Bovine Embryos Research Articles

Effect of the addition of IGF-1 during in vitro culture on the embryonic development speed from different crossbreed bovine embryos.

Supplementation with insulin-like growth factor type 1 (IGF-1) during in vitro culture of bovine embryos has yielded mixed results, likely due to genetic variability among embryos. This work aimed to evaluate the effect of IGF-1 at two concentrations on the development speed embryos from primary F1 crossbreeds used in dual-purpose cattle farming in the Colombian low tropics. Specifically, we investigated the influence of IGF-1 and embryo breed on the blastocyst formation rate. Oocytes were sourced from non-pregnant cows: Bos taurus indicus (20 Brahman and 14 Gyr) and Bos taurus taurus (12 Holstein and 28 Romosinuano). Oocytes were fertilized with semen from specific bulls (Recoil for Holstein, Gabinete for Gyr, and UBER POI 1490 for Brahman). The resulting embryos from each crossbreed group were randomly distributed in three different cultured media with 50 ng/mL IGF-1, 100 ng/mL IGF-1, or no IGF-1 (control) for 7 days. Results showed that 50 ng/mL IGF-1 significantly increased embryo production by day 6 (25.9%±14.56%) compared to control (20.5%±11.84%) and 100 ng/mL IGF-1 (23.0%±9.54%) (p < 0.05). By day 7, both 50 ng/mL (42.6%±26.55%) and 100 ng/mL (49.7%±21.98%) IGF-1 groups exhibited significantly higher production rates compared to the control group (p < 0.001). The embryo breed also influenced development, with Gyr-Holstein (GxH) crossbreeds showing the highest production rates (p < 0.001). In conclusion, IGF-1 supplementation enhances in vitro embryo production, with the effect influenced by both breed and IGF-1 concentration. These findings suggest that breed-specific optimization of IGF-1 conditions is necessary to maximize embryonic development outcomes.

Oviduct epithelial spheroids during in vitro culture of bovine embryos mitigate oxidative stress, improve blastocyst quality and change the embryonic transcriptome

BackgroundIn vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8–16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%).MethodsBovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7–8. Expanded blastocysts on days 7–8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing.ResultsUnder 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted.ConclusionOES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development.

Putrescine supplementation improves the developmental competence of in vitro produced bovine embryos

The aim of this study was to investigate the effect of putrescine, anti-apoptotic, antioxidant, and a cell proliferation stimulant, on embryo development and quality by supplementing it to in vitro culture medium. In this study, oocytes were obtained from the ovaries of Holstein cattle. Following maturation and fertilization, the presumptive zygotes were randomly assigned to two groups. The first group (Putrescine, n = 435) was supplemented with putrescine at a concentration of 0.5 mM to in vitro culture. The second group (n = 407) was maintained under standard culture conditions without any supplementations to the medium. Following the determination of the developmental stages of the embryos, only those in the blastocyst stage were subjected to differential staining and the cell numbers of the embryos were determined. Moreover, the TUNEL assay was employed to ascertain the extent of cell death and the apoptotic index in the embryos. Additionally, the levels of ROS were determined in the embryos. Furthermore, gene expression analyses were conducted on blastocyst-stage embryos to ascertain the potential of putrescine supplementation in embryo development along specific pathways. Following in vitro culture, the blastocyst formation rate was 44.37 % in the putrescine group and 32.97 % in the control group (P < 0.05). The counts of ICM (60.60 ± 15.79 vs 50.73 ± 16.74), TE (117.70 ± 23.67 vs 94.0 ± 22.46), and TCC (178.30 ± 26.15 vs 144.73 ± 26.86) were found to be statistically higher in blastocysts developing after putrescine supplementation compared to the control group. Furthermore, the number of apoptotic cells (7.69 ± 2.17 vs 9.96 ± 3.99) and the apoptotic index (5.07 % vs 8.01 %) were found to be lower in the putrescine group in comparison to the control group. Nevertheless, it was established that the ROS level in the control group was approximately two-fold higher than in the putrescine group (P < 0.05). The findings also revealed that putrescine up-regulated the gene expression of SOD, GPX4, CAT, BCL2, NANOG and GATA3 while simultaneously down-regulating the BAX expression level. In conclusion, the supplementation of putrescine to the culture medium during in vitro bovine embryo production was found to contribute to the improvement of embryo quality and early embryonic development.

Bta-miR-665 improves bovine blastocyst development through its influence on microtubule dynamics and apoptosis.

Extracellular vesicles (EVs) contain microRNAs (miRNAs), which are important regulators of embryonic development. Nevertheless, little is known about the precise molecular processes controlling blastocyst development and quality. In a previous study, we identified bta-miR-665 as one of the miRNAs more abundantly present in extracellular vesicles of embryo-conditioned culture media of blastocysts compared to degenerate ones. Here, we investigated the effect and regulatory roles of bta-miR-665 in blastocyst development by supplementation of bta-miR-665 mimics or inhibitors to the culture media. Supplementation of bta-miR-665 mimics improved cleavage and blastocyst rate (P < 0.01), and blastocyst quality as indicated by increased inner cell mass rates and reduced apoptotic cell ratios (P < 0.01). Furthermore, supplementation of bta-miR-665 inhibitors had the opposite effect on these phenotypes. Low input transcriptome analysis and RT-qPCR revealed that bta-miR-665 acts on genes linked to microtubule formation and apoptosis/cell proliferation. These insights not only elucidate the important role of bta-miR-665 in embryo development, but also underscore its potential in improving reproductive efficiency in bovine embryo culture.

Developmental and Molecular Effects of C-Type Natriuretic Peptide Supplementation in In Vitro Culture of Bovine Embryos.

The use of C-type natriuretic peptide (CNP) in the interaction with the oocyte and in the temporary postponement of spontaneous meiosis resumption has already been well described. However, its action in pre-implantation developmental-stage embryos is yet to be understood. Thus, our study aimed to detect the presence of the canonical CNP receptor (natriuretic peptide receptor, NPR2) in germinal vesicle (GV)-, metaphase II (MII)-, presumptive zygote (PZ)-, morula (MO)-, and blastocyst (BL)-stage embryos and, later, to observe possible modulations on the embryos when co-cultured with CNP. In Experiment I, we detected and quantified NPR2 on the abovementioned embryo stages. Further, in Experiment II, we intended to test different concentrations (100, 200, or 400 nM of CNP) at different times of inclusion in the in vitro culture (IVC; inclusion from the beginning, i.e., day 1, or from day 5). In Experiment III, 400 nM of CNP was used on day 1 (D1) in the IVC, which was not demonstrated to be embryotoxic, and it showed potentially promising results in the blastocyst production rate when compared to the control. Thus, we analyzed the embryonic development rates of bovine embryos (D7) and hatching kinetics (D7, D8, and D9). Subsequently, morula and blastocyst were collected and evaluated for transcript abundance of their competence and quality (apoptosis, oxidative stress, proliferation, and differentiation) and lipid metabolism. Differences with probabilities less than p < 0.05, and/or fold change (FC) > 1.5, were considered significant. We demonstrate the presence of NPR2 until the blastocyst development stage, when there was a significant decrease in membrane receptors. There was no statistical difference in the production rate after co-culture with 400 nM CNP. However, when we evaluated the abundance of morula transcripts, there was an upregulated transcription in ADCY6 (p = 0.057) and downregulated transcripts in BMP15 (p = 0.013), ACAT1 (p = 0.040), and CASP3 (p = 0.082). In addition, there was a total of 12 transcriptions in morula that presented variation FC > 1.5. In blastocysts, the treatment with CNP induced upregulation in BID, CASP3, SOX2, and HSPA5 transcripts and downregulation in BDNF, NLRP5, ELOVL1, ELOVL4, IGFBP4, and FDX1 transcripts (FC > 1.5). Thus, our study identified and quantified the presence of NPR2 in bovine pre-implantation embryos. Furthermore, 400 nM of CNP in IVC, a concentration not previously described in the literature, modulated some transcripts related to embryonic metabolism, and this was not embryotoxic morphologically.

Small extracellular vesicles derived from the crosstalk between early embryos and the endometrium potentially mediate corpus luteum function.

The first interactions among the embryo, endometrium, and corpus luteum (CL) are essential for pregnancy success. Small extracellular vesicles (sEVs) are part of these interactions. We previously demonstrated that sEVs from in vivo- or in vitro-produced bovine embryos contain different miRNA cargos. Herein we show: 1) the presence and origin (in vivo or in vitro) of the blastocyst differentially reprograms endometrial transcriptional profiles; 2) the endometrial explant (EE) cultured with in vivo or in vitro embryos release sEVs with different miRNA contents, and; 3) the luteal explant (CLE) exposed to these sEVs have distinct mRNA and miRNA profiles. To elucidate this, the EE were cultured in the presence or absence of a single Day-7 in vivo (EE-AI) or in vitro (EE-IVF) embryo. After of culture we found, in the EE, 45 and 211 differentially expressed genes (DEGs) associated with embryo presence and origin, respectively. SEVs were recovered from the conditioned media (CM) in which EE and embryos were co-cultured. Four miRNAs were differentially expressed between sEVs from CM-EE-AI and CM-EE-IVF. Luteal explants exposed in culture to these sEVs showed 1360 transcripts, and fifteen miRNAs differentially expressed. The DEGs associated with embryo presence and origin, modulating cells' proliferation, and survival. These results demonstrate that in vivo- or in vitro-produced bovine embryos induce molecular alterations in the endometrium; and that the embryo and endometrium release sEVs capable of modifying the mRNA and miRNA profile in the CL. Therefore, the sEVs-mediated embryo-endometrium-CL interactions possibly regulate the CL viability to ensure pregnancy success.

Effect of the flow-force relationships used in two parameter modelling of freezing and vitrification protocols for bovine embryos

Cells may become damaged by strong volume changes and related intracellular changes during slow freezing or vitrification. These osmotic events can be modelled mathematically, using descriptions of transmembrane flow of solute and water. We compared different variants of an often used 2-parameter (2P) formalism in fitting of an empirical shrink-swell curve of a bovine embryo in 5 vol% glycerol, and in simulations of CPA loading and removal in a vitrification protocol. In its original form, the 2P model uses a flow-force relationship for the flux of CPA that is not analogous to that for water (asymmetrical), but in the other variants used, the flow-force relationships for water and CPA are analogous to each other (symmetrical). The effect of used model on estimated values for Lp and Ps in 5 vol% glycerol was small. Also the effect on shrinking and swelling in vitrification media was small, but the original 2P model predicted stronger swelling of embryos during one-step CPA removal. One variant that we compared simply assumes Raoult's law, i.e. M = m, even in very concentrated solutions We conclude that this simple model is easy and appropriate for simulating osmotic events of embryos. But if a method for correcting for the deviation from Raoult's law is used, a symmetrical model seems more appropriate than the original (asymmetrical) 2P model.

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.

Methodological approaches in vitrification: Enhancing viability of bovine oocytes and invitro-produced embryos.

Cryopreservation of bovine oocytes and embryos is essential for long-term preservation and widespread distribution of genetic material, particularly in bovine invitro embryo production, which has witnessed substantial growth in the past decade due to advancements in reproductive biotechnologies. Among current cryopreservation methods, vitrification has emerged as the preferred cryopreservation technique over slow freezing for preserving oocytes and invitro-produced (IVP) embryos, as it effectively addresses membrane chilling injury and ice crystal formation. Nonetheless, challenges remain and a simple and robust vitrification protocol that guarantees the efficiency and viability after warming has not yet been developed. Furthermore, although slow cooling can easily be adapted for direct transfer, an easier and more practical vitrification protocol for IVP embryos is required to allow the transfer of IVP embryos on farms using in-straw dilution. In addition, the susceptibility of bovine oocytes and embryos to cryoinjuries highlights the need for novel strategies to improve their cryotolerance. This manuscript examines various methodological approaches for increasing the viability of bovine oocytes and IVP embryos during vitrification. Strategies such as modifying lipid content or mitigating oxidative damage have shown promise in improving cryotolerance. Additionally, mathematical modelling of oocyte and embryo membrane permeability has facilitated the rational design of cryopreservation protocols, optimizing the exposure time and concentration of cryoprotectants to reduce cytotoxicity.

A low concentration of choline chloride alters the developmental program of the bovine preimplantation embryo.

Choline is a known developmental programming agent of the bovine preimplantation embryo. Culture of the embryo with 1.8 mmol/L choline, a concentration much higher than in blood, alters development to cause increased weaning weight and other changes during the postnatal period. It was hypothesized here that choline exerts similar effects on the developmental program of the embryo when added at concentrations similar to those in peripheral blood (i.e., 4 mol/L). Oocytes were collected via ovum pick up and embryos were produced in vitro. Embryos were cultured until day 7 after fertilization in medium with 4 mol/L choline chloride, or, as a vehicle control, with an additional 4 mol/L sodium chloride. Blastocysts were transferred into recipients and pregnancy was diagnosed at approximately 28 d of gestation. Subsequent calves (n=37 for vehicle and n=35 for choline) were weighed at birth and at weaning. Addition of choline to culture medium did not affect the proportion of embryos that became blastocysts or the proportion of transferred blastocysts that produced a pregnancy. Birth weight was unaffected by treatment but calves derived from choline-treated embryos were heavier at time of weaning and gained more per day from birth until weaning than calves derived from embryos treated with vehicle. Results demonstrate that choline can act on the preimplantation embryo at a physiologically-relevant concentration to alter postnatal phenotype. Observations are further evidence for the importance of the first days of embryonic development for the phenotype of the resulting calf.

INCREASED IN VITRO PRODUCTION OF BOVINE EMBRYOS RESULTING FROM OOCYTE MATURATION IN THE PRESENCE OF TRICIRIBINE, A SPECIFIC INHIBITOR OF AKT

The aim of this study was to evaluate the effect of different concentrations of triciribine, a selective Akt inhibitor, on various aspects of oocyte maturation and on the IVF of bovine embryos. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with: 0 (control), 1, 5, 10, and 20 μM of triciribine. The nuclear maturation was assessed by staining with acetic orcein, while the cytoplasmic maturation was evaluated by mitochondrial (MitoTracker® Red CMXRos) and lipid droplets distribution (LipidTOX).COCs were fertilizedin vitro and cultured for nine days. Cleavage rates, blastocyst production, and hatching rates were determined on days three, seven, and nine of in vitro culture, respectively. Oocytes from COCs treated with 1μM of triciribine were stained at 3, 6, and 9 hours of IVM to determine the inhibitor's involvement in germinal vesicle breakdown. Analysis of variance (ANOVA) of the data was performed and the means were compared using the SNK test at a 5% significance level. Exposure of COCs to 1, 5, and 10 μM of triciribine did not alter the number of matured oocytes (P<0.05), a concentration of 20 μM reduced the number of oocytes in MII with a consequent increase in oocytes in MI (P<0.05). This concentration markedly reduced the number of oocytes with peripheral cortical granules and the rates of cleavage and blastocysts (P<0.05). On the other hand, when COCs were matured in the presence of 1μM, there was an increase in the blastocyst rate (P<0.05), but without altering the timing of meiosis resumption (P<0.05). It is concluded that the Akt pathway participates in the nuclear and cytoplasmic events of in vitro maturation of bovine oocytes, but through mechanisms that do not interfere with germinal vesicle breakdown. Modulation of Akt activity in bovine COCs during IVM with 1μM of triciribine increases the in vitro production of bovine embryos.

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

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

Folic acids promote in vitro maturation of bovine oocytes by inhibition of ferroptosis via upregulated glutathione and downregulated Fe2+ accumulation

Bovine embryos by in vitro fertilization have become the primary source of commercial embryo transfers globally. However, the developmental capacity of in vitro maturation (IVM) oocytes is considerably lower than that of in vivo maturation (IVO) oocytes, owing to the production of reactive oxygen species (ROS) via mitochondrial metabolism, which was higher in IVM oocytes than in IVO oocytes. To avoid the negative effects of ROS on embryo quality, folic acid (FA) was supplemented directly into the IVM medium to antagonize ROS production, however, the mechanisms remain unknown. In the present study, five levels of FA (0, 25, 50, 100, and 200 µM) were supplemented into the bovine oocyte culture medium. The maturation, cleavage, and blastocyst formation rates increased by 8.95 %, 6.94 %, and 4.36 %, respectively, in the 50 µM group compared to the 0 µM group. Moreover, 7904 differential genes were identified between 0 µM and 50 µM groups by transcriptome sequencing, and they were mainly enriched in 8 pathways. The glutathione, ROS, and Fe2+ levels in oocytes were found to be associated with ferroptosis. Our results revealed that 50 µM FA promoted the IVM of bovine oocytes and affected the expression of genes involved in the ferroptosis pathway. The downregulation of TFR1 and STEAP3 led to a decrease in intracellular Fe2+ accumulation, and the upregulation of GCL increased oocyte GSH levels, thereby reducing the production of ROS in the ferroptosis pathway. Our study provides a new insight into the molecular mechanisms by which FA promotes bovine oocyte development in vitro.

The composition of early lactation milk in recipient dairy cows determines success in bovine embryo transfer.

To identify new criteria for selecting recipient dairy cows for embryo transfer (ET), we retrospectively examined the effects of the composition of early lactation milk on fertility risk in ET. This study investigated the association between milk fat (FAT), milk protein (PRO), and milk urea nitrogen (MUN) levels during early lactation, based on production records, and subsequent fertility risk in ET using contingency table analysis and multivariable logistic regression analysis, which included five confounding variables. The results showed that MUN levels during early lactation were negatively associated with fertility risk in ET, while FAT and PRO levels showed no clear association. A reduction in MUN levels during the peak lactation period suggests a deficiency in dry matter intake, an inadequate protein supply, and an imbalance in the ratio of proteins to fermentable carbohydrates in the rumen, which may have adversely impacted fertility risk in ET.Monitoring MUN levels is crucial for maintaining a proper protein balance. The results obtained in this study suggest that MUN levels in the early lactation phase obtained from production records can be used as a predictor of fertility in recipients to improve the fertility risk in ET. No special techniques or costs are required for using production records, making them easy to use in clinical practice. Our findings provide valuable insights for optimizing cost-effectiveness and fertility risk in ET and their clinical applications.

Replacing sperm with genotyped haploid androgenetic blastomeres to generate cattle with predetermined paternal genomes

Abstract Although meiosis plays an essential role for the survival of species in natural selection, the genetic diversity resulting from sexual reproduction impedes human-driven strategies to transmit the most suitable genomes for genetic improvement, forcing breeders to select diploid genomes generated after fertilization, that is, after the encounter of sperm and oocytes carrying unknown genomes. To determine whether genomic assessment could be used before fertilization, some androgenetic haploid morula-stage bovine embryos derived from individual sperm were biopsied for genomic evaluation and others used to reconstruct “semi-cloned” (SC) diploid zygotes by the intracytoplasmic injection into parthenogenetically activated oocytes, and the resulting embryos were transferred to surrogate females to obtain gestations. Compared to controls, in vitro development to the blastocyst stage was lower and fewer surrogates became pregnant from the transfer of SC embryos. However, fetometric measurements of organs and placental membranes of all SC conceptuses were similar to controls, suggesting a normal post-implantation development. Moreover, transcript amounts of imprinted genes IGF2, IGF2R, PHLDA2, SNRPN and KCNQ1OT1 and methylation pattern of the KCNQ1 DMR were unaltered in SC conceptuses. Overall, this study shows that sperm can be replaced by genotyped haploid embryonic-derived cells to produce bovine embryos carrying a predetermined paternal genome and viable first trimester fetuses after transfer to female recipients.

Comparison of Gene Expression for Preimplantation Genes in Bovine Embryos Fertilized in vitro

Background: In vitro fertilization (IVF) the technique is useful for understanding early mammalian development and maximizing and speeding up genetic improvement in livestock. Genes that function as genetic markers and are involved in the pre-implantation development of embryos may be used to evaluate the quality of an embryo and the expression of these genes is correlated with the timing of embryonic genomic activation. Methods: Maturing the oocytes of bovine, then fertilizing these matured oocytes and finally culturing the fertilized oocytes to develop into embryos. Determined the gene expression of different certain genes HSP70, OCT4, GLUT1, BAX and DNMT1 in these different cell stages of embryos that were produced. Result: The mRNA expression of these genes was estimated after collecting the embryos listed above that were produced by the in vitro technique. The results showed statistically significant differences in the relative abundance between some of the genes mentioned above in each embryo stage. The timing of the zygotic genome activation process (ZGA) can result in differences in gene expression levels between the early embryonic genes in each developmental stage of in vitro-produced bovine embryos.

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

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

229 Awardee Talk: The quest for an ideal in-field embryo evaluation tool in cattle: Opportunities and challenges

Abstract Accurately evaluating bovine embryos to select the most viable ones for transfer and successful pregnancy has been a challenge since embryo transfer began in cattle over six decades ago. Despite more than 3,100 peer-reviewed studies on bovine embryo evaluation published in PubMed from 1964 to 2023, there is still no consensus or gold standard method for assessing their developmental potential. The evaluation “problem” in assisted reproduction extends beyond embryos to gametes as well. Numerous microscopic techniques (e.g., differential interference contrast, electron, fluorescent, time-lapse, and artificial intelligence-based microscopy) and non-microscopic methodologies (e.g., genomics, transcriptomics, epigenomics, proteomics, metabolomics, and nuclear magnetic resonance) have been tested to find an embryo evaluation technique superior to morphological evaluation. While many of these research tools can accurately determine embryo quality and viability, most are invasive, expensive, labor-intensive, technically complex, and time-consuming, making them impractical for in-field embryo evaluation. Employing complex methods like RNA sequencing, SNP chips, mass spectrometry, and multiphoton microscopy at thousands of embryo production and collection facilities is unrealistic, no matter how accurate they may be. Therefore, future research should focus on innovating field-friendly, simple benchtop tests using existing findings, particularly from omics-based research methodologies. Time-lapse monitoring and artificial intelligence-based automated image analysis also potentially provide accurate embryo evaluation. However, further research is necessary to develop economically feasible options for in-field applications. Our recent work has examined possible applications of nuclear magnetic resonance (NMR) imaging, label-free microscopy, particularly holographic microscopy techniques like gradient light interference microscopy (GLIM), spatial light interference microscopy (SLIM), and multiphoton holographic microscopy in IVF labs. The ideal embryo evaluation tool should be accurate, objective, non-invasive, affordable, and simple enough for widespread adoption by embryo production and collection facilities worldwide. Under current circumstances, it is doubtful that transcriptomics, proteomics, metabolomics, GLIM, SLIM, NMR, multiphoton holographic microscopy, and similar techniques can be directly used for in-field embryo evaluation. However, biomarkers identified by these methodologies could be used for in-field embryo evaluation by devising simple, affordable benchtop techniques. Further investigation on time-lapse microscopy (TLM) and AI-based automated image processing may make these methods more affordable and potentially adoptable by the masses. There is great potential for these methodologies to become widely used, provided they can be made more economically viable. In conclusion, the next generation of microscopy capable of providing objective markers for gamete and embryo quality is on the horizon.

439 Bovine embryo epigenetic analysis according to periconceptional conditions and new information on postnatal phenotypes

Abstract In the field of animal reproduction, the environment associated with gametes and embryos refers to the condition of the parents as well as conditions surrounding gametes and embryo in vivo or in vitro. This environment is now known to influence not only the functionality of the early embryo but potentially the future phenotype of the offspring. Using transcriptomic and epigenetic molecular analysis, and the bovine model, recent research has shown that both the female and the male metabolic status, for example age, can affect gene expression and gene programming in the embryo. Evidence demonstrates that milking cows losing body weight at the time of conception generates compromised embryos and offsprings with a unique metabolic signature. A similar phenomenon has been associated with different culture conditions and the IVF procedure. The general common consequence of these situations is an embryo behaving as “economy” mode where translation, cell division and ATP production is reduced, potentially to adapt to the perceived future environment. New epidemiologic studies in bovine to assess if these changes result in a different phenotype especially with animals created by IVF which shows consequences later in life.

Heat shock on bovine embryos from day 2.5–3 selects the most competent for progression to the blastocyst stage

Heat shock can impair embryo formation, while growth factors, such as colony-stimulating factor 2 (CSF2), modulate embryonic development. This study evaluated the effect of heat shock between days 2.5 and 3, as well as the impact of CSF2 at day 5 on bovine embryos cultured in a serum-free in vitro medium. The focus was on blastocyst development, the number of blastomeres, DNA fragmentation (TUNEL-positive cells), and mitochondrial activity. Heat shock reduced the proportion of cleaved embryos that developed into blastocysts (P = 0.0603). The resultant blastocysts exhibited a reduced number and proportion of TUNEL-positive cells in the trophectoderm (P = 0.0270 and P = 0.0240, respectively) and in the entire embryo (P = 0.0029 and P = 0.0031, respectively). Additionally, mitochondrial activity was lower in blastocysts derived from heat-shocked embryos (P = 0.0150) and further reduced in embryos exposed to both heat shock and CSF2 (P = 0.0415). In conclusion, the exposure of cleaved embryos to heat shock reduced their development to the blastocyst stage. However, the resulting blastocysts showed decreased DNA fragmentation and mitochondrial activity.