Cryopreservation of in vitro produced and biopsied bovine embryos

Aujourd’hui, la cryoconservation de l’embryon bovin est primordiale pour assurer sa préservation dans l’attente de sa valorisation. Cependant, la qualité des embryons produits in vitro est inférieure à celle de ceux produits in vivo ne permettant pas l’obtention de taux de gestation équivalents. Lors du processus de congélation, les membranes, majoritairement composées de lipides, subissent des contraintes physiques et chimiques pouvant aboutir à une lyse des cellules embryonnaires. Ainsi, afin d’améliorer la qualité des embryons bovins produits in vitro et biopsiés, deux tâches ont été menées en parallèle : (1) l’acquisition de nouvelles connaissances scientifiques sur le profil lipidique des embryons produits in vitro et la modulation de ce profil par l’ajout d’acides gras de type oméga 3 avec pour objectifs d’augmenter la fluidité membranaire et de diminuer la cryosensibilité des embryons; (2) l’acquisition de nouvelles connaissances scientifiques sur l’intérêt du CRYO3, dans la formulation de milieux de cryoconservation synthétiques, pour la cryoconservation des embryons bovins. Ces travaux ont permis de mettre en évidence que le profil lipidique des embryons est fortement impacté par le processus de production in vitro et les protocoles de cryoconservation. La modulation du profil lipidique par l’ajout d’oméga 3 n’impacte pas le développement et la cryosurvie des embryons mais semble bénéfique à l’obtention d’embryons de meilleure qualité. Enfin, le CRYO3 est un candidat intéressant permettant de se passer des milieux habituels, contenant des dérivés de produits animaux, tout en améliorant la cryosurvie des embryons.Today, cryopreservation of bovine embryo is essential to ensure its preservation while awaiting their valuation. However, the quality of the in vitro produced embryos is inferior to those in vivo produced, which does not allow to obtained equivalent gestation rate. During the freezing process, the membranes, mainly composed of lipids, undergo physical and chemical stress which can lead to lysis of embryonic cells. In this way, in order to improve the quality of in vitro produced bovine embryos, two tasks were carried out in parallel: (1) The acquisition of new scientific knowledge on the lipid profile of in vitro produced bovine embryos and the modulation of the lipid profile by the addition of omega 3 fatty acid with the objectives of increasing membrane fluidity and reducing the embryo cryosensitivity; (2) the acquisition of new scientific knowledge on the interest of CRYO3, in the formulation of synthetic media, for the cryopreservation of bovine embryos in vitro produced and biopsied. This work has demonstrated that the lipid profile of embryos is strongly impacted by the in vitro development and cryopreservation protocol. Modulating the lipid profile by adding omega 3 does not impact the development and cryosurvival of embryos but appears to be beneficial for improving embryonic quality. Finally, CRYO3 is an interesting candidate, making it possible to dispense with the usual media, containing derivatives of animal products, while improving the cryosurvival of the embryos

Cryoconservation d’embryons bovins produits in vitro et biopsiés

Today, cryopreservation of bovine embryo is essential to ensure its preservation while awaiting their valuation. However, the quality of the in vitro produced embryos is inferior to those in vivo produced, which does not allow to obtained equivalent gestation rate. During the freezing process, the membranes, mainly composed of lipids, undergo physical and chemical stress which can lead to lysis of embryonic cells. In this way, in order to improve the quality of in vitro produced bovine embryos, two tasks were carried out in parallel: (1) The acquisition of new scientific knowledge on the lipid profile of in vitro produced bovine embryos and the modulation of the lipid profile by the addition of omega 3 fatty acid with the objectives of increasing membrane fluidity and reducing the embryo cryosensitivity; (2) the acquisition of new scientific knowledge on the interest of CRYO3, in the formulation of synthetic media, for the cryopreservation of bovine embryos in vitro produced and biopsied. This work has demonstrated that the lipid profile of embryos is strongly impacted by the in vitro development and cryopreservation protocol. Modulating the lipid profile by adding omega 3 does not impact the development and cryosurvival of embryos but appears to be beneficial for improving embryonic quality. Finally, CRYO3 is an interesting candidate, making it possible to dispense with the usual media, containing derivatives of animal products, while improving the cryosurvival of the embryos.Aujourd’hui, la cryoconservation de l’embryon bovin est primordiale pour assurer sa préservation dans l’attente de sa valorisation. Cependant, la qualité des embryons produits in vitro est inférieure à celle de ceux produits in vivo ne permettant pas l’obtention de taux de gestation équivalents. Lors du processus de congélation, les membranes, majoritairement composées de lipides, subissent des contraintes physiques et chimiques pouvant aboutir à une lyse des cellules embryonnaires. Ainsi, afin d’améliorer la qualité des embryons bovins produits in vitro et biopsiés, deux tâches ont été menées en parallèle : (1) l’acquisition de nouvelles connaissances scientifiques sur le profil lipidique des embryons produits in vitro et la modulation de ce profil par l’ajout d’acides gras de type oméga 3 avec pour objectifs d’augmenter la fluidité membranaire et de diminuer la cryosensibilité des embryons; (2) l’acquisition de nouvelles connaissances scientifiques sur l’intérêt du CRYO3, dans la formulation de milieux de cryoconservation synthétiques, pour la cryoconservation des embryons bovins. Ces travaux ont permis de mettre en évidence que le profil lipidique des embryons est fortement impacté par le processus de production in vitro et les protocoles de cryoconservation. La modulation du profil lipidique par l’ajout d’oméga 3 n’impacte pas le développement et la cryosurvie des embryons mais semble bénéfique à l’obtention d’embryons de meilleure qualité. Enfin, le CRYO3 est un candidat intéressant permettant de se passer des milieux habituels, contenant des dérivés de produits animaux, tout en améliorant la cryosurvie des embryons

Metabolomic profile of oviductal extracellular vesicles across the estrous cycle in cattle

Oviductal extracellular vesicles (oEVs) have been proposed as key modulators of gamete/embryo maternal interactions. The aim of this study was to examine the metabolite content of oEVs and its regulation across the estrous cycle in cattle. Oviductal EVs were isolated from bovine oviducts ipsilateral and contralateral to ovulation at four stages of the estrous cycle (post-ovulatory stage, early and late luteal phases, and pre-ovulatory stage). The metabolomic profiling of EVs was performed by proton nuclear magnetic resonance spectroscopy (NMR). NMR identified 22 metabolites in oEVs, among which 15 were quantified. Lactate, myoinositol, and glycine were the most abundant metabolites throughout the estrous cycle. The side relative to ovulation had no effect on the oEVs' metabolite concentrations. However, levels of glucose-1-phosphate and maltose were greatly affected by the cycle stage, showing up to 100-fold higher levels at the luteal phase than at the peri-ovulatory phases. In contrast, levels of methionine were significantly higher at peri-ovulatory phases than at the late-luteal phase. Quantitative enrichment analyses of oEV-metabolites across the cycle evidenced several significantly regulated metabolic pathways related to sucrose, glucose, and lactose metabolism. This study provides the first metabolomic characterization of oEVs, increasing our understanding of the potential role of oEVs in promoting fertilization and early embryo development

Metabolomic Profile of Oviductal Extracellular Vesicles across the Estrous Cycle in Cattle

International audienceOviductal extracellular vesicles (oEVs) have been proposed as key modulators of gamete/embryo maternal interactions. The aim of this study was to examine the metabolite content of oEVs and its regulation across the estrous cycle in cattle. Oviductal EVs were isolated from bovine oviducts ipsilateral and contralateral to ovulation at four stages of the estrous cycle (post-ovulatory stage, early and late luteal phases, and pre-ovulatory stage). The metabolomic profiling of EVs was performed by proton nuclear magnetic resonance spectroscopy (NMR). NMR identified 22 metabolites in oEVs, among which 15 were quantified. Lactate, myoinositol, and glycine were the most abundant metabolites throughout the estrous cycle. The side relative to ovulation had no effect on the oEVs' metabolite concentrations. However, levels of glucose-1-phosphate and maltose were greatly affected by the cycle stage, showing up to 100-fold higher levels at the luteal phase than at the peri-ovulatory phases. In contrast, levels of methionine were significantly higher at peri-ovulatory phases than at the late-luteal phase. Quantitative enrichment analyses of oEV-metabolites across the cycle evidenced several significantly regulated metabolic pathways related to sucrose, glucose, and lactose metabolism. This study provides the first metabolomic characterization of oEVs, increasing our understanding of the potential role of oEVs in promoting fertilization and early embryo development

Effect of DHA on the quality of In vitro produced bovine embryos

International audienceDocosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that improves fertility by increasing membrane fluidity. Moreover, embryos produced by donor females supplied with n-3 PUFA did not show any difference in terms of the lipid profile after 7 days of culture. The present study aimed to investigate the effects of DHA (20 and 100 mM) coupled with carnosine (5 mg/mL), an antioxidant, during oocyte maturation and embryo development on the developmental and cryosurvival rates and the number of pluripotent cells. Free fatty acid receptor-4 (FFAR4), which is able to bind DHA, was visualised by immunostaining. The addition of DHA in the in vitro development (IVD) medium decreased the percentage of pluripotent SOX2 positive cells compared with the control (8.4% vs. 10.9%) without affecting the number of cells (196.7 vs. 191.6 cells) or the developmental (20.9% vs. 23.9% blastocysts rate on D7) and cryosurvival rates (86.3% vs 86.2%). Such a decrease in pluripotent cells, relevant to the differentiation of the first lineage within the inner cell mass, represents an improvement in the embryo quality. On the contrary, embryos without any pluripotent SOX2-positive cells would not be able to achieve gestation. Future studies should follow up these results by carrying out embryo transfers to assess the beneficial effects of DHA supplementation

Effects of the donor factors and freezing protocols on the bovine embryonic lipid profile

International audienceAbstract Embryo lipid profile is affected by in vitro culture conditions, that lead to an increase in lipids. Efforts have been made to optimize embryo lipid composition as it is associated with their quality. The objective of this study was to evaluate whether the diet supplementation of donor cows (n-3 or n-6 PUFA), or the slow freezing protocols (ethylene glycol sucrose EG-S vs. glycerol trehalose GLY-TRE), or the physiological stage of the donor (nulliparous heifers vs. primiparous lactating cows) may impact the bovine embryo lipid profile. Lipid extracts of 97 embryos were individually analysed by liquid chromatography-high resolution mass spectrometry, highlighting 246 lipids including 85% being overabundant in cow embryos compared to heifer embryos. Among 105 differential lipids, 72 were overabundant after EG-S protocol, including a single glycerophosphate PA(32:1) representing 27.3% of the significantly modulated lipids, suggesting that it is degraded when GLY-TRE is used. No lipids were different according to the n-3 or n-6 supplementation of the donor cows. In conclusion, the embryonic lipid profile was mainly affected by the physiological stage of the donors and the slow freezing protocols. The overabundance of lipids in lactating cow embryos and the resulting lower quality of these embryos is consistent with the lower pregnancy rate observed in cows compared to heifers. Unlike GLY-TRE protocol, EG-S freezing allowed to preserve glycerophospholipids potentially improving the slow freezing of in vitro-produced embryos. Further studies are required to modulate embryo quality and freezability by modulating the lipidome and integrating all stages of embryonic production

Lipid profile of bovine grade-1 blastocysts produced either in vivo or in vitro before and after slow freezing process

International audienceCurrently, in vitro embryo production (IVP) is successfully commercially applied in cattle. However, the high sensitivity of embryos to cryopreservation in comparison to in vivo (IVD) embryos slows the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to in vitro culture conditions. The objective of this study was to evaluate the lipid composition of biopsied and sexed embryos, produced either in vivo or in vitro from the same Holstein heifers before and after a slow freezing protocol. Lipid extracts were analysed by liquid chromatography-high resolution mass spectrometry, which enabled the detection of 496 features. Our results highlighted a lipid enrichment of IVP embryos in triglycerides and oxidised glycerophospholipids and a reduced abundance in glycerophospholipids. The slow freezing process affected the lipid profiles of IVP and IVD embryos similarly. Lysophosphatidylcholine content was reduced when embryos were frozen/thawed. In conclusion, the embryonic lipid profile is impacted by IVP and slow freezing protocols but not by sex. Lysophosphatidylcholine seemed highly sensitive to cryopreservation and might contribute to explain the lower quality of frozen embryos. Further studies are required to improve embryo freezability by modulating the lipidome