Search

SummaryThe 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.

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 (P0.05) monospermy rate, without alteration (P0.05) of the penetration rate in comparison with the control and OEC groups. When the IVF medium was supplemented with heparin, an overall increase (P0.05) 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.

International audience; High polyspermy is one of the major limitations of porcine in vitro fertilisation (IVF). The addition of oviductal fluid (OF) during IVF reduces polyspermy without decreasing the fertilisation rate. Because extracellular vesicles (EVs) have been described as important OF components, the aim of this study was to evaluate the effect of porcine oviductal EVs (poEVs) on IVF efficiency compared with porcine OF (fresh and lyophilised). OF was collected from abattoir oviducts by phosphate-buffered saline flush, and poEVs were isolated by serial ultracentrifugation. Four IVF treatments were conducted: poEVs (0.2 mg mL(-1)), OF (10%), lyophilized and reconstituted pure OF (LOF; 1%) and IVF without supplementation (control). Penetration, monospermy and IVF efficiency were evaluated. Transmission electron microscopy showed an EVs population primarily composed of exosomes (83%; 30-150 nm). Supplementation with poEVs during IVF increased monospermy compared with control (44% vs 17%) while maintaining an acceptable penetration rate (61% vs 78% respectively) in a similar way to OF and LOF. Western blotting revealed poEVs proteins involved in early reproductive events, including zona pellucida hardening. In conclusion, our finding show that poEVs are key components of porcine OF and may play roles in porcine fertilisation and polyspermy regulation, suggesting that supplementation with poEVs is a reliable strategy to decrease porcine polyspermy and improve in vitro embryo production outcomes.

Remerciements : Pôle STAR; Plateforme CIRE, INRA, UMR PRC 0085, Centre Tours Val de Loire; While cumulus cells are essential for efficient oocyte maturation, the establishment of protocols that support IVD of embryos obtained from denuded oocytes (DOC) is important for optimizing the use of reproductive biotechnologies. Thus, this study aimed to establish a protocol for IVD of goat DOC using different strategies of IVM and methods of oocyte activation. Four experiments were performed. Similar developmental competence of slaughterhouse-DOC was obtained, regardless of maturation media (complex, semi-defined or simplified). However, the ability to reach the blastocyst stage was affected by the activation method. DOC subjected to parthenogenetic activation (PA) had greater (P

International audience; The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification-warming, exposure to P4 improved the proportion of embryos that re-expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI-TOF mass spectrometry, a total of 242 phospholipid masses of 400-1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.

Considerable research has been focused on in vitro production (IVP) of goat embryos to improve its efficiency. In Experiment 1, the effect of the cumulus cells by comparing slaughterhouse-oocytes denuded on purpose (DOP) prior to IVF to intact COC, and the effect of heparin during IVF were assessed. In Experiment 2, oocytes that were already denuded at collection (DOC), DOP and intact COC were studied. Three treatments used oocytes denuded at collection: DOC oocytes were cultured alone for both IVM and IVF; DOC and COC were cultured together for both IVM and IVF or DOC were IVM alone and then mixed with COC for IVF. In other treatments, COC were allocated to four IVF treatments: Intact COC; COC were denuded prior to IVF; COC were denuded and IVF with added cumulus cells; COC were denuded and IVF mixed with intact COC giving two sub-treatments: Denuded oocytes that were IVF with COC; and COC that were IVF with denuded oocytes. After fertilization, all presumptive zygotes were cultured for 8 days. In Experiment 1, the yield of blastocysts as a proportion of total oocytes was greater (P

During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2. Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2. Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2.

This study aimed to verify the effects of cryoprotectant and Open Pulled Straw (OPS) vitrification on ultrastructural changes in bovine oocytes. In experiment 1, the cryoprotectant exposure was analyzed by distributing immature and matured cumulus-oocyte complexes (COCs) in groups: control immature, immature and exposed to one vitrification solution (IVS1), immatured and exposed to two vitrification solutions (IVS1-2), matured and exposed to one vitrification solution (MVS1), matured and exposed to two vitrification solutions (MVS1-2), control matured, IVS1 post in vitro maturation (IVS1 post IVM) and IVS1-2 post in vitro maturation (IVS1-2 post IVM). In experiment 2, immature and matured COCs were distributed in: control and vitrified by OPS using VS1-2. Sample COCs from both experiments were evaluated for mitochondrial distribution, nuclear lamins (immature COCs) and meiotic spindle (matured COCs). In both experiments, COCs were in vitro matured and fertilized. In experiment I, nuclear lamins in immature COCs exhibited higher proportion of structures with irregular shape when treated with VS1 and VS1-2. Immature and matured COCs treated with VS1-2, and matured COCs treated with VS1 had lower (P < 0.05) blastocyst development (29%, 20% and 8%, respectively) than control and immature COCs treated with VS1 (51% and 41%, respectively). In experiment 2, immature oocytes exhibited more (P < 0.05) structures with irregular lamin (72%) than control (9%). Vitrification of matured COCs did not induce abnormalities of meiotic spindle, compared to control matured (25% and 37%, respectively) (P > 0.05). The cleavage rate was higher (P < 0.05) in matured vitrified than immature vitrified group (19% vs. 8%). In conclusion, immature COCs were more tolerant to cryoprotectant exposure. However, matured oocytes showed better embryo development (cleavage) after vitrification.

After insemination in the cow, a sperm reservoir is formed within the oviducts, allowing the storage and then progressive release of spermatozoa toward the ovulated oocyte. In order to investigate the hormonal regulation of these events in vitro, the ovarian steroids 17β-estradiol (E2) and progesterone (P4) were added at various concentrations to monolayers of bovine oviduct epithelial cells (BOEC) before or during co-incubation with spermatozoa. Main findings demonstrate that (1) a 18-h pretreatment of BOEC with 100 pg/mL and 100 ng/mL of E2 decreased by 25% the ability of BOEC to bind spermatozoa after 10 min, and for the highest dose of E2, 60 min of co-incubation; (2) P4 at concentrations of 10, 100 and 1000 ng/mL induced the release within 60 min of 32–47% of bound spermatozoa from BOEC; this sperm-releasing effect was maintained after a 18-h pretreatment of BOEC with 100 pg/mL of E2; (3) E2 in concentrations above 100 pg/mL inhibited the releasing effect of P4 on bound sperm in a dose-dependent manner; (4) spermatozoa bound to BOEC, then released from BOEC by the action of P4-induced higher cleavage and blastocyst rates after in vitro fertilization than the control group. These results support the hypothesis that the dynamic changes in steroid hormones around the time of ovulation regulate the formation of the sperm reservoir and the timed delivery of capacitated spermatozoa to the site of fertilization.

Remerciements :Pôle STAR (Services Techniques d'Appui à la Recherche), Inra, UMR PRC, 37380 Nouzilly, Centre Val de Loire; The positive effect of n-3 poly-unsaturated fatty acids (FA) on fertility in ruminants seems to be partly mediated through direct effects on the oocyte developmental potential. We aimed to investigate whether supplementation with physiological levels of docosahexaenoic acid (DHA, C22:6 n-3 PUFA) during in vitro maturation (IVM) has an effect on oocyte maturation and in vitro embryo development in cattle. We showed that DHA (0, 1, 10 or 100 μM) had no effect on oocyte viability or maturation rate after 22 h IVM. Incubation of oocyte-cumulus complexes (OCC) with 1 μM DHA during IVM significantly increased (p4-cell embryo rate at day 2 after parthenogenetic activation (PA) (39.1% vs. 29.7%, respectively). Supplementation with 1 μM DHA during IVM also induced a significant increase in the blastocyst rate at day 7 after in vitro fertilization (IVF) as compared to control (30.6% vs. 17.6%, respectively) and tended to increase the number of cells in the blastocysts (97.1 ± 4.9 vs. 81.2 ± 5.3, respectively; p = 0.08). On the contrary, 10 μM DHA had no effects, whereas 100 μM DHA significantly decreased the cleavage rate compared to control (69.5% vs.78.8%, respectively) and the >4-cell embryo rate at day 2 after PA (19.5% vs. 29.7%). As was shown by real-time PCR, negative effects of 100 μM DHA were associated with significant increase of progesterone synthesis by OCCs, a three-fold increase in expression level of FA transporter CD36 and a two-fold decrease of FA synthase FASN genes in cumulus cells (CC) of corresponding oocytes. DHA at 1 and 10 μM had no effect on expression of those and other key lipid metabolism-related genes in CC. In conclusion, administration of a low physiological dose of DHA (1μM) during IVM may have beneficial effects on oocyte developmental competence in vitro without affecting lipid metabolism gene expression in surrounding cumulus cells, contrarily to 100 μM DHA which diminished oocyte quality associated with perturbation of lipid and steroid metabolism in CC.

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 × 105 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 × 105 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. Fil: Tavares Pereira Batista, Ribrio Ivan. Centre National de la Recherche Scientifique; Francia. Universidade Estadual Do Ceara; Brasil Fil: Moro, Lucía Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Centre National de la Recherche Scientifique; Francia Fil: Corbin, Emilie. Centre National de la Recherche Scientifique; Francia Fil: Alminana, Carmen. Centre National de la Recherche Scientifique; Francia Fil: Gonçalves Souza Fabjan, Joanna Maria. Universidade Estadual Do Ceara; Brasil Fil: de Figueirêdo Freitas, Vicente José. Universidade Estadual Do Ceara; Brasil Fil: Mermillod, Pascal. Centre National de la Recherche Scientifique; Francia

Previous experiments using in vitro infection have shown that Coxiella burnetii has a strong tendency to adhere to the zona pellucida (ZP) of in vivo-derived goat embryos, and the washing procedure recommended by the International Embryo Technology Society (IETS) for bovine embryos failed to remove it (Alsaleh et al. 2013 Theriogenology 80, 571-575). The aim of this study was, for in vitro-produced caprine embryos infected in vitro, to (1) evaluate the ability of C. burnetii to adhere to intact ZP, (2) test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii, and (3) determine, by confocal microscopy, the bacteria location. One hundred ZP-intact caprine embryos, produced in vitro, at the 8- to 16-cell stage were randomly allocated into 11 batches of 8 to 9 embryos. Nine batches were incubated for 18 h with 109 Coxiella/mL of CbB1 strain (ISP, INRA, Val de Loire, France). The embryos were then recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, 2 batches of embryos were subjected to similar procedures but without exposure to C. burnetii to serve as the control group. One of the 9 batches of infected embryos and 1 of the 2 non-infected control batches were used to perform immunolabelling to localise the bacteria. Coxiella burnetii DNA was detected by conventional PCR in all 8 batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryos of the control group. The first 5 washing media of the infected groups were consistently positive and Coxiella DNA was detected up to the tenth wash in 2 batches. After immunolabelling, the observation of embryos under confocal microscopy allowed us to localise C. burnetti on the external part of the ZP without deep penetration. The presence of C. burnetii was seen on the surface of the ZP, with bacterial loads differing from one embryo to another in the same batch. This study clearly demonstrates that C. burnetii, after in vitro infection at 109 Coxiella mL−1, stick strongly to the external part of the ZP of in vitro-produced early caprine embryos without profound penetration. The 10-washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria in caprine embryos. Nevertheless, the finding of C. burnetii DNA by conventional PCR does not imply that the bacteria found remain infective. Further studies are required to investigate whether enzymatic or antibiotic treatment of caprine embryos infected by C. burnetii would eliminate or inactivate the bacteria from the ZP of in vitro-produced goat embryos.

Previous experiments using in vitro infection have shown that at concentrations of 109 bacteria/mL, Coxiella burnetii strongly adheres to the zona pellucidae (ZP) of caprine embryos produced in vitro or in vivo (Alsaleh et al., 2013). However, spontaneous infection results in bacterial concentrations of between 106 and 107 bacteria/mL (Rodolakis, 2006; Alsaleh et al., 2011). The aim of this study was to determine whether the concentration of Coxiella burnetii affected its ability to bind to the ZP in vitro. A total of 120 ZP-intact 8- to 16-cell embryos, produced in vitro from ovaries collected at slaughter, were infected with Coxiella burnetti (strain CbC1) produced via ovoculture at 109 mL−1 (3 batches of 10 embryos), 107/mL (5 batches of 10 embryos), 105 mL−1 (3 batches of 8 embryos). After overnight incubation at 37°C in 5% CO2, the embryos were recovered and washed in batches, in 10 successive baths of PBS with 5% FCS, in accordance with International Embryo Technology Society guidelines. The 10 wash baths were collected separately and centrifuged for 1 h at 13,000 × g. The presence of C. burnetii was determined by conventional PCR in each batch of embryos and in the pellets of the 10 wash baths (Table 1). As demonstrated previously, Coxiella DNA was detected in embryo batches after 10 washes when a concentration of 109 bacteria/mL was used for in vitro infection, but this binding property did not occur at lower bacterial concentrations. We can conclude that the attachment of Coxiella burnetii to the zona pellucida of in vitro-produced embryos is concentration dependent. This finding illustrates the limitations of in vitro experiments to study the risk of pathogen transmission via embryo transfer. Table 1. Detection of Coxiella burnetii (CB) in successive embryo washing baths and batches of 8 to 10 infected ZP-intact 8- to 16-cell embryos after 10 wash cycles, using conventional PCR (C-PCR), as a function of the concentration of CB used for in vitro infection and determined by quantitative PCR (Q-PCR)

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P

Successful pregnancy requires an appropriate communication between the mother and the embryo(s). Recent studies indicate that exosomes, small (30–100 nm) membrane-bound vesicles of endocytotic origin, could act as intercellular vehicles in this unique communication system in the uterus. However, little is known about the role of these vesicles in the oviduct. Our study aimed at (1) demonstrating the existence of oviducal-embryo communication via exosomes, (2) deciphering the exosomal dialogue between them at the proteomic level, and (3) comparing the exosomal proteomic content to the oviducal fluid proteomic content in order to highlight the key role of exosomes in this dialogue. Cow oviducts (pool of 6 oviducts at different stages of the cycle in 4 replicates) were flushed, and exosomes were isolated by serial ultracentrifugation. Exosomes were measured by dynamic light scattering analysis, resulting in exosomes (63.25–97.03 nm) and microvesicle observations (>100 nm). Bovine embryos were produced in vitro up to the blastocyst and hatching/hatched blastocyst stages. To demonstrate the existence of the oviducal-embryo communication via exosomes, oviducal exosomes were labelled with green fluorescent dye (PKH67), filtered (0.22 µm) to remove microvesicles, and co-incubated with blastocysts and hatching/hatched (H) blastocysts for 20 h, under 5% CO2 and 5% O2 conditions. Subsequently, embryos were washed in exosome-free medium, fixed in 4% paraformaldehyde, and labelled with Hoechst 33342 and Actin Red Phallodin. Confocal microscopy observations confirmed that exosomes were internalized by blastocysts and H-blastocysts and located around the nucleus, demonstrating the existence of an oviducal-embryo communication via exosomes. Moreover, our results showed that the zona pellucida does not represent a barrier for exosomes and they act as natural nanoshuttles bringing oviducal signals into the embryo. Then, proteomic analysis by LC1D-nanoESI-LTQ-Orbitrap was used to decipher oviducal exosomal content, identifying 480 proteins. Gene ontology analysis revealed that a high number of these proteins were involved in metabolism (24.9%), cellular process (19.3%), and 0.8% reproductive processes. Further analysis revealed that more than 56% of exosomal proteins involved in cellular process were associated with cell-to-cell communication. Finally, exosomal proteins were compared with proteins present in oviducal fluid from a pool of samples from cows at Day 0 and Day 10 of the oestrous cycle. Comparative analysis showed that from a total of 607 proteins identified in both oviducal exosomes and fluid sources, 105 were specific to exosomes, 127 were specific to fluid, whereas 375 were common to both sources. Our findings provide the first evidence of oviducal-embryo communication via exosomes, an important first step in furthering the understanding of the oviducal environment and the role of exosomes as early mediators of embryo-maternal cross talk. This research was supported by the EU AgreenSkills fellowship n° 267196 and EU FECUND Project no 312097.

Successful pregnancy requires an appropriate communication between the mother and the embryo(s). Recent studies indicate that exosomes, small (30–200 nm) membrane vesicles of endocytotic origin, could act as intercellular vehicles in this unique communication system. Exosomes have been identified in vivo in all body fluids including follicular, uterine, and oviductal fluids and can be secreted by most cell types in vitro. Bovine oviductal epithelial cells (BOEC) have been thoroughly used to study embryo-maternal communication and to improve embryo development in vitro. Hence, our objective was to provide a morphologic and proteomic characterisation of exosomes secreted by BOEC in vivo in the oviductal fluid and in vitro in the conditioned media. Oviducts from cows were flushed to recover in vivo exosomes and then BOEC were scraped in order to derive primary cultures. In vitro exosomes were collected from conditioned media of BOEC primary cultures after reaching confluence (10 days). Isolation of exosomes from in vivo and in vitro origin was performed by ultracentrifugation. The presence of exosomes was confirmed in oviductal flushings and conditioned media by electron microscopy. Further characterisation of exosomes was carried out based on morphology (transmission electron microscopy), size (dynamic light scattering, DLS), and protein composition (protein profile analysis by SDS-PAGE and Western immunoblotting). Preliminary results by DLS revealed different size distribution profiles in exosome samples (in vivo: mean size of 93.41 nm; in vitro: 433.5 nm). Because exosomes are considered as “micromaps” of the originating cells, protein patterns expressed by in vivo exosomes and in vitro exosomes were compared with scraped and cultured BOEC, respectively. Protein profile analysis by SDS-PAGE showed quantitative and qualitative differences among the exosome samples, their cells of origin, and the milieu (conditioned media or flushing). Exosome-specific protein bands were detected and will be further characterised. In addition, exosomes from in vivo and in vitro origin exhibited distinct proteomic profiles. Western blot analysis demonstrated that (1) both exosomal protein samples were positive for HSP70, a known exosomal protein, and negative for Grp78, an endoplasmic reticulum marker detected in BOEC; (2) in vivo exosomes expressed oviductal glycoprotein (OVGP), heat shock protein A8 (HSPA8), and myosin 9 (MYH9), 3 oviductal proteins with known roles in fertilization and early pregnancy. However, only HSPA8 and MYH9 were detected in in vitro exosomes. Our results provide the first extensive characterisation of oviductal exosomes from in vivo and in vitro origin, an essential step in furthering our understanding of the early embryo-maternal cross talk.

A grade classification (I, II, and III) based on the number of cumulus layers and oocyte morphology is currently used by many laboratories. Oocytes found denuded at collection (grade III) are considered not suitable and routinely discarded. Thus, if a particular strategy could be applied to their use in labour-intensive processes, such as ovum pickup, it would be a benefit. This experiment was performed to examine the effect of IVM medium composition to produce goat embryos in vitro using oocytes already found denuded at collection (DOC). In total, 411 DOC and 141 intact COC (control treatment) obtained by slaughterhouse ovaries were analysed in 4 replicates. The maturation medium consisted in TCM199 supplemented either with (1) 10 ng mL–1 of EGF and 100 µM cysteamine (simplified); (2) 10 ng mL–1 of EGF, 5 IU mL–1 of hCG, 10 IU mL–1 of eCG, 19 ng mL–1 of IGF-1, 2.2 ng mL–1 of FGF, 5 µg mL–1 of ITS, 90 µg mL–1 of l-cystein, 0.1 mM β-mercaptoethanol, 75 µg mL–1 of vitamin C, 720 µg mL–1 of glycine, 0.1 mg mL–1 of glutamine, and 110 µg mL–1 of pyruvate (semidefined); or (3) 10% fetal calf serum (FCS), 100 µM cysteamine, and 50 ng mL–1 of oFSH (complex). Both DOC and COC were subjected to IVM, IVF, and IVD as previously described (Souza-Fabjan et al. 2014, Theriogenology 81, 1021–31). The COC were matured only in simplified medium. On Day 8, all expanded blastocysts were fixed and stained with Hoechst for cell counting. Statistical analysis was performed using all tests with a significant interval of 95%. All variables were compared among treatments using ANOVA and SNK test. The results are described as mean per replicate ± s.e.m. No significant differences were found in simplified, semidefined, or complex medium, respectively, in cleavage rate (52 ± 7.5, 60 ± 9.4, or 51 ± 15.0%), blastocyst from cleaved (36 ± 3.9, 39 ± 9.3, or 41 ± 4.8%), blastocyst from initial DOC (19 ± 5.0, 23 ± 8.1, or 21 ± 3.3%), hatching rate (55 ± 22.9, 55 ± 15.9, or 52 ± 14.8%), or total blastomeres number (184 ± 12.6, 179 ± 12.4, or 190 ± 13.8). The control COC showed no significant differences to any DOC treatment on cleavage (77 ± 3.4%) and blastocyst from cleaved (60 ± 2.2%). However, the blastocyst rate from initial COC was higher (46 ± 0.5%; P < 0.05) than all DOC treatments. Even though the blastocyst yield was lower than COC (~21 v. 46%), it is reasonable to affirm that it is possible to produce embryos from oocytes that would not be utilised, which may represent additional number of embryos. The blastocyst cell numbers in COC (192 ± 13.7) were similar (P > 0.05) to DOC, indicating that the goat embryos produced were of good quality. In conclusion, the inclusion of more complex substances in IVM media did not increase the development rate of DOC and, therefore, more simple IVM media could be used for this purpose. Finally, the goat embryos produced had satisfactorily number of blastomeres, demonstrating that the in vitro development step is able to generate good quality embryos from grade III oocytes. Therefore, some oocytes that in general would be discarded will develop to blastocysts and may represent benefits, especially after ovum pick-up from genetically valuable goats.

Previous experiments demonstrated that co-culture of bovine embryos with bovine oviducal epithelial cells (BOEC) improved blastocyst rate and quality (Cordova et al. 2014). However, the use of primary cell support for improving embryo development in vitro may introduce a higher variability of the results between different BOEC batches used, as well as sanitary risks. The use of well-controlled large batches of frozen BOEC may help to solve these problems. Therefore, the aim of the present study was to characterise the survival and functionality of frozen-thawed BOEC. Bovine oviducts attached to ovaries showing recent ovulation were collected at a local slaughterhouse during 4 replicates (3 oviducts per replicate). Epithelial cells were expelled by gentle squeezing and washed 3 times. Half of the cell pellet was diluted 100-fold in culture medium (TCM199 + 10% FCS) for culture of fresh cells. The other half was diluted 10-fold in cell freezing medium (TCM199 + 20% FCS + 10% dimethyl sulfoxide), allowed to equilibrate in this medium for 10 min, and frozen at –80°C in a container filled with isopropyl alcohol. After 4 h, the tubes were transferred into LN for at least 1 h. The tubes were then thawed (5 min in 37°C water bath), diluted 1 : 1 in cell culture medium, and centrifuged for 10 min at 100 × g. The pellet was then diluted 100× in cell culture medium. Fresh or frozen-thawed cells were seeded in 4-well NUNC plates for 7 days at 38.8°C in a humidified atmosphere with 5% CO2 in air. The medium was renewed every 48 h, and the viability of cells was assessed by calcein-AM and ethidium homodimer labelling. After 7 days of culture, the medium was replaced by SOF medium + 5% FCS, and bovine in vitro-produced zygotes were added the day after and co-cultured for 8 days at 38.8°C in a humidified atmosphere with 5% CO2 in air to evaluate embryo development. Half of the medium was renewed every 48 h. Frozen-thawed cells showed the same viability than fresh ones at Days 0 and 7 of culture and reached confluence at the same time (Day 7). Development results are shown in Table 1. Frozen and fresh cells support early embryo development at the same rate. In conclusion, the present study showed that BOEC frozen on the day of collection are equivalent to fresh BOEC in regards to their survival and proliferation and their ability to support early embryo development. At collection, the cells may face stresses that are just as considerable as freezing/thawing (temperature shock, scrapping, change of environment). This may explain why they are not affected by freezing than at collection. The differentiation status of these cells is now under analysis by immunocytochemistry. Table 1.Cleavage rate and blastocyst rate in 3 different types of culture systems

A total of 3427 goat oocytes were used in this study to identify possible differences during in vitro embryo production from slaughterhouse or laparoscopic ovum pick up (LOPU) oocytes. In experiment 1, one complex, one semi-defined, and one simplified IVM media were compared using slaughterhouse oocytes. In experiment 2, we checked the effect of oocyte origin (slaughterhouse or LOPU) on the kinetics of maturation (18 vs. 22 vs. 26 hours) when submitted to semi-defined or simplified media. In experiment 3, we determined the differences in embryo development between slaughterhouse and LOPU oocytes when submitted to both media and then to IVF or parthenogenetic activation (PA). Embryos from all groups were vitrified, and their viability evaluated in vitro after thawing. In experiment 1, no difference (P > 0.05) was detected among treatments for maturation rate (metaphase II [MII]; 88% on average), cleavage (72%), blastocyst from the initial number of cumulus oocyte complexes (46%) or from the cleaved ones (63%), hatching rate (69%), and the total number of blastomeres (187). In experiment 2, there was no difference of MII rate between slaughterhouse oocytes cultured for 18 or 22 hours, whereas the MII rate increased significantly (P < 0.05) between 18 and 22 hours for LOPU oocytes in the simplified medium. Moreover, slaughterhouse oocytes cultured in simplified medium matured significantly faster than LOPU oocytes at 18 and 22 hours (P < 0.05). In experiment 3, cleavage rate was significantly greater (P < 0.001) in all four groups of embryos produced by PA than IVF. Interestingly, PA reached similar rates for slaughterhouse oocytes cultured in both media, but improved (P < 0.05) the cleavage rate of LOPU oocytes. Slaughterhouse oocytes had acceptable cleavage rate after IVF (∼67%), whereas LOPU oocytes displayed a lower one (∼38%), in contrast to cleavage after PA. The percentage of blastocysts in relation to cleaved embryos was not affected by the origin of the oocytes (P > 0.05). Therefore, slaughterhouse oocytes developed a greater proportion of blastocysts than LOPU ones, expressed as the percentage of total cumulus oocyte complexes entering to IVM. Vitrified-thawed blastocysts presented similar survival and hatching rates between the oocyte origin, media, or method of activation. In conclusion, slaughterhouse and LOPU derived oocytes may have different IVM kinetics and require different IVM and IVF conditions. Although the IVM and IVF systems still need improvements to enhance embryo yield, the in vitro development step is able to generate good quality embryos from LOPU-derived oocytes.

National audience