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Simple Summary: The in vitro culture of mammalian embryos lacks different embryotrophic factors that are present in the female reproductive tract and are essential for correcting embryonic development. It has been reported that supplementation with oviductal fluid (OF) and uterine fluid (UF) during the in vitro culture of mammalian embryos produced by IVF has a positive effect on their development and quality. We investigated the effect of OF–UF supplementation on the balance of reactive oxygen species (ROS), glutathione (GSH), and the development of cloned and parthenogenetic Ovis aries embryos. However, at concentrations of 1 and 2% OF–UF, the blastocyst rate decreased in both groups of embryos. At a concentration of 1% OF–UF, both intracellular ROS and GSH decreased in the blastocysts of parthenogenetic embryos but not in cloned embryos. In cloned sheep embryos, OF–UF had no impact on the embryonic development or redox balance. Somatic cell nuclear transfer (SCNT) has great potential for the replication of high-commercial-value animals, threatened wild species for conservation purposes, and transgenic animals for biomedical purposes. However, SCNT has a low success rate due to intrinsic factors of the technique itself, which leads to low rates of embryonic development and epigenetic alterations in cloned embryos. The objective of this study was to evaluate the effect of OF–UF on the intracellular concentrations of ROS and GSH and the development of cloned and parthenogenetic Ovis aries embryos. The results do not show a beneficial effect on the development of parthenogenetic and cloned embryos at concentrations of 0.5% OF–UF. Furthermore, at 1% OF–UF, an adverse effect was observed in cloned embryos at the blastocyst stage and 2% OF and UF in parthenogenetic embryos during the first divisions. Decreases in ROS and GSH levels were observed in the parthenogenetic blastocysts treated with 1% OF–UF, but not in the clones, in which a higher concentration of GSH and a similar concentration of ROS were observed. No effect of OF–UF was observed on embryonic development and redox balance in sheep embryos cloned via handmade cloning. [ABSTRACT FROM AUTHOR]

Context: Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims: To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods: poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results: poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P < 0.05). Conclusions: poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm–oocyte interaction. Implications: The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm–oocyte interaction. We examined the tiny structures called extracellular vesicles that come from the fluid in pig uterus in order to understand how these vesicles change during the reproductive cycle of female pigs. Those from the late stages of the cycle seemed to have a strong connection with sperm and a possible key role in fertility. These vesicles regulate sperm function and interaction with oocytes, and they could potentially be used in fertility treatments to improve the chances of successful fertilisation. Photograph by SM Toledo-Guardiola. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (SCNT) has great potential for the replication of high-commercial-value animals, threatened wild species for conservation purposes, and transgenic animals for biomedical purposes. However, SCNT has a low success rate due to intrinsic factors of the technique itself, which leads to low rates of embryonic development and epigenetic alterations in cloned embryos. The objective of this study was to evaluate the effect of OF–UF on the intracellular concentrations of ROS and GSH and the development of cloned and parthenogenetic Ovis aries embryos. The results do not show a beneficial effect on the development of parthenogenetic and cloned embryos at concentrations of 0.5% OF–UF. Furthermore, at 1% OF–UF, an adverse effect was observed in cloned embryos at the blastocyst stage and 2% OF and UF in parthenogenetic embryos during the first divisions. Decreases in ROS and GSH levels were observed in the parthenogenetic blastocysts treated with 1% OF–UF, but not in the clones, in which a higher concentration of GSH and a similar concentration of ROS were observed. No effect of OF–UF was observed on embryonic development and redox balance in sheep embryos cloned via handmade cloning.

The seminal plasma (SP) is the liquid component of semen that facilitates sperm transport through the female genital tract. SP modulates the activity of the ovary, oviductal environment and uterine function during the periovulatory and early pregnancy period. Extracellular vesicles (EVs) secreted in the oviduct (oEVs) and uterus (uEVs) have been shown to influence the expression of endometrial genes that regulate fertilization and early embryo development. In some species, semen is composed of well-separated fractions that vary in concentration of spermatozoa and SP composition and volume. This study aimed to investigate the impact of different accumulative fractions of the porcine ejaculate (F1, composed of the sperm-rich fraction, SRF; F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF) on oEVs and uEVs protein cargo. Six days after the onset of estrus, we determined the oEVs and uEVs size and protein concentration in pregnant sows by artificial insemination (AI-sows) and in non-inseminated sows as control (C-sows). We also identified the main proteins in oEVs and uEVs, in AI-F1, AI-F2, AI-F3, and C-sows. Our results indicated that although the size of EVs is similar between AI- and C-sows, the protein concentration of both oEVs and uEVs was significantly lower in AI-sows (p < 0.05). Proteomic analysis identified 38 unique proteins in oEVs from AI-sows, mainly involved in protein stabilization, glycolytic and carbohydrate processes. The uEVs from AI-sows showed the presence of 43 unique proteins, including already-known fertility-related proteins (EZR, HSPAA901, PDS). We also demonstrated that the protein composition of oEVs and uEVs differed depending on the seminal fraction(s) inseminated (F1, F2, or F3). In conclusion, we found specific protein cargo in oEVs and uEVs according to the type of semen fraction the sow was inseminated with and whose functions these specific EVs proteins are closely associated with reproductive processes.

Oviductal extracellular vesicles (oEVs) have been identified as important components of the oviductal fluid (OF) and have been pointed to as key modulators of gamete/embryo-maternal interactions. Here, we determined the functional impact of oEVs on embryo development and the embryonic transcriptome in porcine. Experiment 1 examined the effect of oEVs and OF on embryo development. In vitro-produced embryos were cultured with oEVs or OF for 2 or 7 days using an in vitro sequential system or without supplementation (control). Experiment 2 analyzed transcriptomic alterations of EV-treated embryos versus control and the oEVs RNA cargo by RNA-sequencing. Two days of EV treatment enhanced embryo development over time when compared to other treatments. Different RNA expression profiles between embryos treated with EVs for two or seven days and untreated controls were obtained, with 54 and 59 differentially expressed (DE) genes and six and seven DE miRNAs, respectively. In oEV RNA cargo, 12,998 RNAs and 163 miRNAs were identified. Integrative analyses pointed to specific oEV components that might act as modulators of the embryonic transcriptome, such as S100A11, ANXA2 or miR-21-5p. Overall, the findings suggested that oEVs could be a potential strategy to improve porcine IVP outcomes, particularly by using two days of EV treatment. [ABSTRACT FROM AUTHOR]

Diverse lines of evidence indicate that the mammalian oviduct makes important contributions to the complex process of reproduction other than being simply a conduit for the transport of gametes and embryos. The cumulative synthesis and transport of proteins secreted by oviductal secretory cells into the oviductal lumen create a microenvironment supporting important reproductive events, including sperm capacitation, fertilization, and early embryo development. Among the components that have been identified in the oviductal fluid is a family of glycosylated proteins known collectively as oviduct-specific glycoprotein (OVGP1) or oviductin. OVGP1 has been identified in several mammalian species, including humans. The present review summarizes the work carried out, in various mammalian species, by many research groups revealing the synthesis and secretion of OVGP1, its fate in the female reproductive tract upon secretion by the oviductal epithelium, and its role in modulating biological functions of gametes and embryos. The production and functions of recombinant human OVGP1 and recombinant OVGP1 of other mammalian species are also discussed. Some of the findings obtained with immunocytochemistry will be highlighted in the present review. It is hoped that the findings obtained from recent studies carried out with recombinant OVGP1 from various species will rekindle researchers' interest in pursuing further the role of the oviductal microenvironment, of which OVGP1 is a major component, in contributing to the successful occurrence of early reproductive events, and the potential use of OVGP1 in improving the current assisted reproductive technology in alleviating infertility. [ABSTRACT FROM AUTHOR]

The coordinated interaction between the developing embryo and the maternal reproductive tract is essential for the establishment and maintenance of pregnancy in mammals. An early cross-talk is established between the oviduct/uterus and the gametes and embryo. This dialogue will shape the microenvironment in which gamete transport, fertilisation, and early embryonic development occur. Due to the small size of the gametes and the early embryo relative to the volume of the oviductal and uterine lumina, collection of tissue and fluid adjacent to these cells is challenging in cattle. Thus, the combination of in vivo and in vitro models seems to be the most appropriate approach to better understand this fine dialogue. In this respect, the aim of this review is to summarise the recent findings in relation to gamete/embryo-maternal interaction during the pre-elongation period. [ABSTRACT FROM AUTHOR]

Summary: The present study was designed to evaluate the effect of the combination of oviduct fluid flush (OFF) and oviduct epithelial cells (OEC) in modulating the incidence of polyspermy in pigs. Therefore, for in vitro fertilization (IVF), oocyte and sperm were co-cultured in Tris-buffered medium (TBM) either supplemented with 10% OFF (OFFD group), or in the presence of a bovine OEC monolayer (OEC group), or the oocytes were exposed to OFF for 30 min before IVF (OFFB group), or in the presence of an OEC monolayer (OFFB + OEC group). Regardless of sperm concentration used (0.5, 1.5, and 4.5 × 105 cells/ml), supplementation of IVF medium with 10% OFF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P < 0.05) of the final output of the IVF system in terms of zygotes with two pronuclei (2PN) was observed in the OFFD group, compared with the control and OEC groups, at a sperm concentration of 4.5 × 105 cells/ml. At this concentration, OFFB improved the monospermy rate but decreased the penetration rate, resulting in low efficiency of monospermic zygotes production. Despite this, no major effect was observed in the developmental competence of the presumed zygotes up to the blastocyst stage. The combination of OFFB with OEC improved the penetration rate, while maintaining the high monospermic rate induced by OFFB. In conclusion, the combination of treatment of oocytes by diluted OFF 30 min before IVF, followed by IVF in the presence of OEC, improved monospermic zygote production without reducing the penetration rate, when the IVF medium was supplemented with heparin. [ABSTRACT FROM AUTHOR]

Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida in vitro. Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle (n = 4) or a recent ovulation (n = 4); the samples were kept at −80°C until analysis. After protein extraction and isobaric tags for relative and absolute quantification (iTRAQ) labeling, the samples were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The analysis of the spectra resulted in the identification of a total of 1,173 proteins present in pre-ovulatory and post-ovulatory samples; among these, 691 were unique for Equus caballus. Proteins from post-ovulatory oviductal fluid were compared with the proteins from pre-ovulatory oviductal fluid and were categorized as upregulated (positive log fold change) or downregulated (negative log fold change). Fifteen proteins were found to be downregulated in the post-ovulatory fluid and 156 were upregulated in the post-ovulatory OF compared to the pre-ovulatory fluid; among the upregulated proteins, 87 were included in the metabolism of proteins pathway. The identified proteins were related to sperm–oviduct interaction, fertilization, and metabolism, among others. Our data reveal consistent differences in the proteome of equine OF prior to and after ovulation, helping to increase our understanding in the factors that promote fertilization and early embryo development in horses.

Polyspermic fertilisation has been a continual problem in porcine in vitro fertilisation. Over the years, researchers have tried to resolve this persistent problem, but have not been entirely successful in their endeavour. This can be attributed to several factors. This review discusses the role of sperm capacitation in the in vitro fertilisation and the most recent studies on the capacitation media. This includes the results obtained by testing the effect of various compounds on sperm capacitation. Additionally, the importance of the oviductal fluid on the porcine oocytes prior to fertilisation is also discussed. This is of significance, as the experiments show that oviductal fluid is crucial for the pre-fertilisation zona pellucida hardening. Also, the significance of using high quality oocytes for the in vitro fertilisation is highlighted. Lastly, the different and latest techniques to prevent or reduce polyspermic fertilisation have been reviewed.

Oviductal extracellular vesicles (oEVs) have been identified as important components of the oviductal fluid (OF) and have been pointed to as key modulators of gamete/embryo-maternal interactions. Here, we determined the functional impact of oEVs on embryo development and the embryonic transcriptome in porcine. Experiment 1 examined the effect of oEVs and OF on embryo development. In vitro-produced embryos were cultured with oEVs or OF for 2 or 7 days using an in vitro sequential system or without supplementation (control). Experiment 2 analyzed transcriptomic alterations of EV-treated embryos versus control and the oEVs RNA cargo by RNA-sequencing. Two days of EV treatment enhanced embryo development over time when compared to other treatments. Different RNA expression profiles between embryos treated with EVs for two or seven days and untreated controls were obtained, with 54 and 59 differentially expressed (DE) genes and six and seven DE miRNAs, respectively. In oEV RNA cargo, 12,998 RNAs and 163 miRNAs were identified. Integrative analyses pointed to specific oEV components that might act as modulators of the embryonic transcriptome, such as S100A11, ANXA2 or miR-21-5p. Overall, the findings suggested that oEVs could be a potential strategy to improve porcine IVP outcomes, particularly by using two days of EV treatment.

Global warming is affecting biodiversity; however, the extent to which animal reproductive processes respond to predicted temperature increments remains largely unexplored. The thermal environment has a pronounced impact on metabolic rates of ectotherms; therefore, an interesting question to assess is whether temperature increase might affect specific reproductive mechanisms like sperm performance in ectotherms. Moreover, in many species, oviductal fluid (OF) is known to regulate and maintain sperm quality; however, the role of OF in relation to the effects of high temperature on sperm remains unclear. Our aim was to experimentally test the effect of increased temperature on sperm velocity, swimming path and percentage of motility in neutral conditions at ejaculation (without OF) and in female's reproductive tract fluid (with OF), in a social ectotherm lizard model, Tropidurus spinulosus, which has specific thermal requirements for reproduction. Our results suggest that a rising temperature associated with global warming (+4°C) affects negatively sperm dynamics and survival. However, OF ameliorated the harmful effects of high temperature. This is an important point, as this study is the first to have tested the role of OF in preserving sperm from a warmer pre-fertilization environment. These results contribute to our understanding of how thermal environment changes might affect post-copulatory reproductive mechanisms. This article has an associated First Person interview with the first author of the paper.

Early embryonic development occurs in the oviduct, where an ideal microenvironment is provided by the epithelial cells and by the oviductal fluid produced by these cells. The oviductal fluid contains small extracellular vesicles (sEVs), which through their contents, including microRNAs (miRNAs), can ensure proper cell communication between the mother and the embryo. However, little is known about the modulation of miRNAs within oviductal epithelial cells (OECs) and sEVs from the oviductal fluid in pregnant cows. In this study, we evaluate the miRNAs profile in sEVs from the oviductal flushing (OF-sEVs) and OECs from pregnant cows compared to non-pregnant, at 120 h after ovulation induction. In OF-sEVs, eight miRNAs (bta-miR-126-5p, bta-miR-129, bta-miR-140, bta-miR-188, bta-miR-219, bta-miR-345-3p, bta-miR-4523, and bta-miR-760-3p) were up-regulated in pregnant and one miRNA (bta-miR-331-5p) was up-regulated in non-pregnant cows. In OECs, six miRNAs (bta-miR-133b, bta-miR-205, bta-miR-584, bta-miR-551a, bta-miR-1193, and bta-miR-1225-3p) were up-regulated in non-pregnant and none was up-regulated in pregnant cows. Our results suggest that embryonic maternal communication mediated by sEVs initiates in the oviduct, and the passage of gametes and the embryo presence modulate miRNAs contents of sEVs and OECs. Furthermore, we demonstrated the transcriptional levels modulation of selected genes in OECs in pregnant cows. Therefore, the embryonic-maternal crosstalk potentially begins during early embryonic development in the oviduct through the modulation of miRNAs in OECs and sEVs in pregnant cows.

Background: The current results of in vitro reproduction techniques in pigs, such as in vitro fertilization (IVF) and embryo development, show high performance with both epididymal and ejaculated spermatozoa. However, the results using ejaculated spermatozoa are even better. Ejaculated spermatozoa are exposed to the secretions of the accessory seminal glands: the seminal plasma (SP). It has been reported that exposure of spermatozoa to reproductive fluids, such as SP or periovulatory oviductal fluid (pOF), modulates sperm functionality both in vivo and in vitro. But whether or not this modulating effect of pOF depends on the origin of the spermatozoa being epididymal or ejaculated, is still unknown. Objectives: To determine and compare the effect of pOF on epididymal and ejaculated sperm functionality. Material and methods: The effects of incubating spermatozoa from the epididymis and ejaculate with pOF in capacitating conditions were investigated by analyzing sperm motility, phosphorylation of protein kinase A substrates and proteins in tyrosine (pPKAs and pTyr, respectively), the interaction of the spermatozoa with the oocyte in IVF and intracytoplasmic sperm injection (ICSI), and, finally, the spermatozoa chromatin condensation status. Results: The pOF modified events related to capacitation in epididymal spermatozoa by decreasing motility, pPKAs and pTyr. In the interaction with the oocyte after sperm capacitation, pOF regulated the epididymal and ejaculated spermatozoa differently. While pOF decreased the number of spermatozoa bound to the zona pellucida (Spz/ZP) and increased oocyte activation after ICSI with epididymal spermatozoa, with the ejaculated spermatozoa, it decreased the mean number penetrating each oocyte (Spz/O). Additionally, pOF significantly increased the nuclear decondensation of the epididymal spermatozoa after the fertilization of the oocyte. Conclusion: The modulation of sperm functionality by pOF is conditioned by the origin of the spermatozoa. [ABSTRACT FROM AUTHOR]

The osmolality of mouse oviductal fluid ranges from about 300 mOsmol/kg in the ampulla 0–3 h post coitus (h p.c.) to more than 350 mOsmol/kg in the isthmus 34–36 h p.c. Thus, it has been surprising to find that development of one-cell and cleavage-stage mouse embryos arrests in vitro in media exceeding 300 mOsmol/kg, and they develop best in unphysiological, hypotonic media. The glycine concentration in oviductal fluid can, however, rescue development in hypertonic media, so physiological conditions in vivo and in vitro likely work together to foster embryo well-being. Glycine acts on one-cell and cleavage-stage mouse embryos through the glycine-gated chloride channel, GLRA4, and uptake via the glycine neurotransmitter transporter, GLYT1. Since these processes lead to further signaling in neurons, the presence and function of such signaling in preimplantation embryos also should be investigated. The more we know about the interactions of physiological processes and conditions in vivo , the better we would be able to reproduce them in vitro. Such improvements in assisted reproductive technology (ART) could improve patient outcomes for IVF and potentially help prevent unwanted developmental abnormalities in early embryos, which might include undesirable epigenetic DNA and histone modifications. These epigenetic modifications may lead to transgenerational adult disorders such as metabolic syndrome and related conditions. [ABSTRACT FROM AUTHOR]

The role of non-gametic components of the ejaculate (seminal fluid) in fertility and sperm competitiveness is now well established. Surprisingly, however, we know far less about female reproductive fluid (FRF) in the context of sexual selection, and insights into male-FRF interactions in the context of sperm competition have only recently emerged. Despite this limited knowledge, evidence from taxonomically diverse species has revealed insights into the effects of FRF on sperm traits that have previously been implicated in studies of sperm competition. Specifically, through the differential effects of FRF on a range of sperm traits, including chemoattraction and alterations in sperm velocity, FRF has been shown to exert positive phenotypic effects on the sperm of males that are preferred as mating partners, or those from the most compatible or genetically diverse males. Despite these tantalizing insights into the putative sexually selected functions of FRF, we largely lack a mechanistic understanding of these processes. Taken together, the evidence presented here highlights the likely ubiquity of FRF-regulated biases in fertilization success across a diverse range of taxa, thus potentially elevating the importance of FRF to other non-gametic components that have so far been studied largely in males. [ABSTRACT FROM AUTHOR]

This study investigated the effect of bovine oviductal fluid from late follicular (LF) and early luteal (EL) phases on bull sperm functionality under non-capacitating (NCAP) and capacitating (CAP) conditions. Frozen-thawed semen samples from five bulls were thawed and incubated (0, 1 or 2 h) in NCAP and CAP media supplemented with 1% bovine oviductal fluid (LF and EL groups) and in absence of fluid (C group). Motion parameters were assessed by CASA; sperm viability, acrosomal integrity and membrane lipid disorder parameters were evaluated by flow cytometry; and sperm DNA fragmentation was evaluated by the Comet assay. Finally, in vitro fertilization with sperm treated under CAP conditions was performed and further embryo culture results evaluated. In NCAP medium, addition of LF and EL fluid increased the total and progressive motility, and LF fluid improved the stability of sperm DNA. However, under CAP conditions addition of LF and EL fluid decreased some sperm motion parameters and some parameters of sperm DNA stability. Proportion of viable sperm cells with low lipid disorder was higher in NCAP than CAP medium and addition of LF fluid markedly increased the proportion of viable spermatozoa with high lipid disorder and acrosome alteration (spontaneous acrosome reaction). Under current conditions, incubation of bull sperm with oviductal fluid before insemination did not affect detrimentally the IVF results nor embryo development, being blastocyst rate similar between CAP-LF, CAP-EL and control groups. In conclusion, oviductal fluid positively influences sperm functionality and modulate in vitro capacitation. • Periovulatory bovine oviductal fluid modifies sperm functionality. • Oviductal fluid enhances sperm viability and ability to undergo capacitation and acrosome reaction. • In capacitating conditions, periovulatory fluid decreased sperm motion parameters. • Late follicular fluid provides higher DNA stability. [ABSTRACT FROM AUTHOR]

A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy (1H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF samples were lactate, myoinositol, creatine, alanine and carnitine. Only fumarate and glycine showed significant differences in their concentrations between PRE and PST OF samples, with higher concentrations in the PST samples. In a preliminary study, stallion spermatozoa (n = 3 ejaculates) were incubated with different concentrations of PST OF from one mare (0, 0.0625, 0.125, 0.25, 0.5 or 1%; v:v). After 4 h of sperm incubation, protein tyrosine phosphorylation (PY) by western blotting, sperm motility, and acrosomal status were evaluated. An increase of PY was observed in sperm from two stallions when treated with 0.0625% and 0.125% of OF; however no change in PY was noted in the other stallion. There were no effects of OF on spermatozoa motility or acrosome status. These results provide the first information on the metabolomics of equine OF at different stages of the estrus cycle, and present the possibility that OF may affect PY in stallion spermatozoa. • The metabolome of pre- and post-ovulatory equine oviductal fluid was analyzed. • Differences existed in the metabolome of equine pre- and post-ovulatory fluid. • Sperm capacitation was achieved following incubation with oviductal fluid. • Equine OF induced protein tyrosine phosphorylation in stallion spermatozoa. • OF did not induce hyperactivated motility or acrosome reaction. [ABSTRACT FROM AUTHOR]

N-Acylethanolamines (NAEs) are an important family of lipid-signaling molecules. Arachidonylethanolamide (anandamide) (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) are co-produced from similar phospholipid precursors when neurons are stimulated. AEA is an endogenous agonist (endocannabinoid) for cannabinoid receptors. It binds with higher affinity to type CB1 than to type CB2 cannabinoid receptors. PEA does not bind to CB1, while the hypothesis that it reacts with putative CB2-like receptors has been questioned. OEA does not activate currently known cannabinoid receptors, but it mimics the effects of AEA and cannabinoids in reducing the fertilizing capacity of sea urchin sperm. OEA and PEA also act as entourage compounds by inhibiting the hydrolysis of AEA by fatty acid amide hydrolase. Cannabinoid receptors and/or AEA are present in mammalian reproductive organs including the testis, epididymis, prostate, ovary, uterus, sperm, preimplantation embryo and placenta, as well as prostatic and mammary carcinomas. We now report that analysis by high-performance liquid chromatography/mass spectrometry (HPLC/MS) shows the presence of AEA, PEA, and OEA in human seminal plasma, mid-cycle oviductal fluid, follicular fluid, amniotic fluid, milk, and fluids from malignant ovarian cysts. Previous studies showed that AEA-signaling via cannabinoid receptors regulates capacitation and fertilizing potential of human sperm, early embryonic development and blastocyst implantation into the uterine mucosa of rodents, as well as proliferation of human mammary and prostatic carcinomas. Current results imply that NAEs also may modulate follicular maturation and ovulation, normal and pathological ovarian function, placental and fetal physiology, lactation, infant physiology, and behavior. Collectively, these findings suggest that NAEs in human reproductive fluids may help regulate multiple physiological and pathological processes in the reproductive system, and imply that exogenous cannabinoids delivered by marijuana smoke might impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adolescents and adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.

More suitable and efficient methods to protect gametes from external harmful effects during in vitro handling can be achieved by adding preovulatory porcine oviductal fluid (pOF) to in vitro culture media. The objective of this study was to assess the swim-up procedure’s suitability as a sperm selection method using a medium supplemented with 1mg/mL BSA, 1% preovulatory pOF (v/v), 1% v/v pOF plus 1mg/mL BSA, and 5mg/mL BSA. After selection, various sperm parameters were studied, such as sperm recovery rate, sperm morphology, motility (by CASA), vitality, acrosome status and intracellular calcium (by flow cytometry) and ability to penetrate oocytes in vitro. Around 2% of sperm were recovered after swim-up, and the replacement of BSA by pOF showed a beneficial reduction of motility parameters calcium concentration, resulting in an increased penetration rate. The combination of albumin and oviductal fluid in the medium did not improve the sperm parameters results, whereas a high concentration of BSA increased sperm morphological abnormalities, motility, and acrosome damage, with a reduction of calcium concentration and penetration rate. In conclusion, the replacement of albumin by preovulatory oviductal fluid in the swim-up sperm preparation method modifies boar sperm parameters and improves the in vitro penetration of oocytes.

Polyspermic fertilisation has been a continual problem in porcine in vitro fertilisation. Over the years, researchers have tried to resolve this persistent problem, but have not been entirely successful in their endeavour. This can be attributed to several factors. This review discusses the role of sperm capacitation in the in vitro fertilisation and the most recent studies on the capacitation media. This includes the results obtained by testing the effect of various compounds on sperm capacitation. Additionally, the importance of the oviductal fluid on the porcine oocytes prior to fertilisation is also discussed. This is of significance, as the experiments show that oviductal fluid is crucial for the pre-fertilisation zona pellucida hardening. Also, the significance of using high quality oocytes for the in vitro fertilisation is highlighted. Lastly, the different and latest techniques to prevent or reduce polyspermic fertilisation have been reviewed. [ABSTRACT FROM AUTHOR]

Abstract Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro. [ABSTRACT FROM AUTHOR]

The oviduct plays important roles in reproductive events: sperm reservoir formation, final gamete maturation, fertilization and early embryo development. It is well known that the oviductal environment affects gametes and embryos and, ultimately, the health of offspring, so that in vivo embryos are better in terms of morphology, cryotolerance, pregnancy rates or epigenetic profile than those obtained in vitro. The deciphering of embryo–maternal interaction in the oviduct may provide a better understanding of the embryo needs during the periconception period to improve reproductive efficiency. Here, we perform a comparative analysis among species of oviductal gene expression related to embryonic development during its journey through the oviduct, as described to date. Cross-talk communication between the oviduct environment and embryo will be studied by analyses of the secreted or exosomal proteins of the oviduct and the presence of receptors in the membrane of the embryo blastomeres. Finally, we review the data that are available to date on the expression and characterization of the most abundant protein in the oviduct, oviductin (OVGP1), highlighting its fundamental role in fertilization and embryonic development.

The seminal plasma (SP) is the liquid component of semen that facilitates sperm transport through the female genital tract. SP modulates the activity of the ovary, oviductal environment and uterine function during the periovulatory and early pregnancy period. Extracellular vesicles (EVs) secreted in the oviduct (oEVs) and uterus (uEVs) have been shown to influence the expression of endometrial genes that regulate fertilization and early embryo development. In some species, semen is composed of well-separated fractions that vary in concentration of spermatozoa and SP composition and volume. This study aimed to investigate the impact of different accumulative fractions of the porcine ejaculate (F1, composed of the sperm-rich fraction, SRF; F2, composed of F1 plus the intermediate fraction; F3, composed of F2 plus the post-SRF) on oEVs and uEVs protein cargo. Six days after the onset of estrus, we determined the oEVs and uEVs size and protein concentration in pregnant sows by artificial insemination (AI-sows) and in non-inseminated sows as control (C-sows). We also identified the main proteins in oEVs and uEVs, in AI-F1, AI-F2, AI-F3, and C-sows. Our results indicated that although the size of EVs is similar between AI- and C-sows, the protein concentration of both oEVs and uEVs was significantly lower in AI-sows ( p < 0.05). Proteomic analysis identified 38 unique proteins in oEVs from AI-sows, mainly involved in protein stabilization, glycolytic and carbohydrate processes. The uEVs from AI-sows showed the presence of 43 unique proteins, including already-known fertility-related proteins (EZR, HSPAA901, PDS). We also demonstrated that the protein composition of oEVs and uEVs differed depending on the seminal fraction(s) inseminated (F1, F2, or F3). In conclusion, we found specific protein cargo in oEVs and uEVs according to the type of semen fraction the sow was inseminated with and whose functions these specific EVs proteins are closely associated with reproductive processes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Toledo-Guardiola, Luongo, Abril-Parreño, Soriano-Úbeda and Matás.)

Recent studies have demonstrated that the oviductal environment plays an active role in modulating the epigenetic marks of the preimplantation embryo genome, but the molecular factors that mediate this epigenetic effect are unknown. Folate is a well-known epi-nutrient that can impact on cell epigenetic machinery during embryonic and fetal development. However, the study of this epi-nutrient in the oviduct is still limited. The present study was conducted to confirm the presence and physiological concentration of folate in bovine oviductal fluid (OF) and to determine if bovine oviduct epithelial cells (BOECs) are able to regulate the uptake of this micronutrient. Samples of OF from ipsi- and contralateral oviducts were collected at different stages of the estrous cycle and folate levels were determined using a competitive receptor binding immunoassay. In addition, gene expression of folate receptors ( FOLR1 , FOLR2 ) and transporters ( SLC19A1, SLC46A1 ) were analyzed in BOECs from ampulla and isthmus regions during different stages of the estrous cycle using RT-qPCR. In vitro culture assays were also performed to evaluate whether expression of these genes responds to hormonal stimulation. Our results demonstrated presence of folate in the OF, showing changes of its concentration in the ipsilateral oviduct during the estrous cycle and significantly lower levels at the postovulatory stage. Moreover, gene expression of folate receptors and transporters was detected in BOECs, showing regional and cycle-dependent changes. In particular, differential expression of FOLR1 mRNA was observed in BOECs from the isthmus region, reaching significantly higher levels during the postovulatory stage. Under in vitro culture conditions, gene expression of folate receptors and transporters was maintained in BOEC explants and a particular susceptibility to steroid hormone stimulation was observed. In conclusion, the present study confirms the presence of folate in the bovine oviduct and proves the existence of a fine-tuned regulation of the expression of its receptors and transporters, highlighting the importance to expand the knowledge about this epi-nutrient in the oviductal context. [ABSTRACT FROM AUTHOR]

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability to. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT. [ABSTRACT FROM AUTHOR]

Animal oviducts and human Fallopian tubes are a part of the female reproductive tract that hosts fertilization and pre-implantation development of the embryo. With an increasing understanding of roles of the oviduct at the cellular and molecular levels, current research signifies the importance of the oviduct on naturally conceived fertilization and pre-implantation embryo development. This review highlights the physiological conditions within the oviduct during fertilization, environmental regulation, oviductal fluid composition and its role in protecting embryos and supplying nutrients. Finally, the review compares different aspects of naturally occurring fertilization and assisted reproductive technology (ART)-achieved fertilization and embryo development, giving insight into potential areas for improvement in this technology. [ABSTRACT FROM AUTHOR]

Steroids play an important role in mammalian reproduction and early pregnancy. Although systemic changes in steroid concentrations have been well documented, it is not clear how these correlate with local steroid concentrations in the genital tract. We hypothesised that, in the horse, the preimplantation embryo may be subjected to high local steroid concentrations for several days. Therefore, we measured progesterone, 17-hydroxyprogesterone, 17b-oestradiol, testosterone and 17a-testosterone concentrations in equine oviductal tissue by ultra-HPLC coupled with tandem mass spectrometry, and progesterone, 17b-oestradiol, oestrone and testosterone concentrations in oviduct fluid by radioimmunoassay, with reference to cycle stage and side of ovulation. Progesterone concentrations were high in oviductal tissue and fluid ipsilateral to the ovulation side during dioestrus, whereas other steroid hormone concentrations were not influenced by the side of ovulation. These results suggest that the high ipsilateral progesterone concentration is caused by: (1) contributions from the follicular fluid in the oviduct and diffusion of follicular fluid steroids after ovulation; (2) local transfer of steroids via blood or lymph; (3) local synthesis of progesterone in the oviduct, as evidenced by the expression of steroidogenic enzymes; and (4) a paracrine contribution from follicular cells. These data provide a basis for the study of the importance of endocrine and paracrine signalling during early embryonic development in the horse. [ABSTRACT FROM AUTHOR]

The rat is widely used as a laboratory animal for research. In particular, genetically engineered rats are essential for production of animal models of several diseases. Although embryo manipulation techniques are needed to produce them, such technology for rat preimplantation embryos is not as advanced as it is for mouse embryos. One reason is that in vitro culture systems for preimplantation embryos are limited in rats. Therefore, we intended to develop a new culture system for rat preimplantation embryos focusing on supplementation of amino acids as nutrition to the culture media. First, we found that taurine, glycine, glutamate, and alanine were abundant in the oviductal fluid of Wistar rats. The profile of taurine and these three amino acids was unchanged during the estrous cycle and from Days 0 to 3 of pregnancy (Day 0; vaginal plug was confirmed). Second, we assessed the effect of phosphate and phenol red on the development of rat zygotes and confirmed that they caused two-cell block. Third, we examined the effect of changing the medium on zygote development because addition of amino acids into culture medium causes ammonium accumulation, which is detrimental to embryo development. Blastocyst formation was suppressed in cultures with no medium change (P = 0.004; decreased to approximately one-fourth of that with medium change). Fourth, we examined the effect of supplementation of these three amino acids and taurine to modified potassium simplex optimized medium (KSOM). The zygote development rates were increased by the three amino acids and taurine in a concentration-dependent manner at 48, 72, and 96 hours (P = 0.001, 0.005, and 0.009, respectively) in culture. Finally, we confirmed that blastocysts cultured in modified KSOM had the capacity to develop to full term after implantation. These results showed that not only the supply of nutrients but also removal of wastes and toxicants is important for culture of rat preimplantation embryos. [ABSTRACT FROM AUTHOR]

The present study aimed to examine the oviductal and uterine fluid concentrations of metabolites in normal and metabolic stressed ewes. The study used reproductive tracts collected from mature normal (control group) and metabolically stressed (post parturient and high protein diet) Bellary ewes ( Ovis aries ) after slaughter. The oviductal and uterine fluids collected were analyzed for metabolites which included glucose, triglycerides cholesterol, non-esterified fatty acids (NEFA), β-hydroxybutyrate (β-OHB), ammonia, urea, creatinine, uric acid and total protein. Significant changes were observed in the levels of glucose, total NEFA, β-OHB, ammonia and urea in ewes under stress. β-OHB, NEFA, total protein were significantly higher (P < 0.05)in oviductal fluid while β-OHB and NEFA were significantly higher (P < 0.05) in uterine fluid of metabolically stressed (post parturient) ewes compared to that of normal ewes. Total NEFA and β-OHB levels increased (P < 0.05) in post parturient stress while ammonia and urea levels increased in oviduct and uterine fluids following a high protein diet. The content of β-OHB, total NEFA, ammonia, urea and glucose in oviduct and uterine fluids may be considered as possible biomarkers for metabolically stressed ewes. Our result may provide some basic information regarding the composition of uterine and oviduct fluid in control group and in ewes with metabolic stress. [ABSTRACT FROM AUTHOR]

In vivo , the oviduct provides appropriate microenvironment conditions for monospermic fertilization and early embryo development. In addition, glycosaminoglycans such as heparin are present in the oviduct and have been shown to modulate the activity of oviduct-secreted proteins on the regulation of sperms parameters. Thus, the present study was designed to evaluate the effect of porcine oocytes exposure to oviduct fluid (OF) before in vitro fertilization (IVF; incubation of oocytes in OF for 30 minutes before IVF), during IVF (supplementation of IVF medium with 10% OF), and during IVF in combination with heparin (10% OF + 10-μg/mL heparin) on IVF parameters. Regardless of sperm concentration used (0.5, 1.5, or 4.5 × 10 5 cells/mL), exposure of oocytes to OF led to an increased (P < 0.05) monospermy rate, without alteration (P > 0.05) of the penetration rate in comparison with the control group. This resulted in a general increase (P < 0.05) in the final output of the IVF system in terms of zygotes with two pronuclei in OF-exposed groups: 56 ± 9% (OF before) and 60 ± 7% (10% OF during IVF), compared with control (21 ± 8%), when IVF was performed with 4.5 × 10 5 cells/mL. The combination of 10% OF with heparin during IVF induced a decrease (P < 0.05) of the penetration rate, with no effect (P > 0.05) on the monospermy rate in comparison with 10% OF alone. This resulted in a general reduction (P < 0.05) in the final output of the IVF system (%), which was 33 ± 6% and 52 ± 8%, for 10% OF + heparin and 10% OF, respectively. In conclusion, the OF, used in porcine IVF, exerted a beneficial effect on oocytes by reducing the incidence of polyspermy without decreasing the penetration rate. However, the association of the OF with heparin reduced the efficiency of monospermic zygotes' production. [ABSTRACT FROM AUTHOR]

Knowledge and assessment of the constituents of the oviductal fluid (OF) in camelids is necessary for a correct formulation of specific culture media for the development of reproductive biotechnology. This study is the first describing the biochemical composition and SDS–PAGE protein profile of alpaca oviductal fluid in non-pregnant animals and animals that have completed the first month and second month of gestation. Samples were also classified into oviducts that were ipsilateral or contralateral to the ovary with corpus luteum. No differences were found between both oviducts, whereas pregnant and non-pregnant females displayed significant differences in the biochemical composition and protein profile of the oviductal fluid. Relative albumin content was higher in non-pregnant females. Relative creatinine content in OF from females that have completed the second month of gestation was lower than non-pregnant females and females that have completed the first month of gestation. Ion Na + concentration was higher in OF from non-pregnant females when compared with pregnant ones. The protein profile of non-pregnant females showed five protein bands of 70, 42, 25, 24 and 19 kDa that were significantly more intense compared with pregnant animals. Bands were identified as moesin, actin cytoplasmic 2, hydroxypyruvate isomerase, ferritin light chain and peroxiredoxin-6 with MALDI/MS. Our results encourage more thorough future studies, in order to unravel the complex reproductive processes of the South American camelid oviduct. [ABSTRACT FROM AUTHOR]

The present study was performed to examine the histopathology, cytology, bacteriology and expression pattern of a targeted set of genes of cytokines in the oviduct of cows with inflammation (Experiment 1). In addition, the effects of oviductal fluid from cows with salpingitis on the oocyte maturation and fertilization in vitro were examined (Experiment 2). The most frequent bacterial co-infection was Escherichia coli and Fusobacterium necrophorum , which was always associated with severe histopathologic salpingitis. Out of 15 cows with histologically healthy uterus, only one cow (6.7%) displayed the histologic signs of mild salpingitis, whereas from 50 cows with endometritis, 48 cows (96%) showed histologically different grades of salpingitis. The mRNA expression of IL1β, CD14, IL8 and CASP3 was significantly different among all groups of salpingitis (P < 0.05) with the highest level of mRNA expression in the sever grade of salpingitis. Results of experiment 2 showed a significant decline in the oocytes with peripheral free mitochondria and fertilization rate in the salpingitis group than the no- salpingitis group (P < 0.05). In conclusion, our results showed that histologically detected salpingitis is in most cases associated with histologic and cytologic endometritis. The pattern of the gene expression of chemokines and cytokines was altered in association with different grades of salpingitis. Further, we observed a decline in the peripherally located mitochondria and lower fertilization rate in oocytes following addition of oviductal fluid collected from the cows with sapingitis to the maturation media. • Histologically detected bovine salpingitis is associated with histologic and cytologic endometritis. • The pattern of gene expression of IL1β , CD14 , IL8 and CASP3 was altered in association with different grades of salpingitis. • Addition of oviductal fluid from cows with sapingitis to the maturation media altered the distribution of mitochondria. [ABSTRACT FROM AUTHOR]

This study identified microRNAs (miRNAs) in bovine oviductal fluids (OFs) and examined the effect of miR-17-5p in OFs on embryonic development to the blastocyst stage. Small RNA-seq of extracellular vesicles of OFs revealed 242 miRNAs. Additionally, analyzing expressions of randomly selected OF-miRNAs with RT-qPCR in the culture medium of oviductal epithelial cells indicated that the abundance of miRNAs in OFs increased during the luteal phase. miR-17-5p mimic-treated eight-cell-stage zona pellucida-free embryos showed improved embryonic development to the blastocyst stage. The effect of the miR-17-5p mimic was confirmed using a dual-luciferase assay and immunostaining. In addition, RNA-seq of the miR-17-5p mimic- or control-treated embryos revealed differentially expressed genes (DEGs), suggesting possible pathways that overlapped with the in silico -predicted pathways for miR-17-5p targeting genes. Furthermore, ingenuity pathway analysis of DEG predicted miR-17 to be a significant upstream regulator. Our results suggest that miR-17-5p in OFs regulates embryonic development in bovines. • miR-17-5p presents in OFs of ovulated oviduct at high frequency. • OECs secrete miRNA including miR-17-5p into the culture medium. • RNA-Seq followed by IPA supports the effect of miR-17-5p transfection. • miR-17-5p improves embryonic development to the blastocyst stage. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Is zona pellucida (ZP) resistance to proteolysis, induced by oviductal fluid (OF), a mechanism common to species other than the pig and cow? Summary Answer ZP resistance to proteolysis induced by OF was observed in the mouse, rat, hamster, rabbit, sheep, goat, pig and cow, but not in humans. WHAT IS KNOWN ALREADY Oviductal ZP resistance to proteolysis occurs in the pig and cow where it influences the incidence of fertilization and polyspermy. The effect is observed after incubation of ZP in OFs from pig (pOF), cow (cOF), rabbit (rOF) and sheep (sOF). STUDY DESIGN, SIZE, DURATION Oocytes from nine different species, including ungulates, rodents, lagomorphs and primates were incubated in rOF, sOF, gOF, cOF, pOF and human oviductal fluid (hOF). ZP digestion times for the matured oocytes of these nine species, without any treatment or incubated in 5 (mouse, rat, hamster, rabbit, cow, ewe and goat) or 6 (pig and humans) of the OFs collected were compared using three replicates per treatment and at least three oocytes per replicate. MATERIALS, SETTING, METHODS In vivo matured oocytes from rat, hamster, mouse, rabbit and humans, in vitro matured oocytes from cow, goat, ewe and pig and rOF, cOF, gOF, sOF, pOF and human (hOF) were collected and processed for the study. Oocytes from each species were incubated in the different OFs for 30 min. The resistance of the ZP of the oocytes to enzymatic digestion in a pronase solution (0.5% in PBS) was measured and registered as ZP digestion time. MAIN RESULTS AND THE ROLE OF CHANCE rOF increased ZP resistance to proteolytic digestion in the range of between 96 and 720 h for any of the species tested, whereas the corresponding increase in human ZP was only 1 min. OFs from the remaining species also had a significant effect, with variations among the cross-species experiments (P < 0.05). hOF, which was only tested on human and porcine oocytes, had no effect on ZP chemical hardening. Measurements of ZP digestion times are not of extreme accuracy and errors of a few seconds can be assumed in the experimental data. However, when differences are in the range of hours among treatments, variations measured in seconds do not alter the robustness of the findings. LIMITATIONS, REASONS FOR CAUTION Human oocytes and OF were of limited access, compared with oocytes from species collected in slaughterhouses. OFs from mouse, rat and hamster were not tested due to the small size of the genital tract in these species and the small volume of fluid available. WIDER IMPLICATIONS OF THE FINDINGS Since oviductal modification of ZP resistance to proteolytic digestion has been demonstrated to influence fertilization and this pre-fertilization mechanism is considered to contribute to the control of polyspermy, the apparent absence of this mechanism in humans suggests that the regulation of polyspermy depends mainly on other mechanisms, most probably of cortical granule origin. Investigation into a possible relationship between the lack of oviductal ZP hardening in human oocytes and the existence of tubal ectopic pregnancies in this species is proposed. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Spanish Ministry of Science and Innovation and FEDER, Grant AGL2009-12512-C02-01-02. The authors declare no competing interest. [ABSTRACT FROM AUTHOR]

Abstract: During the egg-laying process, oviductal fluid was collected using a non-invasive procedure from the cloacal vent of the green turtles. Forty-two independent isolates of antibiotic-resistant bacteria from 11 genera were obtained from 20 turtles during nesting. The dominant isolate was Citrobacter (52.4%), followed by Pseudomonas, Proteus, Enterobacter, Salmonella, Escherichia coli, Shigella, Edwardsiella, Morganella, Providencia and Arcomobacter. Most of the isolates were resistant to ampicillin. Ampicillin-resistant isolates showed variations in their resistance for the following classes of β-lactamases: extended-spectrum β-lactamases (EBSLs), AmpC type β-lactamases C (AmpC), and screen-positive β-lactamase. None of the isolates produced metallo β-lactamase. Some ampicillin-resistant genes were detected by multiplex polymerase chain reaction (PCR) only. Inhibitor based test (IBT) categorized some isolates as AmpC β-lactamase producers. β-Lactamase genes were detected from a few strains. The sequencing of those genes revealed the presence of cephamycinase (CMY) and AmpC β-lactamases. The oviductal fluid was used in this study as a source of bacterial antibiotic-resistant determinants for biomonitoring marine turtles exposed to contaminated effluents. This data can be of value in understanding the decline of this endangered species as a result of exposure to marine pollution which is threatening their survival. [Copyright &y& Elsevier]

Abstract: Sperm capacitation takes place in the oviduct and protein tyrosine phosphorylation of sperm proteins is a crucial step in capacitation and acquisition of fertilizing potential. Cryopreserved spermatozoa show altered expression of protein tyrosine phosphorylation in the oviduct. The present study compared two freezing methods (conventional-conventional freezing (CF) and simplified-simplified freezing (SF) methods) for their effect on the ability of boar spermatozoa to undergo protein tyrosine phosphorylation in response to oviductal fluid (ODF). Cryopreserved boar-spermatozoa were incubated with pre- and post-ovulatory ODF for 6 h at 38 °C under 5% CO2. Aliquots of sperm samples were taken at hourly intervals and analyzed for kinematics and protein tyrosine phosphorylation. Global protein tyrosine phosphorylation in spermatozoa was measured using flow cytometry and different patterns of phosphorylation were assessed using confocal microscopy. Immediately after thawing, no significant difference was observed in post-thaw sperm motility, velocity and global tyrosine phosphorylation between the two methods of freezing although the freezing method significantly (P < 0.05) influenced the effect of oviductal fluid on these parameters during incubation. While spermatozoa frozen by the CF method showed a significantly higher (P < 0.001) proportion of phosphorylation in response to preovulatory ODF during incubation, spermatozoa frozen by the SF method did not elicit such significant response as there was no significant difference in the proportion of tyrosine phosphorylated spermatozoa between treatments at any given time during incubation. If the CF method was used, the proportion of spermatozoa displaying either tail or full sperm phosphorylation increased in response to both preovulatory (EODF) and postovulatory oviductal fluid. However, if the SF method was used, a significant increase in these patterns was noticed only in the EODF treated group. The present study demonstrates that preovulatory isthmic ODF induce tyrosine phosphorylation in a higher proportion of boar spermatozoa compared to the post-ovulatory fluid and that the method of freezing significantly influences the response of post-thaw spermatozoa to porcine ODF. [Copyright &y& Elsevier]