Bovine Cumulus-oocyte Complexes Research Articles

Effect of IGFBP-4 during In Vitro Maturation on Developmental Competence of Bovine Cumulus Oocyte Complexes.

Insulin-like growth factors (IGFs) are essential for oocyte maturation. Their bioavailability is regulated by their respective binding proteins (IGFBPs) and proteases. IGFBP-4 blocks the biological effects of IGFs. High IGFBP-4 expression has been associated with follicle atresia. We hypothesized that IGFBP-4 affects oocyte developmental competence during maturation. Therefore, the aim of this study was to examine the effect of IGFBP-4 on the developmental rate of bovine cumulus-oocyte complexes (COCs) during in vitro embryo production. Abattoir-derived COCs were matured with rbIGFBP-4 (2000, 540, and 54 ng/mL) compared to a control. Cumulus expansion, oocyte maturation, cleavage, blastocyst, and hatching rates were evaluated. Furthermore, blastocyst gene expression of SOCS2, STAT3, SLC2A1, SLCA3, BAX, and POU5F1 transcripts were quantified using RT-qPCR. No statistical differences were detected among the groups for cumulus expansion, maturation, cleavage, blastocyst rates, or all gene transcripts analyzed. However, at day 8 and 9, the number of total hatching and successfully hatched blastocysts was lower in 2000 ng/mL rbIGFBP-4 compared to the control (day 8: total hatching: 17.1 ± 0.21 vs. 31.2 ± 0.11%, p = 0.02 and hatched blastocyst 6.7 ± 0.31 vs. 21.5 ± 0.14%, p = 0.004; day 9 total hatching 36.4 ± 0.18 vs. 57.7 ± 0.10%, p = 0.009 and hatched blastocyst 18.2 ± 0.21 vs. 38.1 ± 0.11%, p = 0.004). We concluded that high concentrations of rbIGFBP-4 might negatively affect the subsequent ability of the embryo to hatch and possibly compromise further elongation.

Energy metabolism disorders during in vitro maturation of bovine cumulus-oocyte complexes interfere with blastocyst quality and metabolism

Glucose and fatty acids (FA) metabolism disturbances during oocyte in vitro maturation (IVM) affect their metabolism and surrounding cumulus cells, but only inhibition of glucose metabolism decreases embryo culture efficiency. Therefore, the present experiment aimed to reveal if glucose or FA metabolism inhibition leads to the disruption of embryo developmental potential, and to characterize the metabolic landscape of embryos reaching the blastocyst stage. Inhibitors of glucose (IO + DHEA) or FA (ETOMOXIR) metabolism were applied during IVM, and the control group was matured under standard conditions. Blastocysts obtained from experimental and control groups were analyzed with regard to lipidome and metabolome (mass spectrometry), transcriptome (RNA-Seq) and fluorescence lipid droplets staining (BODIPY). We showed that inhibition of glucose and fatty acid metabolism leads to cellular stress response compromising the quality of preimplantation embryos. The inhibition of energy metabolism affects membrane fluidity as well as downregulates fatty acids biosynthesis and gene expression of trophectoderm cell line markers. Therefore, we conclude that oocyte maturation environment exerts a substantial effect on preimplantation development programming at cellular and molecular levels.

Heat shock interferes with the amino acid metabolism of bovine cumulus-oocyte complexes in vitro: a multistep analysis

By affecting the ovarian pool of follicles and their enclosed oocytes, heat stress has an impact on dairy cow fertility. This study aimed to determine how heat shock (HS) during in vitro maturation affected the ability of the bovine cumulus-oocyte complexes (COCs) to develop, as well as their metabolism of amino acids (AAs). In this study, COCs were in vitro matured for 23 h at 38.5 °C (control; n = 322), 39.5 °C (mild HS (MHS); n = 290), or 40.5 °C (severe HS (SHS); n = 245). In comparison to the control group, the MHS and SHS groups significantly decreased the percentage of metaphase-II oocytes, as well as cumulus cell expansion and viability. The SHS decreased the rates of cleavage and blastocyst formation in comparison to the control and MHS. Compared to the control and MHS-COCs, the SHS-COCs produced significantly more phenylalanine, threonine, valine, arginine, alanine, glutamic acid, and citrulline while depleting less leucine, glutamine, and serine. Data showed that SHS-COCs had the highest appearance and turnover of all AAs and essential AAs. Heat shock was positively correlated with the appearance of glutamic acid, glutamine, isoleucine, alanine, serine, valine, phenylalanine, and asparagine. Network analysis identified the relationship between HS and alanine or glutamic acid, as well as the relationship between blastocyst and cleavage rates and ornithine. The findings imply that SHS may have an impact on the quality and metabolism of AAs in COCs. Moreover, the use of a multistep analysis could simply identify the AAs most closely linked to HS and the developmental competence of bovine COCs.

Plasminogen activation and plasmin inhibition during in vitro fertilization in bovine: implications for fertilization parameters and early embryo development

Components of the plasminogen/plasmin system, known to be present in the oocyte, play a key role in maturation and fertilization. The objective of this study was to examine the effect of plasminogen activation and plasmin inhibition by exogenous supplementation of the IVF medium with streptokinase (SK) or ɛ-aminocaproic acid (ε-ACA), respectively, on fertilization parameters and preimplantation embryo development. After in vitro maturation, bovine cumulus-oocyte complexes (COCs) were inseminated in the presence of SK or ε-ACA. The addition of SK to the IVF medium facilitated the adhesion of the spermatozoa to the zona pellucida without affecting the percentages of monospermy. Cleavage rates and blastocyst yield were similar between the SK and Control groups while they were lower with the ε-ACA treatment. Additionally, we found that the expression levels of embryo quality-related genes (SDHA and DNMT3A) could be modified in blastocysts by the addition of SK or ε-ACA during IVF. The results obtained indicate that supplementation of the IVF medium with SK did not greatly alter the embryonic developmental parameters related to embryo quality in blastocysts. Moreover, we noticed that ε-ACA treatment compromises the success of in vitro embryo development, thus highlighting the importance of the plasminogen/plasmin activity during the early stages of embryogenesis in bovine.

199 Nanoplastics are incorporated by the bovine cumulus–oocyte complex and form a potential treat for oocyte competence

199 Nanoplastics are incorporated by the bovine cumulus–oocyte complex and form a potential treat for oocyte competence

Cytoplasmic granules in bovine oocytes do not affect embryonic or fetal development.

Oocyte cytoplasmic evaluation is based on homogeneity and granular appearance. Our study investigated if a granular cytoplasm, highly heterogeneous, would affect oocyte competence in bovine. In two experiments, bovine cumulus-oocyte complexes (COCs) with homogeneous cytoplasm (control, CC) and granulated cytoplasm (granular, GC) were selected from a regular pool of COCs. Experiment 1 was performed with slaughterhouse ovaries, and Experiment 2 was carried out in Girolando COCs obtained from ovum pick-up. Granular oocytes had higher caspase 3 levels (66.17 ± 11.61 vs 172.08 ± 16.95, P < 0.01) and similar GAP junction activity (5.64 ± 0.45 vs 6.29 ± 0.29). ZAR1 relative mRNA amount was lower in granular oocytes (178.27 ± 151.63 vs 0.89 ± 0.89, P = 0.01) and no effect was detected for MATER, PPP2R1A, ENY2, IGF2R, and BMP15 genes. Despite molecular differences, no detrimental effect was detected on oocyte competence in GC oocytes. Cleavage (Experiment 1: 59.52 ± 7.21% vs 59.79 ± 6.10% and Experiment 2: 68.88 ± 4.82 vs 74.41 ± 5.89%) and blastocyst (Experiment 1: 29.28 ± 4.14% vs 23.15 ± 2.96% and Experiment 2: 21.11 ± 3.28% vs 21.02 ± 6.08%) rates were similar between CC and GC (Experiments 1 and 2, respectively). Post-transfer embryo development revealed that pregnancy (CC: 24.27 ± 9.70% vs GC: 26.31 ± 7.23%) and calving (23.68% vs 33.33%) rates and fetal growth were not affected by the presence of cytoplasmic granules. Our results demonstrated that oocytes with granular cytoplasm present equivalent efficiency for IVF and calf production compared with homogenous cytoplasm oocytes. This could be observed through similar cleavage, blastocyst rates, and fetal growth development. In addition to differences in oocyte gene expression related to oocyte quality, it seems not to affect oocyte developmental competence.

The relationship between miR-21, DNA methylation, and bisphenol a in bovine COCs and granulosa cells.

Introduction: miR-21 is a critical microRNA for the regulation of various processes in oocytes and granulosa cells. It is involved in the modulation of apoptosis and can influence other epigenetic mechanisms. Among these mechanisms, DNA methylation holds significant importance, particularly during female gametogenesis. Evidence has demonstrated that microRNAs, including miR-21, can regulate DNA methylation. Bisphenol A (BPA) is a widespread chemical that disrupts oocyte maturation and granulosa cell function. Recent findings suggested that BPA can act through epigenetic pathways, including DNA methylation and microRNAs. Methods: This study uses anti-miR-21 LNAs to explore the involvement of miR-21 in the regulation of DNA methylation in bovine Cumulus-Oocyte-Complexes (COCs) and granulosa cells, in the presence and absence of BPA. This study investigated 5mC/5hmC levels as well as gene expression of various methylation enzymes using qPCR and western blotting. Results and discussion: Results reveal that BPA reduces 5mC levels in granulosa cells but not in COCs, which can be attributed to a decrease in the methylating enzymes DNMT1 and DNMT3A, and an increase in the demethylating enzyme TET2. We observed a significant increase in the protein levels of DNMT1, DNMT3A, and TET2 upon inhibition of miR-21 in both COCs and granulosa cells. These findings directly imply a strong correlation between miR-21 signaling and the regulation of DNA methylation in bovine COCs and granulosa cells under BPA exposure.

In vitro maturation in synthetic oviductal fluid increases gene expression associated with quality and lipid metabolism in bovine oocytes.

Traditionally, in vitro oocyte and embryo culture progresses through a series of varying culture medium. To investigate simplifying the in vitro production of bovine cumulus-oocyte complexes (COCs), this study used synthetic oviductal fluid (SOF) supplemented with conjugated linoleic acid (CLA). Special interest was placed on gene expression linked to lipid metabolism and oocyte maturation. COCs were matured in different media: Medium 199 (M199 group), M199 with 100 μM CLA (M199 + CLA group), SOF (SOF group), and SOF with 100 μM CLA (SOF + CLA group). COCs matured with SOF showed a higher relative abundance of mRNA of quality indicators gremlin 1 (GREM1) and prostaglandin-endoperoxide synthase 2 (PTGS2) in oocytes, and GREM1 in cumulus cells compared with in the M199 group. SOF medium COCs had a higher relative abundance of fatty acid desaturase 2 (FADS2) compared with the M199 group, which is essential for lipid metabolism in oocytes. Furthermore, the abundance of stearoyl-coenzyme A desaturase 1 (SCD1) in oocytes matured with SOF was not influenced by the addition of CLA, whereas the relative abundance of SCD1 was reduced in M199 medium with CLA. We concluded that maturation in SOF medium results in a greater abundance of genes linked to quality and lipidic metabolism in oocytes, regardless of the addition of CLA.

Ferulic Acid Enhances Oocyte Maturation and the Subsequent Development of Bovine Oocytes.

Improving the quality of oocytes matured in vitro is integral to enhancing the efficacy of in vitro embryo production. Oxidative stress is one of the primary causes of quality decline in oocytes matured in vitro. In this study, ferulic acid (FA), a natural antioxidant found in plant cell walls, was investigated to evaluate its impact on bovine oocyte maturation and subsequent embryonic development. Bovine cumulus-oocyte complexes (COCs) were treated with different concentrations of FA (0, 2.5, 5, 10, 20 μM) during in vitro maturation (IVM). Compared to the control group, supplementation with 5 μM FA significantly enhanced the maturation rates of bovine oocytes and the expansion of the cumulus cells area, as well as the subsequent cleavage and blastocyst formation rates after in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT). Furthermore, FA supplementation was observed to effectively decrease the levels of ROS in bovine oocytes and improve their mitochondrial function. Our experiments demonstrate that FA can maintain the levels of antioxidants (GSH, SOD, CAT) in oocytes, thereby alleviating the oxidative stress induced by H2O2. RT-qPCR results revealed that, after FA treatment, the relative mRNA expression levels of genes related to oocyte maturation (GDF-9 and BMP-15), cumulus cell expansion (HAS2, PTX3, CX37, and CX43), and embryo pluripotency (OCT4, SOX2, and CDX2) were significantly increased. In conclusion, these findings demonstrate that FA supplementation during bovine oocyte IVM can enhance oocyte quality and the developmental potential of subsequent embryos.

Classification of Bovine Cumulus-Oocyte Complexes with Convolutional Neural Networks

Aim: Determining oocyte quality is crucial for successful fertilization and embryonic development, and there is a serious correlation between live birth rates and oocyte quality. Parameters such as the regular/irregular formation of the cumulus cell layer around the oocyte, the number of cumulus cell layers and the homogeneity of the appearance of the ooplasm are used to determine the quality of the oocytes to be used in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) methods. Material and Methods: In this study, classification processes have been carried out using convolutional neural networks (CNN), a deep learning method, on the images of the cumulus-oocyte complex selected based on the theoretical knowledge and professional experience of embryologists. A convolutional neural network with a depth of 4 is used. In each depth level, one convolution, one ReLU and one max-pooling layer are included. The designed network architecture is trained using the Adam optimization algorithm. The cumulus-oocyte complexes (n=400) used in the study were obtained by using the oocyte aspiration method from the ovaries of the bovine slaughtered at the slaughterhouse. Results: The CNN-based classification model developed in this study showed promising results in classifying three-class image data in terms of cumulus-oocyte complex classification. The classification model achieved high accuracy, precision, and sensitivity values on the test dataset. Conclusion: Continuous research and optimization of the model can further improve its performance and benefit the field of cumulus-oocyte complexes classification and oocyte quality assessment.

Effects of cyclic adenosine monophosphate modulating agents during oocyte pre-maturation and the role of melatonin on in vitro maturation of bovine cumulus-oocyte complexes

This study investigated the effects of cyclic adenosine monophosphate modulating during cumulus-oocyte complexes (COCs) pre-maturation and the role of melatonin on in vitro maturation (IVM) of bovine COCs. In experiment one, COCs were pre-matured for 8 h in control medium or with 3-isobutyl-1-methylxanthine (IBMX) and forskolin, IBMX and C-type natriuretic peptide, c-type natriuretic peptide and forskolin or IBMX, forskolin and c-type natriuretic peptide. Then, meiotic progression was evaluated. In experiment two, COCs were pre-matured, followed by IVM in control medium alone or with 10−6, 10−7 or 10−8 M melatonin. After IVM, chromatin configuration, transzonal projections (TZPs), reactive oxygen species, mitochondrial distribution, ultrastructure and mRNA expression for antioxidant enzymes were evaluated. In experiment 1, COCs pre-matured with both C-type natriuretic peptide and forskolin or C-type natriuretic peptide, forskolin and IBMX had lower meiotic resumption rate when compared to control. Considering that IBMX had not an additional effect to potentiate inhibition of meiotic resumption, a combination of C-type natriuretic peptide and forskolin was chosen. In experiment 2, COCs matured with 10−8 M melatonin had greater rates of meiotic resumption when compared to the other treatments (P < 0.05). The COCs matured with 10−7 or 10−8 M melatonin had greater mitochondrial activity (P < 0.05), while those matured with 10−6 or 10−8 M of melatonin had greater levels of TZPs. Ultrastructure of oocyte and cumulus cells after IVM with melatonin was relatively well preserved. COCs matured with 10−8 M melatonin increased mRNA expression for superoxide dismutase (SOD) and catalase (CAT) (P < 0.05), when compared to non-cultured and pre-matured COCs, respectively. In conclusion, bovine COC pre-maturation with C-type natriuretic peptide and forskolin, followed by IVM with 10−8 M melatonin improves meiotic resumption rates, TZPs, mitochondrial distribution and mRNA expression for SOD and CAT.

Translocation of Oocytic HES1 into Surrounding Cumulus Cells in Bovine: Mechanism of Cellular Interaction during IVM?

HES1 (hairy and enhancer of split-1, effector of the NOTCH pathway) plays a role in oocyte maturation and has been detected so far mainly in somatic follicular cells. In this study, we aimed to investigate whether HES1 is present in both compartments of bovine cumulus oocyte complexes (COCs) and whether in vitro maturation itself has an effect on its distribution. We investigated the abundance of HES1 mRNA and protein in bovine COCs characterized by Brilliant-Cresyl-Blue (BCB) stainability by RT-PCR and immunofluorescence before and after in vitro maturation (IVM). To study the interaction of the compartments and the possible translocation of HES1, we injected GFP-HES1 mRNA into oocytes before maturation and analyzed fluorescence recovery after photobleaching (FRAP). The results showed that HES1 mRNA was detectable in oocytes but not in cumulus cells. The number of transcripts increased with maturation, especially in BCB-positive oocytes. In contrast, the protein was mainly visible in cumulus cells both before and after maturation. After GFP-HES1-mRNA injection into oocytes, a signal could be detected not only in the oocytes but also in cumulus cells. Our result shows a nearly exclusive distribution of HES1 mRNA and protein in oocytes and cumulus cells, respectively, that might be explained by the transfer of the protein from the oocyte into cumulus cells.

Supplementation of rapamycin during in vitro maturation promotes oocyte quality and subsequent embryo development in bovine

BackgroundThe developmental competence of oocyte derived from in vitro maturation (IVM) is significantly lower than those of oocyte matured in vivo. Rapamycin is an immunosuppressive substance and widely used to prolong the survival and preserve the cells culture. The objective of this research was to explore whether rapamycin supplementation during IVM enhances meiotic maturation, oocyte quality, and subsequent embryonic development.MethodsDepending on the purpose, bovine cumulus oocyte complexes were matured without (control) or with rapamycin at different concentrations (0, 1, 10, and 100 nM) for 22 h (h) followed by 6 h in vitro fertilization, and zygotes were cultured for 8 days. Following treatment, oocytes developmental competence was assessed by meiotic progression, intra-oocyte GSH synthesis, reactive oxygen species (ROS) levels, and subsequent embryonic development.ResultsThe results showed that addition of 1 nM rapamycin to IVM medium significantly increased the nuclear maturation (90.1 ± 1.5 vs. 81.2 ± 2.5; P < 0.01) and subsequent embryonic development (47.6 ± 2.2 vs. 41.5 ± 1.9; P < 0.05) than that observed in the control group. Consequently, compared to the control group, the relative fluorescence’s intensity of ROS levels in oocyte significantly reduced by rapamycin treatment. More importantly, rapamycin supplementation during maturation significantly increased the reduced glutathione synthesis levels in oocyte compared to that observed in control, indicating the bidirectional communication between cumulus cells and oocyte becomes easy via rapamycin treatment.ConclusionsThis study suggests that the presence of rapamycin during maturation can afford to enhance reduced glutathione synthesized by the oocyte, which is an internal oocyte antioxidant defense and contributes to improve subsequent high-quality embryo production.

Ameliorating Effect of Sodium Selenite on Developmental and Molecular Response of Bovine Cumulus-Oocyte Complexes Matured in Vitro Under Heat Stress Condition.

Selenium (Se), an essential trace element, plays an important role in the antioxidative defense mechanism, and it has been proven to improve fertility and reproductive efficiency in dairy cattle. The present study evaluated the potential protective action of Se supplement of in vitro maturation (IVM) media on the maturation and subsequent development of bovine cumulus-oocyte complexes (COCs) exposed to heat stress (HS). The treatment with Se improved the viability of cumulus cells (CCs) and oocytes (P < 0.05). The proportion of oocytes reached metaphase II (MII) and those arrested at metaphase I (MI) was greater and lower in treatment than control respectively (P < 0.05). Supplementation with Se increased the percentage of cleaved embryos, total blastocysts, and blastocyst/cleavage ratio (P < 0.05). Moreover, the upregulation of CCND1, SEPP1, GPX-4, SOD, CAT, and downregulation of GRP78, CHOP, and BAX in both Se-treated CCs and oocytes were recorded. The upregulation of NRF2 was detected in Se-treated CCs other than in oocytes, which showed upregulation of IGF2R and SOX-2 as the markers of quality as well. Se supplement in IVM media improved the viability, maturation, and the level of transcripts related to antioxidant defense and quality of heat-treated oocytes, which coincided with greater subsequent development outcomes. Se ameliorated the viability of CCs along with upregulation of antioxidative candidate gene expression and downregulation of apoptosis-related ones to support their protective role on restoring the quality of oocytes against compromising effects of HS.

031 Measuring cumulus expansion of bovine cumulus-oocyte complexes: comparing the reliability of three methods

031 Measuring cumulus expansion of bovine cumulus-oocyte complexes: comparing the reliability of three methods

Effects of Vitrification Temperatures and Concentration of Cryoprotective Agents on the Cytoskeleton in Bovine Immature Oocytes

The objective of the research was to evaluate the effect of vitrification temperatures (VTs) and concentration of cryoprotective agents (CPAs) on the cytoskeleton of bovine immature oocytes (BIOs). Bovine cumulus oocyte complexes (COCs) were collected and divided randomly into five groups: (a) fresh control group, (b) oocytes vitrified in liquid helium (LHe; −269◦C with 5.6 M CPAs, (c) oocytes vitrified in LHe (−269◦C with 6.6 M CPAs, (d) oocytes vitrified in liquid nitrogen (LN; −196 ◦C with 5.6 M CPAs, (e) oocytes vitrified in LN (−196◦C with 6.6 M CPAs. The results showed that the developmental level of BIOs in the LHe 5.6 M group was the highest among the four vitrification groups, and the maturation rate and cleavage rate were significantly higher than the other three vitrification groups (P &lt; 0.05). With further experiments, the normal rate of microfilament and spindle in the LHe 5.6 M group was the highest compared with the other three vitrification groups. It was concluded that lower VT (−269◦C and lower CPAs (5.6 M) could significantly reduce damage to the cytoskeleton, which had a positive effect on the development of oocytes after vitrification.

L-carnitine enhances developmental potential of bovine oocytes matured under high lipid concentrations in vitro

Maternal obesity elevates non-esterified fatty acids (NEFA) follicular concentrations. Bovine cumulus-oocyte complexes (COCs) matured in vitro under high NEFA have altered metabolism and reduced quality. Systemically, obesity promotes altered mitochondrial metabolism linked to L-carnitine insufficiency. We hypothesized that L-carnitine supplementation during IVM of bovine COCs in the presence of high NEFA would lessen the negative effects of exposure to excessive lipids on embryonic development and oxidative stress. COCs were collected from abattoir ovaries and matured in four groups: CON (control), LC (3 mM L-carnitine), HN (high NEFA: 200uM oleic, 150uM palmitic and 75uM stearic acid), and HNLC (HN and LC). Mature oocytes were assayed for aerobic and anaerobic metabolism utilizing oxygen and pH microsensors or fertilized in vitro (D0). Cleavage (D3) and blastocyst (D7, D8) rates were assessed. D3 embryos with ≥ 4 cells were stained for cytosolic and mitochondrial ROS. D8 blastocysts were assayed for gene transcript abundance of metabolic enzymes. Oocyte metabolism was not affected by IVM treatment. D3 formation of embryos with ≥ 4 cells were lower in LC or HN than CON or HNLC; blastocyst rates were greater for CON and lower for HN than LC and HNLC. D3 embryo mitochondrial and cytosolic ROS were reduced in HNLC when compared to other groups. IVM in HN altered blastocyst gene transcript abundance when compared to CON, but not LC or HNLC. In conclusion, supplementation with L-carnitine protects oocytes exposed to high NEFA during IVM and improves their developmental competence, suggesting that high lipid exposure may lead to L-carnitine insufficiency in bovine oocytes.

Bovine in vitro oocyte maturation and embryo culture in liquid marbles 3D culture system.

Despite the advances in in vitro embryo production (IVP) over the years, the technique still has limitations that need to be overcome. In cell cultures, it is already well established that three-dimensional culture techniques are more physiological and similar to the in vivo development. Liquid marble (LM) is a three-dimensional system based on the use of a hydrophobic substance to create in vitro microbioreactors. Thus, we hypothesized that the LM system improves bovine in vitro oocyte maturation and embryo culture. In experiment I, bovine cumulus-oocyte complexes (COCs) were placed for in vitro maturation for 22h in two different groups: control (conventional 2D culture) and LM (three-dimensional culture). We found that oocyte nuclear maturation was not altered by the LM system, however it was observed a decrease in expression of genes important in the oocyte maturation process in cumulus cells of LM group (BCL2, EIF4E, and GAPDH). In experiment II, the COCs were conventionally matured and fertilized, and for culture, they were divided into LM or control groups. There was a decrease in blastocyst rate and cell counting, a down-regulation of miR-615 expression, and an increase in the DNA global methylation and hydroxymethylation in embryos of LM group. Therefore, for the bovine in vitro embryo production, this specific three-dimensional system did not present the advantages that we expected, but demonstrated that the embryos changed their development and epigenetics according to the culture system.

Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production

Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 μg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 μg/ml (H60, n = 196), 164 μg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32–76%; median, min-max) in comparison to H60 and H164 (24%,11–33% and 13%, 8–44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P < 0.01). Blastocyst rates (blastocyst/cleaved oocytes) and total cell number were not different among the groups (control 29%, H60 25%, and H164 24%). In a preliminary study, equine oocytes (n = 28) were exposed to PB in vitro for 6 h followed by intracytoplasmic sperm injection (ICSI) and in vitro embryo production. Exposed oocytes showed a numerically lower maturation rate (43% Vs 52%; P > 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained.

Expression profile of key genes involved in DNA repair mechanisms in bovine cumulus cells cultured with bovine serum albumin or fetal calf serum

Cumulus cells from cumulus-oocyte complexes (COC) matured in vitro in serum-free medium show high incidence of apoptosis and DNA double-strand breaks (DSB). This study aimed to characterize the transcript expression profile of selected genes involved in DNA repair mechanisms in bovine cumulus cells cultured with bovine serum albumin (BSA) or fetal calf serum (FCS). Briefly, bovine cumulus-oocyte complexes were in vitro matured with either, 0.4% BSA or 10% FCS for 3, 6, 12 or 24h. The total RNA of cumulus cells was used for real-time PCR analysis. Transcript abundance of XRCC6, XRCC5, DNAPK, GAAD45B, TP53BP1, RAD50, RAD52, ATM and BRCA2 target genes changed as the IVM proceeded (P<0.05). However, an interaction between protein source (FCS or BSA) and time was not detected (P≥0.05). Cumulus cells from COCs matured with BSA presented higher mRNA expression of two genes compared to FCS group: TP53BP1 at 6h and BRCA1 at 3, 6, 12 and 24h (P<0.05). In summary, our results showed for the first time the expression profile of the key genes involved in DSB repair mechanisms in cumulus cells obtained from bovine COCs matured with FCS or BSA. The higher mRNA expression of BRCA1 and TP53BP1 and lower mRNA expression of TNFAIP6 suggests an increase in apoptosis rate and DNA damage in cumulus cells cultured in BSA-supplemented medium and may explain, at least to some extent, the reduced developmental potential of bovine oocytes matured in serum-free medium.