Bovine Embryos Research Articles

Sexually dimorphic gene expression responses of bovine embryos to the maternal microenvironment on day 13 of gestation

BackgroundVarious studies have highlighted significant differences in developmental kinetics and sensitivity to developmental conditions between male and female bovine embryos. These differences are thought to be caused in part by the sexually dimorphic expression of genes located on the sex or autosomal chromosomes. However, little is known about the dimorphic gene expression patterns of bovine embryos at the initiation of elongation, which is one of the critical stages of development. Furthermore, to the best of our knowledge, there is little or no data available on the sexually dimorphic gene expression patterns in bovine embryos in relation to maternal environmental conditions during the initiation of elongation. Therefore, the main objective of this study was to investigate the sexually dimorphic gene expression responses of embryos to the maternal environment at the initiation of elongation in embryos developed in lactating dairy cows and nonlactating nulliparous heifers.ResultsGene expression analysis showed that 159 genes including those involved in steroid biosynthesis and gastrulation were differentially expressed exclusively between male and female embryos developed in cows. Among these, 61 genes including CYP39 A1, CYP2R1 and CYP27B1 were upregulated and 98 genes including HSD17B1, HSD17B10 and aromatase (CYP19 A1) were downregulated in male embryos. Chromosomal analysis showed that 31.2% of the differentially expressed genes (DEGs) including glucose-6-phosphate dehydrogenase (G6PD) were located on the X chromosome, and 96% of those were upregulated in female embryos. Similarly, 254 genes including those involved in female sex differentiation, placenta development, transmembrane transport, and cell adhesion were differentially expressed exclusively between the male and female embryos developed in heifers. Of these, 108 genes including HSD17B11, HSD17B12, and HSD3B1 were upregulated, and 146 genes including SLC16 A9, SLC10 A1, SLC10 A3, SLC16 A5, SLC22 A23, SLC25 A43, SLC35 A2, SLC35 C1, and SLC4 were downregulated in male compared to female embryos. In addition, 17.3% of the DEGs were located on the X chromosome and 75% of the DEGs located on the X chromosome were upregulated in female embryos. On the other hand, 38 genes including SLC30 A10, SLC10 A4, ATP6 AP1, and KDM5 C showed sexually dimorphic expression patterns in day 13 bovine embryos irrespective of the maternal environment. These genes accounted for only 19% and 13% of the genes that showed sexually dimorphic expression in embryos developed in cows and heifers, respectively and the expression difference of these genes in male and female embryos was then likely influenced by the sex of the embryo.ConclusionThis study revealed that embryos developed in lactating cows showed sexually dimorphic expression of genes involved in various functions including steroid biosynthesis and gastrulation. In contrast, embryos developed in heifers displayed sexually dimorphic expression of genes related to placental development, female sex differentiation, and transmembrane transport. This suggests that the reproductive tract environments of cows and heifers differently affect the sex specific expression of genes in bovine embryos. A higher proportion of genes that showed sexually dimorphic expression in cow embryos were located on the X chromosome, and the majority of these genes were upregulated in female embryos. Overall, this study provides insight into genes that exhibit sexually dimorphic expression patterns in day 13 bovine embryos due to the maternal reproductive tract microenvironment or solely due to the sex of the embryo.

The embryonic DPPA3 gene stimulates the expression of pregnancy-related genes in bovine endometrial cells.

Extracellular vesicles (EVs) released by cells contain mRNAs, miRNAs, lncRNAs, lipids, and proteins, playing crucial roles in cell-cell communication. While full-length mRNA transcripts have been documented in EVs secreted by cancer cells, there are no reports on full transcripts secreted by embryos. Our study aimed to identify EV mRNAs in the culture media of bovine embryos and investigate their roles in embryo-maternal communication. Following the isolation of EVs from in vitro fertilization media samples and RNA sequencing, we identified a full mRNA transcript of DPPA3, known to play an essential role in embryo development. To examine the role of DPPA3 in embryo-maternal communication, an in vitro transcribed mRNA of DPPA3 was transfected into bovine endometrial epithelial cells. Transfected and control cells were subsequently analyzed with RNA sequencing and proteomics to assess the effects of DPPA3 on gene expression. A total of 24 genes were found to be upregulated, and one gene was downregulated (FDR <0.01) following DPPA3 transfection, many with known functions in pregnancy recognition. Proteomic analysis revealed 28 differentially expressed proteins, with 19 upregulated and 15 downregulated. Two proteins, ISG15 and MX1, overlapped with the differentially expressed mRNAs. To mimic the natural transfer of EVs from embryos to endometrial cells, we performed coculture with day-8 blastocysts or supplemented the cells with embryo-conditioned culture media. DPPA3 presence was detected in endometrial cells exposed to embryo-conditioned media after just 30 min. Overall, our study highlights the significant role of EVs in cell-cell communication through mRNA signaling from the embryo to the mother.

Calf Fitness Associates with Early Embryo and Recipient Metabolomes and with Calf Epigenetic Marks.

We used metadata to explore the metabolic interplay between culture medium from in vitro-produced bovine embryos transferred fresh or frozen, recipient blood plasma, and calf fitness, alongside gene expression and methylation in calf lymphocytes. Principal component (PC) analysis (PCA) identified covariates that were depicted in Debiased Sparse Partial Correlation networks and analyzed as enriched pathways. Four PCs explained 13.77, 9.58, 7.73 and 5.84% variability. PC1 clustered only mother weight and two embryonic metabolites. PC2, PC3 and PC4 associated 10, 17, and 5 calf features with 10, 6, and 16 embryonic and 2, 20, and 5 recipient metabolites, respectively. Subsequently, gene methylation and expression, and calf fitness were analyzed by PCA. Three PCs covered 100% variability. PC1 associated acid-base balance, protein metabolism, Cl-, and Ca2+ with IGF2 and IL1R1 expression, and IL4 and IL12B methylation. PC2 linked H19 expression and methylation with growth and biochemical traits. PC3 clustered growth, hydration, and redox balance, with IGF2, IGF2R, IL1R1 and IL3 methylation, and H19, IGF2, IGF2R and IL12B expression. Gene methylation connected with embryo metabolites through networks via K+, Cl-, HCO3- and TCO2. Calf fitness parallels the early metabolic fingerprints of the embryo and recipient, allowing embryo transfer decision-making based on calf health.

Establishing Bovine Embryonic Stem Cells and Dissecting Their Self-Renewal Mechanisms

Bovine pluripotent stem cells (PSCs) hold significant potential for diverse applications in agriculture, reproductive biotechnology, and biomedical research. However, challenges persist in establishing stable bovine PSC lines and understanding the mechanisms underlying their pluripotency maintenance. Here, we derived bovine embryonic stem cells (bESCs) from Holstein cattle embryos. These cells exhibited robust differentiation capacity into three germ layers in vitro and in vivo. Transcriptome analysis revealed distinct molecular profiles compared to primed-state bESCs. Notably, bESC proliferation ceased on methanol-treated feeder cells, in contrast to mouse ESCs (mESCs), which proliferated normally. Pathway analysis identified key signaling events critical for bESC survival and proliferation, highlighting species-specific regulatory mechanisms. Furthermore, the derived bESCs demonstrated chimerism capacity in early bovine embryos, underscoring their functional pluripotency. This work provides a foundation for advancing bovine embryology research and stem cell-based biotechnologies in livestock.

A bovine 3D endometrium-on-a-chip reveals the role of conceptus-derived factors CAPG and PDI in conceptus-endometrial communication

Abstract Early embryo loss affects all mammalian species, including humans and agriculturally important food-producing mammals such as cattle. The developing conceptus (embryo and extra-embryonic membranes) secretes factors which modify the endometrium and can be critical for early pregnancy processes such maternal recognition of pregnancy (MRP) and enhancing uterine receptivity to implantation. For example, a competent bovine conceptus secretes IFNT to initiate MRP. The bovine conceptus also secretes other proteins at the time of MRP, including CAPG and PDI, which are highly conserved among placental mammals. We have previously shown that these proteins act upon the endometrium to modulate receptivity, embryo development, and implantation in species with different implantation strategies (humans and cattle). We hypothesise that developing a novel 3D bovine endometrium on a chip system will enhance our understanding of the role of conceptus-derived factors in altering the endometrium and/or ULF secretion. Here we have developed a 3D bovine endometrium on a chip system, comprising both stromal and epithelial cell culture combined with culture medium flow better mimics the in vivo endometrium and exposure to conceptus-derived factors than conventional 2D endometrial cell culture. We have demonstrated that the conceptus-derived proteins CAPG and PDI modulate the endometrial transcriptome and secretory response to promote pathways associated with early pregnancy and alter ULF composition. This work highlights the critical need for more robust and in vivo-like culture systems to study endometrial-conceptus interactions in vitro to further investigate the role of conceptus derived factors for pregnancy success.

Zinc eluted from glassware is a risk factor for embryo development in human and animal assisted reproduction†.

In assisted reproduction, many factors in the culture environment, including light, temperature, pH, and culture media, can reduce preimplantation embryo viability. Laboratory glassware is also a known risk factor for in vitro embryos; however, the underlying mechanisms that disrupt embryonic development remain unclear. We identified Zn eluted from glassware as an embryotoxic substance. In mouse embryos, Zn induced delayed development, abnormalities in chromosome segregation, cytokinesis, zygotic gene activation (e.g. Zscan4a and murine endogenous retrovirus with leucine, also known as MERVL), and aberrantly upregulated developmental gene expression (e.g. Hoxa1, Hoxb9, T, and Fgf8) that could be mediated through metal regulatory transcription factors (e.g. Mtf1). Subsequently, Zn exposure led to significantly reduced blastocyst formation. Post-implantation, Zn-exposed embryos were associated with normal birth rates, however, the birth weight increased by an average of 18% compared with embryos cultured without Zn. Furthermore, Zn exposure affected the development of bovine and human embryos, with species-based variation in the strength and timing of these effects. To mitigate these embryotoxic effects, we identified a method to prevent glass toxicity using chelating agents. This research not only highlights the importance of risk control in embryo culture but also facilitates the development of safe and effective methods for assisted reproduction.

Feeding rumen-protected choline during the periconceptional period programs postnatal phenotype of suckled beef calves

BackgroundSupplementation of choline chloride in culture medium programs the preimplantation bovine embryo to increase weaning weight of the resultant calf. Here, it was hypothesized that similar programming actions of choline can be induced by feeding rumen-protected choline (RPC) to beef cows during the periconceptional period.ResultsA preliminary experiment was conducted to determine changes in circulating concentrations of choline and its metabolites after RPC supplementation. Suckled beef cows were individually fed 0, 30, 60, and 90 g of RPC (i.e., 0, 8.6, 17.3 and 25.9 g choline chloride) and blood samples were collected at random times after feeding. There were no differences in plasma concentrations of choline or its metabolites between groups. In the second experiment, effects of feeding 60 g/d RPC from d −1 to 7 relative to timed artificial insemination were examined for suckled beef cows. Feeding RPC did not affect pregnancy or calving rates, pregnancy losses, plasma concentrations of pregnancy-associated glycoproteins, gestation length or calf birth weight. Calves from RPC fed dams were lighter than control calves at ~118 days of age (range 75–150; age included in the statistical model) and at weaning at ~248 days of age. There was no effect of treatment on hip height at ~118 days of age although there was a trend for RPC calves to be shorter at weaning. Weight/height ratio was lower for RPC than control at both 118 and 248 days of age. Treatment did not affect testis weight at ~118 days of age.ConclusionsSupplementation of RPC during the periconceptional period programmed development to alter calf phenotype in the postnatal period. The net result, reduced body weight, was the opposite of the phenotype caused by the addition of choline to embryo culture medium.

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.

Polarization-independent regulation of the subcellular localization of Yes-associated protein 1 during preimplantation development.

Cell polarization is a crucial developmental process that determines cell differentiation in mouse embryos. During this process, an extensively expressed transcriptional regulator, Yes-associated protein 1 (YAP1), is localized either to the cytoplasm or to the nucleus via HIPPO signaling. In mouse premorula embryos, YAP1 is present in the nuclei of all cells. Thereafter, YAP1 is distributed to the nuclei of outer cells or cytoplasm of inner cells, depending on the establishment of cell polarity and morula formation. However, the dynamics of YAP1 localization in other species, including ruminants, remain unclear. To gain an in-depth understanding of cell differentiation in mammalian embryos, we investigated YAP1 localization changes in bovine embryos. Unlike in mouse morulae, YAP1 displayed cytoplasmic localization in most cells, including the outer cells of bovine morulae, after the 32-cell stage. Next, we analyzed the relationship between cell polarity and nuclear localization of YAP1. Polarization of outer cells in the bovine morula began at the late 16-cell stage and was established by the late 32-cell stage, indicating that polarization preceded the nuclear localization of YAP1 in bovine embryos. To explore the regulation of YAP1 localization in bovine morula, we analyzed zona-free embryos and found that the presence of the zona pellucida significantly enhanced YAP1 cytoplasmic localization. Moreover, we observed ectopic expression of SRY-box transcription factor 2 in zona-free blastocysts, which indicated that cytoplasmic localization of YAP1 was associated with the suppression of pluripotency in the trophectoderm. These findings provide valuable insights into the molecular mechanisms underlying the first cell differentiation in mammalian embryos.

Effect of bovine oviductal epithelial cell lysate on the developmental competence and quality of bovine in vitro fertilized embryos.

Effect of bovine oviductal epithelial cell lysate on the developmental competence and quality of bovine in vitro fertilized embryos.

Revealing the effects of lactate on bovine SCNT embryo development through transcriptome sequencing analyses.

Revealing the effects of lactate on bovine SCNT embryo development through transcriptome sequencing analyses.

Improving Bovine Embryo Development and Quality Using Bovine Oviductal Epithelial Cell-Derived Conditioned Medium (bOEC-CM).

The embryo co-culture systems with the monolayer cultured cells are complex, unreproducible, and have a high probability of biological contamination. Therefore, nowadays, using conditioned media is a suitable alternative to these methods. This study aimed to investigate the impact of utilizing bovine oviductal epithelial cell-derived conditioned medium (bOEC-CM) on subsequent embryo development and quality. Bovine embryos produced in vitro were cultured in a specific medium supplemented with either 5% charcoal-stripped FBS (Charcoled Strip Serum [CSS]) or 10% bOEC-CM from either Days 1 or 3 post-fertilization. Various parameters, such as cleavage rate, blastocyst formation, hatching rate and blastocyst quality, were assessed. The results indicated that adding 10% CM from Day 1 significantly reduced the cleavage rate compared to using CSS on the same day (p<0.05). Furthermore, the CSS and CM from both Days 1 and 3 increased blastocyst formation rates (p<0.05). Notably, the addition of 10% CM on Day 3 significantly improved the hatching rate compared to the other groups (p<0.05). Both CM and CSS were found to enhance the inner cell mass (ICM), trophectoderm (TE) and total cell numbers in blastocysts when used on both Days 1 and 3 (p<0.05). Additionally, CM from Day 3 positively influenced the expression levels of development-specific genes in cultured embryos (p<0.05). Overall, the findings suggest that using bOEC-CM at a 10% concentration may provide a promising supplement even better than serum and traditional co-culture methods during the last 5 days of embryo culture.

Early identification of bovine pregnancy status and embryonic mortality†.

Interferon-tau (IFNT) is produced by the trophectoderm cells in the bovine conceptus as early as Day 12 following fertilization. It was hypothesized that IFNT detection in blood, milk and/or cervical secretions could be used to diagnose pregnancy in lactating cows. Recombinant bovine (rb) IFNT was generated to produce goat and rabbit anti-rbIFNT polyclonal antibodies and an enzyme-linked immunosorbent assay (ELISA) for bIFNT was developed using these reagents. The IFNT ELISA did not cross-react with other type I or II IFNs and had a limit of detection of 50-100pg/ml. The IFNT ELISA detected IFNT in external os cervical swabs from Days 15 to 25 post-AI, but did not detect IFNT in serum, plasma, or milk. The time for most accurately detecting IFNT in cervical fluid was Days 16-19 after AI. A custom bovine swab device used to collect cervical secretions reduced false negative rates to 5.5% (94.5% Sensitivity) in dairy cows on Day 17 and 0 to 3.4% (100 and 96.6% Sensitivity) in beef cows on Days 18 or 16, respectively. In summary, the detection of IFNT in cervical fluid by ELISA provides an accurate indication of pregnancy status in lactating dairy cows. Early identification of the non-pregnant cow allows re-insemination on Day 21 compared to waiting until ultrasound on Day ~32-39. Also, the detection of IFNT on Day 17 followed by loss of pregnancy detected by ultrasound on Day 32 provides a novel research tool for studying pregnancy loss caused by embryonic mortality.

Bovine embryo evaluation with machine learning: A tool for the future of assisted reproduction

In recent years, the integration of machine learning and com­puter vision (ML/CV) technologies has significantly advanced assisted reproductive technologies, and more specifically, the field of embryo analysis. In human in vitro fertilization (IVF), ML/CV has been used on time-lapse imaging systems to evalu­ate embryo morphological features to classify their quality and associated implantation potential. This technology has improved the accuracy and efficiency of embryo grading and selection by evaluating more parameters than the human eye can see under the microscope and removing the subjective na­ture from human evaluation. However, there has been little progress as it relates to ML/CV in the commercial bovine con­ventional embryo transfer and IVF industry. Thus, this study aimed to explore the potential of ML/CV automatic stage and grading techniques on bovine embryos. It was hypothesized that the machine learning model would outperform humans with little to no experience and perform equally or greater than humans with moderate or more experience staging and grading bovine embryos.

Effect of Bovine Follicular Fluid Small Extracellular Vesicles Isolated by Ultracentrifugation and Chromatography on In Vitro Oocyte Maturation and Embryo Development.

Small extracellular vesicles (sEVs) play a crucial role in intercellular communication and have demonstrated significant relevance in reproductive biotechnology, particularly in in vitro maturation (IVM) and bovine embryo production. This study evaluates the effects of bovine follicular fluid-derived extracellular vesicles (ffsEVs) isolated using two methods: ultracentrifugation (UC) and size-exclusion chromatography (SEC) on oocyte maturation and preimplantational embryonic development. Significant differences in the size of ffsEVs obtained by both isolation methods were noted, with UC-derived ffsEVs (UC ffsEVs) being smaller than those isolated by SEC (SEC ffsEVs). UC ffsEVs were more effective in upregulating critical oocyte quality genes, such as HSF1 and CPT1B. However, no significant differences were observed in embryonic developmental rates. Furthermore, the expression of genes associated with preimplantational embryonic quality revealed that only the SEC ffsEVs group exhibited a significant increase in IFNT1 and SOX2 levels, indicating an enhancement in embryonic quality. Notably, blastocysts derived from SEC ffsEVs also showed a higher total cell count compared to those from UC ffsEVs. No differences were found in other critical genes like GLUT1 and CDX2. These results suggest that the use of SEC ffsEVs could improve the in vitro embryo production process, highlighting the importance of the isolation method in determining the functional efficacy of ffsEVs according to research objectives.

Delayed Blastocyst Development is Associated with Altered Metabolism and Proteome in Male and Female Bovine Embryos.

Developmentally delayed embryos are associated with reduced implantation potential and live birth rates; however, inherent causes of delayed development are not well understood. Metabolism during preimplantation development is responsible for the production of energy and biosynthetic material to support growth, and disturbances to these pathways can reduce embryo viability. The present study utilized electrochemical microsensors to determine differences in rates for oxygen consumption, extracellular acidification, and hydrogen peroxide production between normal and slow-growing, male and female bovine blastocysts. In addition, pooled samples of blastocysts were subjected to proteomic analysis to determine differences in the abundance of proteins associated with metabolism between the sexes and developmental timing status. In comparison to blastocysts developing over a normal timespan, blastocysts forming 1 to 2days later had a higher oxygen consumption rate, differences in abundance of electron transport complex proteins, and reduced abundance of biosynthetic enzymes when compared to blastocysts developing during a normal timeline. Embryo sex resulted in unique differences in metabolic enzyme abundance with potentially different contributions to delayed development. In addition, male and female blastocysts had differential protein abundances indicating differences in metabolic pathway activity. Therefore, embryos that took longer to reach the blastocyst stage of development appeared to have an imbalance between energy production and biosynthetic activity, which could differentially impact male and female embryos.

An advanced cytosine base editor enabled the generation of cattle with a stop codon in the β-lactoglobulin gene.

β-Lactoglobulin (BLG) is an allergen present in milk that can induce an acute immune response in certain individuals. The successful use of cytosine base editors (CBEs) can introduce stop codons into premature mRNA, thereby generating animals with disrupted genes that negatively regulate target traits. In this study, we employed a CBE system to target the major milk allergen BLG in bovine embryos, mammary epithelial cells, and live cattle. First, the precise single-base editing of the BLG gene in bovine embryos was achieved by designing an effective sgRNA to induce a c.61C > T substitution in the coding region, converting codon 21Gln (p.21Gln) to a premature stop codon. Sanger sequencing revealed an editing efficiency of 83.3% (20 out of 24 embryos), including two homozygous edits. Second, a bovine mammary epithelial cell line harboring BLG edits was constructed using the same CBE system. Sequencing showed that the designed sgRNA1 enabled the simultaneous conversion of three consecutive cytosines (c.59-61CCC > TTT) to thymines. At position c.61, single-cell clones exhibited monoallelic or biallelic editing (BLGc.61C > T), with monoallelic edits at positions c.59 and c.60 (CC > TT). Gene expression analysis confirmed that the BLGc.61C > T mutation effectively suppressed BLG expression at both the mRNA and protein levels, even in monoallelically edited cells. Finally, we successfully generated a heterozygous BLGc.61C > T single-base-edited dairy cow that despite its heterozygosity, showed significantly reduced BLG expression in the mammary epithelial cells and milk. Collectively, this study demonstrates the feasibility of using CBEs to disrupt BLG expression in dairy cows and provides a foundation for application in generating hypoallergenic dairy products.

Bta-miR-93 regulates the maternal-fetal tolerance in dairy cows via promoting PD-L1-mediated M2 macrophage polarization†.

Bovine embryo resorption is one of the key factors restricting the reproductive efficiency in dairy cattle, which mainly occurs in the early stage of maternal recognition of pregnancy (MRP), causing substantial economic losses to the dairy industry. Macrophages, the most numerous endometrial immune cells in cows during early pregnancy, are critical in developing maternal-fetal immune tolerance. However, the mechanism of their action on the maternal-fetal interface of dairy cows remains unknown. MicroRNAs have important roles in immune tolerance and macrophage polarization, but the underlying mechanisms still need further investigation. In previous laboratory studies, RNA-sequencing revealed that bta-miR-93 was dramatically reduced in bovine endometrial luminal epithelial cells (bEECs) upon IFN-τ stimulation. In current study, it is revealed that the expression of bta-miR-93 was decreased substantially in the endometrium of pregnant cows, and negatively correlated with that of PD-L1. In vitro experiments displayed that suppression of bta-miR-93 promoted M2 Polarization via promoting PD-L1/PD-1/AKT/mTOR signaling pathway in bEECs and bovine macrophages (BoMac) co-culture model, and vice versa. The 3'-UTR of PD-L1 is directly regulated by bta-miR-93. Furthermore, our in vivo studies revealed that overexpression of bta-miR-93 efficiently leads to embryo resorption and suppression of M2 polarization in mice. Overall, the findings suggested that reduced bta-miR-93 levels were found to be implicated in pregnancy immune tolerance by boosting M2 macrophage polarization via promoting PD-L1/PD-1/AKT/mTOR signaling pathway. Bta-miR-93 might be investigated as auspicious identification signal for a successful pregnancy and a potential therapeutic target for reproductive disorders.

Evaluation of Lysophosphatidic Acid Effects and Its Receptors During Bovine Embryo Development.

Lysophosphatidic acid (LPA) is a small bioactive phospholipid which plays an important role during embryonic development and promotes developmental potential of in-vitro-produced (IVP) embryos in several species, including sheep and pigs. In bovines, LPA accelerates IVP blastocyst formation through the Hippo/YAP pathway. However, other LPA effects and its potential receptors during bovine embryo development are less clear. In this study, we used enzyme-linked immunosorbent assay (ELISA) to assess the presence of LPA in bovine oviductal fluid and determine cell apoptosis in embryos after LPA stimulation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and quantitative reverse transcription polymerase chain reaction (qRT-PCR). We further evaluated potential receptors of LPA through molecular docking, RNA-seq data analysis and quantitative RT-PCR. LPA was found to be present in oviductal fluid. An increase in total cell number and a decrease in apoptosis levels were detected in day 7 blastocysts after LPA treatment. Among eight LPA receptors (LPARs), GPR87 and LPAR2 showed the highest affinity with LPA and their transcripts were expressed in embryos after the 16-cell stage in RNA-seq and qRT-PCR analysis. However, only the expression of LPAR2 was significantly increased in day 6 blastocysts after LPA stimulation, indicating its potential role in LPA-mediated signaling pathways. Our data highlight the positive effects of LPA on embryos and enrich information of related signaling mediators of LPA during embryonic development.

TIME-LAPSE КУЛЬТИВИРОВАНИЕ IN VITRO ЭМБРИОНОВ КРУПНОГО РОГАТОГО СКОТА ДО СТАДИИ БЛАСТОЦИСТЫ

The aim of the study is to apply time-lapse observation to monitor the development of bovine embryos in vitro, and to analyze the prognostic potential of morphokinetic parameters of cleavage as a criterion for assessing the competence of the embryo to form a blastocyst. The study was performed on postmortem material; oocyte maturation, fertilization and cultivation of bovine embryos to the blastocyst stage occurred in vitro. The potential ability of the embryo to form a good quality expanded blastocyst can be determined based on the temporal and morphological characteristics of the first and three subsequent divisions of the embryo (from the zygote to the formation of the 4-cell stage). If the embryo division fits into the time intervals we have defined and there are no signs of abnormal multipolar or unequal zygote cleavage, the embryo develops more effectively and more often reaches the blastocyst stage: the first division of the embryo should occur in (27.77 ± 0.28) h after oocyte fertilization; the duration from the moment of formation of the cleavage furrow to the formation of a two-cell embryo should be no more than (0.38 ± 0.05) h; the second and third divisions with the formation of a 4-cell embryo should occur no later than (36.58 ± 0.27) and (37.77 ± 0.32) h after fertilization. Embryos whose development did not fit into the specified time parameters, as a rule, stopped at different stages of development, not developing to the blastocyst stage. Multipolar division into three or more blastomeres and unequal division into blastomeres of different sizes were, according to our data, negative prognostic signs of early embryogenesis. Multipolar division occurred with a frequency of 29.71 % in embryos that did not form a blastocyst, and only 9.26 % in embryos that developed to the blastocyst stage, unequal cleavage – with a frequency of 5.86 and 0.93 %, respectively. Viable and promising embryos follow much stricter time frames of division, and embryos that stop developing, as a rule, go beyond the time range and more often demonstrate division anomalies.