PS12-17. Effects of maternal rate of body weight gain and one-carbon metabolite supplementation on piRNA expression and predicted mRNA targets in bovine fetal muscle.

Abstract Periods of inadequate maternal nutrition during pregnancy and nutritional imbalances during critical windows of fetal development can have lasting consequences on offspring growth, metabolism, and performance. These effects are largely mediated through epigenetic mechanisms. Among them, small non-coding RNAs (sncRNAs), including piwi-interacting RNAs (piRNAs), are suggested to regulate gene expression in somatic tissues through mRNA targeting. One-carbon metabolites (OCM) are key substrates for epigenetic regulation; however, its potential influence on piRNA-mediated regulation remains poorly characterized. We investigated piRNA expression profiles and predicted piRNA-mRNA interactions in bovine fetal muscle using small RNA and mRNA sequencing data. Crossbred Angus heifers were assigned to a 2 × 2 factorial arrangement of maternal body weight gain (Control, C: 0.45 kg/day; Restricted, R: -0.23 kg/day ADG) and OCM supplementation [+OCM daily rumen-protected methionine (7.4 g/d) and choline (44.4 g/d); weekly injections of 320 mg of folate and 20 mg of vitamin B12]; or -OCM (saline solution). Treatments were applied from breeding to day 63 of gestation, after which all animals received a common control diet. At day 161 of gestation, the fetal Longissimus dorsi muscle was collected for total RNA isolation (n = 7 per group, except C+OCM, n = 8) and small RNA-sequencing. Differentially expressed (DE) piRNAs and mRNAs were identified using DESeq2. Predicted piRNA-mRNA interactions were inferred using IntaRNA, with target regions restricted to the 3′UTR of bovine transcripts (Ensembl release 115, ARSUCD2.0) and an energy threshold of ≤ −25 kJ/mol. A total of 9,803 piRNAs were identified after quality control. DE piRNAs (P < 0.05, |LFC| ≥ 0.5) were identified as follows: C+OCM vs C-OCM (n = 368), R-OCM vs C-OCM (n = 571), R+OCM vs C-OCM (n = 425), R-OCM vs C+OCM (n = 555), R+OCM vs C+OCM (n = 509), and R+OCM vs R-OCM (n = 266). Target prediction was performed for contrasts R-OCM vs C-OCM and R+OCM vs C+OCM. Energy-based prediction identified 135 unique interactions involving 85 DE piRNAs and 58 target genes. For R-OCM vs. C-OCM, 73 interactions were detected targeting 40 genes, whereas R+OCM vs C+OCM resulted in 66 interactions targeting 41 genes, with 23 genes shared between contrasts. Genes targeted by piRNAs were also DE at the mRNA level in the same contrasts: 29 genes in (R-OCM vs C-OCM) and 12 in (R+OCM vs C+OCM), providing dual molecular evidence of coordinated regulation. Among them were included THRB, CEBPB, CIDEA, and MAG in the R-OCM vs C-OCM contrast, and LIPT1, MYT1L, NRG4, and TUBB1 in the R+OCM vs C+OCM contrast. These genes were involved in muscle metabolism and transcriptional regulation. These findings suggest that piRNAs may contribute to fetal muscle programming through gene regulation in response to maternal nutrition.