187 Effect of Lactobacillus-conditioned medium supplemented during in vitro maturation of bovine COCs on mRNA expression patterns in cumulus cells

Lipopolysaccharide (LPS) has been detected in the follicular fluid of cows with infections. Administered during IVM, LPS impairs the oocytes’ developmental competence; whereas, Lactobacillus spp. have been associated with increased oocyte quality when in follicular fluid, possibly owing to the secretion of flavonoids (Owens et al. 2020 J. Dairy Sci. 103, 10951–10960). The aim of this study was to determine whether the negative LPS effect could be overcome via Lactobacillus-conditioned medium. Cumulus–oocyte complexes (COCs) from slaughterhouse ovaries were isolated via slicing. Five percent of the IVM medium (5:95) was replaced by L. buchneri-conditioned medium (group 4), and LPS was supplemented during IVM at a concentration of 10 µg mL⁻¹ (group 5). Group 6 was treated with a combination of both supplements. As controls, immature COCs (collected before maturation; group 1) and oocytes after standard IVM with (group 2) or without (group 3) oil overlay were included. COCs of groups 4–6 were also matured without oil overlay. To generate the conditioned medium, L. buchneri was cultured in TCM for 1 day, then the medium was sterile filtered and used as described. In the same experimental setup, the maturation rate was significantly reduced in oocytes of the LPS group compared with all other groups (Granacher et al. 2023 Reprod. Dom. Anim. 58, 13). Total RNA from cumulus cells obtained after denudation of a single COC (n = 5 for each group) was directly used for RT-qPCR to determine the relative abundance of gene transcripts known to be related to hormone function (CYP19, ER, FSHR, LHCGR, HSD3β, PGR, and STAR) and immune function (CD14, IL6, and TLR4). The relative abundance of each transcript was calculated relative to the reference gene RSP9 (Blaschka et al. 2019 Theriogenology 131, 182–192). The data (mean ± s.e.m.) were analyzed via a one-way ANOVA followed by a Tukey test (P ≤ 0.05). Most transcripts (ER, FSHR, LHCGR, HSD3β, PGR, STAR, IL6, and TLR4) were increased due to the LPS supplementation. However, this increase was not detectable when the maturation medium additionally contained the L. buchneri-conditioned medium. These data might suggest that L. buchneri-conditioned medium partly helps to overcome the negative LPS effect on gene expression in bovine cumulus cells, indicating a probiotic effect.