57 Embryo freezing reduces IGF2 methylation in lymphocytes from calves with altered redox status (but normal growth and cytokine gene profiles)

In vitro culture can influence early embryo development, thus affecting the health and phenotype of the offspring. Abnormal offspring syndrome (AOS) was observed in some calves after in vitro-produced (IVP) embryo transfer, and it co-exists with altered expression of IGF2/IGF2R. However, it is unknown whether unbalanced redox status, a condition we previously observed in our calves born from frozen-thawed (F-T) vs fresh embryos (i.e. altered PCO₂, HCO₃⁻, TCO₂, base excess, anion gap [AG] without showing overgrowth), triggers alteration of developmentally imprinted and cytokine coding genes. In this study, we investigated (1) the effect of embryo cryopreservation and calf sex on methylation and expression of imprinted developmental genes and cytokines, and (2) the association between parameters of calf fitness and genes by principal component (PC) analysis (PCA). IVP embryos were obtained from oocytes fertilised and cultured in SOFaaci-bovine serum albumin (BSA) in groups with or without fetal calf serum (FCS) until Day 6, and then singly 24 h without protein. Calf signs (13 features) and analytes (18; Vetscan i-STAT One analyser) were measured in whole blood at birth after first colostrum intake in n = 6 and n = 13 fresh and F-T calves, respectively. Peripheral blood lymphocytes were obtained. After DNA and mRNA extraction, expression of IL1R1, IL12B, IGF2R, H19, IGF2, and methylation of H19, IGF2, IGF2R, ILR1, IL3, IL4, IL12B, was analysed. The effects of cryopreservation and calf sex were analysed by Kruskal-Wallis test. Cryopreservation significantly reduced methylation level (P = 0.029) and tended to reduce mRNA expression of IGF2 (P = 0.0896), which was also affected by calf sex (P = 0.0791). After PCA, three PCs covered 100% variability. PC1 associated acid-base equilibrium (i.e. pH, PCO₂, base excess, HCO₃⁻, TCO₂, lactate, and AG), protein metabolism (creatinine and urea), and Cl⁻ and Ca²⁺, with the expression of IGF2 and IL1R1, and methylation of IL4 and IL12B. PC2 linked expression and methylation of H19 with certain offspring growth parameters (i.e. gestation length, calf size, capillary refill time [CRT], temperature, breaths/min) and fitness (haemoglobin, haematocrit, Ca²⁺, glucose, Na⁺, K⁺, PO₂, and sO₂). PC3 correlated different from PC2 calf growth parameters (i.e. birthweight and chest perimeter), and signs that measure dehydration (i.e. increased CRT with decreased heartbeats) and HCO₃⁻, TCO₂, K⁺, and Cl⁻ with methylation of IGF2, IGF2R, IL1R1, and IL3, and expression of H19, IGF2, IGF2R, and IL12B. The expression level of genes that covariated in PC3 was generally contrary to their methylation degree, and conversely related to the calf covariates. Together with reduced IGF2 methylation at birth in calves from F-T embryos, calf growth, redox, and dehydration are linked to functioning of developmental genes and cytokines.

This research was supported by Horizon 2020 No 952908 – GLOMICAVE-. IG: MINECO BES-2017-082200. FEDER.