277 LIPID ACCUMULATION DURING BOVINE OOCYTES Gyr/HOLSTEIN MATURATION COLLECTED BY OPU IN SPOM SYSTEM

Abstract

In vitro embryo production (IVP) is an important tool for cattle breeding. Brazilian dairy systems are based on Gyr/Holstein crossbreds, which integrates adaptability to tropical conditions and milk production. Oocyte quality is crucial for IVP, and lipid accumulation is a detrimental factor. The aim of this study was to assess the effect of SPOM maturation system (Albuz et al. 2010 Hum. Reprod. 25) on lipid accumulation in bovine oocytes. Oocytes obtained by ovum pick-up (OPU) from heifers without ovarian stimulation were transferred to control media (TCM 199 + sodium pyruvate, ITS, penicillin-streptomycin, BSA, FSH, oestrogen, and hCG) or SPOM (2 h pre-IVM: TCM 199 hepes + sodium pyruvate, ITS, penicillin-streptomycin, BSA, IBMX, and forskolin; followed by 28 h IVM: control media + cilostamide) in 5% CO₂ atmosphere at 38°C. Oocytes were collected after 20, 24, and 28 h (groups: C20, C24, C28; S20, S24, S28) and evaluated for nuclear maturation using HOECHST (experiment 1, n = 301, 35–62 per group) and lipid content using Oil Red (experiment 2, n = 163, 14–42 per group). Oocytes from 4 replicates were fixed with PFA and stored at 4°C. For Oil Red, all structures were stained and evaluated at the same day. After being washed in 50% ethanol, oocytes were incubated in 0.2% Oil Red O solution for 10 min. Stained area fraction in each oocyte cytoplasm was measured using ImageJ (NIH). Nuclear maturation analysis was performed by Chi-squared test and Lipid analysis by Kruskal-Wallis and Dunn's post-test (P = 0.05). Distinct superscript letters indicate statistical difference between groups. In experiment 1, we detected a reduction in the percentage of matured (MII) oocytes only in S20 group (C20 = 64.28%^a, C24 = 74.28%^a, C28 = 60.46%^a, S20 = 25.8%^b, S24 = 59, 01%^a, S28 = 74.13%^a). In experiment 2, we detected an increase in lipid content in both control and SPOM groups from 20 to 28 h of IVM (S20 = 5.72^a ± 4.41, S24 = 15.95^bd ± 7.41, S28 = 37.46^c ± 8.68, C20 = 8.65^a ± 2.58, C24 = 12.14^ad ± 8.08, C28 = 18.50^b ± 8.09). For SPOM groups, the lipid content increased 2.78-fold from S20 to S24, and 6.54-fold from S20 to S28. In the control group, only in C28 wasa lipid increase detected, 2.13-fold higher than C20 content. Comparing control and SPOM groups at each time point, S28 displayed a 2.02-fold increase in lipid content compared to C28. We concluded that despite SPOM system being effective in maintaining meiotic arrest until 20 h of IVM and forskolin being present during pre-IVM in SPOM groups, Gyr/Holstein crossbreed oocytes obtained by OPU presented a significant increase in lipid content when matured in SPOM system, even higher than observed for the control group. During the last 8 h of IVM we observed a huge lipid accumulation in both systems, suggesting this might be a crucial period.