Lead (Pb2+), one of the pervasive and protracted environmental heavy metals, is known to affect the female reproductive system by inducing oxidative stress. The Nrf2 and NF-κB are two key transcriptional factors (TF) known to regulate cellular redox status and response against stress and inflammation, respectively. Evidence showed that these TF undergo functional cross-talk. Although the properties of Pb2+ have been extensively studied, little is known about its effect on bovine granulosa cells with respect to Nrf2/NF-κB interaction. The aim of this study was to investigate the impact of Pb2+ on the response of bovine granulosa cells with regard to the cross-talk between Nrf2 and NF-κB. For this, bovine granulosa cells aspirated from small growing follicles (3-5mm of diameter) from ovaries obtained from a local slaughterhouse were in vitro cultured at 37°C and 5% CO2. Sub-confluent cells were exposed to different doses of lead acetate [Pb (C2H3O2)2] (1, 2, or 3µg mL−1) for 2h. Following this, the accumulation of intracellular reactive oxygen species was measured using the H2DCFDA fluorescent probe (Life Technologies, Darmstadt, Germany). The cell viability was assessed using Cell Counting Kit-8 (CCK-8, Dojindo, Munich, Germany) 24h after treatments. Moreover, cells were harvested and subjected to total RNA isolation using miRNeasy Mini Kit (Qiagen, Hilden, Germany) followed by cDNA synthesis using First Strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and the gene expression was determined using quantitative PCR. Data were analysed using comparative threshold cycle (ΔΔCT) method and normalized against the expression of GAPDH and β-actin genes. Furthermore, the protein lysate was isolated from cells to assess carbonylated protein using OxyBlot protein oxidation detection kit (abcam, Cambridge, MA, USA). Results showed that exposure of granulosa cells to lead acetate provoked intracellular reactive oxygen species accumulation and protein carbonylation. This was accompanied by cell cycle arrest at G0/G1 phase, reduction in cell viability, and decrease in the expression of cell proliferation marker genes (CCND2 and PCNA). Moreover, lead acetate elicited down-regulation of both Nrf2 and NF-κB and their downstream antioxidant genes (SOD and CAT), whereas no significant difference was shown in the level of other genes involved in both TF pathways (Keap-1, HO-1, Trx, IKK, and TNF-α). In addition, lead acetate challenge increased the expression of endoplasmic reticulum stress marker genes (GRP78 and CHOP) and the pro-apoptotic gene (Caspase-3), whereas it reduced the anti-apoptotic gene (BCL-2). Our findings suggest that Pb+2 driven-oxidative stress deregulates granulosa cell viability and proliferation, enhances endoplasmic reticulum stress, induces cell cycle arrest, and mediates apoptosis probably via disruption of Nrf2/NF-κB cross-talk.

Low cryotolerance is considered as the major drawback of in vitro-produced bovine embryos and is frequently associated with a triad encompassing increased cytoplasmic lipid accumulation, enhanced levels of reactive oxygen species (ROS) and mitochondrial dysfunction. The aim of the present study was to explore the role of the AMP-activated protein kinase (AMPK) pathway in the process resulting such phenotypes. Comparative analysis under different environmental conditions revealed downregulation of AMP-activated protein kinase cytalytic subunit 1alpha (AMPKA1), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1A) and carnitine palmitoyltransferase 1 (CPT1) genes and upregulation of acetyl-CoA carboxylase α (ACC). In contrast, the presence of fatty acids within the culture medium resulted in a distinct molecular profile in the embryo associated with enhanced levels of ROS, mitochondrial dysfunction and elevated lipid accumulation in bovine embryos. Because AMPKA1 regulates PGC1A, CPT1 and ACC, the results of the present study reveal that AMPK in active its form is the key enzyme promoting lipolysis. Because AMPK1 activity is, in turn, controlled by the AMP : ATP ratio, it is possible to speculate that excessive uptake of exogenous free fatty acids could increase cellular ATP levels as a result of the disturbed β-oxidation of these external fatty acids and could therefore bypass that molecular feedback mechanism. Subsequently, this condition would cause enhanced generation of ROS, which negatively affect mitochondrial activity. Both enhanced generation of ROS and low mitochondrial activity are suggested to enhance the accumulation of lipids in bovine embryos.

The role of microRNA in oocyte maturation is mostly associated with optimal turnover of the accumulated maternal transcripts during their growth to allow maturation. MiR-20a is a member of the miR-17–92 cluster, which has been found to be differentially expressed in bovine granulosa cells derived from preovulatory dominant and subordinate follicles. Our recent study showed that miR-20a is involved in the regulation of granulosa cell proliferation, differentiation, and progesterone synthesis by targeting PTEN and BMPR2 genes. Here, we aimed to investigate the role of miR-20a in the bovine oocyte maturation processes. For this, cumulus-oocyte complexes (COC) were aspirated from small antral follicles (2–8 mm in diameter) and cultured in groups of 50 in 400 µL of maturation media (TCM-199 media supplemented with 12% oestrus cow serum and 10 µg/ml Follitropin®) at 39°C in a humidified atmosphere with 5% (vol/vol) CO2 in the air for 22 h. The cumulus cells and oocytes before (germinal vesicle) and after maturation (metaphase II) were mechanically separated in 0.1% hyaluronidase (in TCM-199 media). To study whether the presence of cumulus cells or oocyte has an impact on the miR-20a expression, we cultured oocytectomized cumulus cells and oocytes with and without their companion cells. Moreover, COC were co-cultured with miR-20a mimic, inhibitor, or corresponding controls to investigate the role of this miRNA in oocyte maturation. The total RNA from cumulus cells and oocytes was extracted using miRNeasy® mini kit (Qiagen GmbH, Hilden, Germany). Total RNA from respective samples was reverse transcribed for mRNA and microRNA expression analysis. Quantitative expression analysis was performed using StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) and subsequent data were analysed using a comparative cycle threshold method. The progesterone released in the spent media was measured using progesterone enzyme-linked immunosorbent assay kit (ENZO Life Sciences GmbH, Loerrach, Germany). Here, we found that miR-20a expression in cumulus cells increased (P < 0.05) during oocyte maturation. Conversely, miR-20a expression in metaphase II stage oocytes was significantly lower (P < 0.001) compared with the germinal vesicle stage. The absence of oocyte cytoplasm resulted in reduced miR-20a expression in cumulus cells. On the other hand, the absent of cumulus cells increased miR-20a expression in oocytes. The miR-20a expression revealed that the microRNA transduction is restricted in the cumulus cells. The overexpression of miR-20a increased oocyte maturation rate (P < 0.05) by 4.8% (as determined by extrusion of the polar body) and the expression of oocyte maturation-related genes (INHBA, MAPK1, PTGS2, PTX3, and EGFR). The progesterone released in spent media of COC co-cultured with miR-20a mimic and inhibitor showed increasing (P = 0.0936) and decreasing (P = 0.0993) trends, respectively. In this study, we also found that miR-20a modulation altered the expression of PTEN and BMPR2 in cumulus cells. In conclusion, the modulation of miR-20a expression in cumulus cells regulates the oocyte maturation and partially involved in the progesterone synthesis by fine-tuning the expression of PTEN and BMPR2 genes.

Suboptimal culture condition before minor or major genome activation is believed to affect the quality and the transcriptome landscape of the resulting blastocysts. Thus, we hypothesised that exposure of bovine embryos to suboptimal culture condition before minor embryonic genome activation could affect the genome methylation patterns of the resulting blastocysts. Therefore, here we aimed to investigate the genome wide DNA methylation patterns of blastocysts derived from embryos developed up to 2-cell stages in vivo followed by in vitro culture. For this, Simmental heifers were superovulated and artificially inseminated. The 2-cell stage embryos were then flushed using a state-of-the-art nonsurgical endoscopic early-stage embryo flushing technique and in vitro cultured until the blastocyst stage. The DNA methylation patterns of these blastocysts were then determined with reference to blastocysts derived from embryos developed completely under in vivo condition. For this, the genomic DNA isolated from each blastocyst group were fragmented, and unmethylated genomic regions were cleaved using methylation sensitive restriction enzymes. The samples were then amplified using ligation mediated PCR and labelled either with Cy-3 or Cy-5 dyes in a dye-swap design using the ULS Fluorescent genomic DNA labelling kit (Kreatech Biotechnology) and hybridized on an EmbryoGENE DNA Methylation Array as described previously (Saadi 2014 BMC Genomics 15, 451; Salilew-Wondim 2015 PLoS ONE 10, e0140467). Array hybridization was performed for 40 h at 65°C, and 4 hybridizations were preformed to represent 4 biological replicates. The slides were scanned using Agilent’s High-Resolution C Scanner (Agilent Technologies, Santa Clara, CA, USA), and Agilent’s Feature Extraction software (Agilent Technologies) was used to extract data features. Differentially methylated regions with fold change ≥1.5 and P-value < 0.05 were identified using linear modelling for microarray and R software. The results have shown that including imprinted genes (PEG3, IGF1, RASGRF1, IGF2R, GRB10, SNRPN, and PLAGL1) and DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), a total of 10,388 genomic regions were differentially methylated, of which 6393 genomic regions were hypermethylated in blastocysts derived from 2-cell flush compared with the complete vivo group. In addition, comparative analysis of the current DNA methylation data with our previous transcriptome profile data have shown that including DNMT3A, CTSZ, ElF3E, and PPP2R2B, the expression patterns of 603 genes was inversely correlated with the methylation patterns. Moreover, canonical pathways including gap junction, adherens junction, axon guidance, focal adhesion, and calcium signalling were affected by differentially methylated regions. Therefore, this study indicated that exposure of embryos to suboptimal culture condition before embryonic genome activation would lead to a massive dysregulation of methylation pattern of genes involved in developmentally relevant pathways in the resulting blastocysts.

Exosomes are nano-sized (30–100 nm) extracellular membrane vesicles released through exocytosis process in most cells and biological fluids. They contain a cargo of nucleic acids, proteins, lipids and play a vital role in cell-cell communications. Various cell types have been shown to release exosomes into extracellular space as a response to various environmental stress conditions. However, little is known about the response of granulosa cells to oxidative stress, with regard to release of exosomes that may carry mRNA and protein molecules related to cellular oxidative stress response. Here we aimed to investigate the potential release of stress elements by granulosa cells to culture media through exosomes under oxidative stress conditions. For that, bovine granulosa cells from small follicles were aspirated and cultured in DMEM/F-12 media supplemented with exosome free fetal bovine serum (Exo-FBS) and treated with 5 µM H2O2 for 40 min. Granulosa cells were collected 24 h post-treatment to quantify the expression of antioxidants (Nrf2, Keap1, SOD1, CAT1, PRDX1, HOMOX1, TXN1, and NQO1), cell proliferation (PCNA and CNND2), cell differentiation (CYP11A1 and STAR), apoptosis (Casp3), and antiapoptosis (BCL2L1) genes. Reactive oxygen species accumulation, mitochondrial distribution, cell viability, and cell cycle assays were performed in cultured granulosa cells, and the culture medium was used to isolate exosomes using ultracentrifugation procedure. The identity of exosomes was confirmed by immunoblotting of Alix and CD63 proteins, and the expression level of antioxidant was analysed in mRNA isolated from exosomes. Data from 3 independent biological replicates were statistically analysed using the 2-tailed t-test. Results showed that H2O2 treatment increased mRNA and protein level of antioxidants (Nrf2, PRDX1, and TXN1), as well as cell differentiation and apoptosis-related genes compared to untreated controls. However, granulosa cells treated with H2O2 showed lower expression of cell proliferation marker genes (PCNA and CNND2). Cells treated with H2O2 showed increases in reactive oxygen species level, inadequate mitochondrial distribution, and lower cell viability. Cell cycle assay revealed a reduction in G0/G1 proportion and increase in G2 phase in cells treated with H2O2. Higher levels of antioxidant (Nrf2, CAT1, and TXN1) transcripts were detected in exosomes isolated from media with cells under oxidative stress conditions compared to the controls. Labelling and co-transfection of exosomes from stressed cell culture medium with untreated treated recipient granulosa cells resulted in increased abundance of cellular mRNA and protein of Nrf2 and CAT1 in those cells. In conclusion, granulosa cells exposed to oxidative stress could release exosomes that carry molecules related to oxidative stress response, which can be up taken by recipient cells.

The in vitro production (IVP) of bovine embryos is a well-established technique that has been available for nearly 20 years. However, there remain major differences between IVP-derived blastocysts and their in vivo-derived counterparts. Many studies have pointed out that most of these differences are due to the in vitro developmental environment. To circumvent these negative effects due to in vitro culture conditions, a new method – intrafollicular oocyte transfer (IFOT) – was established in the present study. Using modified ovum pick-up (OPU) equipment, in vitro-matured oocytes derived from slaughterhouse ovaries were injected into the dominant preovulatory follicle of synchronised heifers (follicular recipients) enabling subsequent ovulation, in vivo fertilization, and in vivo development. A total of 810 in vitro-matured oocytes were transferred into 14 heifers. Subsequently, 222 embryos (27.3%) were recovered after uterine flushing at Day 7. Based on the number of cleaved embryonic stages, 64.2% developed to the blastocyst stage, which did not differ from the IVP-derived embryos (58.2%). Interestingly, lipid content of IFOT-derived blastocysts did not differ from the fully in vivo-produced embryos, whereas IVP-derived blastocysts showed significantly higher lipid droplet accumulation compared with fully in vivo-derived and IFOT-derived blastocysts (P < 0.05). Accordingly, IFOT blastocysts showed significantly higher survival rates after cryopreservation than complete IVP-derived embryos (77% v. 10%), which might be attributed to a lower degree of lipid accumulation. In agreement, transfer of frozen-thawed IFOT blastocysts to synchronized recipients (uterine recipients) resulted in much higher pregnancy rates compared with transfer of IVP-derived blastocysts (42.1 v. 13.8%) but did not differ from frozen-thawed ex vivo blastocysts (52.4%). Of these presumed IFOT pregnancies, 7 went to term, and microsatellite analysis confirmed that 5 calves were indeed derived from IFOT, whereas 2 were caused by fertilization of the follicular recipient's own oocyte after AI. Taken together, IFOT-derived blastocysts closely resemble in vivo-derived blastocysts, confirming earlier suggestions that the ability to develop to the blastocyst stage is already determined in the matured oocyte, whereas the quality in terms of lipid content and survival rate after cryopreservation is affected by the environment thereafter. However, to the best of our knowledge, this is the first study reporting healthy calves after intrafollicular transfer of in vitro-matured oocytes.

Assessment of the developmental capacity of early bovine embryos is still an obstacle. Therefore, the present paper reviews all current knowledge with respect to morphological criteria and environmental factors that affect embryo quality. The molecular signature of an oocyte or embryo is considered to reflect its quality and to predict its subsequent developmental capacity. Therefore, the primary aim of the present review is to provide an overview of reported correlations between molecular signatures and developmental competence. A secondary aim of this paper is to present some new strategies to enable concomitant evaluation of the molecular signatures of specific embryos and individual developmental capacity.

Transcriptome profiling has been used to identify genes related to developmental competence in bovine embryos and oocytes for several years. However, the direct relationship between the transcriptome profile and developmental potential of the same pre-implantation embryo is missing. Therefore, in the present study, one blastomere of a 2-cell-stage embryo was taken as a biopsy and immediately snap-frozen for transcriptome analysis; the sister blastomere was cultured individually in a well-of-the-well culture system. Frozen individual blastomeres taking the form of 2-cell-stage embryos were pooled together, depending on the developmental destination of the sister blastomeres. Accordingly, three groups were defined: 1) embryos that did not cleave after separation (2CB), 2) embryos arrested before embryonic genome activation (8CB), and 3) embryos that reached the blastocyst stage (BL). On the basis of these developmental phenotypes, blastomeres were pooled and used for transcriptome analysis, using a bovine EmbryoGene microarray platform (Agilent Technologies, Santa Clara, CA, USA). Results revealed 632 genes to be differentially regulated (fold change, P ≥ 1.5, P ≤ 0.05; FDR, P ≤ 0.1) between competent (BL group) and incompetent 2-cell-stage embryos (2CB) as well as 150 genes between the BL group and 8CB. Seventy-seven genes were commonly differentially regulated. Functional annotation analyses of differentially regulated genes indicated those genes to be involved in the protein ubiquitination pathway on the one hand and in the oxidative stress response, including oxidoreductase, peroxidase, and antioxidant activity, as well in oxidative phosphorylation (e.g. NDUFS1, MAPK14, CAT, PRDX1, and PRDX6) on the other hand. Furthermore, selected candidate genes known to function as direct and indirect scavengers of reactive oxygen species (ROS) were analysed for their expression in an independent model for developmental competence; namely, early- and late-cleaved 2-cell-stage embryos. Moreover, ROS detection was performed, showing higher accumulation of ROS in late-cleaved 2-cell-stage embryos, whereas lower ROS levels were detected in early-cleaved 2-cell-stage embryos associated with higher expression of ROS scavengers. The overall findings of the present study indicate the potential of using blastomere biopsies at 2-cell-stage embryos for molecular analysis to understand the molecular mechanisms associated with the further developmental competence of early-stage embryos.

Endometritis is a postparturition infection of endometrium which is known to reduce fertility of dairy cows. However, the molecular and biochemical changes incurred in the endometrium as a result of such inflammations are not yet known. Therefore, the present study was conducted to investigate transcritome (mRNA and microRNA) changes associated with the incidence of clinical endometritis. For this, Holestein Friesian cows were grouped as clinically sick (CS) or healthy (HY) based on clinical symptoms and histological evaluation. Following this, total RNA, including small RNAs, was isolated from endometrial biopsies taken from each group of cows and subsequently used for mRNA and microRNA expression studies. The human miRCURY LNA Universal RT microRNA PCR array system (Exiqon A/S, Vedbaek, Denmark) (with 750 miRNA probes) was used for miRNA expression profiling, while the bovine GeneChip Genome Array (Affymetrix, Santa Clara, CA) was used for expression profiling of mRNAs from the same RNA pools. Following the miRNA PCR array run, data analysis was performed using a comparative threshold cycle (CT) method. A total of 21 miRNAs were found to be differentially expressed (fold change ≥2 and P ≤ 0.05) between CS and HY cows. Among these, 11 miRNAs including mir-526b*, mir-339-3p, mir-608, mir-10a, mir-943 and mir-19b-1* were enriched and 10 miRNAs including mir-1265, mir-658, mir-196b, mir-498 and mir-192 were downregulated in CS compared with the HY animals. The global endometrial mRNA expression analysis between CS and HY cows was performed using 250 ng of total RNA as a starting material. After RNA amplification, fragmented and biotin-labelled cRNA samples were hybridized to Bovine Genome Array (Affymetrix). The microarray data normalization and background correction were performed using GCRMA. A total of 67 genes were found to be differentially expressed (with fold change ≥2 and P ≤ 0.05) between CS and HY cows. Among those, the transcript level of 30 genes including PTHLH, INHBA, P2RY14, MAOB and KCNB2 were upregulated and 37 genes including CHRDL1, PTGDS, F5, ALPL, FOLH1v and TRIB1 were downregulated in CS compared with HY cows. The Ingenuity Pathway Analysis (Ingenuity Systems Inc., Redwood City, CA) showed that some of those genes to be involved in induction of fibrosis (CXCR4, PLOD2, POSTN, PTHLH and TIMP2), growth of ovarian tumour (KLF6), genital tumour (FOLH1, INHBA, KLF6, MGP and PTGDS), proliferation of epithelial cells (EFNA1, INHBA, MAGED1, PTHLH and SNAI2) and chemotaxis of mononuclear leukocytes (CXCR4, INHBA and RGS1). In addition, target prediction of candidate miRNAs using web-based tools (http://mirecords.biolead.org/prediction_query.php) showed that the majority of the differentially expressed genes are potential targets of the miRNA candidates. Therefore, the results of this study showed transcriptome profile changes associated with bovine clinical endometritis and subsequent influence of fertility in cows.

The present study was conducted to determine the predictive value of zona pellucida birefringence (ZPB) measurement for developmental capacity of equine oocytes. In vitro developmental capacity of equine oocytes is related to cumulus morphology as well as glucose-6-phosphate dehydrogenase (G6PD) activity. Therefore, we classified equine oocytes according to cumulus morphology and G6PDH activity into groups of different developmental capacities and analysed the correlation between ZPB measured by polarized light microscopy and developmental competence. Ovaries were obtained from an abattoir and were transported to the laboratory within 2 to 4 h at 25 to 30°C in 0.9% saline. Cumulus–oocyte complexes (COC) were recovered by scraping the granulosa layer from opened follicles using a bone curette and were classified as having an expanded (Ex, n = 86) or compact (Cp, n = 93) cumulus. Other COC were subjected to 26 μM brilliant cresyl blue (BCB) in modified PBS for 90 min at 38.5°C and were categorised into BCB+ (blue cytoplasm, low G6PD activity, n = 89) and BCB– (colourless cytoplasm, high G6PD activity, n = 41) according to their ability to convert the BCB stain from blue to colourless. Following maturation for 28 to 30 h at 38.5°C in 5% CO2 in DMEM-F12 supplemented with 10% FCS, 5 mU mL–1 of FSH and 25 μg mL–1 of gentamicin sulfate, oocytes were imaged individually to assess zona thickness and ZPB by polarized light microscopy using OCTAX polarAIDE software (OCTAX, Bruckberg, Germany). Subsequently, oocytes were subjected to intracytoplasmic sperm injection using frozen-thawed stallion spermatozoa followed by culture in DMEM/F-12 medium (10% FCS and 25 μg mL–1 of gentamicin sulfate) under mineral oil (38.2°C, 5% CO2, 5% O2) for 8 days. Development rates were compared by chi-square test; variations in thickness and birefringence were analysed by Tukey's test and ANOVA. Higher maturation (58.4 vs 40.2%) and blastocyst rates (11.9 vs 3.8%) of Ex oocytes compared with Cp oocytes as well as higher proportions of BCB+ oocytes that matured (57.7% vs 28.1%), cleaved (45.9 vs 29.0%) and developed to the blastocyst stage (9.2 vs 1.4%) compared with BCB– oocytes (all P < 0.05) confirmed cumulus morphology and G6PDH activity as useful predictors for viability. Moreover, Ex oocytes had a thicker zona (18.2 ± 2.2 μm vs 17.3 ± 2.1 μm) and a higher birefringence (64.6 ± 5.2 vs 62.1 ± 4.2) compared with Cp oocytes (both P < 0.05). Concurrently, oocytes classified as BCB+ had a thicker zona (18.8 ± 2.4 μm vs 16.1 ± 2.0 μm) and higher values for birefringence (63.1 ± 4.5 vs 61.3 ± 3.3) compared with BCB– oocytes (both P < 0.05). Taken together, our results revealed that equine oocytes with higher developmental capacity have a thicker zona and greater birefringence scores compared with oocytes of lower developmental competence. Therefore, ZPB measurement provides a new, noninvasive method to assess the developmental competence of equine oocytes.

Efficiencies for in vitro production of equine embryos are still low due to highly variable developmental competences of equine immature oocytes. In contrast to the equine, in vitro developmental competence of immature oocytes has been predicted successfully by the activity of glucose-6-phosphate dehydrogenase (G6PDH) indicated by brilliant cresyl blue (BCB) dye in a range of different species. Therefore, the aim of the present study was to test the association between G6PDH activity in equine oocytes with: (1) cumulus morphology and oocyte properties in terms of diameter and volume; (2) maturational competence; (3) gene expression of certain molecular markers; and (4) in vitro embryo development after intracytoplasmic sperm injection. Equine oocytes were exposed to BCB stain and were classified as BCB+ or BCB− according to their ability to convert the dye from blue to colorless. Additionally, BCB+ and BCB− oocytes were subclassified as having a compact (Cp) or expanded (Ex) cumulus complex. As a result, BCB+ oocytes had a greater proportion of expanded cumulus oocyte complexes compared with BCB− oocytes (71.2% vs. 49.5%). Moreover, we observed a significant difference in oocyte diameter and volume between BCB+ and BCB− oocytes irrespective of cumulus morphology. BCB+ oocytes reached a higher maturation rate compared with BCB− oocytes (59.0% vs. 28.7%). Regarding the analyzed candidate genes, relative transcript abundance was significantly different for nine genes. The expression of eight genes was significantly higher (P < 0.05) for BCB+ oocytes, including ATPV6E, IF-3, TFAM, DNMT1, STAT3, Aurora-A, ODC1, and CKS2 whereas BCB− oocytes showed higher in expression of COX1. These results are in line with the observed developmental competence. Cleavage rate (45.9% vs. 29.0%) and percentage of embryos that reached the blastocyst stage (9.2% vs. 1.4%) were significantly higher for embryos derived from BCB+ oocytes compared with BCB− oocytes. In conclusion, the present study provides evidence that G6PDH-activity in immature equine oocytes is a useful predictor for subsequent in vitro developmental competence.