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Purpose: To investigate the role of dimethyl sulfoxide (DMSO) as a cryoprotectant and solvent in bovine embryogenesis, with a particular focus on its impact on gene expression during early and late developmental stages. Method: Bovine embryos were used to evaluate effects of DMSO on expression of genes critical to apoptosis and developmental processes. Gene expression analysis was performed to assess changes in both pro-apoptotic and anti-apoptotic markers, as well as genes essential for growth and survival. Results: Dimethyl sulfoxide influenced gene expression in a stage-specific manner. During early development, DMSO induced overexpression of the pro-apoptotic gene, BAX, and downregulated the anti-apoptotic gene, BCL2, indicating increased apoptotic activity. Furthermore, the expression of GDF9 and IGF1, which are crucial for growth and survival, was altered, suggesting interference with key developmental pathways. In contrast, late-stage embryos exhibited elevated levels of BCL2 and HSPB1, markers of anti-apoptotic activity, indicating a more complex regulatory role of DMSO at advanced stages of embryogenesis. Conclusion: While DMSO is effective as a cryoprotectant, its impact on gene expression raises concerns about potential developmental consequences. These findings highlight the need for further investigation to better understand the specific effects of DMSO in the context of assisted reproductive technologies (ART). [ABSTRACT FROM AUTHOR]

Integrating in vitro embryo production with embryonic microsurgery facilitates the generation of monozygotic twins. However, despite their potential benefits, these methods have not been widely adopted in commercial settings because of their substantial costs. Hence, there is a need to streamline the bisection procedure while ensuring efficient production of viable demi-embryos. In this study, we investigated the impact of different orientations of microsurgical incisions in relation to inner cell mass on embryonic development, morphology, viability, and expression of cell fate protein markers using a simplified microsurgery approach. Ovaries were transported from the slaughterhouse to the laboratory and aspirated to obtain oocytes that were selected and subjected to in vitro embryo production. The selected expanded blastocysts (n = 204) underwent microsurgery. The blastocysts were immobilized to facilitate incision using an adapted microblade, yielding demi-embryos (vertical incision) and viable embryonic fragments (transverse incision). The structures were then re-cultured for 12 h. Viability was assessed by measuring the re-expansion rate after re-culture, followed by immunofluorescence analysis of proteins (CDX2 and NANOG) and apoptosis analysis using terminal deoxynucleotyl transferase dUTP nick end-labeling (TUNEL). Microsurgically derived embryos exhibited remarkable plasticity, as evidenced by a slight reduction (P < 0.05) in the re-expansion rate (transverse 64.2 % and vertical 57.2 %) compared to that of the control group (blastocysts without microsurgery) (86.7 %). They also demonstrated the ability of morphological reconstitution after culturing. Despite the anticipated decrease (P < 0.05) in the total number of cells and embryo volume, microsurgery did not result in a significant increase (P > 0.05) in the number of apoptotic cells. Furthermore, microsurgery led to higher (P < 0.05) expression of markers associated with pluripotency, indicating its efficiency in preserving regenerative capacity. Moreover, microsurgery, whether followed by immunosurgery or not, made the isolation of embryonic cells easier. In conclusion, both transverse and vertical microsurgery incisions enabled the production of identical demi-embryos and served as tools for isolating embryonic cells without compromising the resumption of development and the apoptotic index. • Different microsurgical incision orientations on embryonic viability. • Cellular and molecular blastocyst reconstitution characterization. • Re-expansion rates after microsurgery demonstrate incredible embryonic plasticity. • Embryonic microsurgery is a tool for obtaining demi-embryos and embryonic cells. [ABSTRACT FROM AUTHOR]

ABSTRACT: Selection of competent recipients before embryo transfer (ET) is indispensable for improving pregnancy and birth rates in cattle. However, pregnancy prediction can fail when the competence of the embryo is ignored. We hypothesized that the pregnancy potential of biomarkers could improve with information on embryonic competence. In vitro-produced embryos cultured singly for 24 h (from d 6 to 7) were transferred to d 7 synchronized recipients as fresh or after freezing and thawing. Recipient blood was collected on d 0 (estrus; n = 108) and d 7 (4–6 h before ET; n = 107) and plasma was analyzed by nuclear magnetic resonance (1H+NMR). Spent embryo culture medium (CM) was collected and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry in a subset of n = 70 samples. Concentrations of metabolites quantified in plasma (n = 35) were statistically analyzed as a function of pregnancy diagnosed on d 40, d 62 and birth. Univariate analysis with plasma metabolites consisted of a block study with controllable fixed factors (i.e., embryo cryopreservation, recipient breed, and day of blood collection; Wilcoxon test and t-test). Metabolite concentrations in recipients and embryos were independently analyzed by iterations that reclassified embryos or recipients using the support vector machine. Iterations identified some competent embryos, but mostly competent recipients that had a pregnancy incompetent partner embryo. Misclassified recipients that could be classified as competent were reanalyzed in a new iteration to improve the predictive model. After subsequent iterations, the predictive potential of recipient biomarkers was recalculated. On d 0, creatine, acetone and l-phenylalanine were the most relevant biomarkers at d 40, d 62, and birth, and on d 7, l-glutamine, l-lysine, and ornithine. Creatine was the most representative biomarker within blocks (n = 20), with a uniform distribution over pregnancy endpoints and type of embryos. Biomarkers showed higher abundance on d 7 than d 0, were more predictive for d 40 and d 62 than at birth, and the pregnancy predictive ability was lower with frozen-thawed (F-T) embryos. Six metabolic pathways differed between d 40 pregnant recipients for fresh and F-T embryos. Within F-T embryos, more recipients were misclassified, probably due to pregnancy losses, but were accurately identified when combined with embryonic metabolite signals. After recalculation, 12 biomarkers increased receiver operator characteristic-area under the curve (>0.65) at birth, highlighting creatine (receiver operator characteristic-area under the curve = 0.851), and 5 new biomarkers were identified. Combining metabolic information of recipient and embryos improves the confidence and accuracy of single biomarkers.

In mouse embryos, inside cells are allocated in 16‐cell embryos through a well‐orchestrated sequence of events involving compaction and polarization. The emergence of inside cells is of great importance as itl later gives rise to the inner cell mass and epiblast. In this study, we report the sequence of critical events in embryology (compaction, inside cells allocation and fragmentation) in bovine 72 h.p.i. 9–16 cell embryos, while also investigating the effects of X‐sorted semen on these events. We found a wide distribution of total cell numbers among embryos, attributed to an asynchronous cleavage pattern and blastomere death. Additionally, 13% of embryos displayed irregular shapes. The establishment of the inside cell compartment increased (p < 0.01) in embryos with more cells. However, only 53.8% of 16‐cell embryos presented inside cells. Compaction was present in 32.4% embryos and was positively correlated (p = 0.03, OR 3.02) with the establishment of inside cells, occurring independently of cell number. Fragmentation was present in 36% embryos, being more frequent (p = 0.01) in embryos with lower cell numbers. A possible association between irregular shape and fragmentation was considered (p = 0.06). The use of X‐sorted semen had no effect on most evaluated parameters. However, it did have a marked effect on cleavage rate (p < 0.01) and the arrest of 2‐ and 4‐ cell embryos. In conclusion, bovine embryos exhibit an asynchronous cleavage pattern, high levels of fragmentation, and demonstrate compaction and inside cell allocation later in development compared to mouse embryos. Semen X‐sorting has major effects on cleavage and embryo arrest. Further studies are needed to elucidate the association between irregularly shaped embryos and fragmentation, as well as the effects of sex on inside cell allocation. [ABSTRACT FROM AUTHOR]

Although embryo transfer is widely applied in cattle, many of the transferred embryos do not result in pregnancy. To determine a new parameter for bovine embryo evaluation, we investigated the relationships between in vitro hatchability and embryo morphological parameters using optical coherence tomography (OCT) that we established recently. Bovine embryos were obtained from Japanese Black cattle by in vitro fertilization (IVF). The quality of the blastocysts was examined under an inverted microscope and confirmed as Codes 1–3 according to the IETS standards for embryo evaluation. The OCT images of the embryos were captured on Day 7 after IVF, and the embryos were cultured until Day 9 to determine their hatchability. During OCT, the embryos were irradiated with near-infrared light for a few minutes to obtain three-dimensional images. In total, 22 parameters were assessed for each of the 42 embryos, of which 25 hatched (H embryos) and 17 did not (NH embryos). The thickness of the trophectoderm (TE) and TE+zona pellucida (ZP) was lesser, and the volumes of the TE, ZP, blastocoel, and whole embryo and blastocoel diameter were greater in the H embryos than in the NH embryos. PCA identified that the increase in the blastocoel-related value along with the decrease in the thickness-related value of the TE and/or ZP could be indicators for evaluating the hatchability of bovine IVF embryos. These results support the idea that OCT-captured structural data of blastocyst-stage embryos can be used as a potential model to predict the quality of bovine embryos.

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

Zinc finger and SCAN domain containing 4 (Zscan4) is a gene that is specifically expressed during zygotic genome activation (ZGA) in mouse preimplantation embryos, and a reduction of Zscan4 transcripts leads to developmental failure. In mouse embryonic stem cells (ESCs), Zscan4 is expressed transiently in as little as 1-5% of the cell population. Zscan4 has also been shown to enhance the efficiency of mouse induced pluripotent stem cells (iPSCs) generation and their quality. Although ZSCAN4 plays important roles in murine embryos and stem cells, its expression and role in bovine embryos is unknown. This study examines ZSCAN4 transcripts in bovine embryos at various developmental stages and attempts to elucidate the functions of ZSCAN4 during bovine preimplantation development. ZSCAN4 transcripts were found to be upregulated at the 8- and 16-cell stages. We next attempted ZSCAN4 downregulation in bovine early embryos by RNA interference and evaluated developmental competency and transcripts levels of genes involved in ZGA and iPSCs generation. Although the bovine embryos injected with ZSCAN4-siRNA could develop to the 8-cell stage, very few were developing beyond the 16-cell stage. PIWIL2 expression was reduced in ZSCAN4 downregulated embryos. It is possible that ZSCAN4 downregulated embryos fail to regulate gene expression during ZGA. Our results indicate that ZSCAN4 is an important factor for the preimplantation development of bovine embryos.

Background: Mexico is innovating in the livestock industry through in vitro generation of bovine embryos with technologies such as well-of-the-well (WOW) and polyester mesh (PM) single-embryo culture systems. These techniques allow to maintain embryos in separate areas of a shared culture medium. Objective: To compare the quantity and quality of bovine embryos produced in WOW and PM culture systems versus the conventional (CG) culture system. Methods: In total, 345 embryos fertilized in vitro were evaluated for blastocyst yield in the three culture systems. To count blastocyst cell numbers, 69 embryos in each system were differentially stained for trophectoderm (TE), inner cell mass (ICM), and apoptotic cells. A qPCR gene expression analysis was performed for embryos in all three systems. Results: The WOW, PM and CG systems developed similar amount of blastocysts (41, 35 and 36%, respectively; p>0.05). Blastocysts in all three systems showed adequate amounts of ICM and apoptotic cells. Blastocysts in the PM system showed a greater number of TE cells [63.7 versus 58.6% in the CG system (p0.05). The ATP5B expression was higher in WOW than in PM (p0.05). The TJP3 expression was higher in PM than in WOW and CG (p

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

Uterine infections cause ovarian dysfunction and infertility. The bacterial endotoxin, lipopolysaccharide (LPS), accumulates in the follicular fluid of dominant follicles of cows with uterine infections. Granulosa cells produce an innate inflammatory response to LPS, altering the follicular microenvironment of the oocyte. We hypothesized that developmental competence and embryo quality would be reduced when oocytes are matured in an inflammatory environment. Bovine mural granulosa cells were exposed to either 1 μg/mL of LPS or medium alone for 24 h to produce conditioned medium. Inflammatory responses of mural granulosa cells were confirmed by increased expression of CXCL8, IL1B, IL6 and TNF. Bovine cumulus-oocyte complexes were matured for 22 ± 1 h in medium supplemented with either 1 μg/mL of LPS, 10% v/v conditioned medium of granulosa cells treated with either LPS (LCM) or medium alone (CCM), or no supplementation (CON). In addition, polymyxin B (20 μg/mL) was added to maturation medium to sequester LPS. Following maturation, cumulus-oocyte complexes were fertilized and cultured for 7.5 days with no further treatment. Oocyte maturation using LPS or LCM impaired development to the blastocysts stage, reduced the number of total and CDX2 negative blastomeres and increased TUNEL positive cells in blastocysts. Polymyxin B could rescue these effects in the LPS group but not in the LCM group, indicating factors produced by granulosa cells and not LPS alone compromised oocyte development. These findings suggest that the inflammatory milieu produced by granulosa cells in response to LPS impairs oocyte competence and quality of resultant blastocyst-stage embryos., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Transcription factor TEA domain family transcription factor 4 (Tead4) is one of the key factors involved in the differentiation of the trophectoderm (TE) in murine embryos. However, knowledge on the roles of TEAD4 in preimplantation development during bovine embryos is currently limited. This study examined the transcript and protein expression patterns of TEAD4 and attempted to elucidate the functions of TEAD4 during bovine preimplantation development using RNA interference. TEAD4 mRNA was found to be upregulated between the 16-cell and morula stages, and nuclear localization of the TEAD4 protein was detected at the morula stage, as well as in subsequent developmental stages. TEAD4 downregulation did not affect embryonic development until the blastocyst stage, and TEAD4-downregulated embryos were capable of forming the TE under both 5% and 21% O2 conditions. Results of gene expression analysis showed that TEAD4 downregulation did not affect the expression levels of POU class 5 transcription factor 1 (OCT-4), NANOG, caudal-type homeobox 2 (CDX2), GATA binding protein 3 (GATA3), and interferon-tau (IFNT). In conclusion, TEAD4 might be dispensable for development until the blastocyst stage and TE differentiation in bovine embryos.

Introduction: Although substantial progress has been made in procedures for in vitro maturation, fertilization, and culture of bovine oocytes, the percentage of embryos that can normally develop is still less in the in vitro produced than in those in vivo produced. Objective: The present work compared two culture systems, which employ different O2 tension, evaluating the in vitro production rate of bovine embryos and their kinetic development. Methods: A total of 1372 bovine cumulus-oocyte complexes (COCs) were obtained and selected by Ovum pick-up (OPU, 703) and slaughterhouse (669) cows, and subsequently in vitro maturated and fertilized. Subsequently, the presumed zygotes of each group were stripped and randomly cultured in one of the two culture systems analyzed. The culture systems evaluated were SOFaa medium and culture for 7 d under 5% CO2 (adjusted) in air, at 38.5 °C (control); SOFaa medium and culture for 7 d under 5% CO2 and 5% O2 (adjusted) at 38.5 °C (treatment). Cleavage rates were evaluated 48 h post-insemination and the blastocyst rates at D6 and D7 post-insemination. The data were analyzed with a GLM and a one-way analysis of variance using Statistica software version 14.0. Results and Discussion: In the cleavage rate, no statistical difference was observed (P>0.05) between the groups of embryos from oocytes obtained by OPU in high (68.1±9.5%) or low tension (78.7±2.2%) respectively and those from the slaughterhouse in high (82.7±3.8%) and low tension (79.5±3.3%). However, in the embryonic development rate, those embryos coming from COCs by OPU had no statistical difference (p>0.05) between high or low tension at day 6 (21.9±9,5% and 22.2±4.3%) and day 7 (25.6±8.5% and 33.33.0%) respectively. On the other hand, embryos from slaughterhouse COCs showed different development kinetics, with a higher (P<0.05) embryo production rate at day 6 in low oxygen tension (26.7±2.4%) compared to those grown in high oxygen tension (15.4±3.8%). However, on day 7 a statistical difference was not shown with low tension: 37.2±5.0% vs. high tension: 21.2±4.2%. Conclusion: The use of low oxygen tension during the in vitro culture of bovine embryos favors the development kinetics of those embryos from oocytes of lower quality, such as those from slaughterhouses. [ABSTRACT FROM AUTHOR]

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Reactive oxygen species (ROS) are one of the factors which reduces oocyte quality and viability of the in vitro produced embryos. Oocyte mitochondria are the major source of ROS production, hence, and the addition of mitochondrion-specific antioxidants could be suggested to minimize the damage caused by ROS during culture. MitoTEMPO, a targeted mitochondrial antioxidant, is formed by conjugating TEMPO to triphenylphosphonium and has an activity like that of superoxide dismutase. It can pass through lipid bilayers easily and accumulate selectively in mitochondria. The goal of this study was to investigate the effects of MitoTEMPO and its non-targeted form, TEMPO, on the developmental competence of bovine oocytes. Accordingly, oocytes were cultured in maturation medium supplemented with either five mM TEMPO (T5) or one μM MitoTEMPO (M1), or T5 + M1 (MT15), or without the antioxidants (C). Nuclear maturation to metaphase II (MII) stage, intracellular glutathione (GSH) content and ROS levels in matured oocytes were analyzed. In addition, cleavage after in vitro fertilization, and blastocyst rates, total cell number in blastocysts as well as the relative abundance of apoptosis-related genes (BAX and BCL2) in blastocysts were determined. Results revealed that the proportion of oocytes at the MII stage, embryos at the blastocyst stage and total cell number in blastocysts increased significantly in the M1 group compared to the C and T5 groups. The levels of intracellular GSH and ROS in oocytes decreased in the M1 group than in the C group (P < 0.05). The expression level of the pro-apoptotic gene (BAX) reduced in blastocysts from the M1 group in comparison to the C and T5 groups (P < 0.05). On the other hand, the expression level of anti-apoptotic gene (BCL2) in obtained blastocysts was not affected by TEMPO and MitoTEMPO. However, the ratio of BAX/BCL2 in blastocysts from the M1 and MT15 groups decreased significantly compared to the C group. These findings suggest that MitoTEMPO can mitigate the adverse effects of oxidative stress on the developmental competence of bovine oocytes. • The mitochondria-targeted antioxidant MitoTEMPO influenced bovine IVC. • MitoTEMPO was more effective than its non-targeted form, TEMPO in oocyte development. • MitoTEMPO prevents intracellular ROS accumulation in bovine oocytes. • Blastocyst production was increased by supplementing IVM medium with MitoTEMPO. • Apoptosis in blastocysts was decreased by MitoTEMPO and a combination of it with TEMPO. [ABSTRACT FROM AUTHOR]

Embryo transfer entails many procedures and techniques, of which embryo freezing is an important component in bovine embryo transfer. Embryo freezing techniques have been developed over the last 40 years, allowing practical availability, and have become essential for cattle reproduction management under field conditions. The direct transfer methods of frozen-thawed, in vivo-derived, and in vitro-produced (IVF) bovine embryos using 1.5 M ethylene glycol (EG) with or without sucrose (SUC) are used widely under on-farm conditions, not only in Japan but also globally. The direct transfer method using 1.5 M glycerol (GLY) and 0.25 M SUC (GLY-SUC) is used mainly in Japan. The pregnancy rate with direct transfer of frozen-thawed bovine embryos in either EG or GLY-SUC has been found to not differ from conventional freezing with GLY and traditional dilution techniques. Pregnancy rates following direct transfer of frozen-thawed bovine embryos were affected by the developmental stage of the embryos and the parity of the recipients. The use of ultrasound-guided on-farm ovum pickup is ushering in a new revolution for the commercial application of IVF embryos. Globally, for the first time more IVF bovine embryos were transferred in 2017 than produced in vivo. More than 60% of IVF embryos were transferred fresh due to a low pregnancy rate of frozen-thawed IVF embryos. Many factors seemed to be involved in improving the survival rate of frozen-thawed IVF embryos. Therefore, further research is needed to improve the freezing tolerance of IVF embryos to develop efficient direct transfer methods analogous to those used for in vivo embryos.

Zinc finger and SCAN domain containing 4 (Zscan4) is a gene that is specifically expressed during zygotic genome activation (ZGA) in mouse preimplantation embryos, and a reduction of Zscan4 transcripts leads to developmental failure. In mouse embryonic stem cells (ESCs), Zscan4 is expressed transiently in as little as 1–5% of the cell population. Zscan4 has also been shown to enhance the efficiency of mouse induced pluripotent stem cells (iPSCs) generation and their quality. Although ZSCAN4 plays important roles in murine embryos and stem cells, its expression and role in bovine embryos is unknown. This study examines ZSCAN4 transcripts in bovine embryos at various developmental stages and attempts to elucidate the functions of ZSCAN4 during bovine preimplantation development. ZSCAN4 transcripts were found to be upregulated at the 8- and 16-cell stages. We next attempted ZSCAN4 downregulation in bovine early embryos by RNA interference and evaluated developmental competency and transcripts levels of genes involved in ZGA and iPSCs generation. Although the bovine embryos injected with ZSCAN4-siRNA could develop to the 8-cell stage, very few were developing beyond the 16-cell stage. PIWIL2 expression was reduced in ZSCAN4 downregulated embryos. It is possible that ZSCAN4 downregulated embryos fail to regulate gene expression during ZGA. Our results indicate that ZSCAN4 is an important factor for the preimplantation development of bovine embryos.

This study elucidated the molecular markers that decrease oocyte quality during in vitro culture, restricting optimal developmental potential. Here, we evaluated the transcriptomic differences between cysteamine-treated and non-treated bovine cumulus oocyte complexes (COCs) after 22 h of co-culture in the maturation media using RNA sequencing. In total, 39,014 transcripts were sequenced between cysteamine-treated and non-treated mature COCs. We evaluated the relative expression of 21,472 genes, with 59 genes showing differential expression between the two COC groups. The cysteamine-treated group had 36 up-regulated gene transcripts and 23 down-regulated gene transcripts. Moreover, gene ontology (GO) enrichment analysis revealed that multiple biological processes were significantly enriched after cysteamine supplementation. Differentially expressed genes appeared to maintain normal oocyte physiology, regulation of apoptosis, differentiation, ossification or bone formation, cardiac and muscle physiology, hormonal secretion, and membrane construction for further embryonic development. In conclusion, cysteamine affects the mRNA level of COCs during oocyte maturation by upregulating potential molecular markers and downregulating genes that affect further embryonic development. [ABSTRACT FROM AUTHOR]

Embryo-derived extracellular vesicles (EVs) may play a role in mediating the embryo-maternal dialogue at the oviduct, potentially carrying signals reflecting embryo quality. We investigated the effects of bovine embryo-derived EVs on the gene expression of bovine oviductal epithelial cells (BOECs), and whether these effects are dependent on embryo quality. Presumptive zygotes were cultured individually in vitro in culture medium droplets until day 8 while their development was assessed at day 2, 5 and 8. Conditioned medium samples were collected at day 5 and pooled based on embryo development (good quality embryo media and degenerating embryo media). EVs were isolated from conditioned media by size exclusion chromatography and supplemented to primary BOEC monolayer cultures to evaluate the effects of embryo-derived EVs on gene expression profile of BOEC. Gene expression was quantified by RNA-seq and RT-qPCR. A total of 7 upregulated and 18 downregulated genes were detected in the BOECs supplemented with good quality embryo-derived EV compared to the control. The upregulated genes included interferon-τ-induced genes, such as OAS1Y, MX1 and ISG15, which have previously been reported as upregulated in the oviductal epithelial cells in the presence of embryos. Of the upregulated genes, OAS1Y and MX1 were validated with RT-qPCR. In contrast, only one differentially expressed gene was detected in BOECs in response to degenerating embryo-derived EVs, suggesting that oviductal responses are dependent on embryo quality. Our results support the hypothesis that embryo-derived EVs are involved in embryo-maternal communication at the oviduct and the oviductal response is dependant on the embryo quality. Key messages: • Extracellular vesicles (EVs) released by individually cultured pre-implantation bovine embryos can alter the gene expression of primary oviductal epithelial cells. • The oviductal response, in terms of gene expression, to the bovine embryo-derived EVs varied depending on the embryo quality. • In vivo, the oviduct may have the ability to sense the quality of the pre-implantation embryos. • The observed effect of embryo-derived EVs on oviductal epithelial cells could serve as a non-invasive method of evaluating the embryo quality. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effect of the force and duration of centrifugation and the impact of cushioned centrifugation on sperm selection by Percoll gradient, on sperm quality and development kinetics of in vitro produced bovine embryos. Two experiments were performed. In Experiment I, a pool of semen was selected by Percoll gradients and the pellet was divided into four groups and distributed in a 2 × 2 factorial, with two forces (2200 × g or 9000 × g) and two durations (1 min or 3 min) of centrifugation. In Experiment II, semen was divided into two groups and selected by Percoll gradient with Cushion Fluid (CF) or without CF (Control) in the second centrifugation. The morphofunctionality, biochemical characteristics and fertilizing capacity of the selected sperms were evaluated. In addition, the development of the resulting bovine embryos was monitored for 48 h post-insemination. Duncan and Chi-square tests (P < 0.05) were used to compare the means. In Experiment I, there was a significant increase in sperm vigor (P < 0.05) after sperm selection in all treatments. The force and duration of centrifugation did not have any effect on sperm motility, vigor, and recovery rate among the different treatments (P > 0.05). In Experiment II, the recovery rate and reactive oxygen species (ROS) production in semen were similar among treatments (P > 0.05) although a higher ROS production was observed in the CF fertilization medium. Total fertilization rate was superior in the CF group (65.4 ± 5.3%) compared to that in Control (39.6 ± 4.9%). However, the normal fertilization and cleavage rate did not differ between the Control (94 ± 6.3% and 58.3 ± 8.3%) and CF (89 ± 7.1% and 75.0 ± 7.3%) groups. The reduction in the force and duration of centrifugation did not decrease the sperm recovery during selection by the Percoll gradient and the use of CF in the second centrifugation did not affect the normal fertilization and development of bovine IVF embryos up to 48 h.

Abstract Background Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. Results The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. Conclusion In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts.

The present study aimed to elucidate the developmental kinetics, growth potential, and viability of bovine embryos produced in vitro with sexed semen. Bovine oocytes were fertilized in vitro using unsorted and X-sorted semen from the same Holstein bulls, and the kinetics of in vitro development were continuously monitored for 10 d through time-lapse cinematography. The blastocyst formation rate was lower in the X-sorted group than in the unsorted group (P < 0.01), whereas the normal fertilization rate did not differ between groups. Morphokinetic evaluation revealed that the incidence of blastomere fusion during the first cleavage division, termed reverse cleavage, was higher in the X-sorted group (P < 0.01). Furthermore, embryos produced with X-sorted semen showed slower growth throughout the developmental period than embryos produced with unsorted semen (P < 0.01). The cell number of the trophectoderm and inner cell mass of blastocysts was reduced in the X-sorted group (P < 0.01). In embryos that developed to the blastocyst stage, the hatchability (P < 0.05), chromosomal normality (P < 0.01), and survivability after the conventional frozen-thawing process (P < 0.05) were reduced in the X-sorted group compared to that in the unsorted group, indicating a compromised viability of embryos derived from X-sorted semen. Taken together, the first cleavage dysmorphism, delayed embryo growth, and impaired viability of embryos developed to the blastocyst stage may explain the mechanism of reduced fertility in embryos derived from sexed semen. The kinetic evaluation of early embryo development and de-selection of embryos presenting the aberrant first cleavage would be valid for clinical application to produce sexed embryos with high implantation potential. • Bovine embryos were produced in vitro with X-sorted semen. • Blastomere fusion incidence increased during the first cleavage division. • X-sperm-derived embryos had slow growth and reduced blastulation rate. • These embryos had reduced hatchability, chromosomal normality, and cryotolerance. [ABSTRACT FROM AUTHOR]

The production of bovine embryos through in vitro maturation and fertilization is an important tool of the genomic revolution in dairy cattle. Gene expression analysis of these embryos revealed differences according to the culture conditions or oocyte donor's pubertal status compared to in vivo derived embryos. We hypothesized that some of the methylation patterns in oocytes are acquired in the last step of folliculogenesis and could be influenced by the environment created in the follicles containing these oocytes. These altered patterns may not be erased during the first week of embryonic development in culture or may be sensitive to the conditions during that time. To quantify the changes related to culture conditions, an in vivo control group consisting of embryos (Day 12 post fertilization for all groups) obtained from superovulated and artificially inseminated cows was compared to in vitro produced (IVP) embryos cultured with or without Fetal Bovine Serum (FBS). To measure the effect of the oocytes donor's age, we also compared a fourth group consisting of IVP embryos produced with oocytes collected following ovarian stimulation of pre-pubertal animals. Embryonic disk and trophoblast cells were processed separately and the methylation status of ten imprinted genes (H19, MEST, KCNQ1, SNRPN, PEG3, NNAT, GNASXL, IGF2R, PEG10, and PLAGL1) was assessed by pyrosequencing. Next, ten Day 7 blastocysts were produced following the same methodology as for the D12 embryos (four groups) to observe the most interesting genes (KCNQ1, SNRPN, IGF2R and PLAGL1) at an earlier developmental stage. For all samples, we observed overall lower methylation levels and greater variability in the three in vitro groups compared to the in vivo group. The individual embryo analysis indicated that some embryos were deviant from the others and some were not affected. We concluded that IGF2R, SNRPN, and PEG10 were particularly sensitive to culture conditions and the presence of FBS, while KCNQ1 and PLAGL1 were more affected in embryos derived from pre-pubertal donors. This work provides markers at the single imprinted control region (ICR) resolution to assess the culture environment required to minimize epigenetic perturbations in bovine embryos generated by assisted reproduction techniques, thus laying the groundwork for a better comprehension of the complex interplay between in vitro conditions and imprinted genes. • Imprinted genes methylation is impacted by culture conditions and the presence of FBS, especially IGF2R, SNRPN, and PEG10. • Oocyte donor's age also impacts Imprinted genes methylation especially KCNQ1 and PLAGL1. • Different assisted reproduction techniques lead to specific imprinted gene methylation signature in bovine embryos. The oocyte donor's age and embryo culture conditions impact on imprinted gene methylation in Day 7 and Day 12 bovine embryos. [ABSTRACT FROM AUTHOR]

Capturing stable embryonic stem cell (ESC) lines from domesticated animals still remains one of the challenges of non-rodent embryology. The stake is high, as stable ESCs derived from species such as cattle present high economic and scientific value. Understanding of the processes leading to the embryonic lineage segregation is crucial to provide species-orientated molecular environment capable of supporting self-renewal and pluripotency. Therefore, the aim of this study was to validate the action of the two core regulatory pathways (WNT and MEK/ERK) during bovine embryo development. I n vitro produced bovine embryos were obtained in the presence of inhibitors (i), which enable activation of the WNT pathway (via GSK3i, CHIR99021) and suppression of MEK signalling by PD0325901 in the 2i system and PD184325 and SU5402 in the 3i system. We have followed the changes in the distribution of the key lineage specific markers both at the transcript and protein level. Our results showed that WNT signalling promotes the expression of key inner cell mass (ICM) specific markers in bovine embryos, regardless of the MEK/ERK inhibitor cocktail used. MEK/ERK downregulation is crucial to maintain OCT4 and NANOG expression within the ICM and to prevent their exclusion from the trophectoderm (TE). At the same time, the classical TE marker (CDX2) was downregulated at the mRNA and protein level. As a follow up for the observed pluripotency stimulating effect of the inhibitors, we have tested the potential of the 2i and the 3i culture conditions (supported by LIF) to derive primary bovine ESC lines. As a result, we propose a model in which all of the primary signalling pathways determining embryonic cell fate are active in bovine embryos, yet the requirement for pluripotency maintenance in cattle may differ from the described standards. WNT activation leads to the formation (and stabilisation of the ICM) and MEK/ERK signalling is maintained at low levels. Unlike in the mouse, GATA6 is expressed in both ICM and TE. MEK/ERK signalling affects HP formation in cattle, but this process is activated at the post-blastocyst stage. With regard to self-renewal, 2i is preferable, as 3i also blocks the FGF receptor, what may prevent PI3K signalling, important for pluripotency and self-renewal. • Signalling pathways determining embryonic cell fate (WNT, MEK/ERK) are active in bovine embryos. • WNT activation leads to the formation and stabilisation of the ICM. • GATA6 expression is not restricted to the bovine hypoblast lineage. • 2i ​+ ​LIF conditions are preferable for the derivation of primary bovine ESCs. [ABSTRACT FROM AUTHOR]

Objective Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. Methods After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense (0.01 μg/mL) and H. japonicus (0.01 μg/mL). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. Results We observed that the blastocysts rate was significantly increased (p

The elevated temperature and high humidity has been known as main reason for heat stress on animals and cause detrimental effects on productivity of organisms and physiological conditions of normal bioactivities. The aims of this study were to evaluate the relationship between time of heat shock simulation during in vitro maturation and developmental competence of subsequent embryo after in vitro fertilization. Heat shocked cumulus-oocyte complexes (COCs) of Korean native cattle were subjected to normal conditions for 22, 21, 18 and 12 h respectively and transferred to heat stress inducing condition at 40.5 °C in other incubator for 0 (control), 1 and 4 h. After maturation for 22 h, the oocytes were fertilized and cultured in mSOF media for 8 d and examined the developmental capacity of embryos. There were no differences in maturation and cleavage rates between 0, 1 and 4 h heat socked oocytes, but blastocysts formation were lower in the 4 h heat stressed oocytes. The apoptotic cells of developed blastocysts were also increased in at day 8 with 4 h heat shocked oocytes. These results indicate that heat shock on oocytes during maturation could cause negative effects on the developmental competence of embryos.

To increase the productivity of in vitro development, the antioxidants have been used for culture system of bovine oocytes and embryos. However, comparative studies on these molecules are rare and direct beneficial effects on blastocyst production cannot be discriminated for best results. The study was conducted to determine the influence of N-acetyl-L-cysteine (NAC), N-acetyl-L-cysteine amide (NACA), glutathione (GSH) and cysteamime (CYS) on maturation competence of COCs from GV to MII stage and productivity of blastocyst formation during in vitro fertilization and culture. There was no difference among maturation rates of oocytes to metaphase II with polar body with antioxidants for any of the treatment groups (p>0.05). However, the significant improvement on the rate of blastocysts (32.3±5.0%) was found in 0.1 mM CYS treatment than 0.3 mM NAC, 0.2 mM NACA or 0.5mM GSH (p0.05). Our study showed that medium supplementation with CYS during IVM and IVC improved the rate of bovine embryo development but not with NAC, NACA and GSH addition.

The behavior of BTV-8 in cattle is different from most other serotypes not only with regards to clinical signs but certainly with respect to virus transmission (transplacental, contact). Therefore, the possibility of virus transmission by means of embryo transfer was examined by in vitro exposure of in vitro produced and in vivo derived bovine blastocysts to BTV-8 followed by different washing protocols, including longer exposure times (up to 120 s) to 0.25% trypsin at room temperature or at 37°C. None of the washing protocols used was successful in removing the viral genome completely from the in vitro produced and in vivo derived embryos as was demonstrated by real-time PCR. Moreover, BTV-8 virus was transmitted to recipient cows after embryo transfer of in vivo derived BTV8-exposed embryos, which had been subjected to routine decontamination as recommended by IETS, consisting of 5 washes in PBS followed by a double treatment of 0.25% trypsin for 45s at 37°C, and an additional 5 washes in PBS with 2% FCS. This study clearly demonstrates the necessity of vigorous application of the directives for screening of potential donors and the collected embryos, especially in regions with BTV-8, to prevent transmission of the disease.

Embryo transfer in cattle is globally becoming more ubiquitous, but the pregnancy rate is lower than that of artificial insemination. The uterus contains its own bacteria, and concentrations of lipopolysaccharides (LPS) from gram-negative bacteria are higher in uteri affected by endometritis than in healthy uteri and they suppress embryogenesis. The purpose of this study was to investigate the morphological characteristics of bovine embryos with a higher viability and implantability, by analyzing the morphology of bovine blastocysts that successfully hatched under challenge of LPS, using an optical coherence tomography (OCT) system. Developing embryos produced by in vitro fertilization that had reached the blastocyst stage on Day 7 were three-dimensionally scanned using an OCT system, then were continued to culture with or without LPS until Day 9, when the presence or absence of hatching was determined. The OCT-captured three-dimensional images were used to quantify 20 different metrics, including inner cell mass (ICM), trophectoderm, blastocoel, and total embryo volume; each of the parameters was compared between the hatched and unhatched embryos. Under the LPS challenge, hatched embryos had higher ICM thickness and volume, and lower trophectoderm thickness than unhatched embryos. Furthermore, hatched embryos under LPS challenge had higher ICM thickness and ICM volume than hatched embryos without LPS challenge. The present results suggest the possibility that ICM thickness and ICM volume calculated by OCT system could be indices for good quality bovine embryos., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

The term “embryokine” has been used to denote molecules produced by the endometrium, oviduct, or by embryo itself that will influence embryo development. Several cytokines have been identified as embryokines in cattle and other mammals. This review will describe how these cytokines function as embryokines, with special emphasis being placed on their actions on in vitro produced (IVP) bovine embryos. Embryokines are being explored for their ability to overcome the poor development rates of IVP embryos and to limit post-transfer pregnancy retention efficiencies that exist in IVP embryos. This review will focus on describing two of the best-characterized cytokines, colony-stimulating factor 2 and interleukin 6, for their ability to modify bovine embryo quality and confirmation, promote normal fetal development, and generate healthy calves. Additional cytokines will also be discussed for their potential to serve as embryokines.

This study assessed the effects of cryoprotectant concentration during equilibration on the efficiency of bovine blastocyst vitrification and the expression of selected developmentally important genes. In vitro produced bovine blastocysts were equilibrated in either 7.5% ethylene glycol (EG) + 7.5% DMSO (Va group) or in 2% EG + 2% DMSO (Vb group) then vitrified on Cryotop® sheets in 16.5% EG + 16.5% DMSO + 0.5M sucrose. After warming, embryos were cultured for 48 hr. Reexpansion, hatching, and the numbers of total and membrane damaged cells were compared among vitrified groups and a control. There was no significant difference between the vitrified groups in survival, cell numbers and the extent of membrane damage. Vitrification increased the number of membrane-damaged cells in both groups, however, in a greater extent in the Vb group. Vitrification increased (p < .05) the expression of the HSP70 gene in Va but not in Vb embryos. The expression of IGF2R, SNRPN, HDAC1, DNMT3B, BAX, OCT4, and IFN-t genes were the same in control and vitrified groups. In conclusion, the concentration of cryoprotectants during equilibration did not affect survival rates; however, normal cell numbers could be maintained only by equilibration in 15% cryoprotectants which was associated with increased HSP70 expression. [ABSTRACT FROM AUTHOR]

Summary: Lumpy skin disease (LSD) is an important transboundary animal disease of cattle with significant economic impact because of the implications for international trade in live animals and animal products. LSD is caused by a Capripoxvirus, LSD virus (LSDV), and results in extensive hide and udder damage, fever and pneumonia. LSDV can be shed in semen of infected bulls for prolonged periods and transmitted venereally to cows at high doses. This study examined the effects of LSDV in frozen‐thawed semen on in vitro embryo production parameters, including viral status of media and resulting embryos. Bovine oocytes were harvested from abattoir‐collected ovaries and split into three experimental groups. After maturation, the oocytes were fertilized in vitro with frozen‐thawed semen spiked with a high (HD) or a lower (LD) dose of LSDV, or with LSDV‐free semen (control). Following day 7 and day 8 blastocyst evaluation, PCR and virus isolation were performed on all embryonic structures. After completing sufficient replicates to reach 1,000 inseminated oocytes, further in vitro fertilization (IVF) runs were performed to provide material for electron microscopy (EM) and embryo washing procedures. Overall, in vitro embryo yield was significantly reduced by the presence of LSDV in frozen‐thawed semen, irrespective of viral dose. When semen with a lower viral dose was used, significantly lower oocyte cleavage rates were observed. LSDV could be detected in fertilization media and all embryo structures, when higher doses of LSDV were present in the frozen‐thawed semen used for IVF. Electron microscopy demonstrated LSDV virions inside blastocysts. Following the International Embryo Transfer Society washing procedure resulted in embryos free of viral DNA; however, this may be attributable to a sampling dilution effect and should be interpreted with caution. Further research is required to better quantify the risk of LSDV transmission via assisted reproductive procedures. [ABSTRACT FROM AUTHOR]

Assisted reproductive technologies (ART) are widely used for both humans and domestic animals. In bovine species, in vitro embryo production is increasingly used and significant efforts are being made to optimize media and culture conditions. Phospholipase A2 (PLA2) are lipolytic enzymes that hydrolyze glycerophospholipids to produce free fatty acids and lysophospholipids that have been found to be critical for many biological processes. Mouse group X secreted PLA 2 (mGX) is abundant in the male reproductive tract and its use during sperm capacitation has been shown to improve in vitro production of viable embryos in a mouse model. Here, we examined its effect in the bovine species, testing the impact of mGX on the three steps involved in vitro production of preimplantation embryos: oocyte maturation, fertilization and preimplantation development. We found that incubating cumulus oocyte complexes (COC) or gametes with mGX resulted in increased blastocyst hatching and blastocyst production, respectively. The increases of embryo production induced by the phospholipase mGX were not observed for the catalytically inactive mutant H48Q-mGX, suggesting that these effects require the enzymatic activity of mGX. We also tested bGIB, a bovine homolog of mGX. bGIB failed to improve blastocyst production, underlining the high specificity of mGX. In conclusion, the results presented show that the effects of mGX are not restricted to the mouse model and that it is potent in the bovine species as well. This result strengthens the potential of mGX as a "pro-fertility drug" for mammalian reproduction. • secreted group X PLA2 applied during oocyte in vitro maturation improves embryo development. • secreted group X PLA2 applied during in vitro fertilization improves embryo development. • Effects of group X sPLA2 include increase of blastocyst formation, expansion and hatching. • Effects of group X sPLA2 is not due to LPA production. • Group IB sPLA2 does not mimic effects of group X sPLA2. [ABSTRACT FROM AUTHOR]

Abstract Culture conditions determine embryo quality, which may be affected on many levels (timing of development, blastomere count, transcripts, metabolite content, apoptosis). Molecular interactions of signalling pathways like MEK/ERK and WNT/β-catenin are critical for cell-to-cell communication and cellular differentiation. Both pathways are important regulators of apoptosis. We have aimed to verify the prolonged effect of MEK/ERK silencing and WNT activation by chemical inhibitors (2i or 3i systems) on bovine IVP embryos. Apoptotic index, total cell count and transcription of embryo quality markers were evaluated. A higher rate of apoptosis was observed in 2i blastocysts, but was not accompanied by changes in transcript content of genes controlling apoptosis (BAX, BCL2, BAK, BAX/BCL2 ratio). Therefore, alternative pathways of apoptotic activation cannot be ruled out. The expression of genes related to embryo quality (HSPA1A, SLC2A1) was not affected. GJA1 transcripts were significantly higher in 3i blastocysts, what indicates a stimulatory effect of the applied inhibitors on cell-to-cell interactions. The lowest mRNA level of the IFNT2 gene was found in 2i embryos. A variation in the SDHA gene transcript was observed (with the highest content in the 3i blastocysts), what may suggest their reduced quality. It may be concluded that the modifications of culture conditions (activation of the WNT and silencing of the MEK/ERK signalling) might alter pathways crucial for embryo development without causing embryonic death. Highlights • WNT and MEK/ERK signalling pathways affect bovine embryo quality. • Activation of pluripotency related signalling reduces apoptosis in preimplantation embryos. • Altering of signalling pathways induce SDHA expression, suggesting oxidative stress conditions. [ABSTRACT FROM AUTHOR]

Abstract The objectives of this study were to determine (i) whether Chlamydia (C.) abortus would adhere to the intact zona pellucida (ZP-intact) of early in vitro produced bovine embryos; (ii) whether the bacteria would adhere to the embryos (ZP-free) after in vitro infection; and (iii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol. The experimentation was made twice. For each replicate 100 (8–16-cell) bovine embryos produced in vitro were randomly divided into 10 batches. Height batches (4 ZP-intact and 4 ZP-free) of 10 embryos were incubated in a medium containing 4 × 107 Chlamydia /ml of AB7 strain. After incubation for 18 h at 37 °C in an atmosphere of 5% CO 2 , the embryos were washed in accordance with the IETS guidelines. In parallel, two batches (1 ZP-intact and 1 ZP-free) of 10 embryos were subjected to similar procedures but without exposure to C. abortus as a control group. The 10 washing fluids from each batch were collected and centrifuged for 1 h at 13,000× g. Each batch of washed embryos and each wash pellets were tested using PCR. C. abortus DNA was found in all ZP-intact and ZP-free batches of 10 embryos after 10 successive washes. For ZP-intact infected embryos, Chlamydia -DNA was also detected in all 10 wash baths for two batches (2/8) of embryos, whereas for ZP-free infected embryos, Chlamydia -DNA was detected in all 10 wash baths for 6/8 batches of embryos. In contrast, none of the embryos or their washing fluids in the control batches was DNA positive. The bacterial load for batches of 10 embryos after the 10 wash baths was significantly higher for batches of ZP-free embryos (20.7 ± 9 × 103 bacteria/mL) than for batches of ZP-intact embryos (0.47 ± 0.19 × 103 bacteria/mL). These results demonstrate that C. abortus adheres to the ZP as well as the early embryonic cells of in vitro produced bovine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS fails to remove it. Highlights • C. abortus adheres to the intact zona pellucida and to the early embryonic cells. • standard washing protocol recommended by the IETS failed to remove it • Finding of C. abortus DNA by PCR did not imply that the bacteria found was infectious. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (scNT) embryos suffer from damage caused by micro-operation (manipulation) and inefficient genome reprograming that hinder their normal development at different levels and in distinct ways. These two effects are inseparable in the nature of the scNT embryo, although methods to separately measure them are needed to improve scNT technology and evaluate incoming reprogramming tools. As an attempt to meet these demands, we made bovine sham nuclear-transfer (shNT) blastocysts, special embryos made with a standard nuclear-transfer procedure at the zygote stage, while retaining an intact genome. We compared their transcriptomes with those of other blastocysts derived by in-vitro fertilization (IVF) or scNT. Correlation analysis revealed a singularity of shNT blastocysts as they separately gathered from the others. Analysis of developmentally important genes revealed that, in shNTs, the stemness-associated differentially expressed genes (DEGs), including OCT4, were mostly underrepresented. Overrepresented epi-driver genes were largely associated with heterochromatin establishment and maintenance. By multilateral comparisons of their transcriptomes, we classified DEGs into three groups: 561 manipulation-associated DEGs (MADs) common to shNTs and scNTs, 764 donor genome-associated DEGs (DADs) specific to scNTs, and 1743 zygote manipulation-associated DEGs (zMADs) specific to shNTs. GO enrichment analysis generated various terms involving “cell-cell adhesion,” “translation,” and “transcription” for MADs and “cell differentiation” and “embryo implantation” for DADs. Because of the transcriptomic specificity of shNTs, we studied zMADs in detail. GO enrichment analysis with the 854 zMADs underrepresented in shNTs yielded terms related to protein and mitochondria homeostasis, while GO enrichment analysis of 889 shNT-high zMADs yielded terms related to endoplasmic reticulum stress and protein transport. We summarized the DEGs, which, with further investigation, may help improve our understanding of molecular events occurring in cloned embryos and our ability to control clonal reprogramming.

Abstract This paper presents the development of a multi-sensor platform capable of simultaneous measurement of dissolved oxygen (DO) concentration, glucose and lactate concentrations in a micro-chamber for real-time evaluation of metabolic flux in bovine embryos. A micro-chamber containing all three sensors (DO, glucose, and lactate) was made to evaluate metabolic flux of single oocytes or embryos at different stages of development in ≤ 120 µL of respiration buffer. The ability of the sensor to detect a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis was demonstrated in embryos by an ablation of oxygen consumption and an increase in lactate production following addition of oligomycin, an inhibitor of mitochondrial adenosine triphosphate (ATP) synthesis. An increased reliance upon glycolysis relative to OXPHOS was demonstrated in embryos as they developed from morula to hatched blastocysts by a progressive increase in the lactate/oxygen flux ratio, consistent with isolated metabolic assessments reported previously. These studies highlight the utility of a metabolic multi-sensor for integrative real-time monitoring of aerobic and anaerobic energy metabolism in bovine embryos, with potential applications in the study of metabolic processes in oocyte and early embryonic development. Highlights • A multi-sensor platform for simultaneous measurement of dissolved oxygen (DO), glucose and lactate concentrations. • DO was measured amperometrically using a three-electrode system. • Glucose and lactate were assessed enzymatically. • Real-time evaluation of metabolic flux in bovine embryos. • An increased reliance upon glycolysis relative to oxidative phosphorylation was demonstrated in embryos with devlopment. [ABSTRACT FROM AUTHOR]

Contents: Antioxidants have been widely used during in vitro production to decrease the negative effect of reactive oxygen species. It was reported that the complex resveratrol–methyl β‐cyclodextrin (RV‐CD) improves resveratrol's stability and bioavailability and increases its antioxidant activity. This study evaluates the effect of RV‐CD during in vitro oocyte maturation (IVM) or in vitro embryo culture (IVC) on developmental competence and quantitative changes in gene expression of developmental important genes. In experiment 1, RV‐CD was added to IVM media and maturation level, embryo development and oocytes, cumulus cells, and blastocysts gene expression by RT‐qPCR were examined. In experiment 2, presumptive zygotes were cultured in SOF supplemented with RV‐CD and embryo development and blastocysts gene expression by RT‐qPCR were studied. A group without RV‐CD (control−) and a group with cyclodextrin (control+) were included. No differences were found in cleavage rate or blastocyst yield between groups. However, the expression of LIPE was higher in blastocysts derived from oocytes treated with resveratrol compared with control groups (p < .05). Blastocysts produced by IVC with resveratrol showed that RV‐CD could modify the expression of genes related to lipid metabolism (CYP51A1, PNPLA2 and MTORC1) compared with control groups (p < .05). RV‐CD in the IVM and IVC media could reduce accumulated fat by increasing lipolysis and suppressing lipogenesis of blastocysts. [ABSTRACT FROM AUTHOR]

Background: Aberrant DNA methylation patterns of genes required for development are common in in vitro produced embryos. In this regard, we previously identified altered DNA methylation patterns of in vivo developed blastocysts from embryos which spent different stages of development in vitro, indicating carryover effects of suboptimal culture conditions on epigenetic signatures of preimplantation embryos. However, epigenetic responses of in vivo originated embryos to suboptimal culture conditions are not fully understood. Therefore, here we investigated DNA methylation patterns of in vivo derived bovine embryos subjected to in vitro culture condition before, during or after major embryonic genome activation (EGA). For this, in vivo produced 2-, 8- and 16-cell stage embryos were cultured in vitro until the blastocyst stage and blastocysts were used for genome-wide DNA methylation analysis. Results: The 2- and 8-cell flushed embryo groups showed lower blastocyst rates compared to the 16-cell flush group. This was further accompanied by increased numbers of differentially methylated genomic regions (DMRs) in blastocysts of the 2- and 8-cell flush groups compared to the complete in vivo control ones. Moreover, 1623 genomic loci including imprinted genes were hypermethylated in blastocyst of 2-, 8- and 16-cell flushed groups, indicating the presence of genomic regions which are sensitive to the in vitro culture at any stage of embryonic development. Furthermore, hypermethylated genomic loci outnumbered hypomethylated ones in blastocysts of 2- and 16-cell flushed embryo groups, but the opposite occurred in the 8-cell group. Moreover, DMRs which were unique to blastocysts of the 2-cell flushed group and inversely correlated with corresponding mRNA expression levels were involved in plasma membrane lactate transport, amino acid transport and phosphorus metabolic processes, whereas DMRs which were specific to the 8-cell group and inversely correlated with corresponding mRNA expression levels were involved in several biological processes including regulation of fatty acids and steroid biosynthesis processes. Conclusion: In vivo embryos subjected to in vitro culture before and during major embryonic genome activation (EGA) are prone to changes in DNA methylation marks and exposure of in vivo embryos to in vitro culture during the time of EGA increased hypomethylated genomic loci in blastocysts. [ABSTRACT FROM AUTHOR]

Somatic cell nuclear transfer (scNT) embryos suffer from damage caused by micro-operation (manipulation) and inefficient genome reprograming that hinder their normal development at different levels and in distinct ways. These two effects are inseparable in the nature of the scNT embryo, although methods to separately measure them are needed to improve scNT technology and evaluate incoming reprogramming tools. As an attempt to meet these demands, we made bovine sham nuclear-transfer (shNT) blastocysts, special embryos made with a standard nuclear-transfer procedure at the zygote stage, while retaining an intact genome. We compared their transcriptomes with those of other blastocysts derived by in-vitro fertilization (IVF) or scNT. Correlation analysis revealed a singularity of shNT blastocysts as they separately gathered from the others. Analysis of developmentally important genes revealed that, in shNTs, the stemness-associated differentially expressed genes (DEGs), including OCT4, were mostly underrepresented. Overrepresented epi-driver genes were largely associated with heterochromatin establishment and maintenance. By multilateral comparisons of their transcriptomes, we classified DEGs into three groups: 561 manipulation-associated DEGs (MADs) common to shNTs and scNTs, 764 donor genome-associated DEGs (DADs) specific to scNTs, and 1743 zygote manipulation-associated DEGs (zMADs) specific to shNTs. GO enrichment analysis generated various terms involving "cell-cell adhesion," "translation" and "transcription" for MADs and "cell differentiation" and "embryo implantation" for DADs. Because of the transcriptomic specificity of shNTs, we studied zMADs in detail. GO enrichment analysis with the 854 zMADs underrepresented in shNTs yielded terms related to protein and mitochondria homeostasis, while GO enrichment analysis of 889 shNT-high zMADs yielded terms related to endoplasmic reticulum stress and protein transport. We summarized the DEGs, which, with further investigation, may help improve our understanding of molecular events occurring in cloned embryos and our ability to control clonal reprogramming. [ABSTRACT FROM AUTHOR]

This study examines the effects of the histone deacetylation inhibitor scriptaid (SCR) on preimplantation embryo development in vitro and on imprinting gene expression. We hypothesized that SCR would increase histone acetylation levels, enhance embryonic genome activation, and regulate imprinting and X-chromosome inactivation (XCI) in in vitro produced bovine embryos. Zygotes were cultured in vitro in presence or absence of SCR added at different time points. We assessed cleavage and blastocyst rates as well as the quality of blastocysts through: (i) differential cell counts; (ii) survival after vitrification/thawing and (iii) gene expression analysis –including imprinted genes. Blastocyst yields were not different in the control and experimental groups. While no significant differences were observed between groups in total cell or trophectoderm cell numbers, SCR treatment reduced the number of inner cell mass cells and improved the survival of vitrified embryos. Further, genes involved in the mechanism of paternal imprinting ( GRB10, GNAS, XIST ) were downregulated in presence of SCR compared with controls. These observations suggest SCR prevents deacetylation of paternally imprinting control regions and/or their up-regulation, as these events took place in controls. Whether or not such reductions in XIST and imprinting gene expression are beneficial for post implantation development remains to be clarified. [ABSTRACT FROM AUTHOR]

The aim of the present study was to compare two different serum-free media, modified synthetic oviduct fluid (mSOF) and modified potassium simplex optimization medium (mKSOM) containing 20% RD (RPMI1640 + DMEM, 1:1 v/v) (RD-mKSOM), for In Vitro culture (IVC) of bovine embryos. After In Vitro maturation and fertilization, the presumptive zygotes were cultured in two different serum-free conditions for 7 days and 9 days to evaluate blastocyst formation and hatching, respectively. Serum supplemented conventional CR2 medium was used as control. After 7 day of culture, there was no significant difference in cleavage and blastocyst formation rates among three groups (mSOF, 59.3 and 30.1%; RD-mKSOM, 65.0 and 41.5%; control, 51.6 and 38.0%, respectively). Hatching rate was significantly higher in control (69.0%) than other experimental groups (mSOF, 22.0%; RD-mKSOM, 39.5%) (P