MEK signalling pathway is required for hypoblast specification and migration in ovine.

In brief: MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. This paper demonstrates that MEK is required for hypoblast specification in the inner cell mass of the ovine blastocyst and that it plays a role during the hypoblast migration occurring following blastocyst hatching. Abstract: Early embryo development requires the differentiation of three cell lineages in two differentiation events. The second lineage specification differentiates the inner cell mass into epiblast, which will form the proper fetus, and hypoblast, which together with the trophectoderm will form the extraembryonic membranes and the fetal part of the placenta. MEK signalling pathway is required for hypoblast differentiation in mouse embryos, but its role in ungulate embryos remains controversial. The aim of this work was to analyse the role of MEK signalling on hypoblast specification at the blastocyst stage and on hypoblast migration during post-hatching stages in vitro in the ovine species. Using well-characterized and reliable lineage markers, and different MEK inhibitor concentrations, we demonstrate that MEK signalling pathway is required for hypoblast specification in the inner cell mass of the ovine blastocyst, and that it plays a role during the hypoblast migration occurring following blastocyst hatching. These results show that the role of MEK signalling pathway on hypoblast specification is conserved in phylogenetically distant mammals.

Metabolomics analysis of human cumulus cells obtained from cumulus-oocyte complexes with different developmental potential.

STUDY QUESTION: Is the abundance of certain biochemical compounds in human cumulus cells (CCs) related to oocyte quality? SUMMARY ANSWER: Malonate, 5-oxyproline, and erythronate were positively associated with pregnancy potential. WHAT IS KNOWN ALREADY: CCs are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Mitochondrial DNA content and transcriptional analyses in CC have been shown to provide a poor predictive value of oocyte competence, but the untargeted analysis of biochemical compounds (metabolomics) has been unexplored. STUDY DESIGN, SIZE, DURATION: CCs were obtained from three groups of cumulus-oocyte complexes (COCs) of known developmental potential: oocytes not developing to blastocyst following ICSI (Bl-); oocytes developing to blastocyst but failing to establish pregnancy following embryo transfer (P-); and oocytes developing to blastocyst able to establish a pregnancy (P+). Metabolomics analyses were performed on 12 samples per group, each sample comprising the CC recovered from a single COC. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human CC samples were obtained from IVF treatments. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Metabolomics analysis was performed by ultra-high performance liquid chromatography-tandem mass spectroscopy. MAIN RESULTS AND THE ROLE OF CHANCE: The analysis identified 98 compounds, five of which were differentially abundant (P < 0.05) between groups: asparagine, proline, and malonate were less abundant in P- compared to Bl-, malonate and 5-oxoproline were less abundant in P- group compared to P+, and erythronate was less abundant in Bl- group compared to P+. No significant association between the abundance of the compounds identified and donor age or BMI was noted. LIMITATIONS, REASONS FOR CAUTION: Data dispersion and the lack of coherence between developmental groups preclude the direct use of metabolic markers in clinical practice, where the uterine environment plays a major role in pregnancy outcome. The abundance of other compounds not detected by the analysis may be associated with oocyte competence. As donors were lean (only two with BMI > 30 kg/m2) and young (<34 years old), a possible effect of obesity or advanced age on the CC metabolome could not be determined. WIDER IMPLICATIONS OF THE FINDINGS: The abundance of malonate, 5-oxyproline, and erythronate in CC was significantly higher in COCs ultimately establishing pregnancy, providing clues on the pathways required for oocyte competence. The untargeted analysis uncovered the presence of compounds that were not expected in CC, such as ß-citrylglutamate and the neurotransmitter N-acetyl-aspartyl-glutamate, which may play roles in chromatin remodeling and signaling, respectively. STUDY FUNDING/COMPETING INTEREST(S): Research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by Madrid Region Government. The authors declare no competing interest. TRIAL REGISTRATION NUMBER: N/A.

The human cumulus cell transcriptome provides poor predictive value for embryo transfer outcome.

RESEARCH QUESTION: Is the transcriptome of cumulus cells a good predictor of the embryo's developmental competence? DESIGN: Cumulus cells were collected from donor oocytes and their transcriptome was analysed by RNA sequencing analysis at >30 × 106 reads in samples grouped according to the developmental potential of their enclosed oocyte: not able to develop to the blastocyst stage (Bl-), able to develop to the blastocyst stage but failing to establish a pregnancy (P-), or able to develop to the blastocyst stage and to establish a clinical pregnancy (P+). RESULTS: The cumulus cell trancriptome was largely independent of the developmental potential as, using a false dscovery rate-adjusted P-value of <0.05, only 10, 11 and 5 genes were differentially expressed for the comparisons P+ versus P-, P+ versus Bl-, and P- versus Bl-, respectively, out of a total of 17,469 genes expressed. Between the differentially expressed genes, those showing little overlap between samples from different groups were CHAC1, up-regulated in the P- and P+ groups compared with the Bl- group, and CENPE, CD93, PECAM1 and HSPA1B, which showed the opposite expression pattern. Focusing on the pregnancy potential, only EPN3 was consistently downregulated in the P+ compared with the P- and Bl- groups. CONCLUSIONS: The cumulus cell transcriptome is largely unrelated to the establishment of clinical pregnancy following embryo transfer, although the expression level of a subset of genes in cumulus cells may indicate the ability to develop to the blastocyst stage.

Transforming growth factor beta (TGFß) pathway is essential for hypoblast and epiblast development in ovine post-hatching embryos.

The developmental failures occurring between blastocyst hatching and implantation in farm ungulates are a major cause of pregnancy losses. At the expanded blastocyst stage, three cell lineages emerge in the embryo: trophoblast, hypoblast and epiblast, the latter being the most vulnerable during post-hatching development. Transforming growth factor beta (TGFß) signaling pathway is involved in hypoblast and epiblast development; however, previous in vitro functional studies are limited to the expanded blastocyst stage. In this study, we have analyzed the effect of TGFß inhibition with 10, 20 or 40 µM SB431542 during ovine post-hatching developmental period using a recently developed culture system able to recapitulate major developmental landmarks. We have found a negative effect of TGFß inhibition on hypoblast and epiblast development that could be partially reverted by Rho-associated protein kinase (ROCK) inhibitor Y-27632. Our findings provide new insights into the molecular networks regulating embryo development beyond the expanded blastocyst and could help to elucidate the causes of early pregnancy losses in farm ungulates.

RNA-sequencing reveals genes linked with oocyte developmental potential in bovine cumulus cells.

Cumulus cells provide an interesting biological material to perform analyses to understand the molecular clues determining oocyte competence. The objective of this study was to analyze the transcriptional differences between cumulus cells from oocytes exhibiting different developmental potentials following individual in vitro embryo production by RNA-seq. Cumulus cells were allocated into three groups according to the developmental potential of the oocyte following fertilization: (1) oocytes developing to blastocysts (Bl+), (2) oocytes cleaving but arresting development before the blastocyst stage (Bl-), and (3) oocytes not cleaving (Cl-). RNAseq was performed on 4 (Cl-) or 5 samples (Bl+ and Bl-) of cumulus cells pooled from 10 cumulus-oocyte complexes per group. A total of 49, 50, and 18 differentially expressed genes (DEGs) were detected in the comparisons Bl+ versus Bl-, Bl+ versus Cl- and Bl- versus Cl-, respectively, showing a fold change greater than 1.5 at an adjusted p value <0.05. Focussing on DEGs in cumulus cells from Bl+ group, 10 DEGs were common to both comparisons (10/49 from Bl+ vs. Bl-, 10/50 from Bl+ vs. Cl-). These DEGs correspond to 6 upregulated genes (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, and SLC1A4), and 4 downregulated genes (GSTA1, PSMB8, FMOD, and SFRP4) in Bl+ compared to the other groups, from which 7 were validated by quantitative PCR (HBE1, ITGA1, PAPPA, AKAP12, ITGA5, PSMB8 and SFRP4). These genes are involved in critical biological functions such as integrin-mediated cell adhesion, oxygen availability, IGF and Wnt signaling or PKA pathway, highlighting specific biological processes altered in incompetent in vitro maturation oocytes.

mtDNA content in cumulus cells does not predict development to blastocyst or implantation.

STUDY QUESTION: Is relative mitochondrial DNA (mtDNA) content in cumulus cells (CCs) related to embryo developmental competence in humans and/or the bovine model? SUMMARY ANSWER: mtDNA content in CCs provides a poor predictive value of oocyte developmental potential, both in vitro and following embryo transfer. WHAT IS KNOWN ALREADY: CCs are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby providing interesting biological material on which to perform molecular analyses designed to identify markers that predict oocyte developmental competence. Previous studies have positively associated oocyte mtDNA content with developmental potential in animal models and women. However, it remains debatable whether mtDNA content in CCs could be used as a proxy to infer oocyte developmental potential. STUDY DESIGN SIZE DURATION: mtDNA content was analyzed in CCs obtained from 109 human oocytes unable to develop to blastocyst, able to develop to blastocyst but failing to establish pregnancy or able to develop to blastocyst and to establish pregnancy. mtDNA analysis was also performed on bovine cumulus samples collected from 120 oocytes unable to cleave, oocytes developing into cleaved embryos but arresting development prior to the blastocyst stage or oocytes developing to blastocysts. PARTICIPANTS/MATERIALS SETTING METHODS: Human CCs samples were obtained from women undergoing IVF. Only unfrozen oocytes and embryos not submitted to preimplantation genetic testing were included in the analysis. Bovine samples were obtained from slaughtered cattle and individually matured, fertilized and cultured in vitro. Relative mtDNA was assessed by quantitative PCR analysis. MAIN RESULTS AND THE ROLE OF CHANCE: mtDNA content in human and bovine CCs did not differ according to the developmental potential of their enclosed oocyte. Moreover, mtDNA content in bovine oocytes did not correlate with that of their corresponding CCs. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: The lack of correlation found between mtDNA content in human CCs and oocytes was also assessed in bovine samples. Although bovine folliculogenesis, mono-ovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, they may not be fully comparable. WIDER IMPLICATIONS OF THE FINDINGS: The use of molecular markers for oocyte developmental potential in CCs could be used to enhance success rates following single embryo transfer. However, our data indicate that mtDNA in CCs is not a good proxy for oocyte quality. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the Industrial Doctorate Project IND2017/BIO-7748 funded by the Madrid Region Government. The authors declare no competing interests.

In vitro culture of ovine embryos up to early gastrulating stages.

Developmental failures occurring shortly after blastocyst hatching from the zona pellucida constitute a major cause of pregnancy losses in both humans and farm ungulates. The developmental events occurring following hatching in ungulates include the proliferation and maturation of extra-embryonic membranes - trophoblast and hypoblast - and the formation of a flat embryonic disc, similar to that found in humans, which initiates gastrulation prior to implantation. Unfortunately, our understanding of these key processes for embryo survival is limited because current culture systems cannot sustain ungulate embryo development beyond hatching. Here, we report a culture system that recapitulates most developmental landmarks of gastrulating ovine embryos: trophoblast maturation, hypoblast migration, embryonic disc formation, disappearance of the Rauber's layer, epiblast polarization and mesoderm differentiation. Our system represents a highly valuable platform for exploring the cell differentiation, proliferation and migration processes governing gastrulation in a flat embryonic disc and for understanding pregnancy failures during the second week of gestation. This article has an associated 'The people behind the papers' interview.

Lineage Differentiation Markers as a Proxy for Embryo Viability in Farm Ungulates.

Embryonic losses constitute a major burden for reproductive efficiency of farm animals. Pregnancy losses in ungulate species, which include cattle, pigs, sheep and goats, majorly occur during the second week of gestation, when the embryo experiences a series of cell differentiation, proliferation, and migration processes encompassed under the term conceptus elongation. Conceptus elongation takes place following blastocyst hatching and involves a massive proliferation of the extraembryonic membranes trophoblast and hypoblast, and the formation of flat embryonic disc derived from the epiblast, which ultimately gastrulates generating the three germ layers. This process occurs prior to implantation and it is exclusive from ungulates, as embryos from other mammalian species such as rodents or humans implant right after hatching. The critical differences in embryo development between ungulates and mice, the most studied mammalian model, have precluded the identification of the genes governing lineage differentiation in livestock species. Furthermore, conceptus elongation has not been recapitulated in vitro, hindering the study of these cellular events. Luckily, recent advances on transcriptomics, genome modification and post-hatching in vitro culture are shedding light into this largely unknown developmental window, uncovering possible molecular markers to determine embryo quality. In this review, we summarize the events occurring during ungulate pre-implantation development, highlighting recent findings which reveal that several dogmas in Developmental Biology established by knock-out murine models do not hold true for other mammals, including humans and farm animals. The developmental failures associated to in vitro produced embryos in farm animals are also discussed together with Developmental Biology tools to assess embryo quality, including molecular markers to assess proper lineage commitment and a post-hatching in vitro culture system able to directly determine developmental potential circumventing the need of experimental animals.

Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.

Embryonic disc formation following post-hatching bovine embryo development in vitro.

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

JUNO protein coated beads: A potential tool to predict bovine sperm fertilizing ability.

Considerable variation in fertility exists between bulls in AI centres, despite passing minimum post-thaw quality control checks. The development of a reliable in vitro test to predict bull fertility could enable the identification and selection of high fertility bulls, without the need to resort to test inseminations. An in-depth knowledge of the molecular basis of fertilization is a prerequisite to the development of such a test or combination of tests. To date, JUNO is the only oocyte plasma membrane receptor described to be involved in gamete binding for which the partner in the sperm, IZUMO1, is known. Despite the fact that this interaction appears to be conserved among mammals, it has not been confirmed yet in some species including cattle. Furthermore, an association between binding and fertility has not been tested. Here, we propose a sperm-binding assay based on magnetic sepharose beads coated with bovine recombinant JUNO protein (BJUNO) to study sperm binding. Bull sperm bound specifically to BJUNO demonstrating that the JUNO-IZUMO1 interaction is conserved in cattle. Moreover, the assay was able to distinguish between epididymal and ejaculated sperm. Lastly, the number of sperm cells bound to BJUNO was significantly lower for frozen-thawed sperm from bulls of low vs high field fertility. In conclusion, our findings document a novel valid sperm-binding assay to predict mammalian sperm function and to investigate the role of specific proteins involved in gamete recognition and fusion.

TMEM95 is a sperm membrane protein essential for mammalian fertilization.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization.

ZP4 confers structural properties to the zona pellucida essential for embryo development.

Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development.

Intergenerational transmission of the positive effects of physical exercise on brain and cognition.

Physical exercise has positive effects on cognition, but very little is known about the inheritance of these effects to sedentary offspring and the mechanisms involved. Here, we use a patrilineal design in mice to test the transmission of effects from the same father (before or after training) and from different fathers to compare sedentary- and runner-father progenies. Behavioral, stereological, and whole-genome sequence analyses reveal that paternal cognition improvement is inherited by the offspring, along with increased adult neurogenesis, greater mitochondrial citrate synthase activity, and modulation of the adult hippocampal gene expression profile. These results demonstrate the inheritance of exercise-induced cognition enhancement through the germline, pointing to paternal physical activity as a direct factor driving offspring's brain physiology and cognitive behavior.

Mitochondrial and metabolic adjustments during the final phase of follicular development prior to IVM of bovine oocytes.

In vitro maturation (IVM) leads to reduced developmental rates compared to the use of in vivo matured oocytes. This reduction can be attributed to the suboptimal environment experienced during IVM, but the use of incompetent oocytes also plays a significant role. The objective of this study has been to characterize the mitochondrial and metabolic differences between competent and incompetent bovine oocytes selected prior to IVM based on Brilliant Cresyl Blue (BCB) staining. BCB selection allowed to sort two populations of cumulus-oocyte complexes (COCs) exhibiting diverse developmental competence despite showing a similar size and thereby being morphologically undistinguishable otherwise. Nuclear maturation rates were similar in both populations, but cleavage and blastocysts rates were significantly higher in BCB+ compared with BCB-. Mitochondrial distribution was similar between both groups, but mtDNA content experienced a 1.9-fold increase between BCB- and BCB+ oocytes, suggesting that a significant mtDNA synthesis must occur at the last stages of follicular development to achieve full competence prior to IVM. Consistently, transcriptional analysis in cumulus cells revealed an upregulation of the mitochondrial transcription factor TFAM in BCB-. Transcriptional analysis also suggested a decrease in both anaerobic glycolysis and pentose phosphate pathway (PPP) in BCB+ COCs, as the anaerobic glycolysis enzymes GAPDH and LDHA and the positive regulator of G6PD activity SIRT2 were upregulated in BCB- cumulus cells. These results suggest that during the final stages of follicular development a significant mtDNA replication must occur to achieve full oocyte developmental competence, and that this replication may be linked to anaerobic glycolysis and PPP activities.

Directions and applications of CRISPR technology in livestock research.

The ablation (KO) or targeted insertion (KI) of specific genes or sequences has been essential to test their roles on a particular biological process. Unfortunately, such genome modifications have been largely limited to the mouse model, as the only way to achieve targeted mutagenesis in other mammals required from somatic cell nuclear transfer, a time- and resource-consuming technique. This difficulty has left research in livestock species largely devoided of KO and targeted KI models, crucial tools to uncover the molecular roots of any physiological or pathological process. Luckily, the eruption of site-specific endonucleases, and particularly CRISPR technology, has empowered farm animal scientists to consider projects that could not develop before. In this sense, the availability of genome modification in livestock species is meant to change the way research is performed on many fields, switching from descriptive and correlational approaches to experimental research. In this review we will provide some guidance about how the genome can be edited by CRISPR and the possible strategies to achieve KO or KI, paying special attention to an initially overlooked phenomenon: mosaicism. Mosaicism is produced when the zygote´s genome edition occurs after its DNA has replicated, and is characterized by the presence of more than two alleles in the same individual, an undesirable outcome when attempting direct KO generation. Finally, the possible applications on different fields of livestock research, such as reproduction or infectious diseases are discussed.

Early sex-dependent differences in response to environmental stress.

Developmental plasticity enables the appearance of long-term effects in offspring caused by exposure to environmental stressors during embryonic and foetal life. These long-term effects can be traced to pre- and post-implantation development, and in both cases, the effects are usually sex specific. During preimplantation development, male and female embryos exhibit an extensive transcriptional dimorphism mainly driven by incomplete X chromosome inactivation. These early developmental stages are crucial for the establishment of epigenetic marks that will be conserved throughout development, making it a particularly susceptible period for the appearance of long-term epigenetic-based phenotypes. Later in development, gonadal formation generates hormonal differences between the sexes, and male and female placentae exhibit different responses to environmental stressors. The maternal environment, including hormones and environmental insults during pregnancy, contributes to sex-specific placental development that controls genetic and epigenetic programming during foetal development, regulating sex-specific differences, including sex-specific epigenetic responses to environmental hazards, leading to long-term effects. This review summarizes several human and animal studies examining sex-specific responses to environmental stressors during both the periconception period (caused by differences in sex chromosome dosage) and placental development (caused by both sex chromosomes and hormones). The identification of relevant sex-dependent trajectories caused by sex chromosomes and/or sex hormones is essential to define diagnostic markers and prevention/intervention protocols.

Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro.

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C- group), in the presence of 3 × 105 EV/mL from ampullary or isthmic OF at either 1 × 104 g (10 K) or 1 × 105 g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7-9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C- and OF-EV groups (12.0-14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5-30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C- group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P < 0.05). In terms of gene expression, blastocysts produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C- being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced.