Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm–Egg Binding,Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus‐associated protein, osteopontin (OPN), lipocalin‐type prostaglandin D synthase (L‐PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg‐associated OPN and L‐PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre‐incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L‐PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 × 10⁵ frozen‐thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre‐treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L‐PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.     

Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre‐exposure of oocytes to Ricinus communis (RCA‐1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm–egg binding and fertilization. In vitro‐matured bovine oocytes were incubated (39°C, 5% CO₂ in air) for 2 h in the following treatments: (i) 500 μl of fertilization medium (FM); (ii) 250 μl of FM with 0.25 ml of non‐luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 μl of FM with 250 μl of NLAUTF and 4 μl of RCA‐1 lectin; (iv) 250 μl of FM with 250 μl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA‐1 lectin; (v) 500 μl of FM and RCA‐1 lectin. Following incubation, oocytes were washed, placed in FM with 2 μg heparin, and incubated with 1 × 10⁵ frozen–thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate‐orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean ± SEM) when oocytes were incubated in treatment 3 (59.0 ± 5.5) than in treatments 2 (46.4 ± 5.6), 4 (18.1 ± 5.4), 5 (33.4 ± 5.6) or 1 (32.5 ± 5.6). More oocytes were fertilized when incubated in treatment 3 (91% ± 3.0) than in 2 (84% ± 3.0), 4 (40% ± 3.0), 5 (77% ± 3.0) or 1 (76% ± 3.0). As in previous studies, this study suggests that RCA‐1 lectin enhances binding of UTF‐derived OPN to bovine oocytes, resulting in increased sperm–egg binding and fertilization in vitro and a possible role in fertilization.     

Interaction of Potential Porcine Sperm Ligands with the Oocyte Plasma Membrane

We previously identified 62, 39, 27 and 7 kDa porcine sperm plasma membrane proteins that demonstrated a predominant affinity for the porcine oocyte plasma membrane by Western ligand blotting. The current experiments were designed to further investigate the potential roles of these molecules in sperm–oocyte plasma membrane interaction. Abilities of these proteins to bind to the oocyte plasma membrane and to inhibit sperm–oocyte interaction were evaluated. Plasma membrane was isolated primarily from the head of ejaculated porcine sperm by nitrogen cavitation and density gradient centrifugation. Fractions containing the 62, 39, 27 and 7 kDa proteins were electroeluted from one dimensional SDS polyacrylamide gels, dialysed and proteins biotinylated. Following incubation with zona‐free porcine oocytes, bound protein was visualized with 20 μg TRITC‐avidin/ml using confocal microscopy. Fractions of the dialysed, electroeluted proteins were added to porcine in vitro fertilization assays. The 62, 39, 27 and 7 kDa proteins all demonstrated binding to the oocyte plasma membrane in contrast to a biotinylated control protein. Addition of unlabelled sperm plasma membrane proteins to the biotinylated protein visibly reduced binding. Addition of each of these protein fractions to in vitro fertilization assays reduced sperm interaction with the porcine oocyte plasma membrane in a concentration‐dependent manner. Binding of these sperm plasma membrane proteins to the oocyte plasma membrane and inhibition of fertilization are consistent with these proteins being involved in sperm–oocyte plasma membrane interaction.     

Function of the Cumulus Oophorus Before and During Mammalian Fertilization

Fertilization encompasses a series of different steps which have to be performed in a well‐orchestrated way to create a new individual. They include sperm capacitation, sperm binding and penetration of the zona pellucida, traversing the perivitelline space, binding and fusion with the oolemma, activation of the oocyte and decondensation of the sperm head to form the male pronucleus. In most mammalian species, cumulus cells surround the oocyte at the time of fertilization. Removal of the cumulus oophorus at this point of time often leads to a drop in fertilization rates. It is not yet known how cumulus cells interact with the oocyte or with spermatozoa to promote fertilization. There are different possibilities:

• 1 cumulus cells cause mechanical entrapment of spermatozoa and guide hyperactivated spermatozoa towards the oocyte, while preventing abnormal spermatozoa to enter the cumulus matrix;
• 2 cumulus cells create a micro‐environment for the spermatozoa which favours their capacitation and penetration into the oocyte;
• 3 cumulus cells prevent changes in the oocyte which are unfavourable for normal fertilization; these changes can be located in the zona pellucida or in the cytoplasm.

In this review, studies in several species are listed to prove the importance of these three cumulus cell functions and the current lines of research are highlighted. Moreover, different ways to improve in vitro fertilization of bovine cumulus‐denuded oocytes are discussed.     

Methyl‐β‐Cyclodextrin Improves Sperm Capacitation Status Assessed by Flow Cytometry Analysis and Zona Pellucida‐Binding Ability of Frozen/Thawed Bovine Spermatozoa

Mammalian sperm undergo a series of biochemical transformations in the female reproductive tract that are collectively known as capacitation. Cyclodextrins added to the sperm culture medium have been described to induce in vitro sperm capacitation, enabling its use in protein‐free media. However, the additive capacitating effect of methyl‐β‐cyclodextrin (MβCD) in the medium containing bovine serum albumin (BSA) is unknown in the bovine species. In this study, we evaluated the effects of incubating frozen–thawed bovine spermatozoa in a BSA‐containing medium supplemented with MβCD on different sperm quality and functional parameters. Sperm viability decreased with the addition of MβCD in a dose‐dependent manner (p < 0.05), and DNA damage could be observed but only with the highest concentration of MβCD. However, pre‐incubation of spermatozoa in MβCD‐supplemented medium improved the capacitation status as assessed by the increase in plasma membrane fluidity, intracellular calcium concentration, induced acrosome reactivity and zona pellucida (ZP)‐binding ability (p < 0.05). Thus, we conclude that MβCD supplementation is able to enhance the capacitation status of frozen–thawed bovine spermatozoa cultured in capacitation medium containing BSA and could result in a valid strategy for its application on artificial reproductive technologies such as in vitro fertilization or intracytoplasmic sperm injection.     

The involvement of N‐glycosylation of zona glycoproteins during meiotic maturation in sperm–zona pellucida interactions of porcine denuded oocytes

The present study was conducted to delineate whether N‐glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation and whether this N‐glycosylation played a role in sperm–ZP interactions of porcine cumulus denuded oocytes (DOs). After mechanical removal of cumulus cells from cumulus oocyte complexes (COCs), DOs were cultured for 44 h in in vitro maturation (IVM) culture. The experiments were carried out to determine the effects of tunicamycin, a specific N‐glycosylation inhibitor, for various intervals during IVM on sperm–ZP interactions in porcine DOs. The results determined that DOs could induce meiotic maturation, although the maturation rate of DOs was earlier than that of COCs. In addition, N‐glycosylation of ZP glycoproteins occurred during meiotic maturation and was crucial in sperm–ZP interactions, was responsible for sperm penetration, sperm binding to ZP and induction of acrosome reaction in ZP‐bound sperm. However, the inhibition of N‐glycosylation by tunicamycin during IVM did not influence ZP hardness and male pronuclear formation, indicating that this N‐glycosylation was involved in the initial stage of fertilization. We conclude that 24–44 h of N‐glycosylation of ZP glycoproteins during meiotic maturation was crucial in sperm penetration and sperm binding to ZP and the induction of acrosome reaction in sperm bound to ZP of porcine DOs.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

Boar Sperm Encapsulation Reduces In Vitro Polyspermy

A boar sperm encapsulation technology in barium alginate has been developed to enhance reproductive performances and spermatozoa preservation time; aim of this work was to evaluate the effect of in vitro sperm encapsulation on polyspermy as a function of storage time at 18°C. A total number of 40 in vitro fertilization (IVF) tests were performed using encapsulated or diluted spermatozoa (20 IVF each treatment). Overall, 1288 in vitro matured oocytes were fertilized with spermatozoa stored at 24, 48 or 72 h at 18°C for both treatments polyspermy and normospermy, and the non‐penetration rates were assessed by optical microscopy. Results indicate a significant reduction in risk of polyspermic oocytes when spermatozoa are preserved in barium alginate membranes (incidence risk ratio: 0.766 with respect to diluted); such enhancement could be explained by lesser damage of sperm membranes achieved by encapsulation technology.     

Prognostic Value of Various Spermatological Attributes as Predictors of Zona Binding and Zona Penetration of Buffalo (Bubalus bubalis) Semen

Twenty‐four ejaculates from six (four ejaculates each) Surti buffalo bulls aged 4–8 years were used to assess various attributes of spermatozoa influencing the zona‐binding and zona‐penetration tests. Ejaculates from each bulls were subjected to in vitro sperm‐‐zona binding and sperm‐‐zona penetration tests (four replicates per bull) using immature buffalo oocytes. The average number of spermatozoa bound per oocyte was 27.79 ± 5.90. The average number of spermatozoa penetrated per oocyte was 3.35 ± 0.64. The average number of zona‐bound and ‐penetrated spermatozoa differed significantly between animals. Significant difference (p < 0.05) was observed between the plasmalemma integrity as assessed by eosin‐‐nigrosin stain and hypo‐osmotic swelling (HOS) test. Furthermore, the percentage of cells positive for the HOS test, i.e. functional membrane integrity (51.25 ± 2.32) was significantly (p < 0.05) higher than hypo‐osmotic swelling‐Giemsa (HOS‐G) test, i.e. the subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (42.87 ± 4.56). The HOS test had significant correlations with plasmalemma integrity as measured by the vital stain, eosin‐‐nigrosin (r = 0.85, p < 0.05). The HOS‐G test also had significant correlation with plasmalemma integrity measured by vital stains such as eosin‐‐nigrosin (r = 0.90, p < 0.05) and fluorogenic stains [carboxyfluorescein diacetate (CFDA) and propidium iodide (PI); r = 0.92, p < 0.01] and HOS test (r = 0.93), acrosomal integrity (r = 0.86, p < 0.05) and mitochondrial membrane potential (r = 0.99, p < 0.01). The plasmalemma integrity (fluorogenic stain), functional membrane integrity (HOS test), subpopulation of spermatozoa positive for functional membrane and acrosomal integrities (HOS‐G test) and mitochondrial membrane potential had significant (p < 0.05) correlation with sperm zona binding and penetration. The present study indicates that these parameters could represent important determinants of sperm quality influencing zona binding and penetration.     

Effect of Protease Inhibitors on the Acrosome Reaction and Sperm‐Zona Pellucida Binding in Bovine Sperm

Acrosomal proteases participate in several events during fertilization process and are necessary during the acrosome reaction (AR) and sperm‐zona pellucida (ZP) binding process. In this study, the participation of sperm trypsin‐like, chymotrypsin‐like, and metalloprotease enzymes in the AR and ZP binding in cattle was investigated using protease inhibitors. Motile bovine sperm were obtained by a swim‐up method (4 × 10⁶ cells / ml) in Brackett and Oliphant medium. The sperm were capacitated and then incubated with Antithrombin III (trypsin and chymotrypsin inhibitor); N‐α‐p‐tosyl‐l ‐lysine‐chloromethyl‐ketone (trypsin inhibitor); Trypsin inhibitor (I‐S Type from soybean); N‐tosyl‐l ‐phenylalanine‐chloromethyl‐ketone (chymotrypsin inhibitor); or disodium salt from the hydrated ethylenediaminetetraacetic acid (metalloprotease inhibitor). Then, the AR was induced with lysophosphatidylcholine and evaluated with the double stain technique. Sperm‐zona binding capacity was evaluated using cumulus cell‐free oocytes. A significant decrease (p < 0.05) in the percent of true acrosome‐reacted sperm was observed only in cells incubated with trypsin (10.2 ± 1%) and chymotrypsin inhibitors (18.5 ± 1%) in relation to the control (52.2 ± 1%). Treatment with the metalloprotease inhibitor did not affect the AR percentage (51.8 ± 1%). On the contrary, there was no significant change in the number of sperm bound to the ZP with any of the inhibitors used. The results suggest a role for trypsin and chymotrypsin proteases, but not metalloproteases, in the AR in bovine sperm. In addition, these proteases do not seem to be involved in the binding of bovine sperm to the ZP.     

Evaluation of Zona Pellucida Function for Sperm Penetration During In Vitro Fertilization in Pigs

In porcine oocytes, the function of the zona pellucida (ZP) with regard to sperm penetration or prevention of polyspermy is not well understood. In the present study, we investigated the effects of the ZP on sperm penetration during in vitro fertilization (IVF). We collected in vitro-matured oocytes with a first polar body (ZP+ oocytes). Some of them were freed from the ZP (ZP− oocytes) by two treatments (pronase and mechanical pipetting), and the effects of these treatments on sperm penetration parameters (sperm penetration rate and numbers of penetrated sperm per oocyte) were evaluated. There was no evident difference in the parameters between the two groups. Secondly, we compared the sperm penetration parameters of ZP+ and ZP− oocytes using frozen-thawed epididymal spermatozoa from four boars. Sperm penetration into ZP+ oocytes was found to be accelerated relative to ZP− oocytes. Thirdly, we evaluated the sperm penetration of ZP+ and ZP− oocytes at 1−10 h after IVF (3 h gamete co-incubation). The proportions of oocytes penetrated by sperm increased significantly with time in both groups; however, the number of penetrated sperm per oocyte did not increase in ZP− oocytes. Finally, we performed IVF using ZP− oocytes divided into control (3 h) and prolonged gamete co-incubation (5 h) groups. Greater numbers of sperm penetrated in the 5 h group than in the control group. These results suggest that the ZP and oolemma are not competent factors for prevention of polyspermy in our present porcine IVF system. However, it appears that ZP removal is one of the possibilities for reducing polyspermic penetration in vitro in pigs.     

Effect of Ca Ionophore On Blastocyst Production Following Intracytoplasmic Sperm Injection in Caprine Oocytes

The aim of the present investigation was to study the effect of calcium ionophore activation on blastocyst production following intracytoplasmic sperm injection (ICSI) in in vitro‐matured Caprine oocytes. A total of 470 in vitro‐matured oocytes were selected and randomly divided in to three groups. Cumulus oocyte complexes (COCs) recovered by slicing the Caprine ovaries were matured in TCM199 supplemented with 10% foetal bovine serum (FBS) + 10% follicular fluid + FSH (5 μg/ml) + LH (10 μg/ml) + estradiol (1 μg/ml) + EGF (10 ng/ml) + BSA (3 mg/ml) for 27 h in humidified atmosphere at 38.5°C with 5% CO₂ in CO₂ incubator. After 27 h of culture, selected COCs (n = 470) were separated from cumulus cells by treating with 0.1% hyaluronidase enzyme and passing repeatedly through a fine pipette and randomly divided into three groups. In group 1, (n = 168) matured oocytes were injected with injection micropipette without sperm as control. In group 2, (n = 152) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette. In group 3, (n = 150) capacitated spermatozoa were injected into cytoplasm of in vitro‐matured oocytes through injection micropipette and then activated with 5 μm Ca ionophore for 5 min. The oocytes of all groups were then culture in RVCL media for embryo development. The cleavage rate was observed after 48–72 h of injection. The cleavage rate and blastocyst production in group 1, 2 and 3 were 0.00 and 0.00, 18.42 and 3.57 and 61.33% and 16.30%, respectively. The result indicated that mechanical activation failed to induce cleavage in in vitro‐matured Caprine oocytes, whereas chemical activation of intracytoplasmic sperm‐injected in vitro‐matured Caprine oocytes showed significantly higher cleavage rate and blastocyst production as compare to non‐activated oocytes.     

Effects of Melatonin Implants During Non‐Breeding Season on Sperm Motility and Reproductive Parameters in Rasa Aragonesa Rams

The effect of melatonin implants administered during non‐breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer‐assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46–75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona‐pellucida binding assays, using spermatozoa from experiment 1, obtained 60–70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen‐thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non‐breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46–60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non‐breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained.     

In vitro production of porcine zygotes using intracytoplasmic injection of vitrified sperm

The objectives of this study were to evaluate if vitrified porcine spermatozoa are able to maintain their capacity to produce zygotes in vitro using intracytoplasmic sperm injection (ICSI) and to evaluate the zygote development in two in vitro atmospheric conditions: 5% CO₂ and tri‐gas. A group of porcine oocytes maturated in vitro were injected with vitrified‐warmed sperm (treatment group) and another group, with sperm diluted and conserved at 17°C (control group). To evidence parthenogenetic activation, some oocytes were submitted to a Sham test. The injected oocytes were cultured in G1 medium at 38°C, 100% humidity and 5% CO₂ or tri‐gas. No significant differences (p > .05) were observed in embryo development between the oocytes injected with vitrified‐warmed sperm (31.8%; 36/113), and those injected with semen diluted and conserved at 17°C (35.5%; 32/90), when cultured in 5% CO₂ or under tri‐gas atmosphere (42.9%; 39/91 vs. 34.2%; 26/76, respectively). No significant differences (p > .05) were observed in the percentage of pronuclei (PN) obtained between 5% CO₂ and tri‐gas, within each treatment either. Of the 52 oocytes submitted to the Sham test, only two presented a female PN (activation) indicating that the PN observed in the treatment group were a product of fertilization and not parthenogenetic activation. To conclude, porcine sperm vitrified using spheres, at a concentration of 5 × 10⁶ spermatozoa/ml in TALP medium with 1% bovine serum albumin (BSA), conserve condensed and intact chromatin capable of producing early embryo development up to the pronuclear stage.     

Heterologous In Vitro Fertility Evaluation of Cryopreserved Iberian Red Deer Epididymal Spermatozoa with Zona-intact Sheep Oocytes and its Relationship with the Characteristics of Thawed Spermatozoa

A heterologous in vitro system, using zona‐intact sheep oocytes, was used to evaluate the relationship between sperm factors of Iberian red deer thawed epididymal sperm and the percentage of cleaved oocytes. Epididymal spermatozoa were recovered from six males, diluted with freezing extender and cryopreserved. After thawing sperm motility (SM) and acrosome and membrane integrities were evaluated. Again, these parameters were assessed after incubation in freezing extender at 37°C for 2 h. After cryopreservation the values for SM and acrosome and membrane integrities were high (～80, 80 and 70% respectively). However, these values significantly decreased after incubation (～59, 62 and 47% respectively). Red deer thawed epididymal sperm fertilized zona‐intact sheep oocytes, although the percentage of cleaved oocytes was low (～22%). No relationship was found between sperm parameters assessed after thawing and the percentage of cleaved oocytes. Likewise, any sperm parameter evaluated after incubation was assessed in relation to the percentage of cleaved oocytes. However, acrosome and membrane integrities were near to significance (p = 0.06 and p = 0.09 respectively). Then, we conducted a reduced model with these two variables and both were related to the percentage of cleaved oocytes (p = 0.02 and p = 0.04 respectively). Thus, acrosome and membrane integrities were related to the percentage of cleaved oocytes negatively and positively respectively. It was concluded that the classical parameters assessed in deer thawed sperm samples can be good predictors of the ability to fertilize zona‐intact sheep oocytes.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

Interaction of Intact Porcine Spermatozoa with Epithelial Cells and Neutrophilic Granulocytes during Uterine Passage

New insemination techniques allow a tremendous sperm reduction for successful artificial insemination (AI) if highly diluted semen is deposited in the tip of the uterine horn and close to the utero‐tubal junction. High sperm losses are known to occur during uterine passage and it was the general question whether specific binding mechanisms are involved. Upon arrival in the uterus, spermatozoa are confronted with mainly two different cell types: uterine epithelial cells (UEC) and neutrophilic granulocytes (polymorphonuclear neutrophil, PMN). As cell–sperm interactions can hardly be observed in vivo, an ex vivo system was established to study the interaction between spermatozoa and the UEC. Uterine segments (10 cm) from freshly slaughtered synchronized juvenile gilts were inseminated for 60 min at 38°C. Thereafter spermatozoa were recovered, counted flow cytometrically and examined for changes in viability and mitochondrial membrane potential (MMP). Significantly less spermatozoa with a functioning MMP and intact plasma membranes could be retrieved (55 ± 7%), while the number of damaged spermatozoa hardly changed (93 ± 12%), indicating retention of viable sperm cells in the uterine lumen. The interactions between porcine PMN and spermatozoa (motile, immotile, membrane‐damaged) were studied in coincubation assays in vitro. The binding of membrane‐damaged sperm cells to PMN was virtually non‐existent (3 ± 2%). Viable and motile spermatozoa attached to PMN without being phagocytosed within 60 min (45 ± 3%), whereas binding to sodium fluoride (NaF)‐immobilized spermatozoa was reduced to 20 ± 2%. The binding of viable sperm to PMN is most likely not lectin‐dependent; although both viable cell types were shown to express a broad range of different lectin‐binding sugar residues, none of the lectins tested was able to selectively block PMN‐sperm binding significantly. The results of the study suggest that viable spermatozoa are already subject to selective processes within the uterus before further selection is initiated at the utero‐tubal junction and in the oviductal isthmus.     

Effects of (−)‐Epigallocatechin Gallate on the Motility and Penetrability of Frozen–Thawed Boar Spermatozoa Incubated in the Fertilization Medium

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea (Camellia sinensis) and is known for its antioxidant effects. The objective of the present study was to examine the effects of EGCG during in vitro fertilization (IVF) on the sperm quality and penetrability into oocytes. In the first experiment, the effects of concentration and incubation period of EGCG on the motility and penetrability of spermatozoa were examined. When frozen–thawed spermatozoa were incubated in IVF medium supplemented with 0 (control), 1, 50 and 100 μm EGCG for 1, 3 and 5 h, supplementation with 50 and 100 μm EGCG improved motility of the spermatozoa (p < 0.05), but not viability, as compared with the control group. When frozen–thawed spermatozoa were co‐incubated with in vitro‐matured (IVM) oocytes in IVF medium supplemented with 50 and 100 μm EGCG for 5 h, supplementation of EGCG had positive effects on sperm penetration rates. In the second experiment, the effects of supplementation of EGCG in IVF medium on penetrability of sperm from different boars and development of fertilized oocytes were evaluated. When frozen–thawed spermatozoa from six boars were co‐incubated with IVM oocytes in IVF medium supplemented with 50 μm EGCG, the effect of EGCG on sperm penetration and development of oocytes after fertilization was found to vary with individual boar. Our results indicate that motility and penetrability of boar spermatozoa are improved by co‐incubation with 50 μm EGCG, but the effects vary with individual boars.     

Assessment of Different Sperm Quality Parameters to Predict in vitro Fertility of Bulls

Frozen‐thawed semen from six bulls with high (> 60%) and low (20–35%) in vitro fertility was used for studying the predictive value of simple sperm quality tests with respect to in vitro fertilization (IVF) outcome as assessed by pronucleus (PN) formation ability. Sperm quality parameters, such as sperm concentration, motility, progressive motility, live‐dead sperm ratio, morphology, membrane integrity, mitochondrial activity and acrosomal status were analysed using both conventional and automatic techniques at three time points during the IVF process, namely after sperm thawing, Percoll differential gradient centrifugation and IVF. Associations between the sperm quality parameters before and after IVF, and PN formation ability were assessed by using linear regression analyses. The percentages of motility, progressive motility and normal morphology determined after sperm thawing, and the percentage of live spermatozoa assessed after Percoll preparation by using nigrosin‐eosin (N‐E) staining showed a good correlation with PN formation ability, but the regression parameters were borderline not significant. These parameters formed the most reliable basis for predicting IVF outcome. After IVF, the percentage of live spermatozoa determined by using N‐E staining was the only sperm quality parameter showing a significant association with the PN formation ability of a given bull. This sperm quality test can be used as a non‐invasive method to estimate the PN formation ability of oocytes which are further cultured to assess embryonic development.     

A Scanning Electron Microscopy Study of Frozen / Thawed Dog Sperm During In Vitro Gamete Interaction

The aim of this work was to study the effects of cryopreservation on the binding and penetration of dog spermatozoa to the zona pellucida (ZP) by scanning electron microscopy (SEM). The sperm-rich fraction of six ejaculates from five dogs was divided into two aliquots and washed by centrifugation. One aliquot was processed as fresh control sample and the other aliquot frozen in Tris-fructose extender. Gamete interaction was assessed using in vitro matured bitch oocytes, which were co-incubated for up to 3 h. At hourly intervals after the start of co-incubation, in vitro fertilized (IVF) oocytes were processed by SEM. The results were analysed statistically using the anova test. Differences in binding and penetration of the spermatozoa to the ZP occurred; a lower proportion of oocytes with spermatozoa bound to ZP was observed using frozen sperm (p < 0.05) than with fresh sperm (61%, 57% and 53% vs 42%, 40% and 44% at 1, 2 and 3 h, respectively). The percentage of ZP penetration by fresh sperm was directly proportional to the time of co-incubation (9%, 25% and 34%; p < 0.05); in contrast, no differences were observed in the penetration rate with frozen-thawed sperm (21%, 17% and 21%). More acrosome reacted sperm were observed in frozen sperm than in fresh sperm on the surface of the ZP. The differences in the percentage of binding and penetration between fresh and frozen sperm during the co-culture could indicate that the time course of penetration is faster in frozen-thawed dog spermatozoa than in fresh sperm, but that fresh spermatozoa can penetrate more oocytes over a given period of time, which may be related to their reacted or non-reacted initial status.