222 Influence of complex proteins and L-carnitine during in vitro maturation on COC and blastocyst metabolism and blastocyst cryosurvival

In vitro oocyte maturation (IVM) media generally contain undefined complex proteins (CP) such as serum and albumin, which are also a source of lipids. While lipids provide energy, excess can influence in vitro embryo production and cryopreservation outcomes. L-carnitine is added to media to regulate/increase the utilisation of lipids. The current study assessed the effect of L-carnitine (L) during IVM, in the presence or absence of CP, on oocyte and embryo metabolism and blastocyst cryopreservation. Eight heifers donated oocytes in six consecutive cycles of ovum pickup in a 2 × 2 factorial IVM study (CP vs CP-free; L-carnitine supplemented [L+] vs free [L−]). Medium for IVM was TCM199 previously described (Tutt et al. 2021 Theriogenology 161, 108–19), supplemented with either 10% (v/v) FBS + 4 mg/mL BSA (CP) or 4 mg/mL PVP40 (D). L-carnitine was added at 2.5 mM. RNAseq (QuantSEqn 3′ mRNA-Seq) was performed on cumulus cells (CC) following IVM, and gene ontology analysis (ShinyGo; http://bioinformatics.sdstate.edu) applied to differentially expressed genes. Glucose, lactate, and pyruvate were measured in spent IVM media using an auto-analyser (RX Imola, Randox Laboratories Ltd); glucose was measured in spent culture media between Days 6–8 using Glucose-Glo assay (Promega Ltd); and Stage 7–9 Grade 1 blastocysts were control-rate frozen in ethylene glycol and sucrose, and post-thaw recovery assessed. Proportions were analysed by logistic regression and metabolism by ANOVA. KEGG pathway analysis of CC genes indicated maturation with CP downregulated lipid/steroid metabolism/biosynthesis related genes, and upregulated apoptosis, immune/inflammation, carbohydrate metabolism, and amino acid biosynthesis related genes. Genes upregulated with addition of L in the presence of CP related to carbon metabolism and amino acid synthesis; when CP was absent, genes downregulated related to inflammation and apoptosis. The presence of CP during IVM increased glucose uptake (CP 1.90 vs D 1.60 nM/COC/h; SED 0.141; P = 0.028) and lactate release (CP 3.90 vs D 2.92 nM/COC/h; SED 0.276; P = 0.001). An interaction (P = 0.016) between CP and L indicated that the addition of CP increased pyruvate consumption when L was present but decreased consumption when L was absent (CP 2.83 vs CP + L 42.00 vs D 4.61 vs D + L 9.92 p.m./COC/h; SED 11.13). The interaction between CP and L during IVM affected blastocyst glucose consumption (P = 0.034) which decreased with the addition of L in the presence of CP but increased when CP was absent (CP 17.6 vs CP + L 13.0 vs D 11.3 vs D + L 15.9; SED 2.37 p.m./h). Postcryopreservation recovery of blastocysts from L groups was slower (P = 0.044) with fewer re-expanded by 6 h (+L 0.679 ± 0.0645 vs −L 0.798 ± 0.0662). However, re-expansion by 24 h was similar for CP and L. Addition of L during IVM alters COC metabolism. However, these effects depend on the presence or absence of CP, with metabolic differences persisting to the blastocyst stage. Despite these different metabolic responses, the addition of L during IVM had negligible effects on embryo post-thaw recovery.

This research was supported by BBSRC BB/R007985/1.