of the HpaII sites reduced transformation by the virus two- to sixfold, while methylation at HhaI sites increased transformation two- to fourfold. DNA methylated at both HpaII and HhaI sites did not differ detectably from unmethylated DNA in its efficiency of transformation. These results indicate that specific methylation sites, rather than the absolute level of methylated cytosine residues, are important in determining the effects on transformation and that the negative effects of methylation at some sites can be compensated for by methylation at other sites. BPV molecules in cells transformed by methylated BPV DNA contained little or no methylation, indicating that the pattern of methylation was not faithfully retained in these extrachromosomally replicating molecules. Methylation at the HpaII sites (but not the HhaI sites) in the cloned BPV plasmid or in pBR322 also inhibited transformation of the plasmids into Escherichia coli HB101 cells.     

Heparin and Ca2+-free medium can enhance release of bull sperm attached to oviductal epithelial cell monolayers

The success of assisted reproductive techniques, such as IVF, could be enhanced by being able to select the most competent spermatozoa in a sample. Attachment and subsequent release of spermatozoa from oviductal epithelial cells (OEC) could provide populations of functionally superior spermatozoa for use in these protocols. The objective of the present study was to investigate the ability of heparin and Ca2+-free medium to induce spermatozoa release from bovine OEC. Epithelial cells were grown to confluence in 24-well plates and pooled frozen bull semen was added to a final concentration of 1 x 10(6) spermatozoa/well. Spermatozoa were allowed to bind to OEC for 2 h. Medium with unbound spermatozoa was removed and replaced by Sperm-TALP, only (control), with heparin (5, 10, or 15 IU/mL), or Ca2+-free with 2 mM EGTA. Treatments were left on sperm-OEC co-cultures for 0.5, 1, 2, 3, or 5 h. At each time, the media were recovered and spermatozoa from each treatment were counted and evaluated for acrosome integrity and motility. The total number of spermatozoa attached to OEC after 2 h of co-culture was considered 100%. Spermatozoa release is expressed as percentage of the total number of sperm cells bound to OEC after 2 h of co-culture. Data were analyzed by ANOVA and results are expressed as mean +/- SEM from three independent replicates. Beginning at 0.5 h, more sperm cells (P < 0.05) were released from OEC in the heparin groups (10 and 15 IU/mL, 77.3 +/- 6.2% and 84.0 +/- 6.2%, respectively) as compared to the control (46.4 +/- 6.2%). The Ca2+-free medium also induced spermatozoa release when compared with the control, but the effect was not significant until 3 h (38.2 +/- 1.9% vs 59.5 +/- 6.9%; P < 0.05). The percentage of acrosome reacted spermatozoa was not affected by heparin treatment. Heparin at 10 IU/mL increased (P < 0.05) the percentage of motile spermatozoa, whereas Ca2+-free medium caused the opposite effect at 0.5 h after addition of treatments. We conclude that both heparin and Ca2+-free medium are able to promote spermatozoa displacement from OEC attachment. Based on motility and acrosome status data, we predict that released sperm cells may be used for IVF and other assisted reproductive techniques.     

Effect of co-culture of porcine sperm and oocytes with porcine oviductal epithelial cells on in vitro fertilization

This study was designed to determine the effect of co-culture with porcine oviductal epithelial cell (POEC) monolayers on in vitro fertilization of pig oocytes. The in vitro penetrability of mature (experiment 1) or immature (experiment 2) oocytes was studied in presence or absence of POEC during IVF with fresh semen. In experiment 3, boar and POEC effects were analyzed but in this case with frozen-thawed spermatozoa. In experiment 4, the spermatozoa were pre-incubated before IVF with or without POEC in order to assess their effect on IVF sperm-related parameters. In experiment 5, the effect of POEC was studied by co-culturing them with oocytes before IVF to determine if monospermy was improved. The results showed that high sperm concentration and POEC increase oocyte penetrability (P<0.01) and decrease monospermy rate (P<0.01), in both mature and immature oocytes (P<0.01) with fresh semen and a 18 h culture time. With frozen semen was detected a boar and POEC effect (P<0.01) on penetration rate. The sperm pre-culture 2 h with POEC also resulted in an increase of sperm penetration in terms of number of sperm per oocyte (P<0.01) and this treatment did not increase monospermy when contact time between gametes was limited to 6 h although monospermy was higher when POEC were present during IVF. Finally, exposure of oocytes to POEC for 4 h before IVF facilitated monospermic penetration to over 70% (P<0.01). In conclusion, the use of POEC in porcine IVF systems provides the possibility of working with low sperm concentrations and the effect of POEC on monospermy depends on sperm concentration, boar and contact time between gametes. Moreover, the exposure of oocytes to POEC before IVF improves the rate of monospermy.     

Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm

Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. The aim of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro. In vitro matured bovine oocytes were inseminated with fresh semen stored in a caprogen-based diluent, with or without prior Percoll separation. Irrespective of sire, storage of fresh semen at ambient temperature for up to 7 d post collection had no effect on cleavage rate or blastocyst development after IVF. In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Higher numbers of fresh sperm migrated through artificial mucus on Day 0 (day of semen collection) compared with frozen-thawed sperm. On Day 1 and 2 postcollection there was no difference in the number of sperm migrating through the mucus, but storage of sperm at ambient temperature for longer than 2 d resulted in a significant decline in their ability to penetrate mucus compared with frozen sperm from the same ejaculate. In conclusion, bovine sperm retain the ability to fertilize oocytes in vitro for up to 7 d following storage at ambient temperature. However, the ability of sperm to migrate through artificial mucus in vitro is severely depressed after 2 d storage which may have significant implications for the ability of these sperm to reach the site of fertilization in vivo after AI.     

In vitro capacitation and fertilizing ability of ejaculated rabbit sperm treated with lysophosphatidylcholine

Four experiments were replicated 1) to establish dose-response relationships between lysophosphatidylcholine (LPC), sperm motility, and the acrosome reaction (AR), 2) to evaluate the influence of rabbit serum (RS) on these endpoints, 3) to compare buck differences in induction of the AR, and 4) to examine fertilizing ability in vitro of sperm tested under the first three objectives. Semen was collected from Dutch-belted rabbits, washed once by centrifugation, resuspended, and preincubated for 2 or 4 hr in a chemically defined medium (DM), DM plus 20% RS, or BSA-free DM plus 20% RS at 37°C. At the end of preincubation LPC was added to the preincubated sperm at concentrations of from 0 to 100 μg/ml. Sperm were examined .5–4 hr later for AR and sperm motility. For in vitro fertilization, sperm and ova were coincubated in DM up to 24 hr after insemination and in a more complex medium for another 24 hr. Addition of LPC to 4-hr-preincubated sperm was more effective for inducing the AR than addition to 2-hr-preincubated sperm. A significant increase (P < .05) in the AR occurred in 15 and 30 min following exposure to 100 and 80 μg of LPC per ml, respectively, but the higher concentration of LPC decreased sperm motility. Addition of 20% RS to DM with or without BSA surprisingly inhibited the AR but maintained sperm motility, as expected. Bucks differed (P < .05) in the initial percentage and the induced percentage of AR sperm. For the AR the optimal concentration of LPC per ml was 80 μg, but for in vitro fertilization 60 μg tended to be superior.     

Reactive oxygen species-mediated loss of bovine sperm motility in egg yolk Tris extender: protection by pyruvate,metal chelators and bovine liver or oviductal fluid catalase

Improvement of bovine semen cryopreservation requires a better understanding of the properties of the currently used extenders. At present, about half of the spermatozoa die or become immotile following cryopreservation. The implication of an oxidative stress during or following the process of cryopreservation has been suspected to alter sperm functions. However, insufficient information is available on the effect of oxidative stress on sperm functions in their surrounding environment, the extender, such as the one based on egg yolk, Tris and glycerol. In this study, we investigated the effects of hydrogen peroxide (H2O2) and superoxide anion (O2*-) on bovine sperm motility in a widely used egg yolk Tris glycerol (EYTG) extender in comparison to a reference medium, the Tyrode's albumen lactate pyruvate (TALP). Bovine sperm were incubated for 6 h with or without concentrations of H2O2 ranging from 12.5 microM to 1.25 mM and with the hypoxanthine/xanthine oxidase system (X/XOD) that generates O2*-. Sperm motility was established by computer assisted semen analysis (CASA) in four similar experiments using the same frozen pool of semen. We have found that sperm motility was reduced significantly by H2O2 concentrations 20-fold lower in EYTG than in TALP medium. The differential resistance of the two media was explained by pyruvate present in TALP that acts as an antioxidant and metals ions, chelated by ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DETAPAC), found in egg yolk that might react with H2O2. Addition of only 5 U/ml of bovine liver catalase or oviductal fluid catalase (OFC) were sufficient to overcome the loss of sperm motility caused by 100 microM H2O2 in both EYTG and TALP. However, OFC was the most effective of the two catalases in EYTG. In addition to maintain sperm motility, catalase (5 U/ml) and pyruvate (5 mM) increased the intracellular sperm ATP level in comparison to sperm incubated alone for 6 h at 38.5 degrees C in EYTG. Moreover, EDTA, pyruvate and catalase prevented sperm ATP loss in presence of 100 mM of H2O2 in EYTG. These results indicated that EYTG has a very limited capacity to neutralize H2O2, and the addition of low amounts of catalase and millimolar concentrations of pyruvate greatly improved the antioxidant properties of a commonly used extender.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.     

Development of in vitro matured and fertilized bovine embryos cocultured with bovine oviductal epithelial cells obtained from oviducts ipsilateral to cystic follicles.

The present experiment was conducted to clarify the effect of bovine oviductal epithelial cells (BOEC) collected from oviducts ipsilateral to cystic follicles (CFs) using an in vitro coculture system on the development of in vitro matured/fertilized (IVM/IVF) bovine embryos. In the first comparison, the effect of the presence of CF on the development of the embryos cocultured with BOEC derived from the cows with CF (n = 18) and corpus hemorrhagicum (CH, n = 10) was examined. In the second comparison, the effect of the type of cyst [progesterone (P4)-dominant; n = 9, estradiol-17beta (E2)-dominant; n = 5] on the development of the embryos cocultured with BOEC derived from the cystic cows was examined. No difference was observed between CF and CH (control) groups in the mean developmental rates of embryos developed to > or =2-cell (86.3% vs. 86.4%), 8-16 cells (53.0% vs. 56.2%), blastocyst (24.2% vs. 24.8%) and hatched blastocyst (12.0% vs. 14.6%). However, the blastocyst production rate was significantly different (P<0.05) between the P4-dominant (19.8%) and E2-dominant (32.6%) groups. The rate of development from cleavage stage embryo to blastocyst was significantly different between P4-dominant (22.9%) and E2-dominant (37.9%) groups. Moreover, the blastocyst rate from 8-16 cells of E2-dominant group (61.6%) was significantly higher than that of P4-dominant one (39.5%). These results indicate that the effects of BOEC collected from oviduct ipsilateral to CFs on embryo development are variable, and the variability is closely associated with the steroid hormone profiles of the follicular fluid.     

Effects of freeze-drying on cytology, ultrastructure, DNA fragmentation, and fertilizing ability of bovine sperm

Freeze-drying sperm is an alternative to cryopreservation. Although sperm from various species has been freeze-dried, there are few reports for bovine sperm. The primary objective of this study was to evaluate the protective effect of various freeze-drying media on the structural and functional components of bovine sperm. The media tested were composed of TCM 199 with Hanks salts supplemented with 10% fetal calf serum (FCS) and TCM 199 with Hanks salts supplemented with 10% FCS and 0.2 M trehalose and EGTA solution. The efficiency of each medium on the preservation of freeze-dried sperm structures was evaluated with conventional and electron microscopy, DNA integrity was analyzed by a TUNEL assay, and fertilizing ability of lyophilized sperm was determined with ICSI. Although the plasma membrane was damaged in all media tested, mitochondria were similarly preserved in all freeze-drying treatments. The acrosome was best preserved in the media that contained trehalose (other treatments also conserved this structure). In contrast, media containing EGTA or trehalose most effectively preserved the nuclei in freeze-dried sperm, with only 2 and 5%, respectively, of cells with fragmented DNA. Furthermore, sperm conserved with these media also had higher (P<0.05) rates of sperm head decondensation (32.5 and 27.5%), pronucleus formation (37.5 and 45.0%) and blastocyst formation (19.4 and 18.3%) than medium supplemented with FCS (15.0, 20.0 and 10.2%, respectively). In conclusion, media with EGTA and trehalose adequately protected bovine sperm during freeze-drying by preserving the viability of their nuclei.