Despite significant progress in vitro maturation (IVM) and in vitro culture (IVC) of oocytes and embryos, their developmental competence remains low. To address this issue, we used buffalo oocytes as a model system to investigate the effects and mechanisms of oxygen concentration on IVM and IVC. Our findings demonstrated that culturing buffalo oocytes with 5% oxygen significantly enhanced the efficiency of IVM and developmental competence of early embryos. Immunofluorescence results suggested that HIF1α played a critical role in these progresses. RT-qPCR results showed that maintaining a stable expression of HIF1α in cumulus cells with 5% oxygen concentration enhanced glycolysis, expansion, and proliferation abilities, up-regulated the expression of development-related genes, and suppressed apoptosis level. Consequently, it improved the maturation efficiency and quality of oocytes, leading to improve developmental capacity of buffalo early embryos. Similar outcomes were also observed when embryos were cultured with 5% oxygen. Collectively, our study provided insights into the role of oxygen regulation during oocytes maturation and early embryo development, and could potentially improve the efficiency of human assisted-reproduction technology.

Neural tube (NT) defects arise from abnormal neurulation and result in the most common birth defects worldwide. Yet, mechanisms of primate neurulation remain largely unknown due to prohibitions on human embryo research and limitations of available model systems. Here, we establish a three-dimensional (3D) prolonged in vitro culture (pIVC) system supporting cynomolgus monkey embryo development from 7 to 25 days post-fertilization. Through single-cell multi-omics analyses, we demonstrate that pIVC embryos form three germ layers, including primordial germ cells, and establish proper DNA methylation and chromatin accessibility through advanced gastrulation stages. In addition, pIVC embryo immunofluorescence confirms neural crest formation, NT closure, and neural progenitor regionalization. Finally, we demonstrate that the transcriptional profiles and morphogenetics of pIVC embryos resemble key features of similarly staged in vivo cynomolgus and human embryos. This work therefore describes a system to study non-human primate embryogenesis through advanced gastrulation and early neurulation.

The in vitro production (IVP) of cattle embryos requires that germinal-vesicle stage oocytes undergo a period of maturation in vitro prior to fertilization and culture to the blastocyst stage. Success of IVP in taurine cattle is enhanced following ovarian stimulation prior to oocyte retrieval (OPU), particularly if preceded by a short period of FSH withdrawal (\'coasting\'). However, evidence regarding the importance of progesterone (P4) support during OPU-IVP is equivocal. The current study, therefore, determined the effects of increased peripheral P4 concentrations during FSH-stimulated (\'coasted\') cycles of OPU. Progesterone support was provided by either an active corpus luteum (CL) and/or one of two intravaginal P4 releasing devices (i.e., CIDR® [1.38 g P4] or PRID® Delta [1.55 g P4]). Expt. 1 established an initial estrus prior to OPU, allowing CL formation (single luteal phase) spanning the first two of five cycles of OPU; the remaining three cycles were supported by either a CIDR® or PRID® Delta. Expt. 2 commenced with two cycles of dominant follicle removal (including prostaglandin F2α) undertaken seven days apart prior to six cycles of OPU. The absence of a CL meant that these cycles were supported only by a CIDR® or PRID® Delta. As each experiment involved several sequential cycles of OPU, the cumulative effects of device use on vaginal discharges were also assessed. Each experiment involved 10 sexually mature Holstein heifers. In the absence of a CL, peak plasma P4 concentrations were greater (P = 0.002) for the PRID® Delta (4.3 ± 0.22) than for the CIDR® (2.9 ± 0.22). In Expt. 1 there was an interaction (P < 0.05) between CL presence at OPU and P4 device on Day 8 blastocyst yields, indicating an effect of P4 device only when the CL was absent. The percentage hatching/hatched blastocysts of matured oocytes for the CIDR® and PRID® Delta was 44.3 ± 5.04 and 41.0 ± 5.40 in the presence, and 17.1 ± 3.48 and 42.2 ± 3.76 in the absence, of a CL (P = 0.018). Combined analyses of data from Expt. 1 and 2, when no CL was present, confirmed that Day 8 blastocyst yields were greater (P = 0.022) for the PRID® Delta than the CIDR®. Vaginal discharge scores were higher (P < 0.001) for the PRID® Delta than the CIDR® in Expt. 1 but not in Expt 2; however scores were low, did not increase with repeated use, and thus were deemed of no clinical or welfare concern. In conclusion, enhanced P4 support during FSH-stimulated cycles of OPU-IVP can improve in vitro embryo development.

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.

The aim of this study was to evaluate the effect of multilamellar vesicles (MLVs) of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in co-culture with in vitro-produced bovine embryos (IVPEs). The stability of five concentrations of MLVs (1.0, 1.25, 1.5, 1.75, and 2.0 mM) produced using ultrapure water or embryonic culture medium with 24 or 48 h of incubation at 38.5 °C with 5% CO2 was assessed. In addition, the toxicity of MLVs and their modulation of the lipid profile of the plasma membrane of IVPEs were evaluated after 48 h of co-culture. Both media allowed the production of MLVs. Incubation (24 and 48 h) did not impair the MLV structure but affected the average diameter. The rate of blastocyst production was not reduced, demonstrating the nontoxicity of the MLVs even at 2.0 mmol/L. The lipid profile of the embryos was different depending on the MLV concentration. In comparison with control embryos, embryos cultured with MLVs at 2.0 mmol/L had a higher relative abundance of six lipid ions (m/z 720.6, 754.9, 759.0, 779.1, 781.2, and 797.3). This study sheds light on a new culture system in which the MLV concentration could change the lipid profile of the embryonic cell membrane in a dose-dependent manner.

The in vitro embryo\'s competence is lower than in vivo counterparts and miRNAs found to affect the developmental competence, affecting pluripotency, stress level and apoptosis in embryos. We aimed to investigate the effect of miRNA-155 on parthenogenically activated early development porcine embryos at the preimplantation blastocyst stage. We designed miRNA-155 mimics and inhibitors and microinjected them into post-activated oocytes. The embryonic cleavage rate, blastocyst rate, and embryo development quality in terms of stress and apoptosis levels were investigated by confocal microscopy. Furthermore, we selected target genes, analyzed gene interaction and prediction networks, and compared the gene expression level in treatments and controls. miRNA-155 inhibition improved in vitro developmental competence by increasing cell numbers and reducing stress and apoptosis levels. The cleavage rate in the miRNA-155 inhibitor group was significantly higher (P < 0.05) than that in the miRNA-155 mimic group, but not in the control, whereas the blastocyst rate of the miRNA-155 inhibitor group was statistically significantly higher (P < 0.05) than in both control and miRNA-155 mimic groups. The relative gene expression level analysis showed downregulation of mRNAs related to stress and apoptosis, BAX, and the stress-induced autophagy gene ATF4, and TNF-ɑ in the miRNA-155 inhibitor group. Moreover, miRNA-155 inhibition showed upregulation of the relative expression of OCT4, ZEB2, BCL2, and IL-1 mRNA compared to control and mimic groups. However, the miRNA-155 mimic-injected group showed lower cleavage rates and blastocyst development rates than the other two groups. In conclusion, miRNA-155 inhibition in porcine in vitro embryos improved their preimplantation developmental competence and in vitro embryo production.

Energy is essential for early embryogenesis, and fertilized eggs can successfully develop to blastocyst in in vitro culture medium with an appropriate energy supply. Conversely, embryonic development is negatively affected by a suboptimal energy supply. We previously observed that a low level of pyruvate greatly arrests mouse embryos at the 2-cell stage. However, how methylation modifications are affected at this specific stage remains unknown. In this study, we found that mouse embryos could timely develop to the 4-cell stage in K+simplex optimized medium (KSOM) with control level of pyruvate, but embryos were significantly arrested at the 2-cell stage when pyruvate was reduced to 0.2-fold of the control level. Moreover, the fluorescence intensities of 5 mC, H3K4me2, H3K9me2 and H3K27me2 in the 2-cell stage embryos of the 0.2-fold pyruvate group were notedly lower than those of the control group, but N6-methyladenosine (m6A) fluorescence intensity was higher, suggesting that global genomic DNA, histone and m6A methylation modifications are disrupted with low levels of pyruvate. Consistently, the mRNA levels of genes related to DNA methylation, histone methylation and m6A modifications were also disturbed in the 2-cell stage embryos cultured with low levels of pyruvate. In summary, our findings demonstrate that insufficient pyruvate in culture medium results in mouse embryonic developmental arrest, at least in part due to defects in methylation modifications.

The aim of this study was to evaluate the effect of 1 μmol/L zearalenone (ZEN) and 1 μmol/L enterolactone (ENL), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 10 sheep were collected at a local abattoir and fragmented, and the ovarian pieces were submitted to in vitro culture for 3 days in the presence or absence of the test compounds. The morphology of primordial and primary follicles was impaired by ZEN, whereas that of cultured secondary follicles was improved by ENL. However, the combination of ENL with ZEN impaired the quality of primary and secondary follicles. Both ZEN and ENL induced apoptosis, but only ZEN was responsible for oocyte autophagy. None of these xenoestrogens affected endoplasmic reticulum stress as observed by the unaltered expression of ERP29. Differently from ZEN, ENL increased the expression of the efflux transporter ABCG2. In conclusion, although ENL can counteract the negative effects of ZEN on primordial and primary follicles, this positive effect is not similar to that observed in ovarian tissue cultures in the presence of ENL alone.

The present study was carried out to compare the proteomic profiles of spermatogenic cells of crossbred and zebu cattle in an effort to understand the possible reasons for a higher incidence of sub-fertility in crossbred bulls. The spermatogenic cells collected from the testes of pre-pubertal (6 mo) and adult (24 mo) crossbred and zebu males through fine needle aspiration were proliferated in vitro, and proteomic profiling was done using a shotgun proteomics approach. The age- and species-specific variations in the expression level of proteins were identified in spermatogenic cells. The number of differentially expressed proteins (DEPs) identified in pre-pubertal zebu and crossbred was 546, while 579 DEPs were identified between adult zebu and crossbred bulls. Out of these, 194 DEPS were common to these groups and 40 DEPs displayed a fold change ≥2. However, only 20 proteins exhibited similar expression variation trends (upregulated or downregulated) among pre-pubertal as well as adult zebu and crossbred bulls. Out of these 20 DEPs, 13 proteins were upregulated, and 7 proteins were downregulated in spermatogenic cells of zebu compared to crossbred bulls. Among the upregulated proteins were RPLP2, PAXIP1, calumenin, prosaposin, GTF2F1, TMP2, ubiquitin conjugation factor E4A, COL1A2, vimentin, protein FAM13A, peripherin, GFPT2, and GRP78. Seven proteins that were downregulated in zebu bulls compared to crossbred included APOA1, G patch domain-containing protein 1, NAD P transhydrogenase mitochondrial, glutamyl aminopeptidase, synaptojanin 1 fragment, Arf GAP with SH3 domain ANK repeat and PH domain-containing protein 1, and protein transport protein sec16B. It was inferred that the proteins associated with sperm function and fertilization processes, such as calumenin, prosaposin, vimentin, GRP78, and APOA1 could be studied further to understand the precise cause of subfertility in crossbred bulls.

Early embryonic development may be affected by adrenal hyperactivity in stressful situations which may lead to endocrine changes in the embryo environment. A sensitive period in porcine embryo development is the 4-cell stage when the embryo genome activation occurs. A mixed in vivo-in vitro system was implemented to test whether an altered milieu around this stage could affect embryo development and blastocyst quality in the porcine model. After in vitro maturation and fertilisation, presumptive zygotes were exposed for 24 h to plasma collected after ovulation from adrenocorticotropic hormone (ACTH)-treated, non-ACTH-treated sows; and, medium without plasma, supplemented with bovine serum albumin. Subsequently, embryo development and differences in gene expression were tested among treatments. Cleavage and blastocyst rates did not differ between treatments. Blastocyst quality by morphology assessment was similar when all the resulting blastocysts were included in the analysis. However, when only expanded blastocysts (and onwards) were included in the analysis, the blastocysts from the non-ACTH plasma group showed better quality score. Blastocyst quality by morphological assessment was not mirrored by the transcription levels of various important genes for embryo development whose gene expression profile did not significantly differ among groups. It is likely that the effect of the altered environment provided by plasma from ACTH-treated sows was too short to affect embryo development. Therefore, a brief exposure to an altered endocrine environment may not have harmful consequences for the embryo once fertilisation occurs.