Quality Of Bovine Embryos Research Articles

Nobiletin-induced partial abrogation of deleterious effects of AKT inhibition on preimplantation bovine embryo development in vitro†.

During preimplantational embryo development, PI3K/AKT regulates cell proliferation and differentiation and nobiletin modulates this pathway to promote cell survival. Therefore, we aimed to establish whether, when the AKT cascade is inhibited using inhibitors III and IV, nobiletin supplementation to in vitro culture media during the minor (2- to 8-cell stage, MNEGA) or major (8- to 16-cell stage, MJEGA) phases of EGA is able to modulate the development and quality of bovine embryos. In vitro zygotes were cultured during MNEGA or MJEGA phase in SOF + 5% FCS or supplemented with: 15μM AKT-InhIII; 10μM AKT-InhIV; 10μM nobiletin; nobiletin + AKT-InhIII; nobiletin + AKT-InhIV; 0.03% DMSO. Embryo development was lower in treatments with AKT inhibitors, while combination of nobiletin with AKT inhibitors was able to recover their adverse developmental effect and also increase blastocyst cell number. The mRNA abundance of GPX1, NFE2L2, and POU5F1 was partially increased in 8- and 16-cell embryos from nobiletin with AKT inhibitors. Besides, nobiletin increased the p-rpS6 level whether or not AKT inhibitors were present. In conclusion, nobiletin promotes bovine embryo development and quality and partially recovers the adverse developmental effect of AKT inhibitors, which infers that nobiletin probably uses another signaling cascade that PI3K/AKT during early embryo development in bovine.

Well-of-the-well (WOW) versus polyester mesh (PM): a comparison of single-embryo culture systems in bovines

Background: Mexico is innovating in the livestock industry through in vitro generation of bovine embryos with technologies such as well-of-the-well (WOW) and polyester mesh (PM) single-embryo culture systems. These techniques allow to maintain embryos in separate areas of a shared culture medium. Objective: To compare the quantity and quality of bovine embryos produced in WOW and PM culture systems versus the conventional (CG) culture system. Methods: In total, 345 embryos fertilized in vitro were evaluated for blastocyst yield in the three culture systems. To count blastocyst cell numbers, 69 embryos in each system were differentially stained for trophectoderm (TE), inner cell mass (ICM), and apoptotic cells. A qPCR gene expression analysis was performed for embryos in all three systems. Results: The WOW, PM and CG systems developed similar amount of blastocysts (41, 35 and 36%, respectively; p>0.05). Blastocysts in all three systems showed adequate amounts of ICM and apoptotic cells. Blastocysts in the PM system showed a greater number of TE cells [63.7 versus 58.6% in the CG system (p<0.05)]. Relative mRNA expression of the embryonic genes POUF5F1, GNAS and TP53 did not differ significantly among systems (p>0.05). The ATP5B expression was higher in WOW than in PM (p<0.05), but similar to CG (p>0.05). The TJP3 expression was higher in PM than in WOW and CG (p<0.05). Expression of ID2 and CLDN4 was higher in WOW than in PM and CG (p<0.05). The biplot graphic from Principal Component Analysis (PCA) revealed that CG was located near degenerated embryos, whereas PM was located near arrested embryos, larger ICM and TE, and TJP3 expression. The WOW was located toward blastocysts, morulae, and expression of CLDN4, ID2 and GNAS. Conclusion: Compared with CG, both the PM and WOW systems are good options for culturing single embryos in the bovine model. Moreover, the PCA results suggest that embryos developed in the WOW system have greater capacity for generating blastocysts with increased ability to form TE and ICM layers, which might improve implantation.

Cytokines That Serve as Embryokines in Cattle.

Simple SummaryThis review will explore how some cytokines also influence early embryonic development. We term these types of molecules as embryokines. Understanding how cytokines serve as embryokines could offer new opportunities to improve embryo development and the overall health of the embryo so that pregnancies will be retained after embryo transfer and so that viable offspring are produced. At least two cytokines may offer these benefits to bovine embryos produced in vitro. Additional cytokines also are identified in this review that may contain beneficial activities on bovine embryos.The term “embryokine” has been used to denote molecules produced by the endometrium, oviduct, or by embryo itself that will influence embryo development. Several cytokines have been identified as embryokines in cattle and other mammals. This review will describe how these cytokines function as embryokines, with special emphasis being placed on their actions on in vitro produced (IVP) bovine embryos. Embryokines are being explored for their ability to overcome the poor development rates of IVP embryos and to limit post-transfer pregnancy retention efficiencies that exist in IVP embryos. This review will focus on describing two of the best-characterized cytokines, colony-stimulating factor 2 and interleukin 6, for their ability to modify bovine embryo quality and confirmation, promote normal fetal development, and generate healthy calves. Additional cytokines will also be discussed for their potential to serve as embryokines.

Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

English

The study was conducted from December 2019 to May 2020 to evaluate the efficiency of local convectional semen and imported sexed semen on in vivo produced bovine embryo quality in Boran and HB cross breed donor cows in Bishoftu, Ethiopia. In this study, it was hypothesized that the breed of donor cows and types of semen can influence the quality of in vivo produced embryo as well as the number of transferable embryos. Randomized experimental design was employed, and a total of 20 donor cows (10 Boran and 10H*B cross) were superovulated and flushed excluding non-responsive donor cows. Donor cows were randomly assigned to two groups: Group-1- five donors inseminated with sexed semen and group-2- five other cows inseminated with convectional semen from each breed. The results of study showed that the embryo recovery rate was 68% in H*B crossbreds and 53% in Boran donor cows. A total of 121 embryos were produced of which, 60 were transferable embryos (36 from H*B crossbreds and 24 from Boran), 39 UFOs (31 from H*B crossbreds and 8 from Boran) and 22 degenerated embryos (12 from HB cross and 10 from Boran). The results of this study showed that the number of transferable embryos produced from donors inseminated with sexed semen and convectional semen was nearly similar. However, the number of defective embryos tended to be higher with sexed semen and the number of unfertilized oocytes is significantly (p<0.05) higher in crossbred cows. This finding suggests that farmer can use sexed semen to get sex specific dairy calves in both cattle genotypes. Key words: Breed, donor cows, superovulation.

Extracellular Vesicles from Follicular and Ampullary Fluid Isolated by Density Gradient Ultracentrifugation Improve Bovine Embryo Development and Quality.

Extracellular vesicles (EVs) have been isolated from follicular (FF) and ampullary oviduct fluid (AOF), using different isolation methods. However, it is not clear whether different purification methods can affect the functionality of resulting EVs. Here, we compared two methods (OptiPrep™ density gradient ultracentrifugation (ODG UC) and single-step size exclusion chromatography (SEC) (qEV IZON™ single column)) for the isolation of EVs from bovine FF and AOF. Additionally, we evaluated whether the addition of EVs derived either by ODG UC or SEC from FF or AOF during oocyte maturation would yield extra benefits for embryo developmental competence. The characterization of EVs isolated using ODG UC or SEC from FF and AOF did not show any differences in terms of EV sizes (40–400 nm) and concentrations (2.4 ± 0.2 × 1012−1.8 ± 0.2 × 1013 particles/mL). Blastocyst yield and quality was higher in groups supplemented with EVs isolated from FF and AOF by ODG UC, with higher total cell numbers and a lower apoptotic cell ratio compared with the other groups (p < 0.05). Supplementing in vitro maturation media with EVs derived by ODG UC from AOF was beneficial for bovine embryo development and quality.

52 Extracellular vesicles from oviduct and uterus in sequential invitro culture affects mitochondrial activity and lipid metabolism transcripts in bovine embryos

Oviducal fluid (OF) and uterine fluid (UF) improve the quality of embryos during invitro culture, and their extracellular vesicles (EV) may be involved in such an effect. We aimed to evaluate the effect of EV from OF and UF in sequential invitro culture on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid supplemented either with 3mg mL−1 BSA (n=1584) or 5% EV-depleted fetal calf serum (dFCS, n=1594) in absence or presence (BSAEV, n=1853 and dFCSEV, n=1473) of 3×105 EV mL−1 from OF (Day 1 to Day 4) and UF (Day 5 to Day 8), mimicking invivo conditions. EV from oviducts (early luteal phase) and uterine horns (mid luteal phase) from slaughtered heifers were isolated by size exclusion chromatography; size and concentration were assessed by nanotracking analysis (NTA) and morphology by transmission electron microscopy (TEM). Blastocyst rate was recorded on Days 7–8 and their quality was assessed for mitochondrial activity by staining with Mitotracker Deep Red (ThermoFisher Scientific), survival rate after vitrification/warming by invitro culture for up to 72h, and relative mRNA abundance of lipid metabolism-related transcripts by quantitative PCR. Housekeeping genes were H2AFZ and ACTB. Data were analysed by one-way ANOVA and Tukey test. TEM confirmed the presence and morphology of EVs, and NTA indicated mode size and concentration of particles (137.2 and 151.2 nm; 2.97×1010 and 7.98×1010 particles mL−1, for OF and UF, respectively). Blastocyst yield was lower (P&amp;lt;0.05) in BSA groups compared with dFCS groups (BSA: 16.2±1.5 and 31.0±1.9; BSAEV: 14.1±1.6 and 26.2±2.0% vs. dFCS: 30.5±2.0 and 40.6±2.4; dFCSEV: 31.1±2.5 and 39.8±2.7%, Day 7 and Day 8, respectively), irrespective of EV supplementation. Blastocyst mitochondrial activity was increased (P&amp;lt;0.05) by EV in dFCSEV compared with the other groups. No differences in survival rate after vitrification/warming were found (range at 72 h: 67.1±8.1 to 87.8±5.7%). PPARGC1B was downregulated and ACC upregulated by EV, irrespective of protein source in medium (P&amp;lt;0.05). In contrast, EV affected some transcripts depending on the protein source in the medium (CD36 upregulated in dFCSEV, downregulated in BSAEV; PLIN2 downregulated in dFCSEV and ATGL downregulated in BSAEV, P&amp;lt;0.05). In conclusion, mimicking physiological conditions using EV from OF and UF in sequential IVC does not affect development but improves embryo quality by increasing blastocysts’ mitochondrial activity and favours the expression of specific lipid metabolism transcripts. Functional effects of EV may be influenced by the protein source in the medium. This research was funded by MINECO-Spain AGL2015-70140-R, PID2019-111641RB-I00, RTI2018-093548-B-I00; YN Cajas, SENESCYT-Ecuador; CLV Leal, FAPESP-Brazil 2017/20339-3, CNPq-Brazil 304276/2018-9.

Three-dimensional live imaging of bovine embryos by optical coherence tomography

While embryo transfer (ET) is widely practiced, many of the transferred embryos fail to develop in cattle. To establish a more effective method for selecting bovine embryos for ET, here we quantified morphological parameters of living embryos using three-dimensional (3D) images non-invasively captured by optical coherence tomography (OCT). Seven Japanese Black embryos produced by in vitro fertilization that had reached the expanded blastocyst stage after 7 days of culture were transferred after imaged by OCT. Twenty-two parameters, including thickness and volumes of the inner cell mass, trophectoderm, and zona pellucida, and volumes of blastocoel and whole embryo, were quantified from 3D images. Four of the seven recipients became pregnant. We suggest that these 22 parameters can be potentially employed to evaluate the quality of bovine embryos before ET.

TET3 overexpression facilitates DNA reprogramming and early development of bovine SCNT embryos.

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

Inverse relationship between autophagy and CTSK is related to bovine embryo quality.

Improving the quality and the developmental competence of in vitro produced (IVP) embryos is an indispensable goal for assisted reproductive technology. Autophagy is a major protective mechanism for intracellular degradation of unnecessary cytoplasmic components. Autophagy ends by the fusion between autophagic vacuoles and lysosomes, allowing the degradation of the cargo by lysosomal enzymes, especially the cathepsins (CTSs). However, it is still unclear how autophagy and cathepsin K (CTSK) relate to embryo development. This study evaluated (1.) the activities of autophagy and CTSK in relation to bovine embryo quality and (2.) the effect of autophagy induction and/or CTSK inhibition on preimplantation embryo development and quality. We show here that good-quality embryos exhibited a greater autophagic activity and less CTSK activity compared to poor-quality embryos. Blastomeres of an individual embryo may vary in their quality. Good quality blastomeres showed an increased autophagic activity and decreased CTSK activity compared to poor-quality blastomeres within the same embryo at different developmental stages. Importantly, induction of autophagy and/or inhibition of CTSK improved the developmental rate (increased blastocyst and hatching rates) and the quality (increased total cell number and decreased the percentage of apoptotic cells) of IVP bovine embryos. These results demonstrate a promising approach to selectively isolate good-quality embryos and improve the efficiency of IVEP of cattle embryos.

Anethole Supplementation During Oocyte Maturation Improves In Vitro Production of Bovine Embryos

Oxidative stress is one of the most detrimental factors that affect oocyte developmental competence and embryo development in vitro. The impact of anethole supplementation to in vitro maturation (IVM) media on oocyte maturation and further bovine in vitro embryo production was investigated. Oocytes of slaughterhouse-derived bovine ovaries were placed in IVM with anethole at different concentrations of 30 (AN30), 300 (AN300), and 2000 μg/mL (AN2000), or without (control treatment). The oocytes were assessed for maturation rates, and for reactive oxygen species (ROS) and ferric reducing antioxidant power (FRAP) levels, and mitochondrial membrane potential. Embryo development was assessed by cleavage and blastocyst rates, and embryo cell number. The percentage of metaphase II oocytes were similar among the treatments (range, 77%-96%). Anethole at 300 μg/mL was the only treatment that yielded higher cleavage and embryo development (morula and blastocyst) rates compared to the control treatment. The ROS production in the oocytes after maturation did not differ among treatments. However, oocytes treated with anethole at 300 μg/mL had higher (P < .05) FRAP and mitochondrial membrane potential compared to the control treatment. Furthermore, AN300 treatment increased (P < .05) the average number of total cells in blastocysts compared to the control and AN30 treatments. The use of anethole at 300 μg/mL during IVM is suggested to improve the quantity and quality of bovine embryos produced in vitro. The beneficial effects of anethole on embryonic developmental competence in vitro seems to be related to its capacity to regulate the redox balance and improve mitochondrial function in oocytes and embryos.

Supplementation of insulin-transferrin-sodium selenite in culture medium improves the hypothermic storage of bovine embryos produced in vitro

Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations. After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P < 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P < 0.05) total cell number per blastocyst and lesser apoptotic cells number. Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P < 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P < 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P < 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P < 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage.

Low-dose Roundup induces developmental toxicity in bovine preimplantation embryos in vitro.

Roundup is a widely used glyphosate-based herbicide worldwide. Roundup residues can be detected in the organs and urine of animals. However, its toxicity on mammalian preimplantation embryos has not been well investigated. Here, we show Roundup impairs the development and quality of bovine preimplantation embryos in a dose-dependent manner. Exposure to the agricultural recommended doses of Roundup caused in vitro developmental arrest and quick death of bovine embryos. Furthermore, even a very low concentration (0.9ppm) of Roundup was harmful to bovine preimplantation development. In addition, Roundup increases intracellular calcium levels and induces oxidative stress and apoptosis in bovine embryos. Even if the embryos developed to morphologically normal blastocysts when cultured with low concentrations of Roundup, abnormal intracellular calcium and oxidative stress could be detected inside the embryos and led to an increased incidence of apoptosis in the blastocysts. These data suggest Roundup residues from the agricultural application are potentially dangerous to mammalian preimplantation embryos.

76 Extracellular vesicles from oviduct and uterus in sequential culture improve the quality of bovine embryos produced invitro

Extracellular vesicles (EVs) are released by cells and transport cargo that affect functions of other cells. Oviductal fluid (OF) and uterine fluid (UF) have been shown to improve quality of embryos during invitro culture (Hamdi et al. 2017 Reprod. Fertil. Dev. 30, 935-945) which may be due to their content of EV (Lopera-Vásquez et al. 2017 Reproduction 153, 461-470). Thus, the aim of this study was to evaluate the effect of EVs from OF and UF on a sequential invitro culture system on the development and quality of bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF) supplemented with 3mgmL−1 bovine serum albumin (BSA; n=1228) or 5% EV-depleted fetal calf serum (dFCS, n=1261) in the presence (BSAEV, n=1265 and dFCSEV, n=1253) or absence of 3×105 EVmL−1 from OF (Day 1 to Day 4) and UF (Day 4 to Day 9), mimicking invivo conditions. The EVs pooled from 5 oviducts (early luteal phase) and 5 uterine horns (middle luteal phase) from slaughtered heifers were isolated by a Size Exclusion Chromatography kit (Hansa BioMed). The EV size and concentration were assessed by the nanotracking analysis system and morphology by transmission electron microscopy. Embryo development was recorded on Days 7/9. Day 7/8 blastocysts were assessed for quality by staining with (a) Hoechst 33342 (10 µgmL−1, 30min) for total cell number, (b) Bodipy 493/503 (20 µgmL−1, 1h) for lipid content (lipid droplet area in µm2), and (c) for survival rate after vitrification/warming. Data were analysed by one-way ANOVA and Tukey test. The EV concentration was 2.97 and 7.98×1010 particlesmL−1, and mode size 137.2 and 151.2nm for OF and UF, respectively. Transmission electron microscopy confirmed EV presence and size, showing typical cup-shaped morphology. Blastocyst yield was lower (P&amp;lt;0.05) on Day 7 in the BSA groups (BSA: 15.7±1.9 and BSAEV: 15.2.4%) compared with serum groups (dFCS: 28.1±2.6 and dFCSEV: 30.1±2.9%) irrespective of EV supplementation; however, these differences were compensated at Days 8 and 9 (range: 30.0±3.2-40.8±3.9%). The EVs increased (P&amp;lt;0.05) blastocyst total cell number in dFCSEV (152.6±2.9) and BSAEV (140.5±1.5) compared with dFCS (117.9±2.0) and BSA groups (122.4±1.1). However, lipid content was decreased (P&amp;lt;0.05) in the presence of EVs only in dFCSEV (0.231±0.05µm2) compared with BSA (0.393±0.03µm2) and BSAEV (0.379±0.03µm2) groups. The dFCS did not differ from any group (0.371±0.05µm2; P&amp;gt;0.05). Blastocyst survival after vitrification/warming was high in all groups up to 72h (range: 80.0±3.8-100%; P&amp;gt;0.05). In conclusion, mimicking physiological conditions using EV from OF and UF during invitro culture does not affect development but improves embryo quality by increasing blastocyst total cell numbers and decreasing lipid contents. These results provide evidence of the association of the reproductive tract environment and developing embryo, confirming embryo-maternal communication. Funding was provided by MINECO-Spain AGL2015-70140-R; Y. N. Cajas, SENESCYT-Ecuador; C. L. V. Leal, FAPESP-Brazil 2017/20339-3.

65 Effect of oviductal fluid extracellular vesicle supplementation during invitro culture on development and quality of bovine embryos

Recently, it has been postulated that oviductal extracellular vesicles (oEV) might act as natural nanoshuttles bringing key components (small noncoding RNAs and proteins) of the oviduct into gametes and embryos. Furthermore, co-incubation of frozen-thawed oEV with invitro-produced bovine embryos was reported to increase blastocyst rate and quality (Almiñana et al. 2017 Reproduction 154, 153-168). The objective of this study was to determine the dose-dependent effect of oEV supplementation of embryo culture medium on the invitro development and cryotolerance of embryos. Briefly, oEV were isolated by ultracentrifugation from a pool of oviductal fluids (8 cows/sample) collected at the slaughterhouse at the post-ovulatory stage and ipsilateral to ovulation and stored at −80°C until used. Slaughterhouse-derived bovine oocytes were invitro matured and fertilised with frozen-thawed semen from one bull (4 replicates; 194 presumptive zygotes per group), according to our standard procedures. After IVF, groups of presumptive zygotes (n=20/drop) were cultured under humidified air with 5% CO2, 5% O2 at 38.8°C for 7 days in 30µL of synthetic oviductal fluid-bovine serum albumin supplemented with oEV at different protein concentrations: 0.5, 0.05, or 0.005mgmL−1 and without (control). Cleavage rates were evaluated on Day 2 and blastocyst rates were assessed on Days 6 and 7 (IVF as Day 0). At Day 7, expanded grade 1 blastocysts were evaluated (International Embryo Technology Society classification) and embryos at the expanded grade 1 blastocyst stage were slow frozen in 1.5M ethylene glycol + 0.1M sucrose and stored in liquid nitrogen. For cryotolerance evaluation, embryos were thawed and cultured for 48h in synthetic oviductal fluid-bovine serum albumin + 1% estrous cow serum. Hatching rates were assessed at 48h post-thawing. Data were analysed by a logistic regression mixed model (SAS, SAS Institute Inc.; Glimmix procedure) followed by post-hoc Tukey for multiple comparisons. Differences were considered significant at P&amp;lt;0.05. No differences were observed among the different oEV concentrations tested for cleavage and Day 6 blastocysts. A tendency (P=0.0535) was observed for Day 7 blastocyst rates (19.1±2.8, 29.4±3.3, 16.0±2.6, and 20.6±2.9 for 0.5, 0.05, 0.005mgmL−1, and control, respectively) in favour of the 0.05mgmL−1 group. However, a significant difference (P&amp;lt;0.0288) for Day 7 grade 1 expanded blastocyst rates in favour of the 0.05mgmL−1 group was observed (5.2±1.6, 12.9±2.4, 3.1±1.2, and 9.8±2.2 for 0.5, 0.05, 0.005mgmL−1, and control, respectively). For cryopreserved embryos, hatching rates of frozen-thawed embryos were not significant among experimental groups (81.6±10.2 (n=19), 89.6±6.6 (n=27), 77.2±12.2 (n=10), and 60.2±13.6 (n=23) for 0.5, 0.05, 0.005mgmL−1, and control, respectively). In conclusion, under our experimental conditions, the supplementation of the embryo culture medium with frozen-thawed post-ovulatory oEV at the protein concentration of 0.05mgmL−1 increased the Day 7 grade 1 expanded blastocyst rate. Moreover, we showed a tendency to improve Day 7 blastocyst rates but with no apparent effects on the cryotolerance of embryos. This work was supported by APIS GENE.

70 Trolox during invitro maturation of bovine oocytes protects developing embryos from palmitic acid-induced lipotoxicity

Maternal metabolic disorders are associated with elevated concentrations of palmitic acid (PA), which is known to jeopardize bovine oocyte and embryo development and quality. Analyses of PA-exposed bovine oocytes and embryos point toward oxidative stress (OS)-related pathways. Previous research has shown that the detrimental effects of PA exposure during oocyte IVM cannot be alleviated by antioxidant supplementation, e.g. Trolox (TR, water-soluble vitamin E), during IVM or invitro culture (IVC). In contrast, supplementing TR during IVM could protect developing zygotes from PA-induced lipotoxicity by increasing their development into 4-cell embryos and Day 8 blastocysts. In the present study, we examined the effects of TR during IVM or IVC on PA-exposed oocytes and embryos by evaluating OS and mitochondrial membrane potential (MMP) of the produced ≥2-cell Day 2 embryos (Table 1). Bovine cumulus-oocyte complexes (COCs) were matured, fertilised, and cultured in 2 experiments. In Experiment 1, COCs were exposed to pathophysiological follicular PA concentrations (150 µM), and subsequent embryos were cultured under solvent control (ethanol) conditions (PA-SC). The TR treatment was applied during IVM or IVC (100 µM; PATR-SC, PA-TR). In Experiment 2, COCs were matured under SC conditions, and subsequent embryos were exposed to pathophysiological oviductal PA concentrations (230 µM; SC-PA). The TR treatment was applied during IVM or IVC (100 µM; TR-PA, SC-PATR). In each experiment, a SC was included (SC-SC). A total of 126 and 137 Day 2 embryos were stained with JC-1 and CellRox Deep Red in Experiment 1 and Experiment 2 (3 repeats), respectively. The MMP was evaluated as active/total mitochondria and OS as OS/total mitochondria and analysed by one-way ANOVA. Exposure of oocytes and embryos to PA significantly increased OS and MMP in Day 2 embryos compared with controls. Regardless of the moment of PA exposure, TR treatment during IVM increased MMP even more. The increased MMP levels in PA-exposed oocytes and embryos were not influenced by TR treatment during IVC. Regardless of the antioxidant effect of TR, increased embryonic OS levels observed in PA-exposed oocytes and embryos could not be reversed to control levels by TR treatment during IVM or IVC. However, when OS levels were expressed on active mitochondria, we found that TR treatment before the PA insult (TR-PA) generated Day 2 embryos that displayed significantly lower OS/active mitochondria rates compared with SC-PA-treated embryos. Taken together, we conclude that the combination of relatively low OS levels with highly active mitochondria may be a mechanism implicated in the protective effect of TR before the PA insult on embryo developmental competence. Table 1.Effects of TR during IVM or IVC on PA-exposed oocytes and embryos in Experiments 1 and 2 Controls PA exposure TR during IVM TR during IVC SC-SC PA-SC PATR-SC PA-TR Exp. 1 MMP 0.39±0.02a 0.53±0.02b 0.65±0.02c 0.50±0.02b OS 0.55±0.04a 0.68±0.04b 0.78±0.03b 0.59±0.03ab SC-SC SC-PA TR-PA SC-PATR Exp. 2 MMP 0.39±0.02a 0.50±0.02b 0.65±0.02c 0.58±0.02b OS 0.55±0.04a 0.69±0.04b 0.70±0.03b 0.71±0.02b abcDifferent letters in the same row indicate significant differences (P&amp;lt;0.05).

The PPARδ Agonist GW501516 Improves Lipolytic/Lipogenic Balance through CPT1 and PEPCK during the Development of Pre-Implantation Bovine Embryos.

The PPARs (peroxisome proliferator-activated receptors) play critical roles in the regulation of lipid and glucose metabolism. PPARδ, a member of the PPARs family, is associated with decreased susceptibility to ectopic lipid deposition and is implicated in the regulation of mitochondrial processes. The current study aimed to determine the role of PPARδ in fatty acid β-oxidation and its influence on PEPCK for the lipogenic/lipolytic balance during in vitro bovine oocyte maturation and embryo development. Activation of PPARδ by GW501516, but not 2-BP, was indicated by intact embryonic PEPCK (cytosolic) and CPT1 expression and the balance between free fatty acids and mitochondrial β-oxidation that reduced ROS and inhibited p-NF-κB nuclear localization. Genes involved in lipolysis, fatty acid oxidation, and apoptosis showed significant differences after the GW501516 treatment relative to the control- and 2-BP-treated embryos. GSK3787 reversed the PPARδ-induced effects by reducing PEPCK and CPT1 expression and the mitochondrial membrane potential, revealing the importance of PPARδ/PEPCK and PPARδ/CPT1 for controlling lipolysis during embryo development. In conclusion, GW501516-activated PPARδ maintained the correlation between lipolysis and lipogenesis by enhancing PEPCK and CPT1 to improve bovine embryo quality.

Hyaluronic acid and epidermal growth factor improved the bovine embryo quality by regulating the DNA methylation and expression patterns of the focal adhesion pathway.

Focal adhesion pathway is one of the key molecular pathways affected by suboptimal culture conditions during embryonic development. The epidermal growth factor (EGF) and hyaluronic acid (HA) are believed to be involved in the focal adhesion pathway function by regulating the adherence of the molecules to the extracellular matrix. However, regulatory and molecular mechanisms through which the EGF and HA could influence the embryo development is not clear. Therefore, this study aimed to investigate the effect of continued or stage specific supplementation of EGF and/or HA on the developmental competence and quality of bovine preimplantation embryos and the subsequent consequences on the expression and DNA methylation patterns of genes involved in the focal adhesion pathway. The results revealed that, the supplementation of EGF or HA from zygote to the blastocysts stage reduced the level of reactive oxygen species and increased hatching rate after thawing. On the other hand, HA decreased the apoptotic nuclei and increased blastocyst compared to EGF supplemented group. Gene expression and DNA methylation analysis in the resulting blastocysts indicated that, combined supplementation of EGF and HA increased the expression of genes involved in focal adhesion pathway while supplementation of EGF, HA or a combination of EGF and HA during the entire preimplantation period changed the DNA methylation patterns of genes involved in focal adhesion pathway. On the other hand, blastocysts developed in culture media supplemented with EGF + HA until the 16-cell stage exhibited higher expression level of genes involved in focal adhesion pathway compared to those supplemented after the 16-cell stage. Conversely, the DNA methylation level of candidate genes was increased in the blastocysts obtained from embryos cultured in media supplemented with EGF + HA after 16-cell stage. In conclusion, supplementation of bovine embryos with EGF and/or HA during the entire preimplantation period or in a stage specific manner altered the DNA methylation and expression patterns of candidate genes involved in the focal adhesion pathway which was in turn associated with the observed embryonic developmental competence and quality.

Frozen-thawed ampullary cell monolayer improves bovine embryo in vitro development and quality.

The aim of this study was to evaluate the effects of different timing for frozen-thawed bovine ampullary epithelial cell (BAEC) and bovine oviductal epithelial cell (BOEC) co-culture on the development and quality of bovine embryos produced in vitro. Embryo development was assessed by day 8 blastocyst yield, whereas embryo quality was determined using blastocyst differential cell count, cryotolerance and the expression of selected genes related to embryo quality. The results showed that the presence of BAECs during the last 6 h of in vitro maturation (IVM) increased blastocyst yield and survival of the vitrified-warmed blastocysts. In addition, embryos produced in the presence of BAECs during the last 6 h of IVM or in the presence of BOECs during the first 4 days of in vitro culture (IVC) showed a greater number of trophectoderm cells and a greater inner cell mass. In terms of gene expression, IFN-T was downregulated and PLAC8, AQP3 and ATP1A1 were upregulated in the presence of the BAECs during the last 6 h of the IVM and/or in the presence of BOECs during the first 4 days of IVC. In conclusion, co-culturing bovine oocytes with a frozen-thawed ampullary cell monolayer during the last 6 h of maturation increased blastocyst yield and quality.

Antioxidant activity of oily extract obtained from Lippia origanoides improves the quality of bovine embryos produced in vitro

ABSTRACT The aim of this study was to evaluate the supplementation of embryo culture medium with antioxidant obtained from oily extract of Lippia origanoides on in vitro blastocyst development and quality. Oocytes collected from slaughterhouse ovaries were matured and fertilized in vitro following standard laboratory procedures. Zygotes were cultured in SOF medium supplemented according to the following treatments: T1 embryo culture medium without antioxidant supplementation; T2)50μM/mL Cysteamine; T3)2.5μg/mL; T4)5.0μg/mL and T5)10.0μg/mL of antioxidant obtained from oily extract of Lippia origanoides. On the seventh day of culture, the blastocysts were fixed and evaluated for apoptosis rates, number of total cell and inner cell mass cells by means of the TUNEL Test. The use of antioxidants during cultivation did not increase (P&gt; 0.05) the final blastocyst production rate. The treatments T2, T3, T4 and T5 had the lowest (P&lt; 0.05) apoptotic indexes (4.5±1.1%, 8.4±2.5%, 3.4±1.1% and 5.5±0.9%, respectively) when compared to T1 treatment (10.0±1.4%). The number of inner cell mass did not differ (P&gt; 0.05) among embryos from different treatments. The addition of antioxidant obtained from oily extract of Lippia origanoides reduces the apoptosis rate and improves the quality without increasing the total in vitro production of bovine embryos.