217 Effects of mature - recombinant GDF9 and BMP15 on in vitro maturation of bovine oocytes

In vitro maturation (IVM) is one of the most important components for successful in vitro embryo development, and has shown improvement with the use of recombinant growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15). These proteins are oocyte secreting factors (OSFs) within the transforming growth factor-β (TGF-β) superfamily and are vital for female fertility. They exist as homodimers with pro and mature domains that interact to form a highly potent heterodimer called cumulin and can activate latent GDF9 (Stocker et al. 2020 J. Biol. Chem. 295, 7981–7991). Now, mature versions of GDF9 (Sigma) and BMP15 (R&D Systems) are commercially available. We hypothesised that cumulus-oocyte complexes (COCs) matured for 23 h in maturation media supplemented with GDF9 and BMP15 would increase blastocyst development and quality. IVP embryos were produced in three treatment groups, including a commercial IVM media (J, VitroMat Protect, Laboratory Art Solutions), control (T, TCM 199 supplemented with gonadotrophins), and control supplemented with GDF9 (200 ng/µL) and BMP15 (100 ng/µL) (TGB). Embryos were produced in six replicates by aspirating COCs (n = 753) from 2 to 8 mm follicles from abattoir ovaries, matured at 38.5°C and 6.2% CO₂ in air, fertilised with frozen-thawed semen from one of two bulls, and cultured in SCF1 at 38.5°C, 6.2% CO₂, 7% O², and 86.8% N₂. Stage 5–8 blastocysts were stained with 1 µg mL⁻¹ Nile Red or 300 nM Mitotracker Red CMX-Rosamine to evaluate lipid content and mitochondrial polarity, respectively, utilising confocal microscopy at ×40. Five images per embryo were evaluated for fluorescence with ImageJ (National Institutes of Health). Cleavage and blastocyst rates, blastocyst stage and grade, Nile Red (sqrt transformed), and Mitotracker (log₁₀ transformed) data were analysed by an ANOVA and means separated by l.s.d. Results indicate that there was no difference for cleavage and blastocyst rates (J: 82.0 ± 4.3, 26.8 ± 3.5%, n = 178; T: 78.6 ± 3.2, 26.8 ± 5.8%, n = 335; TGB: 79.6 ± 5.5, 24.0 ± 7.5%, n = 377, respectively; P > 0.05), and stage and grade (J: 6.72 ± 0.12, 1.21 ± 0.09; T: 6.77 ± 0.11, 1.19 ± 0.08; TGB: 6.97 ± 0.1, 1.16 ± 0.07, respectively; P > 0.05). Nile Red fluorescence intensity was similar (P > 0.05) for TBG (6.3 ± 2.2) and J (7.2 ± 2.7) and different (P < 0.05) from T (13.2 ± 2.6). MitoTracker fluorescence was similar among all treatments (P > 0.05). These results suggest that the mature forms of GDF9 and BMP15 lower lipid content of resulting embryos; however, they do not increase developmental competence of oocytes. Cryopreservation studies are ongoing.