Increasing litter size is of great interest to the pig industry. DNA methylation is an important epigenetic modification that regulates gene expression, resulting in livestock phenotypes such as disease resistance, milk production, and reproduction. We classified Berkshire pigs into two groups according to litter size and estimated breeding value: smaller (SLG) and larger (LLG) litter size groups. Genome-wide DNA methylation and gene expression were analyzed using placenta genomic DNA and RNA to identify differentially methylated regions (DMRs) and differentially expressed genes (DEGs) associated with litter size. The methylation levels of CpG dinucleotides in different genomic regions were noticeably different between the groups, while global methylation pattern was similar, and excluding intergenic regions they were found the most frequently in gene body regions. Next, we analyzed RNA-Seq data to identify DEGs between the SLG and LLG groups. A total of 1591 DEGs were identified: 567 were downregulated and 1024 were upregulated in LLG compared to SLG. To identify genes that simultaneously exhibited changes in DNA methylation and mRNA expression, we integrated and analyzed the data from bisulfite-Seq and RNA-Seq. Nine DEGs positioned in DMRs were found. The expression of only three of these genes (PRKG2, CLCA4, and PCK1) was verified by RT-qPCR. Furthermore, we observed the same methylation patterns in blood samples as in the placental tissues by PCR-based methylation analysis. Together, these results provide useful data regarding potential epigenetic markers for selecting hyperprolific sows.

Litter size is among the most important traits in swine breeding. However, information on the genetics of litter size in pigs is lacking. In this study, we identified single nucleotide polymorphisms (SNPs) in the insulin-like growth factor binding protein 2 and 3 (IGFBP2 and IGFBP3) genes in Berkshire pigs and analyzed their association with litter size traits. The IGFBP2 SNP was located on chromosome 15 intron 2 (455, A > T) and the IGFBP3 SNP was on chromosome 18 intron 2 (53, A > G). The AT type of IGFBP2 and the GG type of IGFBP3 had the highest values for all litter size traits including total number born (TNB), number of pigs born alive, and breeding value according to TNB. Homozygous GG pigs expressed higher levels of IGFBP3 mRNA in the endometrium than pigs of other genotypes, and a positive correlation was observed between litter size traits and IGFBP3 but not IGFBP2 expression level. These results suggest that SNPs in the IGFBP2 and the IGFBP3 gene are useful biomarkers for increasing the reproductive productivity of Berkshire pigs.

In pigs, litter size is typically defined as the total number of piglets born (TNB) or the number of piglets born alive (NBA). Increasing pig litter size is of great economic interest as a means to increase productivity. The capacity of the uterus is a critical component of litter size and may play a central role in prolificacy. In this study, we investigated litter-size-related epigenetic markers in uterine tissue from Berkshire pigs with smaller litter size groups (SLGs) and larger litter size groups (LLGs) using genome-wide bisulfite sequencing (GWBS). A total of 3269 differentially methylated regions (DMRs) were identified: 1566 were hypermethylated and 1703 hypomethylated in LLG compared to SLG. The zona pellucida binding protein (ZPBP) gene was significantly hypomethylated in the LLG promoter region, and its expression was significantly upregulated in uterine tissue. Thus, the methylation status of ZPBP gene was identified as a potential indicator of litter size. Furthermore, we verified its negative correlation with litter size traits (TNB and NBA) in whole blood samples from 172 Berkshire sows as a blood-based biomarker by a porcine methylation-specific restriction enzyme polymerase chain reaction (PMP) assay. The results suggest that the methylation status of the ZPBP gene can serve as a valuable epigenetic biomarker for hyperprolific sows.

In mammals, Squalene epoxidase (SQLE) is an enzyme that converts squalene to 2,3-oxidosqualene, in the early stage of cholesterol generation. Here, we identified single nucleotide polymorphisms (SNPs) in the SQLE gene (c.2565 G > T) by RNA Sequencing from the liver tissue of Berkshire pigs. Furthermore, we found that homozygous GG pigs expressed more SQLE mRNA than GT heterozygous and TT homozygous pigs in longissimus dorsi tissue. Next, we showed that the SNP in the SQLE gene was associated with several meat quality traits including backfat thickness, carcass weight, meat colour (yellowness), fat composition, and water-holding capacity. Rates of myogenesis and adipogenesis induced in C2C12 cells and 3T3-L1 cells, respectively, were decreased by Sqle knockdown. Additionally, the expression of myogenic marker genes (Myog, Myod, and Myh4) and adipogenic marker genes (Pparg, Cebpa, and Adipoq) was substantially downregulated in cells transfected with Sqle siRNA. Moreover, mRNA expression levels of ROS scavengers, which affect meat quality by altering protein oxidation processes, were significantly downregulated by Sqle knockdown. Taken together, our results suggest the molecular mechanism by which SNPs in the SQLE gene can affect meat quality.

Upload this completed form to website with submission ARTICLE INFORMATION Fill in information in each box below Article Type Research article Article Title Investigation of Immunostimulatory Effects of Heat-treated Lactiplantibacillus plantarum LM1004 and its Underlying Molecular Mechanisms Running Title (within 10 words) Immunostimulatory Effects of Heat-treated Lactiplantibacillus plantarum LM1004 Author Won-