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.

In vitro maturation (IVM) of oocytes is a pivotal step in assisted reproductive technologies for livestock. However, oxidative stress (OS) and mitochondrial dysfunction during in vitro culture often lead to oocyte aging, thereby limiting the efficiency of the technologies. To address these challenges, this study investigated the regulatory effects of 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) on bovine oocyte IVM, aging, and developmental competence to determine the optimal concentration and explore underlying mechanisms. Cumulus–oocyte complexes (COCs) were collected from abattoir-derived bovine ovaries and cultured in IVM medium supplemented with 0 (control), 50, 100, 150, or 200 μmol/mL of POPC (n = 300 per group) at 38.5 °C under 5% CO₂ for 22 h. The optimal concentration was determined based on the first polar body extrusion rate, followed by in vitro fertilization (IVF), fluorescence staining, Smart-seq2 transcriptome sequencing, and quantitative PCR (qPCR) analysis. The results demonstrated that 150 μmol/mL of POPC yielded the highest maturation rate, significantly exceeding the control group (p < 0.05), and enhanced 2-4-cell cleavage rates after IVF. Furthermore, POPC markedly reduced intracellular reactive oxygen species (ROS) levels, increased glutathione (GSH) content, improved mitochondrial function, and restored normal spindle morphology. Transcriptomic analysis identified 350 upregulated and 280 downregulated differentially expressed genes (DEGs), which were enriched in pathways related to OS. qPCR validation confirmed upregulation of SIRT1/2 and BCL-2, along with downregulation of BAX and Caspase-1/3. Collectively, these findings suggest that 150 μmol/mL of POPC alleviates OS and activates the “SIRT–antioxidant–antiapoptotic” signaling axis, thereby providing valuable insights for optimizing assisted reproductive technologies in livestock.

Oxidative stress is a major factor limiting oocyte quality during in vitro maturation (IVM) and subsequent embryonic developmental competence. This study investigated the effects of combined melatonin (MLT) and glutathione (GSH) supplementation on sheep oocyte quality and the potential regulatory mechanisms involved. Sheep oocytes were assigned to Control, MLT, GSH, and MLT+GSH groups, and oocyte maturation, redox homeostasis, and mitochondrial function were evaluated by assessing cumulus expansion, first polar body extrusion, F-actin organization, ROS- and GSH-associated fluorescence, JC-1 staining, and MitoTracker signals, while subsequent developmental competence was assessed following in vitro fertilization (IVF). SMART-seq2 transcriptomic analysis, Nrf2 immunofluorescence, antioxidant-related gene expression analysis, and pharmacological inhibition of Nrf2 with ML385 were further performed to explore the underlying mechanisms. MLT and GSH, either alone or in combination, improved multiple indices related to oocyte maturation, redox status, and mitochondrial function, with the MLT+GSH group generally showing the most favorable numerical changes. MLT+GSH treatment was associated with greater cumulus expansion, a higher first polar body extrusion rate, improved F-actin organization, reduced ROS accumulation, increased GSH-associated fluorescence, a higher JC-1 red-to-green fluorescence ratio, and enhanced mitochondrial-associated fluorescence. However, for some endpoints, the numerical improvement observed with MLT+GSH did not differ significantly from the corresponding single treatment. Following IVF, MLT+GSH treatment increased cleavage and blastocyst formation rates and improved subsequent developmental outcomes. Mechanistically, MLT+GSH treatment was accompanied by increased Nrf2 immunofluorescence intensity and altered expression of NFE2L2 and several Nrf2-related antioxidant genes, including NQO1, HMOX1, GCLC, GCLM, and GPX4, suggesting the involvement of an Nrf2-associated antioxidant regulatory network. Addition of the Nrf2 inhibitor ML385 to the MLT+GSH treatment increased ROS levels and reduced JC-1 and other redox- and mitochondria-related indices, while partially attenuating improvements in oocyte maturation and embryonic development. Overall, MLT and GSH co-supplementation was associated with improved redox homeostasis, antioxidant capacity, mitochondrial function, and subsequent developmental competence in sheep oocytes, with Nrf2-associated antioxidant regulation potentially contributing to these beneficial effects under standard IVM conditions.

The intensification of global climate warming exacerbates the issue of heat stress in dairy cows, making the SLICK mutation in the prolactin receptor (PRLR) gene a critical target for enhancing heat tolerance in these animals. This study aims to investigate the effects of CRISPR/Cas9-mediated editing of the PRLR gene on the biological characteristics of bovine fibroblasts and early embryonic development following somatic cell nuclear transfer (SCNT). Using the CRISPR/Cas9 system, we targeted and edited a 20 bp–150 bp region within exon nine of the PRLR gene. After conducting off-target predictions and activity screenings, we identified optimal guide RNA (sgRNA) sequences and established stable transgenic cell lines. Transcriptome sequencing was performed on edited cells to identify key genes and validate their expression profiles. Edited cells were utilized as donor cells for SCNT, during which we assessed oocyte levels of reactive oxygen species (ROS), glutathione (GSH), and mitochondrial function to analyze embryonic developmental performance. We constructed a cellular stress resistance network aimed at mitigating damage transmission while maintaining embryonic developmental homeostasis. This research provides technical support and theoretical reference for genetic editing breeding programs aimed at improving heat tolerance in dairy cattle.