50 Effects of lipoic acid, L-carnitine, and vitamin C on oocyte maturation and cryopreservation of in vitro-produced bovine embryos

Antioxidants have been used in maturation and cryopreservation media to increase oocyte developmental competence and post-thaw survival rates of bovine in vitro-produced (IVP) embryos. We hypothesised that the use of a combination of lipoic acid (5 μM), L-carnitine (0.5 mM), and vitamin C (0.5 mM) (LLC) in maturation and cryopreservation media would increase blastocyst rates and embryo viability post-thawing. The experimental design was a 2 × 2 factorial for re-expansion rates (maturation [Control, T] and the addition of LLC to the control [TLLC]; and cryopreservation [Control, C], and LLC added to the control [CLLC] media) with six replicates. Oocytes (n = 649) were aspirated from 2–8 mm follicles of abattoir ovaries, randomly assigned to maturation media, matured for 23 h at 38.5°C in 6% CO₂ in air and fertilised with frozen-thawed semen from one of two bulls and cultured in synthetic oviductal fluid for conventional freezing 1 (SCF1) medium (Owen et al. 2022 J. Anim. Sci. 100, skac043) at 38.5°C in 6.2% CO₂, 7% O₂, and 86.8% N₂. On Days 7 and 8, post-fertilisation embryos stage 6–8, grade 1–2 were slow frozen using conventional methods C (Adapt EG with sucrose, ABT 360) or CLLC. Embryos were thawed using conventional methods and assessed for re-expansion after 24 h of culture. Embryonic development was analysed with ANOVA with replicates and bulls in the model for cleavage and blastocyst rates; and for re-expansion, maturation, and cryopreservation, media factors were used and means were separated by least significant difference. Cleavage and blastocyst rates were similar (T: 86.2 ± 0.8, 30.1% ± 2.9, n = 357; TLLC: 88.7 ± 0.9, 28.8% ± 3.4, n = 292, respectively; P > 0.05). Main effects for re-expansion rates were T: 41.8 ± 8.0 n = 103, TLLC: 36.3 ± 9.5 n = 85 and C: 34.5 ± 8.6 n = 94, CLLC: 48.5% ± 8.6 n = 94, respectively; P > 0.05. No interactions were found between antioxidant treatments (T-C: 30.6 ± 11.3 n = 54, T-CLLC: 53.1 ± 11.3 n = 49, TLLC-C: 38.5 ± 12.9 n = 40, TLLC-CLLC: 43.9% ± 12.9 n = 45). These results suggest that the addition of antioxidants LLC in maturation or cryopreservation media does not influence oocyte developmental competence or embryo cryotolerance. While there is a 1.7-fold numerical difference between the mean re-expansion rates for T-C and T-CLLC (30.6 and 53.1%), more embryos should be evaluated to better assess the results found in this study.