Search Results (120)

In vitro maturation (IVM) is a critical step affecting the efficiency of bovine in vitro embryo production; however, oxidative stress during in vitro culture can impair oocyte quality and subsequent developmental competence. This study investigated the effects of cysteine supplementation on bovine oocyte IVM, redox homeostasis, mitochondrial status, and transcriptomic changes. Bovine cumulus-oocyte complexes were cultured in IVM medium supplemented with 0, 25, 50, 75, 100, or 125 μM cysteine, and 75 μM was identified as the optimal concentration. Compared with the control group, 75 μM cysteine increased the first polar body extrusion rate from approximately 78% to 81% and improved the fertilization/cleavage rate from approximately 74% to 82%. It also significantly increased the proportions of 2-cell, 4-cell, and 8-cell embryos, whereas morula and blastocyst rates were not significantly affected. At the cellular level, 75 μM cysteine significantly reduced ROS levels and increased GSH content, as indicated by changes in relative fluorescence intensity. JC-1 staining showed that the JC-1 monomer signal decreased from approximately 16.0 to 13.5, whereas the JC-1 aggregate signal increased from approximately 13.2 to 14.8, indicating improved mitochondrial membrane potential status. In addition, lipid droplet fluorescence intensity increased from approximately 11.8 to 13.4, mitochondrial fluorescence intensity increased from approximately 6.0 to 7.0, and cytoskeletal fluorescence intensity showed no significant difference between groups. Smart-seq2 transcriptomic analysis identified 1935 differentially expressed genes, including 1778 upregulated and 157 downregulated genes, which were mainly enriched in translation, ribosomal structural components, RNA binding, oxidative phosphorylation, and metabolism-related pathways. qRT-PCR further confirmed the upregulation of key genes, including NDUFS2, VDAC3, ANXA2, MTHFD1L, and SCD. Overall, 75 μM cysteine improves bovine oocyte IVM quality by enhancing antioxidant capacity, improving mitochondrial membrane potential, increasing lipid-derived energy substrate storage, and regulating genes related to energy metabolism and developmental competence. Full article

While early embryonic loss affects the conception rate in lactating cows, the underlying mechanisms remain unknown. Here, we examined whether the developmental morphokinetics and transcriptomic profiles of cleaved embryos are associated with developmental competence. Developing bovine embryos were produced in vitro, and their morphokinetics were monitored through 190 h using a time-lapse system. The proportion of embryos that developed to the blastocyst stage was lower following abnormal cleavage, i.e., reverse, direct, or unequal, relative to normally cleaved embryos (p < 0.05). In a second set of experiments, exploratory RNA-seq analysis was performed on first-cleaved embryos, which were individually collected immediately after the first division; embryos were defined by the time-lapse system as normally, directly, or unequally cleaved (n = 6 per group), or reverse-cleaved (n = 5). Analysis revealed 672 genes that were differentially expressed between normally and abnormally cleaved embryos (adjusted p < 0.05). Abnormally cleaved embryos differed in pathways associated with energy production and metabolism. The most profound difference in gene expression (n = 632) was between unequally and normally cleaved embryos, mainly in genes affiliated with the oxidative phosphorylation pathway. These findings support the concept that the morphokinetic pattern of early embryonic cleavage is associated with developmental competence. We therefore suggest that the observed differential expression profiles in abnormally cleaved embryos might be involved in the mechanism underlying early embryonic loss. Full article

Haploid embryos constitute a valuable model for genetic and epigenetic studies; however, their developmental competence is reduced compared with diploid counterparts. This study evaluated whether supplementation of the culture medium with specific small molecules could improve developmental competence and outgrowth establishment of parthenogenetic haploid embryos. The effects of TGF-β inhibition (A83-01), WNT pathway modulation (CHIR99021 and IWR-1), and activin A (AA) supplementation were assessed from the morula stage onward under serum-free conditions. A83-01 treatment did not improve blastocyst formation or morphology and was associated with reduced total cell numbers relative to IVF controls. CHIR99021 supplementation increased the number of SOX2-positive cells compared with IWR-1 and vehicle-treated embryos, suggesting partial support of pluripotency; however, overall developmental progression remained inferior to diploid controls. In contrast, activin A significantly increased the proportion of haploid morulae developing into blastocysts and improved hatching rates. Nevertheless, AA supplementation did not restore CDX2-positive cell numbers or total cell counts to diploid levels. Furthermore, neither CHIR99021 nor AA affect DNA fragmentation levels, although a tendency toward increased TUNEL-positive cells was observed. Activin A treatment also failed to improve embryonic outgrowth formation. Collectively, these findings demonstrate that although activin A enhances blastocyst yield and hatching in bovine haploid embryos, modulation of TGF-β or WNT signaling alone is insufficient to restore diploid-like proliferative developmental competence. Full article

Sperm quality influences bovine in vitro embryo production (IVEP). Linear regression is a statistical tool that models the relationship between a dependent variable and one or more independent variables. It can be used to predict outcomes, analyze trends, and understand the impact of variables. These models are useful for indicating which sperm variables most influence IVEP results, facilitating the selection of superior samples to enhance IVEP. Using early IVEP indicators, such as cleavage rate, can assist in scheduling recipient preparation. This work aimed to construct linear regression models to study the influence of a comprehensive set of sperm variables and cleavage rate on IVEP yields. A dataset comprising 51 semen batches from 23 Nellore bulls was compiled, including 26 sperm variables from computer-assisted sperm analysis (CASA) and flow cytometry per batch, with 184 IVEP procedures. The most robust predictive model had a coefficient of determination of 0.6358; furthermore, the BULL variable was the most influential predictor, yielding an independent coefficient of determination of 0.5218. Models that were exclusively founded on sperm analysis yielded meager coefficients of determination (<0.04). However, to predict the best batch from a bull, individual models achieve coefficients of determination ranging from 0.58 to 0.99. Contributions, impacts, and positive or negative correlations of various sperm variables with in vitro performance were influenced by the bull. We conclude that the BULL variable was the dominant predictor of in vitro performance, with cleavage rates serving as an early estimator of blastocyst rates. The predictive utility of analyzed sperm traits remains limited. Nonetheless, individualized models offer a valuable tool for selecting optimal batches for preferred bulls within IVEP laboratories, culminating in heightened blastocyst rates. Full article

Endogenous heat shock cognate 73 kDa protein (HSC70) plays a role in early embryonic development and cellular stress regulation. This study evaluated the effects of exogenous recombinant HSC70 supplementation on bovine embryo development and the expression of apoptosis-related genes under in vitro conditions. Expression analyses of HSPA1A, HSPA8, BCL-2, and BAX were performed on Day 7 bovine embryos produced in vivo and in vitro. In vitro embryos exhibited higher basal expressions of HSPA8, BAX and BCL-2 compared with in vivo embryos (p ≤ 0.001). Supplementation with 500 or 1000 ng/mL HSC70 was associated with increased expression of HSPA1A, HSPA8, BCL-2, and BAX relative to control embryos (p ≤ 0.01). The 1000 ng/mL group showed significantly higher HSPA8 expression compared with both the control and 500 ng/mL groups. Morphological evaluation indicated that embryos supplemented with 500 ng/mL were associated with improved blastocyst yield and quality compared with control and 1000 ng/mL groups.In conclusion, supplementation with 500 ng/mL recombinant HSC70 was associated with modulation of apoptosis-related gene expression and improved morphological developmental parameters under in vitro conditions. These findings indicate dose-dependent regulatory effects of HSC70 on apoptosis-related signaling pathways; however, as apoptosis was assessed at the transcriptional level, the results should be interpreted as molecular associations rather than direct confirmation of altered apoptotic activity. Full article

To improve the vitrification efficiency of bovine germinal vesicle (GV) oocytes, the use of hydroxyapatite (HA) nanoparticles as a novel cryopreservation additive represents a promising approach. This study aimed to investigate the effects of HA nanoparticles and permeable cryoprotective agents (CPAs) on the ultrastructure, developmental competence, and gene expression of bovine GV oocytes following vitrification. Oocytes were vitrified in vitrification solutions containing HA nanoparticles of different sizes (20, 40, or 60 nm) and concentrations (0.01%, 0.05%, or 0.1%) to determine the optimal conditions based on survival rate, mitochondrial membrane potential (MMP) level, and developmental competence. Subsequently, the synergistic effects of HA nanoparticles and permeable CPAs (VS: 20% EG + 20% DMSO; VS1: 17.5% EG + 17.5% DMSO) were further evaluated. The optimal treatment (40 nm 0.05% HA nanoparticles) significantly increased MMP level, and improved developmental competence compared with the vitrified control group (p < 0.05). Among the vitrified groups, vitrified oocytes in the VS1-HA group (combining HA nanoparticles with reduced concentrations of permeable CPAs) exhibited the highest MMP level (1.89), maturation rate (50.39%), cleavage rate (27.07%), and blastocyst rate (10.53%) (p < 0.05). Ultrastructural analysis further revealed that the VS1-HA group maintained more intact zona pellucida structures and showed reduced mitochondrial swelling compared with the vitrified control group. Moreover, the expression levels of genes associated with zona pellucida formation (ZP3), mitochondrial fusion (MFN1), and chromatin remodeling (NPM2) were significantly upregulated in the VS1-HA group relative to the vitrified control group. Overall, these findings indicate that the combination of HA nanoparticles with lower concentrations of permeable CPAs enhances MMP level, alleviates vitrification-induced ultrastructural damage, and upregulates the expression of key developmental genes, thereby improving the developmental competence of vitrified bovine GV oocytes. Full article

In bovine embryo transfer (ET) using in vitro-produced (IVP) embryos, recipient factors and embryo grade are well-established predictors of pregnancy success, but the impact of the laboratory-level developmental competence of IVP embryos remains insufficiently characterized. This retrospective study evaluated factors affecting pregnancy rates following the transfer of IVP beef embryos to dairy recipients. Medical records from 462 ETs were analyzed across three categories: (1) recipient-related factors (parity, body condition, estrus synchronization, corpus luteum characteristics); (2) laboratory factors (cleavage, blastocyst formation, degeneration, embryo grade, developmental stage, cryopreservation); and (3) environmental factors (temperature–humidity index, transport time). Mean comparison and chi-square analyses revealed significant differences in pregnancy rates based on corpus luteum volume, cleavage rates, blastocyst formation rates, degeneration rates, and embryo grade. In binary logistic regression, categorized increases in blastocyst formation rate, degeneration rate, and embryo grade were associated with a 1.45-fold increase, 0.74-fold decrease, and 0.56-fold decrease in pregnancy odds, respectively; no recipient or environmental variables were independent predictors. These findings indicate that developmental competence of IVP embryos is more critical for pregnancy success than recipient or environmental factors, suggesting that optimizing IVP systems to maximize embryo quality is the most effective strategy to improve reproductive efficiency in ET. Full article

Short-term hypothermic storage at 4 °C represents a promising non-freezing alternative for transporting bovine embryos and synchronizing assisted reproductive procedures. However, chilling induces oxidative stress, mitochondrial dysfunction, and apoptosis, which markedly impair post-preservation embryonic viability. Glutathione (GSH), a key intracellular antioxidant, may mitigate these damaging effects, yet its protective mechanisms during bovine blastocyst hypothermic preservation remain unclear. Here, we investigated the impact of exogenous GSH supplementation on the survival, hatching ability, cellular integrity, mitochondrial function, and developmental potential of bovine blastocysts preserved at 4 °C for seven days. Optimization experiments revealed that 4 mM GSH provided the highest post-chilling survival and hatching rates. Using DCFH-DA, TUNEL, and γ-H2AX staining, we demonstrated that 4 °C preservation significantly increased intracellular reactive oxygen species (ROS), DNA fragmentation, and apoptosis. GSH supplementation markedly alleviated oxidative injury, reduced apoptotic cell ratio, and decreased DNA double-strand breaks. MitoTracker and JC-1 staining indicated severe chilling-induced mitochondrial suppression, including decreased mitochondrial activity and membrane potential (ΔΨm), which were largely restored by GSH. Gene expression analyses further revealed that chilling downregulated antioxidant genes (SOD2, GPX1, TFAM, NRF2), pluripotency markers (POU5F1, NANOG), and IFNT, while upregulating apoptotic genes (BAX, CASP3). GSH effectively reversed these alterations and normalized the BAX/BCL2 ratio. Moreover, SOX2/CDX2 immunostaining, total cell number, and ICM/TE ratio confirmed improved embryonic structural integrity and developmental competence. Collectively, our findings demonstrate that exogenous GSH protects bovine blastocysts from chilling injury by suppressing ROS accumulation, stabilizing mitochondrial function, reducing apoptosis, and restoring developmental potential. This study provides a mechanistic foundation for improving 4 °C embryo storage strategies in bovine reproductive biotechnology. Full article

Dimethyl sulfoxide (DMSO) is widely utilized in the vitrification of oocytes, but DMSO exhibits concentration-dependent toxicity, which can compromise oocyte developmental potential by disrupting key cellular processes. This study reports the first successful use of cold shock protein B (CspB protein) as a substitute for DMSO in vitrification solutions for oocyte vitrification. Combining dynamics simulations and experimental validation, we demonstrated CspB’s ability to inhibit ice crystallization and recrystallization by stabilizing its position at the ice–water interface and reducing ice formation rates. Recombinant CspB was successfully expressed and shown to bind to the oolemma. In vitrification solutions, CspB (1–2 mg/mL) effectively reduced ice crystal size and enabled a significant reduction or complete replacement of DMSO. This strategy markedly improved the post-thaw survival rates of both mouse and bovine metaphase II (MII) oocytes. Furthermore, oocytes vitrified with an optimized formulation (15% ethylene glycol + 2 mg/mL CspB) exhibited developmental competence (cleavage and blastocyst rates), oxidative stress markers (ROS, GSH), mitochondrial function (membrane potential and content), and apoptosis levels (Caspase-3/9) comparable to those treated with a standard DMSO-containing system. Transcriptomic analysis revealed that CspB’s cryoprotection involves the modulation of the mTOR signaling pathway. This role was functionally confirmed, as activation of mTOR abolished CspB’s beneficial effects, reinstating oxidative damage, mitochondrial dysfunction, and apoptosis. Thus, the CspB protein replaces DMSO with direct ice crystal formation suppression and mTOR-mediated oxidative stress regulation. This study offers a protein-based alternative to conventional permeable cryoprotectants. This approach holds promise for improving reproductive biotechnologies across species. Full article

Cytosine base editors (CBEs) enable precise C-to-T (G-to-A) conversions in genomic DNA, offering significant potential for specific gene editing. This study compared the prototypical Base Editor 3 (BE3) and a modified variant, BE3-Y130F, which utilizes an hA3A deaminase with the Y130F mutation, focusing on their editing efficiency and editing window characteristics using bovine zygotes. Following in vitro fertilization (IVF), sgRNA and Cas9 mRNA were injected as a targeting efficiency control, which resulted in 100% editing with no wild-type sequence. Then, either BE3 or BE3-Y130F mRNA, synthesized via in vitro transcription, and an sgRNA targeting exon 4 of the MYO7A gene was injected into zygotes. Genomic DNA was extracted from both blastocysts and developmentally arrested embryos, and Sanger sequencing was performed to evaluate C-to-T conversion efficiency and editing window. Both BE3 and BE3-Y130F achieved 100% C-to-T conversion efficiency at the primary target cytosine. BE3 displayed a defined editing window, primarily affecting cytosines at positions 7 and 8, indicating a predictable profile. In contrast, BE3-Y130F maintained high efficiency but had a less clearly defined editing window, resulting in incomplete editing and a remaining cytosine on the target sequence. Full article

The developmental competence of oocytes is a critical limiting factor in bovine in vitro embryo production (IVEP). Our study aimed to investigate the relationship between the intrafollicular concentrations of interleukin-10 (IL-10) and progesterone (P4), follicle characteristics, and the subsequent developmental success of bovine oocytes. Follicular fluid (FF) and corresponding cumulus–oocyte complexes (n = 314) were collected from FSH-stimulated heifers. A novel, high-sensitivity Surface Plasmon Resonance Imaging biosensor was used to quantify IL-10, while P4 was measured by an enzyme-linked fluorescent assay. Oocytes were individually cultured to assess cleavage (Day 3) and blastocyst formation (Day 7). Statistical analysis revealed that intrafollicular IL-10 concentration was a significant positive predictor of developmental success, significantly correlating with blastocyst rate (ρ = 0.29, p = 0.016). Oocytes from follicles with IL-10 concentrations above an optimized cutoff of 142.16 pg/mL had a 16.33-fold greater chance of developing into a blastocyst (p = 0.006). A predictive model combining IL-10 and oocyte morphology demonstrated the highest accuracy for predicting blastocyst success (AUC = 0.724). Conversely, poor oocyte morphology (Grade 4) and large follicular volume (>1200 µL) were significantly associated with developmental failure. Intrafollicular P4 concentration was not directly correlated with embryo development but rather with follicle size. Our findings identify intrafollicular IL-10 as a potent biomarker for predicting bovine oocyte competence and suggest that its quantification using sensitive biosensor technology could enhance the efficiency of IVEP programs. Full article

This study aimed to evaluate the effects of melatonin supplementation during bovine in vitro embryo production (IVEP) on embryonic development and quality, oxidative stress, lipid metabolism, mitochondrial activity, gene expression, DNA methylation patterns, and cryotolerance. Four treatments were tested: control (without melatonin), melatonin at maturation (IVM + Mlt), culture (IVC + Mlt), and both treatments (IMV/IVC + Mlt). Melatonin significantly improved blastocyst rate and developmental kinetics on D7, reduced ROS and intracellular lipid levels, and increased mitochondrial activity. The most significant effects were observed in the IVC + Mlt group. Melatonin modulated antioxidant (SOD1, Cat, and GSS) and epigenetic (TET1, TET3, and DNMT3A) genes, and although it did not alter lipid gene expression, it reduced lipid content. Methylation analysis showed hypomethylation patterns in repetitive regions (Satellite I and LINE-1), which were even more pronounced in the melatonin-treated groups. However, no significant differences were observed between treatments in terms of cryotolerance or apoptosis rates. These findings suggest that melatonin exerts positive multifactorial effects, regardless of the supplementation stage. In particular, its addition during the IVC phase appears to provide greater benefits to embryos by improving their quality. Full article

High non-esterified fatty acids (NEFAs) during negative energy balance in dairy cattle can impair reproduction. While their effects on oocyte maturation and preimplantation embryo development are known, their impact during fertilisation is largely unexplored. This study examined the effects of high NEFA exposure exclusively during in vitro fertilisation (IVF). Bovine oocytes were matured in vitro and fertilised under physiological or high NEFA concentrations. High NEFA concentrations decreased fertilisation, cleavage, and blastocyst rates. Reactive oxygen species production in zygotes was not affected, but blastocysts derived from the High-NEFA group had fewer cells. Spermatozoa exposed to high NEFA concentrations exhibited increased plasma membrane and acrosome damage, higher DNA fragmentation, and reduced mitochondrial membrane potential. The expression of H3K27me3, a repressive histone mark normally erased from fertilisation to embryonic genome activation, was higher in 2-cell than in 4-cell embryos on day 2 after IVF, but only in the High-NEFA group. This delayed H3K27me3 loss, along with increased DNA damage, could partially explain the reduced blastocyst formation observed. In conclusion, high NEFA concentrations can impair pre-implantation embryo development during zygote formation, potentially via effects on both the oocyte and spermatozoon. The latter warrants further investigation using an intracytoplasmic sperm injection model. Full article

Retinoic acid receptor gamma (RARγ) plays a critical but poorly understood role in early embryo development and stem cell pluripotency. Here, we investigated the effects of the RARγ-specific inhibitor LY2955303 (LY) on in vitro-produced bovine embryos. Treatment with LY significantly increased blastocyst rates and quality, demonstrating its potential to enhance IVF outcomes in cattle. Single-embryo RNA sequencing using Smart-seq indicated that LY promotes metabolic reprogramming (upregulating glycolysis and TCA cycle activity) while suppressing apoptosis and inflammatory responses. LY treatment starting from the 16-cell stage led to enhanced glycolysis in blastocysts compared with exposure from the 2-cell stage. Furthermore, LY treatment also upregulated oxidative phosphorylation in bovine embryonic stem cells (ESCs). These findings establish RARγ as an important regulator of bovine embryo development, with its inhibition offering a novel strategy to optimize IVF embryo culture systems for livestock production. Full article

The in vitro production of embryos has significant potential to enhance animal productivity. However, further refining of this technology is required for its widespread adoption and cost-effectiveness. The objectives were to evaluate the effects of L-carnitine (LC) on the maturation, lipid content, and Hippo signaling of oocytes, and the cryotolerance of the resulting embryos. Abattoir-derived oocytes were in vitro matured using fetal bovine serum (FBS), bovine serum albumin (BSA), or FBS + 1.5 or 3.0 mM LC. The maturation rates did not differ among FBS (83%) and FBS with LC (1.5 or 3.0 mM; 82 and 80%, respectively). In contrast, the BSA group exhibited a significantly lower maturation rate of 71% compared to the other groups. The lipid content of matured oocytes (assessed using Nile red staining) was significantly reduced in the BSA group and in the FBS + LC groups, compared to the FBS group. The blastocyst-stage embryos were cryopreserved, and their cryotolerance was evaluated by assessing their ability to re-expand and hatch after thawing. The embryos from the FBS + LC groups showed a numerically higher re-expansion rate at 24 h (78.8%), compared to the BSA (74.0%) and FBS groups (57.7%). The expression of Hippo signaling pathway genes was not significantly affected by LC, indicating that LC enhanced cryotolerance and reduced lipid content without impacting oocyte maturation or the Hippo signaling pathway. Full article