Apigenin alleviates lipopolysaccharide-exposed oocyte dysfunction during in vitro maturation in bovine

Lipopolysaccharide (LPS) is a large molecule found on the outer membrane of most Gram-negative bacteria. LPS exposure impaired the quality of oocytes and led to defective maturation through oxidative stress (OS) and apoptosis induction. Apigenin (APG) is a naturally occurring, non-toxic flavonoid with potent antioxidant, anti-apoptotic, and anti-inflammatory properties. This study investigated whether APG supplementation during in vitro maturation (IVM) can mitigate LPS-induced toxicity on bovine oocyte maturation and subsequent embryo development. To determine the optimal LPS concentration, bovine cumulus oocyte complexes (COCs) were exposed to 5, 10 and 20 μg/mL LPS during IVM for 24 h. Among the tested concentrations, 10 μg/mL LPS was selected for subsequent experiments because it induced a significant and reproducible impairment in oocyte maturation for preserving a sufficient number of viable mature oocytes for downstream analyses. Then, we assessed the impact of adding APG at three different concentrations (1, 10, and 50 μM) in the IVM medium on polar body extrusion (PBE) in LPS-treated oocytes (10 μg/mL) during maturation. Accordingly, the 10 μM concentration of APG was used to evaluate its effects on cumulus cell expansion (CCE) and viability (CCV), intracellular reactive oxygen species (ROS) and GSH levels, mitochondrial distribution, apoptosis, and the relative expression of antioxidant enzyme genes (Sod, Cat, and Gpx) and apoptosis-related genes (Caspase-3, Bax, and Bcl-2), as well as the subsequent embryonic developmental competence of oocytes matured under LPS challenge in vitro. Experiments were conducted in three to five independent biological replicates, and data were analyzed using one-way ANOVA followed by Fisher's protected least significant difference (LSD) post hoc test (P < 0.05). The findings indicate that LPS exposure impaired oocyte maturation, CCE, and CCV, whereas supplementation with 10 μM APG significantly mitigated these deleterious effects (81.79% ± 2.49, 3.29 ± 0.07 and 90.85% ± 2.03 respectively; P < 0.001). Furthermore, APG alleviated the LPS-induced impairments in oocyte maturation by reducing ROS levels (20.75 ± 0.85; P < 0.001), increasing GSH content (38.38 ± 1.45; P < 0.0001), preserving mitochondrial mass (7.45 ± 0.31) and membrane potential (1.63 ± 0.04) (P < 0.0001), and decreasing apoptosis (11.55% ± 2.21; P < 0.001). Ultimately, APG improved cleavage (77.36% ± 0.80) and blastocyst (36.04% ± 1.56; P < 0.0001) rates and quality of the resulting embryos. In addition, APG upregulated the expression of Sod, Cat, Gpx, and Bcl-2 genes and downregulated Caspase-3, Bax, TLR4, and NF-κB genes (P < 0.05). In conclusion, APG co-treatment significantly mitigated LPS-induced toxicity, accompanied by reduced OS, enhanced mitochondrial integrity, suppression of apoptosis, and coordinated alterations in gene expression.