131. Genome-wide DNA Methylation Profiling of in Utero Heat Stressed Gilts Genomically Selected for Heat Stress Tolerance or Sensitivity in the F3 Generation.

Abstract In utero heat stress (IUHS) impacts postnatal phenotypes leading to a compromised stress response and reduced productivity relative to in utero thermoneutral (IUTN) exposed pigs. The postnatal effects of IUHS may be mediated by epigenomic modifications, defined as chemical changes in DNA that regulate gene expression without altering the underlying DNA sequence. The study objective was to assess the interaction between divergent genomic selection for heat stress tolerance (TOL) and sensitivity (SEN), and intergenerational IUHS exposure on the whole-genome methylation profile of F3 replacement gilt offspring. Twenty-eight F2 replacement gilts were divided by genetic line, in utero environment, and gestation environment forming eight groups. The TOL and SEN gilts were bred to TOL (n = 3) or SEN (n = 3) sires, respectively, and exposed to thermoneutral (TN; 23.32 ± 1.51 °C) or cyclical heat stress (HS; 27.50 ± 1.07 °C to 36.06 ± 2.00 °C) from gestation d 6.5 ± 1.71 to 59.5 ± 1.71. All pregnant gilts were then maintained at 24.80 ± 1.90 °C after d 60.5 ± 1.71 until farrowing. At weaning, blood samples were collected from 32 median weight F3 replacement gilt offspring (n = 4/treatment combination) representing all litters for whole-genome enzymatic methylation sequencing on Illumina platforms. Bismark workflows were used for bioinformatic analyses. Raw reads quality control and trimming of adapter sequence were performed using TrimGalore, followed by alignment to the Sus scrofa v11.2 reference genome using Bowtie2. Differentially methylated loci were identified using the MethylKit R package, with the threshold FDR < 0.05 and methylation difference ≥ 10%. Differentially methylated cytosine (DMC) analysis was performed between genetic line (GL), F2 dam in utero environment (DIU), and F3 offspring in utero environment (OIU) differences. Genetic line differences yielded 1,140 DMCs (548 hypermethylated and 592 hypomethylated with respect to TOL), F2 dam in utero exposure resulted in 793 DMCs (463 hypermethylated, 330 hypomethylated with respect to IUTN), while F3 offspring in utero exposure produced 901 DMCs (426 hypermethylated, 475 hypomethylated with respect to IUTN). A total of 403 differentially methylated genes (DMGs) were identified across all three scenarios, with 257, 212, and 234 DMGs for GL, DIU, and OIU, respectively. Genes were primarily enriched in signal transduction, cell communication, steroid hormone biosynthesis, and retinol metabolism pathways, among others. The identified genes play a role in the behavioral stress response and glucocorticoid production (MC2R), central nervous system function (EGFR), lipid metabolism (SMPD3), reactive oxygen species generation (CYP2E1), and innate immune defense (PR39). In summary, these observations improve our understanding of the interplay between IUHS and genomic selection for TOL, highlighting the role of epigenomic mechanisms in pigs’ HS responses.