Transcriptomic profiling of caruncular and cotyledonary tissues of 83 days fetuses in response to early maternal nutrient supplementation

We measured the gene expression profiles of maternal (caruncular; CAR) and fetal (cotyledonary; COT) portions of the placenta to identify differentially expressed genes (DEG), biological processes (BP), and pathways underlying placental development and function in response to early maternal nutrition. To this end, we used the RNA-Seq-based differential expression analysis of bovine placental tissues. The factors examined included vitamin and mineral supplementation (VTM or NoVTM) and rate of gain (low gain – LG or moderate gain – MG). The treatments were arranged as follows: (1) no vitamin and mineral supplementation and low gain (NoVTM_LG, n = 9); (2) vitamin and mineral supplementation and low gain (VTM_LG, n = 9); (3) no vitamin and mineral supplementation and moderate gain (NoVTM_MG, n = 9), and (4) vitamin and mineral supplementation and moderate gain (VTM_MG, n = 8). On day 83 ± 0.27 of gestation the uteroplacental tissues were collected through ovariohysterectomy and the largest placentome closest to the fetus was collected. Then, maternal (CAR) and fetal (COT) portions were manually dissected, snap-frozen, and stored at -80 °C. After RNA quality control, read mapping was performed with STAR aligner to the bovine ARS-UCD1.2 reference genome. Genes with expression values lower than 1 count per million in 50% of the samples were filtered out. After filtering, the genes of CAR and COT tissues were analyzed using the DESeq2 v.1.22.1 software to identify DEGs. The DESEq2 model was used to measure the treatment effect while controlling for batch effect differences that included the surrogated variables (sva) and the heifer's birthplace (farm of origin). Genes were identified as differentially expressed for each one of the contrasts when the adjusted p-value (padj) cutoff < 0.1 and classified as up- or down-regulated based on the sign of the log2 fold change. We identified 267 unique DEGs throughout all tissues and group contrasts. The over-represented functions are essential to fetal growth and development, and included fatty acid metabolism, hormone synthesis, nutrient transport, energy metabolism, and biosynthesis of amino acids. Overall design: Angus-cross heifers (n = 35) were randomly assigned by initial body weight (X ¯ = 359.5 ± 7.1 kg) to a 2×2 factorial arrangement of treatments. The factors examined included vitamin and mineral supplementation (VTM or NoVTM) and rate of gain (low gain – LG or moderate gain – MG). The treatments were arranged as follows: (1) no vitamin and mineral supplementation and low gain (NoVTM_LG, n = 9); (2) vitamin and mineral supplementation and low gain (VTM_LG, n = 9); (3) no vitamin and mineral supplementation and moderate gain (NoVTM_MG, n = 9), and (4) vitamin and mineral supplementation and moderate gain (VTM_MG, n = 8). Diets were delivered once daily via total mixed ration and consisted of triticale hay, corn silage, modified distillers grains plus solubles, ground corn, and if indicated by treatment, mineral premix. To achieve MG (0.79 kg/d), heifers were fed the total mixed ration with the addition of the starch-based protein/energy supplement (a blend of ground corn, dried distillers grains plus solubles, wheat midds, fish oil, urea, and ethoxyquin). The LG heifers were maintained on the basal total mixed ration and targeted to gain 0.28 kg/d. The VTM treatment started 71 to 148 days before artificial insemination by providing 0.45 kg/heifer daily of a ground corn and vitamin and mineral premix (Purina Wind & Rain Storm All-Season 7.5 Complete, Land O'Lakes, Inc., Arden Hills, MN). Based on the VTM starting date, heifers were assigned to one of seven breeding groups so that the supplementation period was at least 60 days for all. At breeding, heifers were randomly assigned to either LG or MG treatments within their respective VTM treatment. Heifers were bred by artificial insemination using female-sexed semen from a single sire. The VTM and rate of gain treatments were carried out until day 83 ± 0.27 of gestation, when uteroplacental tissues were collected through ovariohysterectomy. After QC, 29 and 31 samples (seven or eight samples per group) remained for further analyses from CAR and COT, respectively. To make all pair-wise comparisons between the four treatment groups, six contrasts were created as follows: (1) VTM_MG vs. NoVTM_LG, (2) VTM_MG vs. VTM_LG, (3) VTM_MG vs. NoVTM_MG, (4) VTM_LG vs. NoVTM_LG, (5) VTM_LG vs. NoVTM_MG, (6) NoVTM_MG vs. NoVTM_LG. Multiple testing adjustment of the p-values (padj) was adopted using the Benjamini–Hochberg procedure for false discovery rate (FDR).