en <p>ckground: Reactive oxygen species (ROS) are critical factors for oocyte maturation and early embryogenesis; however, excessive ROS can induce oxidative stress, impairing mitochondrial function, DNA integrity, and embryo competence. The role of oxidative status in buffalo follicular fluid (FF) remains underexplored. This study assessed the relationship between total oxidant status (TOS) in buffalo FF and the developmental competence of oocytes retrieved by ovum pick-up (OPU) for subsequent <em>in vitro</em> fertilization (OPU-IVF).<br /> Methods: Follicular fluid and cumulus&ndash;oocyte complexes (COCs) were collected from 62 healthy buffaloes. Oocytes were matured and fertilized <em>in vitro</em>, and the animals were classified based on blastocyst yield: G1 (no blastocysts), G2 (&ge;2 blastocysts), and G3 (&gt;3 blastocysts). TOS was measured spectrophotometrically. The relationship between TOS and oocyte competence was analyzed by ROC (G1 <em>vs.</em> G3) and Spearman correlation (G1 <em>vs.</em> G2), with p&lt;0.05 considered statistically significant.<br /> Results: G2 group with &gt;2 blastocyst exhibited lower TOS levels (1.10&plusmn;0.51 <em>&micro;mol </em>H₂O₂ Eq/L) than G1 (2.15&plusmn;0.92; p=0.004), with higher follicle counts, oocyte yield, cleavage rate, and blastocyst production (p&lt;0.05). ROC analysis identified a TOS threshold of 1.12 <em>&micro;mol</em> H₂O₂ Eq/L (area under the curve [AUC]=0.851), and TOS inversely correlated with the proportion of high-quality blastocysts (r=-0.553, p=0.021).<br /> Conclusion: Elevated oxidative stress in FF compromises oocyte developmental competence and embryo quality. TOS may serve as a predictive biomarker, supporting antioxidant-based optimization of assisted reproductive technology (ART) in buffalo.</p> Biomarker, Buffalo, Follicular fluid, IVF, Oocyte competence, Total oxidant status 144 152 https://www.jri.ir/article/140280 /documents/fullpaper/en/140280.pdf SaraBorjian-BoroujeniDepartment of Clinical Sciences, Faculty of Veterinary Medicine, Shahrekord University, Shahrekord, Iran122926NaserShams-EsfandabadiResearch Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Irandrn_shams@yahoo.com122927AbolfazlShiraziReproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iranابوالفضلشيرازیa.shirazi2023@gmail.com795EbrahimAhmadiResearch Institute of Animal Embryo Technology, Shahrekord University, Shahrekord, Iranابراهيماحمدی798KambizGilanyDepartment of Integrative Oncology, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iranكامبيزگيلاني932