In Vitro Culture Medium Research Articles

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1%, fibroblasts: 33.1%, control: 27.3%). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(-). In Expt. 3, addition of 10% fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2%; 10% FCS, 43.4%; 1% ADSC lysate, 34.2%; 10% ADSC lysate, 48.1%). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10% addition, corresponding to 1 x 105 cells/mL, is as effective as 10% FCS without a decrease in cryotolerance due to the increased lipid content.

PSXIII-14 Effect of melatonin supplementation during in vitro embryo culture on cleavage rates following in vitro maturation and fertilization of bovine oocytes

Abstract In vitro embryo production (IVEP) is an important advanced reproductive technology and has great potential in enhancing livestock production. However, in vitro culture (IVC) of embryos exposes them to stress resulting in compromising quality and efficiency of embryo production. Melatonin has been reported in several studies to reduce oxidative stress and improve the efficiency of IVEP. Our objective was to evaluate the effect of melatonin supplementation in IVC media on cleavage rates of bovine embryos. Bovine ovaries (n = 22) were obtained from local abattoir with ovarian weight of 7.29 ± 3.31 g, and size of 3.65 ± 0.69 cm. Cumulus-oocyte complexes (COCs) were recovered from 2 to 8 mm size follicles by aspiration using an 18-gauge needle attached with a 10 mL syringe. COCs were matured, fertilized, and cultured in vitro using IVF Bioscience bovine media and their protocols. Oocytes were in vitro matured for 23 h at 38.8°C, 5.5% CO2 in a humidified atmosphere and assessed for maturation by assessing COC expansion. Fully expanded and partially expanded COCs were fertilized for 20 h in a 4-well dish with frozen/thawed bull semen. Following IVF, the cumulus cells were removed by vertexing, and the presumptive zygotes were cultured in vitro with and without 1 µM melatonin in 4-well dishes in triplicates under the oil for embryo development at 38.8°C and 5.5% CO2. Cleavage rates were determined on d 3 of culture. The percentage of cleaved embryos was calculated as (number of zygotes cleaved/total number of fertilized oocytes) x100. Data were analyzed by student’s unpaired t- test with Welch’s correction using GraphPad Prism software. The cleavage rate was greater in melatonin supplemented group (54.17 ± 11.24%) than the control (49.83 ± 7.36%) but the difference was not significant (P > 0.05). In conclusion, under our experimental conditions, melatonin supplementation during embryo culture, following IVM/IVF of the bovine oocytes, did not significantly improve cleavage rates.

Diosmetin Promotes Early Embryonic Development in Pigs by Alleviating Oxidative Stress.

Diosmetin (DIOS), a natural flavonoid monomer derived from lemons and present in various plants such as spearmint and spider moss, exhibits antioxidant, anti-inflammatory, and antiaging properties. Nonetheless, its impact on early embryonic development in pigs remains unexplored. This study aimed to determine the influence of DIOS supplementation in an in vitro culture (IVC) medium on porcine embryo development and to elucidate the underlying mechanisms. Findings revealed that embryos cultured in IVC medium with 0.1 μM DIOS demonstrated an increased blastocyst formation rate, higher total cell number, reduced LC3B and CASPASE3 levels, elevated Nrf2 levels, decreased ROS, and enhanced GSH and mitochondrial membrane potential at the 4-cell embryonic stage. Additionally, the expression of proapoptotic genes (CAS3, CAS8, and BAX) and autophagy-related genes (BECLIN1, ATG5, LC3B, and P62) was downregulated, whereas the expression of embryonic development-related genes (CDK1 and CDK2), antioxidant-related genes (SOD1 and SOD2), and mitochondrial biogenesis-related genes (NRF2) was upregulated. These findings suggest that DIOS promotes early embryonic development in pigs by mitigating oxidative stress and enhancing mitochondrial function, thereby reducing autophagy and apoptosis levels.

PAPP-A enhances the antioxidative effects of IGF-1 during bovine in vitro embryo production

We investigated whether exogenous pregnancy-associated plasma protein-A (PAPP-A) enhances the antioxidant role of insulin-like growth factor-1 (IGF-1) in bovine in vitro embryo production (IVP). We performed standard in vitro maturation (IVM) and in vitro culture (IVC) or added menadione to promote an oxidative stressed microenvironment and evaluated the antioxidant effect of IGF-1 alone or in combination with PAPP-A (IGF-1/PAPP-A). In IVM, the treatments did not affect oocyte nuclear development, total GSH content, cumulus cell gene expression, and blastocyst yield. Nevertheless, IGF-1/PAPP-A treatment prevented an increase in reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) levels. In IVC, the treatments did not affect the total GSH content on blastocysts and IVC media, but IGF-1 and IGF-1/PAPP-A treatments increased blastocyst yield compared to the menadione group. In addition, IGF-1/PAPP-A treatment had lower ROS levels and regulated genes related to embryonic quality compared to the control and menadione groups. Overall, we showed that PAPP-A could enhance the antioxidant role of IGF-1 during IVP in cattle by avoiding higher ROS levels in oocytes and blastocysts and modulating the transcriptional abundance of genes involved in oxidative protection and embryonic quality.

P-343 Assessing the effect of adipose tissue-derived stem cell-conditioned medium on follicles and stromal cells in cultured ovarian tissue

Abstract Study question Does addition of adipose tissue-derived stem cell-conditioned medium (ASC-CM) enhance follicle survival and development and stromal cell viability in ovarian tissue culture? Summary answer Adding ASC-CM to ovarian tissue in vitro culture (IVC) promotes follicle survival, development, estradiol (E2) production, and stromal cell viability. What is known already Despite extensive efforts to optimize ovarian tissue IVC conditions, outcomes of follicle in vitro development remain suboptimal, mainly showing poor follicle survival and growth. IVC conditions that best resemble the in vivo environment are likely to generate less cellular distress and yield better outcomes. ASCs are known for promoting cell proliferation and survival through secretion of growth factors and cytokines. CM obtained from ASC culture contains these beneficial biomolecules and could potentially be a valuable supplement for ovarian tissue IVC. Study design, size, duration This prospective experimental study used 8 fragments of bovine ovarian cortex, which were cultured for 8 days in two different groups: (i) standard ovarian tissue culture (OT Ctrl D8); and (ii) ovarian tissue cultured with ASC-CM supplementation (OT+CM D8). One fragment per sample served as a non-cultured control (D0). Half of the culture medium was replaced every other day, and medium from days 2 and 8 was stored for further analyses. Participants/materials, setting, methods E2 production was evaluated on days 2 and 8 by ELISA. Follicle classification was established using hematoxylin and eosin (H&E) staining. Follicle and stromal cell apoptosis rates were assessed by TUNEL assays, while growth differentiation factor-9 (GDF-9) labeling served as a follicle quality marker. ASC-CM was analyzed to identify and measure soluble factors like vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF-β1), associated with protective effects. Main results and the role of chance The OT+CM D8 group showed significantly lower primordial follicle rates (p = 0.03) and higher secondary follicle rates (p = 0.02) than the OT Ctrl D8 group. Concerning apoptosis, the OT+CM D8 group demonstrated significantly lower follicle (p = 0.014) and stromal cell (p = 0.001) rates than the OT Ctrl D8 group. Furthermore, follicles in the OT+CM D8 group exhibited a significant increase (p = 0.002) in GDF-9 expression compared to those in the OT Ctrl D8 group. E2 production was also significantly higher (p = 0.04) after 8 days of culture in the OT+CM D8 group. All studied paracrine factors (VEGF, IL-6 and TGF-b1) were found to be present in the ASC-CM. Limitations, reasons for caution This study focuses only on improving follicle outcomes during the first step of ovarian tissue IVC, where follicles remain in situ within the tissue, and not on subsequent steps. Wider implications of the findings Our study demonstrates that adding ASC-CM to ovarian tissue IVC medium enhances follicle survival, development and E2 secretion, and promotes stromal cell viability. Moreover, our data suggest that ASC-derived factors VEGF, IL-6 and TGF-β1 could be possible paracrine mediators underlying the beneficial effects. Trial registration number not applicable

A 3D “sandwich” co-culture system with vascular niche supports mouse embryo development from E3.5 to E7.5 in vitro

BackgroundVarious methods for ex utero culture systems have been explored. However, limitations remain regarding the in vitro culture platforms used before implanting mouse embryos and the normal development of mouse blastocysts in vitro. Furthermore, vascular niche support during mouse embryo development from embryonic day (E) 3.5 to E7.5 is unknown in vitro.MethodsThis study established a three-dimensional (3D) “sandwich” vascular niche culture system with in vitro culture medium (IVCM) using human placenta perivascular stem cells (hPPSCs) and human umbilical vein endothelial cells (hUVECs) as supportive cells (which were seeded into the bottom layer of Matrigel) to test mouse embryos from E3.5 to E7.5 in vitro. The development rates and greatest diameters of mouse embryos from E3.5 to E7.5 were quantitatively determined using SPSS software statistics. Pluripotent markers and embryo transplantation were used to monitor mouse embryo quality and function in vivo.ResultsEmbryos in the IVCM + Cells (hPPSCs + hUVECs) group showed higher development rates and greater diameters at each stage than those in the IVCM group. Embryos in the IVCM + Cells group cultured to E5.5 morphologically resembled natural egg cylinders and expressed specific embryonic cell markers, including Oct4 and Nanog. These features were similar to those of embryos developed in vivo. After transplantation, the embryos were re-implanted in the internal uterus and continued to develop to a particular stage.ConclusionsThe 3D in vitro culture system enabled embryo development from E3.5 to E7.5, and the vascularization microenvironment constructed by Matrigel, hPPSCs, and hUVECs significantly promoted the development of implanted embryos. This system allowed us to further study the physical and molecular mechanisms of embryo implantation in vitro.

Ochratoxin A triggers endoplasmic reticulum stress through PERK/NRF2 signaling and DNA damage during early embryonic developmental competence in pigs

Ochratoxin A (OTA), a mycotoxin found in foods, has a deleterious effect on female reproduction owing to its endocrine-disrupting activity mediated through endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production. However, the mechanisms of OTA-induced ER stress in pig embryos during in vitro culture (IVC) are not yet fully understood. In the present study, porcine embryos were cultured for two days in an IVC medium supplemented with 0.5, 1.0, and 5.0 μM OTA, which led to an OTA-induced reduction in the developmental rate of blastocysts. The mRNA-seq transcriptome analysis revealed that the reduced blastocyst development ability of OTA-exposed porcine embryos was caused by ER stress, ultimately resulting in the accumulation of ROS and the occurrence of apoptosis. The expression levels of some UPR/PERK signaling-related genes (DDIT3, EIF2AK3, EIF2S1, NFE2L2, ATF4, EIF2A, and KEAP1) were found to differ in OTA-exposed pig embryos. OTA induces DNA damage by triggering an increase in RAD51/γ-H2AX levels and suppressing p-NRF2 activity. This effect is mediated through intracellular ROS and superoxide accumulation in the nuclei of porcine embryos. The cytotoxicity of OTA increased the activation of the PERK signal pathways (p-PERK, PERK, p-eIF2α, eIF2α, ATF4, and CHOP) in porcine embryos, with abnormal distribution of the ER observed around the nucleus. Collectively, our findings indicate that ER stress is a major cause of decline in the development of porcine embryos exposed to OTA. Therefore, OTA exposure induces ER stress and DNA damage via oxidative stress by disrupting PERK/NRF2 signaling activity in the developmental competence of porcine embryos during IVC.

Mito-Q supplementation of in vitro maturation or in vitro culture medium improves maturation of buffalo oocytes and developmental competence of cloned embryos by reducing ROS production

Mito-Q is a well-known mitochondria-specific superoxide scavenger. To our knowledge, the effect of Mito-Q on buffalo oocyte maturation and developmental competency of cloned embryos has not been examined. To investigate the effects of Mito-Q on the in vitro maturation (IVM) of buffalo oocytes and the developmental competence of cloned embryos, different concentration of Mito-Q were supplemented with IVM (0, 0.1, 0.5, 1, 2 μM) and in vitro culture (IVC) medium (0, 0.1 μM). Supplementation of IVM medium with 0.1 μM Mito-Q significantly (P ≤ 0.05) increased the cumulus expansion, nuclear maturation, mitochondrial membrane potential (MMP) and antioxidants genes (GPX1 and SOD2) expression and effectively reduced ROS production leading to a significant improvement in the maturation rate of buffalo oocytes. Further, the supplementation of 0.1 μM Mito-Q in IVC medium promotes the cleavage and blastocyst rate significantly over the control. Mito-Q supplementation improves (P ≤ 0.05) MMP, antioxidant gene (GPX1) expression and reduced the ROS level and apoptosis related genes (caspase 9) expression in cloned blastocysts. In conclusion, the present study demonstrated that the supplementation of 0.1 μM Mito-Q in IVM and IVC media exerts a protective role against oxidative stress by reducing ROS production and improving MMP, fostering improved maturation of buffalo oocytes and enhanced developmental competence of cloned embryos. These findings contribute valuable insights into the optimization of assisted reproductive technologies protocols for buffalo breeding and potentially offer novel strategies to enhance reproductive outcomes in livestock species.

Effects of niacin supplementation during in vitro culture on the developmental competence of porcine embryos.

Niacin is a water-soluble vitamin belonging to the vitamin B complex. It has been found to possess various biological activities, including antioxidant and lipid modification capacities. This study aimed to elucidate the effects of niacin treatment in porcine in vitro culture (IVC) medium on embryo developmental competence after parthenogenetic activation. IVC medium was supplemented with different concentrations of niacin (0 [control], 300, 600 and 900 μM). The results showed that embryos cultured in an IVC medium supplemented with 300 and 600 μM niacin had an increased cleavage rate (p < .05). In addition, 300 μM niacin treatment resulted in a higher blastocyst formation rate than the control and other niacin-treated groups. However, the total cell number did not differ significantly among the experimental groups. Niacin supplementation at 600 μM decreased reactive oxygen species, whereas treatment with 300, 600 and 900 μM increased glutathione levels in day two embryos. On day seven, 300 μM niacin exhibited improved fatty acid levels and fewer lipid droplets than the control group. Furthermore, gene expression at the mRNA level was performed on day two and day seven embryos, treated with or without 300 μM niacin. The expression of anti-apoptotic BCL2 and lipid metabolism PLIN2-related genes were upregulated, whereas the pro-apoptotic BAX and CASPASE3 were downregulated with niacin supplementation compared with the control group. However, SIRT1, a gene related to energy and the oxidative state, was up-regulated in niacin-treated day two embryos (p < .05). Overall, the results indicate that niacin has a beneficial effect on pre-implantation embryo development by modulating lipid metabolism and reducing oxidative stress and apoptosis. The expression patterns of PLIN2 and SIRT1 reported here suggest that these transcripts may be involved in the mechanism by which niacin affects the developmental capacity of IVC embryos.

Alpha-lipoic acid improves bovine preimplantation blastocyst quality and cryotolerance

In vitro embryo production has grown in recent decades due to its great potential for cattle production. However, the quality of in vitro-produced embryos is lower compared with those produced in vivo. The postfertilization culture environment has a major influence on bovine embryo quality. We hypothesize that the inclusion of the inclusion of alpha-lipoic acid (ALA) in the in vitro culture (IVC) medium during the first 24 h would have positive effects on embryo development in vitro and cryotolerance. The aims of this study were to evaluate the antioxidant effect of ALA in IVC medium for 24 h on bovine zygotes (21 h post in vitro fertilization, IVF), day 2 cleaved embryos (46 h post-IVF), and to assess embryo quality, developmental competence, and cryotolerance after vitrification. In all experiments, IVC medium was the Control, and 2.5 μM ALA was the treatment implemented. Viability and reactive oxygen species (ROS) levels in zygotes and day 2 embryos did not differ from the Control (P > 0.05). Supplementation with ALA increased total blastocyst and hatching rates (P < 0.05). It also improved embryo quality, evidenced by the increased blastocyst total cell number and the percentage of excellent-quality embryos observed (P < 0.05). In embryos cultured with ALA and then vitrified, ALA reduced intracellular ROS levels in warmed blastocysts (P < 0.05). In conclusion, ALA supplementation to IVC medium during 24 h is a new advantage in improving embryo quality for assisted bovine reproduction.

Interleukin-7 enhances in vitro development and blastocyst quality in porcine parthenogenetic embryos.

Interleukin-7 (IL-7), a vital factor that affects cell development, proliferation, and survival, plays an important role in oocyte maturation. However, its role in embryonic development remains unknown. Therefore, in this study, we aimed to investigate the effects of IL-7 supplementation on in vitro culture (IVC) of porcine embryos after parthenogenetic activation (PA) based on characteristics such as cleavage, blastocyst formation rate, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels in cleaved embryos, total cell number, apoptosis rate, and cell lineage specification in blastocysts. Immunofluorescence revealed that IL-7 and its receptor, IL-7Rα (IL-7R) localized in the cytoplasm of porcine parthenote embryos. By supplementing the IVC medium (PZM5) with various concentrations of IL-7, an optimal concentration that enhanced embryonic development, promoted intracellular GSH, and decreased ROS levels in the cleavage stage during porcine embryo IVC was determined. Investigation of mRNA expression patterns via qRT-PCR suggested that IL-7 possibly regulated maternal mRNA clearance and zygotic genome activation. Furthermore, IL-7 supplementation reduced blastocyst apoptosis, enhanced the expression of the inner cell mass marker SOX2, and phosphorylated STAT5 levels in the blastocysts. Moreover, it altered the transcription patterns of genes that regulate apoptosis, IL-7 signaling, and development. Thus, we demonstrated the localization of IL-7 and IL-7R in porcine preimplantation embryos in vitro for the first time. Furthermore, we suggest that IL-7 supplementation can be employed to enhance embryonic development and blastocyst quality based on the activation of the transcripts of genes that are involved in developmental competence and IL-7 signaling during in vitro porcine embryo development following PA.

Oct-4 activating compound 1 (OAC1) could improve the quality of somatic cell nuclear transfer embryos in the bovine

Previous studies described aberrant nuclear reprogramming in somatic cell nuclear transfer (SCNT) embryos that is distinctly different from fertilized embryos. This abnormal nuclear reprogramming hampers the proper pre- and/or post-implantation development. It has been demonstrated that SCNT blastocysts aberrantly expressed POU5F1 and POU5F1-related genes. With regard to this, it has been postulated that promoting the expression of POU5F1 in SCNT embryos may enhance reprogramming in SCNT embryos. In this study, we treated either fibroblast donor cells or SCNT embryos with OAC1 as a novel small molecule that has been reported to induce POU5F1 expression. Quantitative results from the MTS assay revealed that lower concentrations of OAC1 (1, 1.5, and 3 μM) are non-toxic after 2, 4, and 6 days, but higher concentrations (6, 8, 10, and 12 μM) are toxic and reduced the proliferation of cells after 6 days. No enhancement in the expression of endogenous POU5F1 was observed when both mouse and bovine fibroblast cells were treated with 1.5 and 3 μM OAC1 for up to 6 consecutive days. Subsequently, we treated either fibroblast as donor cells in the SCNT procedure (BFF-OAC1 group) or SCNT embryos [for 4 days (IVC-OAC1: D4-D7 group) or 7 days (IVC-OAC1: D0-D7 group)] with 1.5 μM OAC1. We observed that neither treatment of fibroblast donor cells nor SCNT embryos improved the cleavage and blastocyst rates. Interestingly, we observed that treatment of SCNT embryos all throughout the in vitro culture (IVC) (IVC-OAC1: D0-D7) with 1.5 μM OAC1 improves the quality of derived blastocyst which was indexed by morphological grading, blastomere allocation, epigenetic marks and mRNA expression of target genes. In conclusion, our results showed that supplementation of IVC medium with 1.5 μM OAC1 (D0-D7) accelerates SCNT reprogramming in bovine species.

Oroxin A reduces oxidative stress, apoptosis, and autophagy and improves the developmental competence of porcine embryos in vitro.

Oroxin A (OA) is a flavonoid isolated from Oroxylum indicum (L.) Kurz that has various biological activities, including antioxidant activities. This study aimed to examine the viability of using OA in an in vitro culture (IVC) medium for its antioxidant effects and related molecular mechanisms on porcine blastocyst development. In this study, we investigated the effects of OA on early porcine embryo development via terminal deoxynucleotidyl transferase dUTP nick-end labeling, 5-ethynyl-2'-deoxyuridine labeling, quantitative reverse transcription PCR, and immunocytochemistry. Embryos cultured in the IVC medium supplemented with 2.5μM of OA had an increased blastocyst formation rate, total cell number, and proliferation capacity, along with a low apoptosis rate. OA supplementation decreased reactive oxygen species levels while increasing glutathione levels. OA-treated embryos exhibited an improved intracellular mitochondrial membrane potential and reduced autophagy. Moreover, levels of pluripotency- and antioxidant-related genes were upregulated, whereas those of apoptosis- and autophagy-related genes were downregulated by OA addition. In conclusion, OA improves preimplantation embryonic development by reducing oxidative stress and enhancing mitochondrial function.

Lycopene Improves In Vitro Development of Porcine Embryos by Reducing Oxidative Stress and Apoptosis

In vitro culture (IVC) for porcine embryo development is inferior compared to in vivo development because oxidative stress can be induced by the production of excessive reactive oxygen species (ROS) under high oxygen tension in the in vitro environment. To overcome this problem, we investigated the effect of lycopene, an antioxidant carotenoid, on developmental competence and the mechanisms involved in mitochondria-dependent apoptosis pathways in porcine embryos. In vitro fertilized (IVF) embryos were cultured in IVC medium supplemented with 0, 0.02, 0.05, 0.1, or 0.2 μM lycopene. The results indicate that 0.1 μM lycopene significantly increased the rate of blastocyst formation and the total cell numbers, including trophectoderm cell numbers, on Day In terms of mitochondria-dependent apoptosis, IVF embryos treated with 0.1 μM lycopene exhibited significantly decreased levels of ROS, increased mitochondrial membrane potential, and decreased expression of cytochrome c on Days 2 and Furthermore, 0.1 μM lycopene significantly decreased the number and percentage of caspase 3-positive and apoptotic cells in Day-6 blastocysts. In addition, Day-2 embryos and Day-6 blastocysts treated with 0.1 μM lycopene showed significantly reduced mRNA expression related to antioxidant enzymes (SOD1, SOD2, CATALASE) and apoptosis (BAX/BCL2L1 ratio). These results indicate that lycopene supplementation during the entire period of IVC enhanced embryonic development in pigs by regulating oxidative stress and mitochondria-dependent apoptosis.

45 Embryonic disc development invitro in ovine embryos

Embryonic mortality during the second week of pregnancy has an important economic impact on farming. At this time, the embryo undergoes critical developmental events that cannot be recapitulated invitro, limiting our understanding of these pregnancy losses. After the blastocyst stage, the hypoblast migrates to cover the inner surface of the embryo and the epiblast forms a flat embryonic disc (ED) that initiates gastrulation. The aim of this study was to develop an invitro culture system to support sheep embryo development after the blastocyst stage. Day 6/7 invitro-produced blastocysts were cultured over agarose gels to prevent attachment and allocated to different media: synthetic oviductal fluid with 10% fetal bovine serum (SOF-FBS, n=52), an invitro culture medium (hIVC, n=35) supporting ED formation in human embryos (Deglincerti et al. 2016 Nature 533, 251-254; https://doi.org/10.1038/nature17948), and chemically defined N2B27 medium (n=38) supporting ED formation in bovine embryos (Ramos-Ibeas et al. 2020 Reproduction 160, 579-589, https://doi.org/10.1530/REP-20-0243). At Day 14, survival and embryo area were recorded, the abundance of transcripts encoding interferon Tau (TP1) and metabolic enzymes was analysed by RT-qPCR, and the development of epiblast and hypoblast was assessed by immunostaining for SOX2 and SOX17. Embryo survival and size and the percentage of embryos achieving complete hypoblast migration were significantly reduced in SOF-FBS (Chi-squared test and one-way ANOVA; P&amp;lt;0.05). Only N2B27 medium supported epiblast survival in 11/28 embryos. TP1 expression increased at Day 14 in all culture conditions and metabolism-related genes revealed a shift from anaerobic glycolysis to oxidative phosphorylation after culture in hIVC and N2B27. Next, to promote epiblast development, we allocated blastocysts to N2B27 medium supplemented with activin A (n=45), rho-associated protein kinase (ROCK) inhibitor (ROCKi, n=42), or insulin growth factor 1 (IGF1, n=29). IGF1 reduced significantly the percentage of embryos showing an ED-like structure, whereas activin A supplementation significantly increased epiblast survival (Chi-squared test; P&amp;lt;0.05) and SOX2+ cell number was higher in embryos cultured with ROCK inhibitor. When we combined activin A and ROCKi supplementation (N2B27+A+R, n=151), SOX2+ cell number and the percentage of embryos showing an ED-like structure increased significantly (165.1±53.1 and 31/35 embryos with SOX2+ epiblast cells; ∼89%) compared with N2B27 alone (49.4±12.7 and 5/11; ∼45%) (one-way ANOVA and Chi-squared test; P&amp;lt;0.05). Moreover, 18/31 (∼58%) ED-like structures developed in N2B27+A+R lost the Rauber’s layer (polar trophoblast), and BRACHYURY expression, denoting the onset of gastrulation, was observed in 3/14. In conclusion, we have developed a culture system that supports complete hypoblast migration and ED development invitro, which represents a valuable tool to explore early embryo mortality in livestock species without the need for experimental animals.

66 Effect of the co-culture system and the culture medium on invitro embryo development in alpacas (Vicugna pacos)

The aim was to evaluate 4 co-culture systems and 4 culture medium types to produce alpaca embryos by IVF. Gametes were obtained from ovaries and testes collected from a slaughterhouse. Oocytes were recovered by follicle aspiration using a 5-mL syringe. Oocytes with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured for 26h in an incubator (5% CO2 in air at 38.5°C), in TCM-199 supplemented with 10% fetal calf serum (FCS), 0.02IU mL−1 FSH, 1µg mL−1 oestradiol 17β, 0.2mM sodium pyruvate, and 50µg mL−1 gentamicin sulphate. After maturation, oocytes were placed in FERT-TALP (fertilization- Tyrode’s medium with albumin, lactate, and pyruvate) solution for 30min before IVF with epididymal sperm. Motile sperm were selected by swim-up method. Oocytes were co-cultured with 3×106 spermatozoa mL−1 for 18 to 20h under the same atmospheric conditions mentioned above. In Experiment 1, we evaluated 4 co-culture systems: ear fibroblasts (T1, n=224), fetal fibroblasts (T2, n=154), granulosa cells (T3, n=225), oviduct cells (T4, n=169) and synthetic oviductal fluid (SOF) invitro culture (IVC) (T5, n=116). As culture base, we used 0.5mL of SOF IVC medium supplemented with 10% FCS in all treatments. In Experiment 2, we evaluated 4 culture media: TCM-199+10% FCS (T1, n=137), CR1aa+3mg of bovine serum albumin (BSA) mL−1 (T2, n=85), KSOM medium+3mg of BSA mL−1 (T3, n=110), and SOF IVC+10% FCS (T4, n=66). The volume of culture medium used was 0.5mL in 4-well Nunc plates for each treatment. The time (8 days) and culture (38.5°C, 5% CO2 in air and high humidity) conditions, and change of medium (each 24h) were the same in both experiments. Statistical significance was determined using ANOVA. The mean and s.d. were calculated from the average of the percentages obtained in each repetition. In experiment 1, the cleavage rates were higher (P&amp;lt;0.05) in co-culture with fetal fibroblasts (46%±0.17), oviduct cells (50%±0.09), and ear fibroblasts (58%±0.17) than with granulosa cells (28%±0.12) and SOF IVC (30%±0.18). Also, the morula rates were higher (P&amp;lt;0.05) in co-culture with fetal fibroblasts (35%±0.16), oviduct cells (31%±0.01), and ear fibroblasts (35%±0.14) than with granulosa cells (11%±0.01) and SOF IVC (27%±0.17). In contrast, there were no differences in blastocyst rates between co-culture with granulosa cells (10%±0.04), SOF IVC (12%±0.09), and fetal fibroblasts (24%±0.14). However, there were differences between co-culture with oviduct cells (19%±0.06) and ear fibroblasts (32%±0.14). In Experiment 2, there were no differences in cleavage rates between TCM-199+FCS (28%±0.12), CR1aa+BSA (44%±0.24), KSOM+BSA (38%±0.19), and SOF-IVC+FCS (50%±0.20). However, there were differences in morula rates between CR1aa+BSA (7%±0.09) and SOF IVC+FCS (35%±0.21), TCM-199+FCS (23%±0.09), and KSOM+BSA (27%±0.15). We obtained a higher blastocyst rates in SOF IVC+BSA (24±0.12) and KSOM+BSA than with CR1aa+BSA (3±0.06) and TCM-199+FCS (9±0.03). In conclusion, KSOM and SOF-IVC were the best media for culture, and oviduct cells and ear and fetal fibroblasts were the best cells to produce alpaca embryos by IVF.

Procyanidin B1 promotes in vitro maturation of pig oocytes by reducing oxidative stress.

Oxidative stress negatively affects the in vitro maturation (IVM) of oocytes. Procyanidin B1 (PB1) is a natural polyphenolic compound that has antioxidant properties. In this study, we investigated the effect of PB1 supplementation during IVM of porcine oocytes. Treatment with 100 μM PB1 significantly increased the MII oocytes rate (p <0.05), the parthenogenetic (PA) blastocyst rate (p <0.01) and the total cell number in the PA blastocyst (p < 0.01) which were cultured in regular in vitro culture (IVC) medium. The PA blastocyst rate of regular MII oocytes activated and cultured in IVC medium supplemented with 100 and 150 μM PB1 significantly increased compared with control (p < 0.01 and p < 0.05). We also evaluated the reactive oxygen species (ROS) levels, mitochondrial membrane potential (Δψm) levels, glutathione (GSH) levels, and apoptotic levels in MII oocytes and cumulus cells following 100 μM PB1 treatment. The results showed that the PB1 supplementation decreased ROS production and apoptotic levels. In addition, PB1 was found to increase Δψm levels and GSH levels. In conclusion, PB1 inhibited apoptosis of oocytes and cumulus cells by reducing oxidative stress. Moreover, PB1 improved the quality of oocytes and promoted PA embryo development. Taken together, our results suggest that PB1 is a promising antioxidant additive for IVM of oocytes.

Leonurine improves in vitro porcine embryo development competence by reducing reactive oxygen species production and protecting mitochondrial function

Leonurine (LEO) is pseudoalkaloid that has been isolated from motherwort. It has been found to have various biological activities, including an antioxidant capacity. This study aimed to confirm whether LEO could be used in porcine in vitro culture (IVC) medium for its antioxidant effect and related molecular mechanisms. The results showed that embryos in IVC medium supplemented with 40 μM LEO had an increased blastocyst formation rate, total cell number, and proliferation capacity and a low apoptosis rate. LEO supplementation decreased reactive oxygen species levels and increased glutathione levels. Moreover, LEO-treated embryos exhibited improved intracellular mitochondrial membrane potential and reduced autophagy. In addition, pluripotency related gene was up-regulated while apoptosis and autophagy related genes were down-regulated with LEO supplementation. These results suggest that LEO has a beneficial effect on pre-implantation embryo development by reducing oxidative stress and enhancing mitochondrial function.

L-Carnitine improves in vitro maturation of feline oocytes and subsequent development of embryos generated by parthenogenesis

The objective of the current study was to assessment the effect of L-Carnitine as a stimulant of lipid catabolism in vitro maturation (IVM) and in vitro culture (IVC) media on IVM rate (nuclear maturation) and embryonic development. A total of 495 oocytes were assigned into two groups; the control group, oocytes matured in TCM-199 medium; L-Carnitine group oocytes matured in TCM-199 medium supplemented with L-Carnitine (0.5mg/ml). After that 250 oocytes were stained with Hoechst 33342 then with Nile Red dye. The nuclear maturation and oocyte lipid content were determined under a fluorescence microscope. The other matured oocytes were incubated for cell activation by Thimerosal and DL- Dithiothreitol (DDT). Potential zygotes were incubated in vitro culture 1 and 2, the stained embryos were counted and evaluated for morula and blastocyst stages. The Metaphase II rates were 16.2% and 42.1% in the control group and L-carnitine group, respectively. Overall, 9.3% and 14.9% of the oocytes reached at least the MI stage in the control group and L-carnitine group respectively. The addition of L-carnitine in IVM media resulted in a significant decrease in the amount of lipid in cat oocyte. Moreover, the incubation of matured oocytes of the L-carnitine group in IVC1 lead to significant increase in cleaved cells 29.4% in comparison with the control group 15.4%. The incubation of cleaved cell of the L-carnitine group in IVC2 lead to decrease significantly in degenerated cells 12.5 % compared to the control group 42.9% while caused an increase significantly in morula 50% compared to the control group 33.3% and blastocyst 37.5% comparison with the control group 23.8%. In conclusion, the use of L-Carnitine as a supplement in IVM and IVC media decrease lipid content of cat oocyte and subsequent enhance the nuclear maturation, cell activation of cat's embryo, and embryonic development.

197 The use of photostimulation to enhance oocyte cytoplasmic maturation

Phototherapy uses monochromatic light from low-power lasers and light-emitting diodes (LEDs) to modulate biological processes. It has been proposed that the red-to-near infrared optical region (~600-1000nm) enhances cellular metabolic activity by activation of the mitochondrial respiratory chain. However, photostimulation induces the generation of oxide free radicals and could create oxidative stress in exposed cells. The main objective was to use photostimulation to affect the cumulus-oocyte complex metabolic state, aiming to enhance cytoplasmic maturation rates and subsequent embryonic development. A secondary objective was to determine the toxicity of the proposed photostimulation protocol. Abattoir-derived ovaries were used. All media was from IVF Biosciences (Falmouth, Cornwall, UK). Follicles 2 to 6mm in diameter were aspirated. Oocytes with compact cumulus and homogeneous cytoplasm were selected, and 50 oocytes/well were placed in invitro maturation medium (0h) and incubated at 38.5°C in 5% CO2 in air with high humidity in the presence (treatment=exposure for 2min to super-bright LED 1 and 2h after the beginning of maturation; LED wavelength of 660-665 nm; NTE30041; NTE Electronics Inc.) or absence (Control) of light. After maturation (22h), oocytes were split into two wells (25 oocytes/well) and subjected to IVF with semen from two different bulls for 18 to 20h. Cumulus cells were separated by vortexing, zygotes were placed in invitro culture medium, and incubated at 38.5°C in 5% CO2 in air with high humidity. Culture medium was renewed every 48h. Cleavage, morula, and blastocyst rates were recorded as a percentage of the number of oocytes subjected to IVF per treatment. The experiment was replicated 4 times. Statistical analysis was conducted using the Mixed procedure (SAS 9.4, SAS Institute Inc.) with repeated-measures and autoregressive covariance. The model's random effect was well within treatment. Fixed effects were bull, stage of development, and treatment. There was no difference (P=0.8) between treatments for any stage of development measured (cleavage: 76.4±2.7 vs. 74.8±4.1; morula: 36.1±4.8 vs. 35.9±5.8; blastocyst: 20.8±3.2 vs. 20.6±4.4 for control and treatment respectively; mean±s.e.). Sire affected development: bull 1 had a greater percentage (P&amp;lt;0.05) cleavage (82.9±0.02 vs. 68.3±0.02), morula (42.6±0.05 vs. 29.4±0.04), and blastocyst (27.8±0.04 vs. 13.6±0.01) development than bull 2. There was no treatment×bull interaction (P=0.9). In conclusion, there were no stimulatory or toxic effects of this preliminary photostimulation protocol. Further research is needed to develop an optimal protocol that shows a metabolic effect and, potentially, an enhancement of invitro cytoplasmic maturation rates and subsequent embryonic development.