Development Of Bovine Embryos Research Articles

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.

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.

61 The role of CIDEA and dynamics of lipid droplets in bovine embryo development

61 The role of CIDEA and dynamics of lipid droplets in bovine embryo development

90 Effect of sediment from spontaneously immortalized oviduct epithelial cells on the development and fertility of bovine OPU-IVF embryos

90 Effect of sediment from spontaneously immortalized oviduct epithelial cells on the development and fertility of bovine OPU-IVF embryos

50 Ploidy correction during pre-implantation development of bovine triploid embryos

50 Ploidy correction during pre-implantation development of bovine triploid embryos

200 Effect of coenzyme Q10 supplementation on bovine oocyte maturation and embryo development

200 Effect of coenzyme Q10 supplementation on bovine oocyte maturation and embryo development

Beta-hydroxybutyrate alters bovine preimplantation embryo development through transcriptional and epigenetic mechanisms†.

Developing embryos are susceptible to fluctuations in the nutrients and metabolites concentrations within the reproductive tract, which can lead to alterations in their developmental trajectory. Ketotic dairy cows have diminished fertility, and elevated levels of the ketone body beta-hydroxybutyrate (BHB) have been associated with poor embryonic development. We used an in vitro model based on either in vitro fertilization (IVF) or parthenogenesis to investigate the effects of BHB on the preimplantation bovine embryo development, epigenome, and transcriptome. Embryo culture medium was supplemented with BHB at a similar concentration to that present in the blood of cows suffering with severe ketosis, followed by analysis of blastocysts formation rate, diameter, total number of cells, levels of H3K9 beta-hydroxybutyrylation (H3K9bhb), apoptosis, and transcriptional alterations. As a result, we observed that BHB reduced the blastocysts rates, the diameter and the total number of cells in both parthenotes and IVF embryos. Exposure to BHB for either 3 or 7days greatly increased the H3K9bhb levels in parthenotes at the 8-cells and blastocyst stages, and affected the expression of HDAC1, TET1, DNMT1, KDM6B, NANOG, and MTHFD2 genes. Additionally, culture of IVF embryos with BHB for 7days dramatically increased H3K9bhb and reduced NANOG in blastocysts. RNA-seq analysis of IVF blastocysts revealed that BHB modulated the expression of 118 genes, which were involved with biological processes such as embryonic development, implantation, reproduction, proliferation, and metabolism. These findings provided valuable insights into the mechanisms through which BHB disrupts preimplantation embryonic development and affects the fertility in dairy cows.

Molecular cloning of PRD-like homeobox genes expressed in bovine oocytes and early IVF embryos.

Embryonic genome activation (EGA) is a critical step in early embryonic development, as it marks the transition from relying on maternal factors to the initiation of transcription from embryo's own genome. The factors associated with EGA are not well understood and need further investigation. PRD-like (PRDL) homeodomain transcription factors (TFs) are considered to play crucial roles in this early event during development but these TFs have evolved differently, even within mammalian lineages. Different numbers of PRDL TFs have been predicted in bovine (Bos taurus); however, their divergent evolution requires species-specific confirmation and functional investigations. In this study, we conducted molecular cloning of mRNAs for the PRDL TFs ARGFX, DUXA, LEUTX, NOBOX, TPRX1, TPRX2, and TPRX3 in bovine oocytes or in vitro fertilized (IVF) preimplantation embryos. Our results confirmed the expression of PRDL TF genes in early bovine development at the cDNA level and uncovered their structures. For each investigated PRDL TF gene, we isolated at least one homeodomain-encoding cDNA fragment, indicative of DNA binding and thus potential role in transcriptional regulation in developing bovine embryos. Additionally, our cDNA cloning approach allowed us to reveal breed-related differences in bovine, as evidenced by the identification of a high number of single nucleotide variants (SNVs) across the PRDL class homeobox genes. Subsequently, we observed the prediction of the 9aa transactivation domain (9aaTAD) motif in the putative protein sequence of TPRX3 leading us to conduct functional analysis of this gene. We demonstrated that the TPRX3 overexpression in bovine fibroblast induces not only protein-coding genes but also short noncoding RNAs involved in splicing and RNA editing. We supported this finding by identifying a shared set of genes between our and published bovine early embryo development datasets. Providing full-length cDNA evidence for previously predicted homeobox genes that belong to PRDL class improves the annotation of the bovine genome. Updating the annotation with seven developmentally-important genes will enhance the accuracy of RNAseq analysis with datasets derived from bovine preimplantation embryos. In addition, the absence of TPRX3 in humans highlights the species-specific and TF-specific regulation of biological processes during early embryo development.

Comparative analysis of Piezo-ICSI and conventional ICSI in bovine embryo development

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique (ART) mainly used to overcome severe male factor infertility problems in humans and animals. However, in cattle, one of the most demanded species for its meat and milk, the efficiency of this technique is low. The present study compared the effect of the piezoelectric and conventional injection systems on the preimplantational development and quality of bovine embryos generated by ICSI. Evaluations of the conditions for performing the Piezo-ICSI procedure showed that the application of a strong pulse (I4S7) was more effective in damaging the sperm plasma and acrosomal membranes prior to injection, compared to a soft pulse (I2S2, P < 0.05). In addition, Piezo-ICSI embryos without the application of exogenous activators achieved similar levels of development as Piezo-ICSI embryos activated with ionomycin and anisomycin (P > 0.05). When comparing conventional and piezoelectric injection systems, no significant differences in embryo development were observed (P > 0.05). However, embryos generated by Piezo-ICSI showed a higher embryo quality in terms of total cell number (P < 0.05). In addition, Piezo-ICSI embryos showed an expression profile of genes essential for embryonic development similar to IVF embryos (P > 0.05), in contrast to conventional ICSI-derived embryos, which presented overexpression of CASP3 and IFNT2 (P < 0.05). In conclusion, we confirmed that Piezo-ICSI is a more convenient approach than traditional ICSI, since does not require exogenous activation and generate embryos of better quality, regarding the total number of blastomeres and the pattern of gene expression observed.

Elucidation of the pluripotent potential of bovine embryonic lineages facilitates the establishment of formative stem cell lines.

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cellnuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding.

Can C-type natriuretic peptide during in vitro maturation or culture influence the development of bovine embryos?

The C-type natriuretic peptide (CNP) plays a central role in regulating the meiotic progression of oocytes into growing follicles in mammals. However, there are few reports examining the relationship between CNP and embryonic development. In our study, different concentrations (50, 100, or 150 nM) of CNP were added during in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) or in vitro culture (IVC) of the bovine embryos (B. taurus indicus). The effects on embryo production and transcript abundance of the 20 genes of greatest interest that are related to metabolism, oocyte maturation, follicular development, cell signaling, oxidative and thermal stress, maternal-fetal interaction, and epigenetic regulation were evaluated. The blastocyst rate was influenced by CNP treatment (P = 0.049). Blastocyst rates were 31.05% (136/438) in the control group, 33.47% (162/484) in the 50 nM treatment group, 35.24% (179/508) in the 100 nM treatment group, and 32.53% (162/498) in the 150 nM treatment group for IVM. Furthermore, with IVC CNP supplementation, blastocyst rates were 28.49% (100/351) at 50 nM, 27.67% (119/430) at 100 nM, and 26.92% (112/416) at 150 nM. Moreover, the expression of RE1 silencing transcription factor (REST), a gene related to pluripotency and to embryonic development, was greater (P = 0.028) in response to 150 nM CNP supplementation in IVM. Finally, we observed for the first time the expression of the CNP receptor (NPR2) in embryos and the possible action of CNP at this stage. In conclusion, our data provide a reference for the improvement of IVM results in the in vitro production of bovine embryos with supplementation with 100 nM CNP, and this is the first study to demonstrate the expression of the CNP receptor (NPR2) in bovine embryos.

Human recombinant interleukin-6 improves the morphological quality of cryopreserved in vitro produced bovine blastocysts

This work explored whether a well-characterized recombinant human interleukin-6 (hIL6) protein will influence in vitro produced (IVP) bovine embryo development and survival after cryopreservation. Cumulus oocyte complexes were collected from abattoir derived ovaries, matured for 24 h, and fertilized using pooled semen from Holstein bulls. Embryos were treated with 0, 25, 50, or 100 ng/mL hIL6 on day 5 post-fertilization. An increase in ICM cell numbers was observed in each hIL6 treatment, with the lowest hIL6 treatment having the same magnitude of response as the middle and highest hIL6 concentration. No effects on TE cell numbers were observed. The second study involved cryopreserving (via slow freezing) of hIL6-treated blastocysts, then examining post-thaw blastocyst survival by incubating for 24 h in the absence of hIL6 treatments. Blastocyst re-expansion and hatching rates were unaffected by any of the IL6 treatments, however, increases in both ICM and TE cell numbers were detected at 24 h post-thawing in blastocysts exposed to 100 ng/mL hIL6 but not lower concentrations before freezing. A reduction in the percentage of TUNEL-positive TE cells was observed after thawing in blastocysts exposed to 25, 50 and 100 ng/mL hIL6 before cryopreservation. No treatment-dependent changes in TUNEL-positive ICM cells were observed. In summary, hIL6 supplementation improves ICM cell numbers in bovine blastocysts to a degree that is commensurate with what has been observed when using bovine recombinant IL6. This positive effect of hIL6 on ICM cell numbers is maintained after freezing and thawing, and a novel improvement in post-thaw TE cell numbers occur in hIL6 treated embryos. This positive effect on TE cell numbers is attributed, at least in part, to an hIL6-dependent reduction in TE cell apoptosis.

Impact of Mycotoxin Metabolites Deepoxy-Deoxynivalenol and Beta-Zearalenol on Bovine Preimplantation Embryo Development in the Presence of Acetonitrile.

The quality of animal feed is increasingly affected by weather conditions, high humidity, and damage to grains, which have led to various mycotoxin-producing moulds. The aim of this study was to determine the effects of the combination of deepoxy-deoxynivalenol and beta-zearalenol on the development of preimplantation bovine embryos, the extent to which the presence of both mycotoxin metabolites affects the development of in vitro cultured bovine embryos, or whether the effect of both toxins enhances embryotoxicity. Ovaries were transported from the abattoir to the laboratory and, after maturation and fertilisation, zygotes were placed in an embryo culture medium (IVC) with different mycotoxin metabolite concentrations diluted in acetonitrile. It was found that the blastocyst rate of cleaved embryos was affected by 1 μL acetonitrile in 400 μL medium (0.25%) compared to the group without acetonitrile. For this reason, it was decided to use acetonitrile as a control group, and the desired mycotoxin metabolite concentrations were diluted in the lowest possible amount of acetonitrile (0.5 μL) that could be accurately added to the study groups. There was no statistical difference when the higher mycotoxin metabolite concentrations were added.

Enhancing Bovine Embryo Development In Vitro Using Oil-in-Water Nanoemulsions as Specific Carriers for Essential Lipids.

Worldwide meat consumption and production have nearly quintupled in the last 60 years. In this context, research and the application of new technologies related to animal reproduction have evolved in an accelerated way. The objective of the present study was to apply nanoemulsions (NEs) as carriers of lipids to feed bovine embryos in culture media and verify their impact on the development of embryos produced in vitro. The NEs were characterized by particle size, polydispersity, size distribution, physical stability, morphology using atomic force microscopy (AFM), surface tension, density, pH, and rheological behavior. The NEs were prepared by the emulsification/evaporation technique. A central composite rotatable design (CCRD) was used to optimize the NE fabrication parameters. The three optimized formulations used in the embryo application showed an emulsion stability index (ESI) between 0.046 and 0.086, which reflects high stability. The mean droplet diameter analyzed by laser diffraction was approximately 70-80 nm, suggesting a possible transit across the embryonic zona pellucida with pores of an average 90 nm in diameter. AFM images clearly confirm the morphology of spherical droplets with a mean droplet diameter of less than 100 nm. The optimized formulations added during the higher embryonic genome activation phase in bovine embryos enhanced early embryonic development.

PPARG is dispensable for bovine embryo development up to tubular stages†.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine invitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of invitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve invitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Preimplantation development of in vitro-produced bovine embryos treated with hydroxychloroquine

Hydroxychloroquine (HCQ) is a safe antimalarial drug but its overdosage or inappropriate use, such as during the pandemic, may cause adverse effects once this drug is considered a potent inhibitor of autophagy. Information about HCQ's effects on the reproductive field, including gametes and initial embryos, is limited. In this study, we evaluated the effect of HCQ (1, 6, 12, and 24 μM) on pre-implantation embryo development, autophagy, and apoptosis of bovine embryos produced in vitro. A dose-response experiment showed a reduction (p < 0.05) in cleavage only at the highest concentration. Blastocyst rate was gradually reduced (p < 0.05) with the increase of HCQ dosage starting at 6 μM, with no embryo formation occurring at 24 μM. Further analysis showed that embryos treated with 12 μM of HCQ had a higher (p < 0.05) accumulation of acidic autophagic vesicles on Days 5 and 7 of development and a higher (p < 0.01) apoptotic index on Day 7. To our knowledge, this is the first study to evaluate the effects of HCQ on embryo pre-implantation development in mammals. The results contribute with more information related to the study of autophagy in embryology as well as add some discussion on HCQ toxicology and its effects on reproductive cells.

Effects of Regulating Hippo and Wnt on the Development and Fate Differentiation of Bovine Embryo.

The improvement of in vitro embryo development is a gateway to enhance the output of assisted reproductive technologies. The Wnt and Hippo signaling pathways are crucial for the early development of bovine embryos. This study investigated the development of bovine embryos under the influence of a Hippo signaling agonist (LPA) and a Wnt signaling inhibitor (DKK1). In this current study, embryos produced in vitro were cultured in media supplemented with LPA and DKK1. We comprehensively analyzed the impact of LPA and DKK1 on various developmental parameters of the bovine embryo, such as blastocyst formation, differential cell counts, YAP fluorescence intensity and apoptosis rate. Furthermore, single-cell RNA sequencing (scRNA-seq) was employed to elucidate the in vitro embryonic development. Our results revealed that LPA and DKK1 improved the blastocyst developmental potential, total cells, trophectoderm (TE) cells and YAP fluorescence intensity and decreased the apoptosis rate of bovine embryos. A total of 1203 genes exhibited differential expression between the control and LPA/DKK1-treated (LD) groups, with 577 genes upregulated and 626 genes downregulated. KEGG pathway analysis revealed significant enrichment of differentially expressed genes (DEGs) associated with TGF-beta signaling, Wnt signaling, apoptosis, Hippo signaling and other critical developmental pathways. Our study shows the role of LPA and DKK1 in embryonic differentiation and embryo establishment of pregnancy. These findings should be helpful for further unraveling the precise contributions of the Hippo and Wnt pathways in bovine trophoblast formation, thus advancing our comprehension of early bovine embryo development.

Zinc improves the developmental ability of bovine in vitro fertilization embryos through its antioxidative action

Zinc, an essential trace mineral, exerts a pivotal influence in various biological processes. Through zinc concentration analysis, we found that the zinc concentration in the bovine embryo in vitro culture (IVC) medium was significantly lower than that in bovine follicular fluid. Therefore, this study explored the impact of zinc sulfate on IVC bovine embryo development and investigated the underlying mechanism. The results revealed a significant decline in zygote cleavage and blastocyst development rates when zinc deficiency was induced using zinc chelator N, N, N′, N′-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) in culture medium during embryo in vitro culture. The influence of zinc-deficiency was time-dependent. Conversely, supplementing 0.8 μg/mL zinc sulfate to culture medium (CM) increased the cleavage and blastocyst formation rate significantly. Moreover, this supplementation reduced reactive oxygen species (ROS) levels, elevated the glutathione (GSH) levels in blastocysts, upregulated the mRNA expression of antioxidase-related genes, and activated the Nrf2-Keap1-ARE signaling pathways. Furthermore, 0.8 μg/mL zinc sulfate enhanced mitochondrial membrane potential, maintained DNA stability, and enhanced the quality of bovine (in vitro fertilization) IVF blastocysts. In conclusion, the addition of 0.8 μg/mL zinc sulfate to CM could enhance the antioxidant capacity, activates the Nrf2-Keap1-ARE signaling pathways, augment mitochondrial membrane potential, and stabilizes DNA, ultimately improving blastocyst quality and in vitro bovine embryo development.

GDF-8 improves in vitro implantation and cryo-tolerance by stimulating the ALK5-SMAD2/3 signaling in bovine IVF embryo development.

Transforming growth factor-beta (TGF-β) plays a critical role in regulating trophoblast invasion and proliferation. Growth differentiation factor-8 (GDF-8) is a member of the TGF-β superfamily and is categorized as a myostatin subtype. It is primarily a secreted protein synthesized in skeletal muscle cells. It is expressed in the placenta, reproductive tissues, and cells. In this study, we investigated the role of GDF-8 in the development and hatching rate of bovine embryos. We noted a notable elevation (p < 0.05) in the development and hatching rates compared to the control embryos. Furthermore, the GDF-8 group showed a significantly improved total cell number (p < 0.05) and an increase in trophectoderm ratio inner cell mass (trophectoderm: inner cell mass) cells (p < 0.001) compared to the control group. Additionally, blastocysts treated with GDF-8 exhibited significantly higher mRNA levels of caudal-type homeobox 2 (CDX2) (p < 0.05). The trophoblast invasion area was significantly larger in the GDF-8 group than in the control group (p < 0.01). Furthermore, qRT-PCR analysis revealed significantly higher mRNA levels (p < 0.05) of matrix metalloproteinases 9 (MMP9) and follistatin-like 3(FSTL3), both of which are associated with the ALK5-SMAD2/3 signaling pathway, in the GDF-8 group than those in the control group. The mRNA expression levels of genes related to tight junctions (TJ) and adherent junctions were higher in the GDF-8 group than those in the control group (p < 0.05). After 24h of thawing, blastocysts were analyzed using 4-kDa FITC-dextran, which revealed a higher TJ integrity in the GDF-8 group (p < 0.01). Thus, GDF-8 plays a crucial role in bovine embryonic development, in vitro implantation, and cryotolerance.