Effects and mechanisms of action of dietary n-3 polyunsaturated fatty acids, in dairy cattle, on oocyte quality in terms embryo production

Ce travail a pour objectif d'étudier les effets d’une supplémentation en acides gras polyinsaturés (AGPI) n-3 à longues chaines sur la production d’embryons chez les vaches laitières. Le régime enrichi en AGPI n-3 a eu tendance à augmenter le nombre moyen des complexes ovocyte-cumulus récupérés par vache par session d’OPU par rapport au groupe témoin AGPI n-6. Une tendance à augmenter le nombre moyen par vache par session d’OPU de blastocystes et de blastocystes expansés avec une augmentation significative de blastocystes de qualité 1 et 2 ont été notés dans le groupe n-3. De même, les taux de blastocystes, de blastocystes expansés et de blastocystes de qualité 1 et 2 ont été significativement augmentés dans le groupe n-3. Nous avons aussi montré que l’addition d’acide docosahexaénoïque (DHA, C22:6 n-3) à une faible concentration (1μM) pendant la maturation in vitro des ovocytes bovins augmente significativement le taux de clivage et le taux et la qualité de blastocyste. La stimulation de FFAR4 (free fatty acid receptor 4), au cours de la MIV, par un agoniste spécifique (TUG 891), a entrainé des effets bénéfiques similaires à ceux du DHA (1μM) sur la compétence ovocytaire. Cela suggère que l’effet bénéfique du DHA pourrait passer, au moins partiellement, par l’activation du FFAR4.We aimed to investigate whether dietary n-3 PUFA, in Prim‘Holstein dairy cattle in lactation, could influence reproductive performance in terms of embryo production. We showed that n-3 PUFA-enriched diet had a tendency to increase the average number of oocyte-cumulus complexes recovered per cow per OPU session. A tendency to increase the average numbers per cow per OPU session of blastocysts and expanded blastocysts with significantly increase of blastocysts of quality 1 and 2 were noted in the n-3 group compared to the n-6 group. Similarly, the rate of blastocysts, expanded blastocysts and blastocysts of quality 1 and 2 were significantly increased in the n-3 group compared to the n-6 group. We also showed that the addition of docosahexaenoic acid (DHA, C22:6 n-3) at low concentration (1μM) during IVM of bovine oocytes increased cleavage rate, blastocyst rate and embryo quality. DHA and free fatty acid receptor 4 (FFAR4) agonist TUG- 891 (1 or 5 μM) supplied during IVM similarly improved in vitro embryo development, suggesting that FFAR4 may, at least partially, mediate DHA beneficial effects on OCC, through activation of signaling pathways

Effets et mécanismes d'action d'une supplémentation en acide gras polyinsaturés n-3 sur la qualité ovocytaire, dans le contexte de la production d'embryons chez les vaches laitières

We aimed to investigate whether dietary n-3 PUFA, in Prim‘Holstein dairy cattle in lactation, could influence reproductive performance in terms of embryo production. We showed that n-3 PUFA-enriched diet had a tendency to increase the average number of oocyte-cumulus complexes recovered per cow per OPU session. A tendency to increase the average numbers per cow per OPU session of blastocysts and expanded blastocysts with significantly increase of blastocysts of quality 1 and 2 were noted in the n-3 group compared to the n-6 group. Similarly, the rate of blastocysts, expanded blastocysts and blastocysts of quality 1 and 2 were significantly increased in the n-3 group compared to the n-6 group. We also showed that the addition of docosahexaenoic acid (DHA, C22:6 n-3) at low concentration (1µM) during IVM of bovine oocytes increased cleavage rate, blastocyst rate and embryo quality. DHA and free fatty acid receptor 4 (FFAR4) agonist TUG-891 (1 or 5 µM) supplied during IVM similarly improved in vitro embryo development, suggesting that FFAR4 may, at least partially, mediate DHA beneficial effects on OCC, through activation of signaling pathways.Ce travail a pour objectif d'étudier les effets d’une supplémentation en acides gras polyinsaturés (AGPI) n-3 à longues chaînes sur la production d’embryons chez les vaches laitières. Le régime enrichi en AGPI n-3 a eu tendance à augmenter le nombre moyen des complexes ovocyte-cumulus récupérés par vache par session d’OPU par rapport au groupe témoin AGPI n-6. Une tendance à augmenter le nombre moyen par vache par session d’OPU de blastocystes et de blastocystes expansés avec une augmentation significative de blastocystes de qualité 1 et 2 ont été notés dans le groupe n-3. De même, les taux de blastocystes, de blastocystes expansés et de blastocystes de qualité 1 et 2 ont été significativement augmentés dans le groupe n-3. Nous avons aussi montré que l’addition d’acide docosahexaénoïque (DHA, C22:6 n-3) à une faible concentration (1μM) pendant la maturation in vitro des ovocytes bovins augmente significativement le taux de clivage et le taux et la qualité de blastocyste. La stimulation de FFAR4 (free fatty acid receptor 4), au cours de la MIV, par un agoniste spécifique (TUG 891), a entrainé des effets bénéfiques similaires à ceux du DHA (1μM) sur la compétence ovocytaire. Cela suggère que l’effet bénéfique du DHA pourrait passer, au moins partiellement, par l’activation du FFAR4

Effet d’un acide gras oméga-3 (DHA) sur la compétence de l’ovocyte bovin

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Addition of omega-3 DHA during <em>in vitro</em> maturation affected embryo development

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FFAR4 is involved in docosahexaenoic acid effects on oocyte developmental potential during in vitro maturation

Besides affecting uterine environment, a direct effect of n-3 poly-unsaturated fatty acids (PU-FA) on the oocyte could enhance fertility. We previously showed that docosahexaenoic acid (DHA, C22:6 n-3, Sigma), when provided during in vitro maturation (IVM), improved oocyte developmental competence through possible effects on cytoplasm but not nuclear maturation and without affecting lipid metabolism gene expression in cumulus cells (CC) (Oseikria et al Theriogenology 85:1625-1634. 2016). DHA could act through several mechanisms of action: i.e. via surface fatty acid receptors (free fatty acid receptor 1 or 4, FFAR1 and 4) or sensors involving PPAR or NFkB pathways; via changes in composition of cell membrane phospholi-pids; via production of eicosanoids… The aim of the present work was to investigate whether the FFAR4 was involved in the DHA effects previously reported on oocyte quality. We there-fore investigated the effect of a specific agonist of the FFAR4, TUG-891, on embryo deve-lopment after IVF. The response of surrounding CC to DHA or TUG treatment was also stu-died by gene expression analyses. Oocyte cumulus complexes were collected from slaughtered cows. The protein FFAR4 was first localized by immunohistochemistry, by using a cus-tomized antibody produced specifically against the bovine protein. FFAR4 is expressed in CC and localized close to the cellular membrane, as expected. After 22h IVM with or without DHA 1 µM or TUG 1 and 5 µM oocytes were subjected to in vitro fertilization (IVF) and in vitro development in modified synthetic oviduct fluid supplemented with 10% fetal calf serum for 7 days. At day 7, both blastocyst and expanded blastocyst rates were significantly increased with either DHA 1µM or TUG 1 or 5 µM (logistic regression, P < 0.05). In order to decipher the DHA mechanisms linked to oocyte developmental competence, we then investi-gated the common pathways of DHA and TUG actions. Microarray hybridization of CC after 4h IVM in the presence or absence of 1µM DHA was performed (n = 4 samples per condi-tion). A customized 60K bovine microarray (Agilent technology) including 97.4% of Ensembl Bos taurus transcripts was used (GEO accession: GPL21724). Only 14 differentially expres-sed genes varied more than two-fold and were enriched in gene ontologies related to regula-tion of translation, RNA splicing and spliceosome formation, oxidation/reduction, actin cy-toskeleton organization and vesicle-mediated transport. The kinetic of expression of these genes is currently characterized by qRT-PCR analysis on CC samples at 0, 4, 10 and 24h IVM with or without DHA 1 µM, TUG 1 or 5 µM. Altogether the IVF data suggest that DHA exert its effect partly through FFAR4 on oocyte developmental competence. Also, we are studying the common transcriptomic modulation between DHA and TUG to provide insights on its detailed mechanism of action

N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle

Remerciements :Pôle STAR (Services Techniques d'Appui à la Recherche), Inra, UMR PRC, 37380 Nouzilly, Centre Val de LoireThe 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 (p&lt;0.05) oocyte cleavage rate as compared to control (86.1% vs. 78.8%, respectively) and the &gt;4-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 &gt;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

Beneficial effect of low dose docosahexaenoic acid during IVM on oocyte developmental potential and impact on cumulus cell transcriptome in cattle

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Risk of Chlamydia abortus transmission via embryo transfer using in vitro produced early bovine embryos

International audienceThe objectives of this study were to determine (i) whether Chlamydia (C) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (LETS) washing protocol.The experimentation was made twice. For each replicate 100 (8-16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 x 10(7) Chlamydia/ml of AB7 strain. After incubation for 18 h at 37 degrees C in an atmosphere of 5% CO2, the embryos were washed in accordance with the LETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000 xg. Each batch of washed embryos and each wash pellets were tested using PCR.C abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia-DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia-DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 +/- 9 x 10(3) bacteria/mL) than for batches of ZP-intact embryos (0.47 +/- 0.19 x 10(3) bacteria/mL).These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the LETS fails to remove it

Effects of an enriched n-3 polyunsaturated fatty acid diet on in vitro embryo production in dairy cowsEffects of an enriched n-3 polyunsaturated fatty acid diet on in vitro embryo production in dairy cows

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