189 In vitro prematuration of summer bovine cumulus–oocyte complexes in winter follicular fluid increases oocyte competence and changes cumulus cell microRNA profile

During high temperature-humidity index (THI) summers in northern Uruguay, IVM and IVF of cumulus–oocyte complexes (COCs) from grazing cows often result in low embryo development rates. We previously found that prematuration of winter COCs for 6 h in winter follicular fluid (WFF) improved blastocyst rates. Given that miRNAs participate in the communication between cumulus cells and the oocyte, we examined the effect of WFF prematuration on summer and winter oocyte developmental capacity and their cumulus cells miRNA profile. A total of 150 ovaries were obtained from Hereford, Angus, and crossbred cows from a local slaughterhouse during the Austral winter of 2021 (July–August; average THI: 54 ± 7). Three independent collections of WFF were obtained from 2- to 8-mm follicles and stored at −80°C. Then, in 2023, at least 400 COCs per group were collected in summer (February–March; THI: 74 ± 4) and winter (June–August; THI: 55 ± 7). Half of COCs were prematured in 10% WFF for 6 h, followed by a 22-h maturation period; control COCs only underwent a 24-h maturation. A portion of matured COCs were fertilized, and presumptive zygotes (~130 per group) were cultured for 8 days. Seven expanded blastocysts per group were used for cell number and apoptotic index evaluation through TUNEL assay. Data from four replicates were analyzed using one-way ANOVA with Tukey’s post-hoc test. Additionally, 50 mature COCs per group were collected for oocyte meiotic progression assessment by Hoechst staining and cumulus cell small RNAs isolation. Small RNA libraries were prepared with the SMARTer smRNA-Seq Library Prep Kit (Illumina) and sequenced (Macrogen SK). Bioinformatic analysis, performed with CLC Genomics Workbench v24, included mapping and annotating filtered reads against bovine miRNAs (miRBase 22). Prematuration tended to enhance meiotic progression (P = 0.06) in summer oocytes, significantly improving blastocyst rates (P = 0.03) and reducing blastocyst apoptosis (P = 0.04) compared with controls. Small RNA sequencing revealed that 3% of mapped reads corresponded to known bovine miRNAs, identifying an average of 386 miRNAs in each sample. The top five miRNAs in all samples were miR-2887, miR-11972, miR-12034, miR-11976, and let-7a-5p. In prematured summer cumulus cells, five miRNAs (miR-222, miR-23a, miR-23b-3p, miR-328, and miR-10174-3p) were upregulated (>2.0-fold change; P < 0.05 FDR); among these, miR-23a and miR-23b-3p are associated with apoptosis reduction and glycolysis. In winter cumulus cells, only miR-2424 and miR-222 were consistently upregulated. Interestingly, miR-222, present in both comparisons, has been linked to bovine oocyte maturation and fertilization. In conclusion, 6-h prematuration in WFF for summer and winter COCs enhanced oocyte meiotic progression and improved blastocyst yield and quality while upregulating miRNAs in cumulus cells that might play a role in oocyte maturation and developmental capacity.