Bovine Embryos Research Articles

Dimethyl sulfoxide-induced DNA demethylation during vitrification of early cleavage-stage embryos and possible countermeasures.

Dimethyl sulfoxide (DMSO) alters DNA methylation in vitrified-warmed embryos and potentially affects subsequent development. This study aimed to examine possible countermeasures against DMSO-induced demethylation. In vitro-produced bovine embryos (8-cell stage) were vitrified using a combination of DMSO and ethylene glycol (EG) or propylene glycol (PG) + EG. After warming, the lipid content and expression levels of 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), DNMTs, and TETs were examined. In addition, RNA-sequencing was performed on blastocysts derived from the vitrified embryos. Furthermore, the effect of supplementation with a vitrification medium containing DMSO and N-acetyl-L-cysteine (NAC, 5mM) on the levels of 5mC in embryos was examined. Vitrification decreased the levels of 5mC and increased the levels of 5hmC in 8-cell stage embryos. Low levels of 5mC persisted until the blastocyst stage in the DMSO group but increased in the PG group. The expression level of TET3A was higher in the DMSO group than in the fresh group, but not in the PG group. Both cryoprotectants reduced the lipid levels in post-warmed 8-cell stage embryos. The addition of NAC ameliorated DMSO-induced demethylation at both the 8-cell and blastocyst stages. RNA-seq analysis revealed that PG-specific pathways included ribosomes and mitochondria and that both DMSO and PG affected cGMP-PGK, MAPK, Wnt, and insulin secretion-related signaling. The K-medoids method predicted that DMSO affected cell adhesion molecules and that MAPK signaling was affected the most. PG and NAC may antagonize DMSO-induced demethylation; however, PG exerts adverse effects on embryos.

Effect of Alpha-Lipoic Acid on the Development, Oxidative Stress, and Cryotolerance of Bovine Embryos Produced In Vitro

Oxidative stress (OS) induced by an imbalance in reactive oxygen species (ROS) levels in vitro impairs embryonic development. Here, we assessed the effects of alpha-lipoic acid (ALA) in in vitro production media on OS reduction, embryonic development, and cryotolerance of bovine embryos. We evaluated the effects of adding different concentrations of ALA (2.5, 5, 10, and 25 μM) to in vitro maturation (IVM) or in vitro culture (IVC) medium on embryonic development. We also determined the effects of adding ALA (25 μM) to the IVM and IVC medium in the same routine on the development and quality of embryos, ROS levels, and cryotolerance. Embryos were produced in vitro using conventional protocols for each treatment. The inclusion of ALA in the IVM and IVC media did not affect the development or quality of embryos; however, it reduced ROS levels in grade II embryos and increased hatching after 12 h on day 7 in grade I embryos and on day 8 in grade II embryos after warming. These findings prompt questions regarding the potential of ALA in improving embryo metabolism, considering the initial embryo recovery in the first few hours of embryo warming.

Developmental stage specific effect of Mito-TEMPO on the expression outline of antioxidant genes, ROS balance and cryo-resilience of bovine IVP embryos

Developmental stage specific effect of Mito-TEMPO on the expression outline of antioxidant genes, ROS balance and cryo-resilience of bovine IVP embryos

METTL7A improves bovine IVF embryo competence by attenuating oxidative stress.

In vitro fertilization (IVF) is a widely used assisted reproductive technology to achieve a successful pregnancy. However, the acquisition of oxidative stress in embryo in vitro culture impairs its competence. Here, we demonstrated that a nuclear coding gene, methyltransferase-like protein 7A (METTL7A), improves the developmental potential of bovine embryos. We found that exogenous METTL7A modulates expression of genes involved in embryonic cell mitochondrial pathways and promotes trophectoderm development. Surprisingly, we discovered that METTL7A alleviates mitochondrial stress and DNA damage and promotes cell cycle progression during embryo cleavage. In summary, we have identified a novel mitochondria stress eliminating mechanism regulated by METTL7A that occurs during the acquisition of oxidative stress in embryo in vitro culture. This discovery lays the groundwork for the development of METTL7A as a promising therapeutic target for IVF embryo competence.

Effects of endometrial embryokines on the preimplantation bovine embryo to create a gene expression signature consistent with a high competence phenotype†.

Optimal embryonic development depends upon cell-signaling molecules released by the maternal reproductive tract called embryokines. Identity of specific embryokines that enhance competence of the embryo for sustained survival is largely lacking. The current objective was to evaluate effects of three putative embryokines in cattle on embryonic development to the blastocyst stage. The molecules tested were vascular endothelial growth factor A (VEGFA), C-X-C motif chemokine ligand 12 (CXCL12), and interleukin-6 (IL6). Molecules were added from day 4 to 7.5 of culture at 50ng/mL (VEGFA and CXCL12) or 100ng/mL (IL6). Endpoints were development to the blastocyst stage, and transcript abundance for 94 specific genes involved in lineage commitment, epigenetic regulation, and other functions. Among the genes examined were eight whose transcript abundance have been related to embryo competence for survival after embryo transfer. None of the molecules increased the proportion of putative zygotes or cleaved embryos becoming blastocyst at day 7.5 of development. An embryo competence index based on a Bayesian multiple regression formula to weigh transcript abundance of the eight biomarker genes was not affected by treatment with VEGFA but was increased by both CXCL12 and IL6. Transcript abundance of five genes were modified by VEGFA, 19 by CXCL12 and 19 by IL6. A total of 11 genes were modified in a similar manner by CXCL12 and IL6. Most differentially-expressed genes for CXCL12 and IL6 were downregulated, suggesting that the embryokines may promote a less energetically-demanding metabolic state than would be the case in their absence.

Utilization of oocytes from closely related domestic species for interspecies somatic cell nuclear transfer in gaur embryo production

Context The limited availability of recipient oocytes poses significant challenges in increasing the population of endangered species. Aims This study demonstrates the use of oocytes from closely related domestic species (cattle, buffalo, and goat) in the production of gaur embryos via interspecies somatic cell nuclear transfer (iSCNT). The developmental abilities of embryos from various combinations – gaur–bovine, gaur–buffalo, gaur–goat, and others – are analyzed. Previous achievements are retrospectively reviewed and summarized. Methods The methodologies for iSCNT and in vitro gaur embryo production are outlined. Key results Blastocyst rates among gaur–bovine, gaur–buffalo, and gaur–goat embryos showed no significant differences (P < 0.05). Gaur–bovine embryo development was comparable to intraspecies embryos (bovine–bovine), indicating successful developmental compatibility. Pregnancy rates after transferring gaur–bovine embryos were reported for the first time, with rates of 4.76%, 42.86%, 19.05% and 4.76% at Days 45, 60, 90 and 120, respectively. Conclusions Bovine, buffalo, and goat oocyte cytoplasm is effective for producing gaur iSCNT embryos, capable of initiating early pregnancies. Implications Laboratory techniques and methodologies can save costs and time, contributing to the conservation of endangered species.

Unraveling the elongating bovine conceptus microenvironment: identification of gene transcripts and proteins along the conceptus-maternal interface in cattle.

The bovine conceptus elongates near Day 16 of development and releases interferon-tau (IFNT), disrupting the endometrial luteolytic mechanism to sustain luteal P4 and pregnancy. Conceptus factors other than IFNT modify local endometrial activities to support pregnancy; however, the microenvironment is largely uncharacterized. We utilized a bovine conceptus-endometrial culture system to elucidate the microenvironment in the form of RNA and protein. Estrus synchronized heifers remained cyclic (13) or were inseminated (9) to produce Day 16 cyclic endometrium and elongating conceptuses, respectively. Conceptus sections and endometrium were then used to generate tissue cultures in 1mL of medium: (1) no tissue (Control Med; n = 7), (2) mono-cultured conceptus (Conceptus; n = 9, 3) mono-cultured endometrium (Endo; n = 13), or (4) Endo-Conceptus Co-culture (n = 15). After 12h, tissue RNA was sequenced (RNA-Seq) and media underwent proteomic analysis (LC-MS/MS). Compared to Conceptus and Endo, co-cultured conceptus and endometrial tissue contained 3400 and 4575 differentially expressed genes (DEG), respectively (P ≤ 0.01). More abundantly expressed endometrial DEG were associated with interferon signaling whereas more abundantly expressed conceptus DEG were associated with protein homeostasis and metabolism (FDR < 0.001). When Co-culture media where compared to Endo media, 288 more abundant protiens were identified (P < 0.05). Biological processes related to these proteins included antigen presentation via MHC Class Ib and keratinization (FDR < 0.001). Within the mono-cultured conceptus and endometrial media, folate receptor alpha (FOLR1) (P < 0.001) was identified as the most abundant secreted protein suggesting the reproductive tissues elicit a microenvironment supportive of conceptus growth involving folate metabolism.

Supplementation with serine-enriched non-essential amino acids from minimum essential medium promotes blastocyst development of in vitro-fertilized bovine embryos

To produce an embryo, a high conception rate must be complied along with four evaluation criteria based on the timing of early cleavage and proper embryo morphology (hereafter, these blastocysts will be referred to as "four-criteria-compliant blastocysts"). Therefore, it is necessary to construct a culture system for high efficiency production of embryos meeting these four criteria. Non-essential amino acids (NEAAs) are widely used for the culture of bovine embryos fertilized in vitro; however, the necessity and optimal concentration of individual NEAA must be verified to produce four-criteria-compliant blastocysts. DNA methylation is a critical event for blastocyst formation in bovines, and serine is a common NEAA that serves as a methyl donor and participates in DNA methylation. Serine is generally added at a concentration of 100 µM in bovine embryo culture medium. However, the rate of formation of four-criteria-compliant blastocysts was significantly improved when 1,000 µM of serine was added. Analysis of endogenous serine synthases gene expression in oocytes and embryos revealed that phosphoglycerate dehydrogenase, the rate-limiting enzyme in the serine synthesis pathway, is expressed at the morula stage and beyond. The addition of serine at 1,000 µM increased the amount of methyl donors; moreover, the addition of an inhibitor of serine-metabolizing enzymes decreased the number of methyl donors and markedly inhibited blastocyst formation. These results indicate that the addition of serine at an optimal concentration of 1,000 µM favors production of four-criteria-compliant blastocysts, and that methyl donor synthesis may be involved in this effect.

Ultrastructural Characteristics of Bovine Embryos Produced In Vitro And Vitrified Using the Cryotop Method

BACKGROUND: Despite advancements in bovine embryos cryopreservation techniques, challenges remain, warranting further investigation into their impact on embryo morphology and viability so that outcomes can be improved. OBJECTIVE: To analyze, through transmission electron microscopy (TEM), in vitro-produced bovine embryos vitrified using the Cryotop method. MATERIALS AND METHODS: Groups of embryos were transferred to a stabilization solution (SS) containing 7.5% EG, 7.5% DMSO in maintenance medium (TCM-199 supplemented with 20% FBS) for 2 min, and then transferred to a vitrification solution (VS) containing 15% EG, 15% DMSO, and 0.5 M sucrose in maintenance medium. Warming was performed in five stages with decreasing concentrations of sucrose. After warming, the blastocysts were cultured for 24 h for subsequent survival analysis and ultrastructural evaluation. In vitro-produced bovine embryos that did not undergo the vitrification process were used as a fresh control. RESULTS: Blastocoel reestablishment was observed in 52.3% (66/126) of vitrified embryos 24 h after warming, demonstrating the method's effectiveness in postcryopreservation survival. Ultrastructural analysis of embryos from the fresh control group showed flattened trophoectodermal cells with prominent nuclei, well-preserved mitochondria, and Golgi complexes were also evident. Microvilli were observed in some regions near the zona pellucida. Embryos vitrified using the Cryotop method exhibited lesions consistent with the cryopreservation process, such as intracellular disorganization, mitochondrial injuries, and dispersion of microvilli. CONCLUSIONS: Ultrastructural evaluation of in vitro-produced bovine embryos vitrified using the Cryotop method is an effective tool for increased understanding of the injuries caused to embryonic cells during the cryopreservation process.

Reactive oxygen species attenuation improves the quality of vitrified-warmed bovine embryos

Reactive oxygen species attenuation improves the quality of vitrified-warmed bovine embryos

METTL7A improves bovine IVF embryo competence by attenuating oxidative stress.

In vitro fertilization (IVF) is a widely used assisted reproductive technology to achieve a successful pregnancy. However, the acquisition of oxidative stress in embryo in vitro culture impairs its competence. Here, we demonstrated that a nuclear coding gene, methyltransferase-like protein 7A (METTL7A), improves the developmental potential of bovine embryos. We found that exogenous METTL7A modulates expression of genes involved in embryonic cell mitochondrial pathways and promotes trophectoderm development. Surprisingly, we discovered that METTL7A alleviates mitochondrial stress and DNA damage and promotes cell cycle progression during embryo cleavage. In summary, we have identified a novel mitochondria stress eliminating mechanism regulated by METTL7A that occurs during the acquisition of oxidative stress in embryo in vitro culture. This discovery lays the groundwork for the development of METTL7A as a promising therapeutic target for IVF embryo competence.

Controlled Dynamic Microfluidic Culture of Murine, Bovine, and Human Embryos Improves Development: Proof-of-Concept Studies.

Classical preimplantation embryo culture is performed in static fluid environments. Whether a dynamic fluid environment, like the fallopian tube, is beneficial for embryo development remains to be determined across mammalian species. Objectives of these proof-of-concept studies were to determine if controllable dynamic microfluidic culture would enhance preimplantation murine, bovine, and human embryo development compared to static culture. This prospective randomized controlled trial tested static versus controlled dynamic culture of preimplantation mouse (n = 397), bovine (n = 242), and human (n = 512) zygotes to blastocyst stages with outcome measures of embryo cleavage, cellular fragmentation, apoptosis, and blastocyst conversion rates. Dynamic culture of mouse and bovine zygotes with microfluidics significantly improved embryo development. Mouse placental imprinted gene expression was significantly different between embryos derived in vivo, by static culture, and by dynamic culture. Using human sibling zygotes, this dynamic microfluidic culture system increased the number of blastomeres per cleavage-stage embryo, reduced cellular fragmentation or apoptosis, improved blastocyst conversion rates, and enhanced blastocyst developmental stages. In conclusion, species-specific longitudinal studies demonstrated that dynamic microfluidic culture significantly improved embryo development, independent of culture media composition, temperature, and gaseous environment. These cellular indicators represent improved embryo development that can translate into higher pregnancy rates in transgenics, domestic livestock and endangered species and treating human infertility.

Establishment of bovine extraembryonic endoderm cells.

Understanding the mechanisms of hypoblast development and its role in the implantation is critical for improving farm animal reproduction, but it is hampered by the lack of research models. Here we report that a chemical cocktail (FGF4, BMP4, IL-6, XAV939, and A83-01) enables de novo derivation and long-term culture of bovine extraembryonic endoderm cells (bXENs). Transcriptomic and epigenomic analyses confirmed the identity of bXENs and revealed that they are resemble hypoblast lineages of early bovine peri-implantation embryos. We showed that bXENs help maintain the stemness of bovine ESCs and prevent them from differentiation. In the presence of a signaling cocktail sustaining bXENs, the growth and progression of epiblasts are also facilitated in the developing pre-implantation embryo. Furthermore, through 3D assembly of bXENs with bovine ESCs and TSCs, we developed an improved bovine blastocyst like structure (bovine blastoid) that resembles blastocyst. The bovine XENs and blastoids established in this study represent accessible in vitro models for understanding hypoblast development and improving reproductive efficiency in livestock species.

EFFECT OF LOW-TEMPERATURE ARGON PLASMA ON THE VIABILITY AND PROLIFERATION OF FIBROBLASTS IN VITRO

Background: Controlled generation of active forms of oxygen and nitrogen stimulates wound healing processes. One of the sources of controlled generation of active forms of oxygen and nitrogen is low-temperature argon plasma. Low-temperature plasma activates various forms of cellular activity. However, the subtle mechanisms of such stimulation remain unclear. Thus, there is a need for a detailed study of the biological effect of low-temperature argon plasma on human cells.Objective: to evaluate the effect of low-temperature argon plasma on the structure and proliferative activity of human fibroblasts in vitro.Materials and methods. Human fibroblast culture (line M-21, passage 25, Chumakov Institute of Poliomyelitis and Viral Encephalitis) was used in the work. After exposure to low-temperature argon plasma, the cells were cultured for 3 days in DMEM/F12 medium supplemented with 10% Gibco bovine embryo serum, then morphofunctional analysis of the cells was performed using vital staining. After 3 days, the cells were passaged (reseeded in other Petri dishes with a 1:2 dilution), then the cells were cultured for 3 days, and a repeated morphofunctional analysis was performed. All work with cell cultures was carried out in accordance with the international GMP standard.Results. Stimulation of the proliferative activity of fibroblasts was noted in the second passage after treatment with low-temperature argon plasma from a distance of 10 cm for 30 and 45 seconds and 15 cm for 45 seconds.The use of a collagen bandage screen significantly increases cell survival when exposed to plasma from a distance of 10 cm for 15 seconds. With screening, the decrease in the integrity of cell membranes after plasma exposure is less pronounced than with exposure without screening. When reseeding, cells treated in the presence of a collagen screen exhibit high proliferative activity and restore membrane integrity.

Influence of thermal stress during in vitro maturation on the developmental competence of oocytes and embryos and the expression of Sirtuins in cumulus oocyte complexes in cattle

Sirtuins are of central importance in many cellular functions and promote cell survival under stress. However, little information is available regarding the relationship between sirtuins and female reproductive biology, especially in response to thermal stress. This study investigated the influence of moderately high (40°C) and low (37°C) thermal stress during in vitro maturation on the development competence of bovine oocytes and embryos. The expression and abundance of sirtuins and other proteins involved in stress response were also studied. The cumulus-oocyte complexes (COCs) of Simmental (Bos taurus) cows underwent in vitro maturation (IVM) at different temperatures (37°C, 38.5°C and 40°C). Before maturation, the oocytes were stained with Brilliant Cresyl Blue (BCB) and categorized as labeled (BCB+) or unlabeled (BCB-). Embryo production was analyzed at the different IVM temperatures. Polar body extrusion was evaluated following IVM, and the mRNA and protein abundance of sirtuins and P53 in oocytes and cumulus cells were analyzed. The differing temperatures during IVM did not significantly alter polar body extrusion and cleavage rates; however, significant differences in blastocyst production were observed. COCs matured at 38.5°C (control, 37.3%) had the highest blastocyst rate, in contrast to those matured at 37°C (33.2%) and 40°C (21.5%). In all groups, the blastocyst rates were higher for BCB+ oocytes than for BCB- oocytes. In BCB+ oocytes, the expression of SIRT1, SIRT2, SIRT3, and SIRT5 genes was higher after maturation than that before maturation and in most of the cases, the expression was higher when IVM was performed at 38.5°C. In the cumulus cells of BCB+ COCs, only SIRT2 remained unaffected by the maturation temperature. In summary, the temperature change of ±1.5°C for 24 h during bovine oocyte maturation impaired in vitro embryo development. This lead to several cellular biochemical alterations in oocytes and granulosa cells from COCs with higher developmental competence (BCB+). Thus, SIRT1 is important for in vitro embryonic development and may protect against cold and heat stress.

74 Impact of tetraploid complementation on trophectoderm and inner cell mass in hybrid (Bubalus bubalis × Bos taurus) and bovine embryos

74 Impact of tetraploid complementation on trophectoderm and inner cell mass in hybrid (Bubalus bubalis × Bos taurus) and bovine embryos

151 Assessing the efficiency of cytosine base editors targeting MYO7A in bovine embryos

151 Assessing the efficiency of cytosine base editors targeting MYO7A in bovine embryos

91 Haptoglobin addition during in vitro culture influences gene expression related to oxidative stress and lipid metabolism in bovine embryos

91 Haptoglobin addition during in vitro culture influences gene expression related to oxidative stress and lipid metabolism in bovine embryos

What we can learn from the bovine embryo and mouse models to enable in vitro gametogenesis in cattle

What we can learn from the bovine embryo and mouse models to enable in vitro gametogenesis in cattle

102 Expression pattern of cytoplasmic polyadenylation element binding protein 1 in bovine oocytes and embryos

102 Expression pattern of cytoplasmic polyadenylation element binding protein 1 in bovine oocytes and embryos