Oxidative stress critically affects cellular viability and function under in vitro culture conditions, often compromising physiological integrity of somatic cells used in livestock biotechnology. This study aimed to investigate hydrogen peroxide (H₂O₂)-induced oxidative stress in buffalo fibroblasts and evaluated the cytoprotective effects of melatonin, focusing on redox homeostasis, mitochondrial function, apoptosis, and antioxidant defence. Fibroblasts were exposed to graded concentrations of H₂O₂ (100–1000 µM) for 2 h, followed by treatment for 72 h in culture media with and without melatonin (10⁻⁹ M). Oxidative stress markers, including GSSG/GSH ratio, ROS generation, mitochondrial membrane potential (MMP), and apoptosis, were assessed using flow cytometry and biochemical assays, while antioxidant (GPx, SOD, CAT) and apoptotic (BAX, Caspase 9) gene expression was analyzed by qPCR. H₂O₂ exposure induced a dose-dependent increase in oxidative stress, evidenced by elevated ROS, redox imbalance, mitochondrial depolarization, and enhanced apoptosis. Severe oxidative damage was observed at higher H₂O₂ (500–1000 µM) concentrations. Melatonin (MT) significantly (p ≤ 0.05) alleviated oxidative stress under mild to moderate conditions (100–200 µM H₂O₂) by restoring redox homeostasis, preserving mitochondrial integrity, suppressing ROS accumulation, enhancing antioxidant defence, and reducing apoptosis. However, its protective efficacy was lost under severe oxidative stress, indicating a defined redox threshold beyond which cellular damage becomes irreversible. These findings suggest that melatonin exerts cytoprotective effect against oxidative stress within a limited oxidative window and provide mechanistic insights for improving fibroblasts culture systems in livestock biotechnology and regenerative applications.

Somatic cell nuclear transfer (SCNT) holds promise for animal cloning but remains limited by low efficiency and phenotypic abnormalities, often attributed to incomplete nuclear reprogramming. This study presents an integrative genomic and epigenomic analysis of cloned buffaloes and their respective donors using long-read Oxford Nanopore sequencing. Our results showed a high degree of genomic similarity between clones and donors, with most variations located in non-coding regions and structural variants (SV) distributions highly correlated at the chromosomal level. Gene and protein level overlap of SV-affected loci revealed 70.9–73.3% gene-level and 69.7–72.5% protein-level similarity. Despite this genetic similarity, DNA methylation analysis identified differentially methylated regions (DMRs), particularly in intergenic and promoter regions. Clones exhibited slightly lower CpG methylation than the donors. The DMRs in donor vs. clone comparisons indicated higher hypomethylated regions than hypermethylated regions. Functional enrichment of DMR-associated genes highlighted pathways linked to mitochondrial function, oxidative phosphorylation, and reproductive processes. Although clones showed moderate genome-wide methylation correlation with donors, key differences in methylation suggest incomplete epigenetic reprogramming. Despite these epigenetic differences, all clones were phenotypically normal and healthy into adulthood. This study offers the first comprehensive SV and methylome profile of SCNT-derived buffaloes and emphasizes the role of epigenetic mechanisms in clone development and health, providing valuable insights to enhance cloning efficiency.

Background: The Kachchhi-Sindhi horse is a native Indian breed, mainly found in the Gujarat and Rajasthan regions of India, with an estimated population of about 4,000 horses. A comprehensive genetic assessment to understand the genetic diversity and population structure of this breed is essential for conservation strategies and for maintaining the health and vitality of the breed. Methods: To determine the genetic diversity and population structure of the Kachchhi-Sindhi breed, we genotyped 50 unrelated, healthy adult horses using 30 microsatellite DNA loci. The genetic data were analyzed to calculate the number of alleles, observed and expected heterozygosity and the inbreeding coefficient (FIS). We also tested for Hardy-Weinberg equilibrium deviations and marker neutrality. We used Structure analysis to assess the admixture within the breed. Besides, population structure analysis was done with principal coordinates analysis (PCoA). Alongside mode shift tests, as well as Sign, Standardized differences and Wilcoxon sign rank tests, were made for assessing signs of a potential population bottleneck recently. Result: Our study disclosed substantial genetic diversity in the Kachchhi-Sindhi breed. Key genetic parameters averaged an average number of alleles of 10.10±3.18, with an effective number of alleles of 5.15±1.88 and observed heterozygosity of 0.90±0.11, indicating significant genetic variation in the population. The inbreeding coefficient (FIS) was very low at -0.17±0.19, indicating limited inbreeding. While 14 of the 30 microsatellites showed deviations from Hardy-Weinberg equilibrium, most markers were considered neutral, with only four showing non-neutral behaviour, according to the Ewens-Watterson neutrality test. Population analysis by PCoA revealed a single large but fragmented population of Kachchhi-Sindhi horses. Further tests, such as the mode-shift test and M ratio value, showed no evidence of a recent bottleneck in the breed. These are crucial for guiding future breeding and conservation efforts for the Kachchhi-Sindhi breed.