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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.

Litter size is an important trait in the pig industry. Therefore, a lot of effort has been put into improving this trait. DNA methylation is an essential epigenetic modification present in unique DNA sequences. Alterations in methylation can affect transcription and phenotypic variation. The purpose of this study was to investigate the effect of DNA methylation on litter size. Methylation-specific restriction enzymes are simple and useful tools for detecting DNA methylation status. We used a pair of methylation-sensitive isoschizomers, which have the same recognition site, HpaII and MspI. Insulin-like growth factor binding protein 4 (IGFBP4) is a key regulator of ovarian follicular development and fetal growth in eutherian mammals. In this study, we discovered that IGFBP4 was hyper-methylated in the uterus tissue of a larger litter size group using bisulfite sequencing, and validated the positive relationship between the methylation status of IGFBP4 and the total number born of pigs using the porcine methylation-specific restriction enzyme polymerase chain reaction (PMP) assay. We suggest that the IGFPB4 gene can be used as a prognostic biomarker for hyperprolific sows and that the PMP assay is a useful tool for methylation status screening.

Litter size is an economically important trait in the pig industry. We aimed to identify genetic markers associated with litter size, which can be used in breeding programs for improving reproductive traits. Single-nucleotide polymorphisms (SNPs) of Berkshire pigs in the N-acetyltransferase 9 (NAT9) and Mitogen-activated protein kinase kinase kinase 3 (MAP3K3) genes were from RNA sequencing results, and already exist in the databank (NCBI), and were confirmed by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). A total of 272 Berkshire sows were used to examine the genotype, and their association with litter size traits was analyzed. The NAT9 SNP was located in chromosome 12 exon 640 mRNA (A > G) and the MAP3K3 SNP was located in chromosome 12 intron 11 (80, C > T). Association analysis indicated that the GG genotype of NAT9 and the CT genotype of MAP3K3 had the highest values for litter size traits. The GG genotype expressed higher levels of NAT9 mRNA in the endometrium than the other genotypes did, and a positive correlation was found between litter size traits and NAT9, but not MAP3K3 expression level. These results indicate that the NAT9 and MAP3K3 can be used as candidate genes applicable in breeding program for the improvement of litter size traits in Berkshire pigs.

The electron transfer flavoprotein α subunit gene (ETFA) encodes a protein that forms part of the ETF enzyme. This enzyme is normally active in mitochondria, the energy-producing centres of cells, to donate electrons derived from fatty acid oxidation to ETF- ubiquinone oxidoreductase. In the present study, we identified a non-synonymous single-nucleotide polymorphism (nsSNP) (rs321948383, c.569G > A) in the porcine ETFA gene and analyzed the association between this nsSNP and meat quality traits in 405 Berkshire pigs. The pigs with rs321948383 G/G had a higher meat quality from the aspects of meat colour (L*, p = 0.013), water-holding capacity (p = 0.001), drip loss (p = 0.000), carcass temperatures (T1, p = 0.000; T4, p = 0.000; T12, p = 0.000; T24, p = 0.001), and pH24 (p = 0.014). Therefore, this nsSNP (c.569G > A) could play an important role in improving the meat quality of pigs.

Corticosteroid-binding globulin (CBG) is a plasma transport protein that has glucocorticoid-binding activity. In the present study, we identified CBG gene expression in several tissues of four pig breeds: Berkshire, Duroc, Landrace, and Yorkshire. Expression of CBG mRNA was detected in the liver of all four breeds, and was the highest in Berkshire pigs. We also found single nucleotide polymorphisms (SNPs) in the CBG gene from Berkshire pigs, including SNP c.919G>A, which corresponds to 307G>R. We analyzed the relationships between this CBG variant and various meat-quality traits. The SNP was significantly associated with backfat thickness, post-mortem pH24h, meat color [CIE a* (redness), CIE b* (yellowness)], water-holding capacity, fat content, moisture content, protein content, cooking loss, and shear force. However, the effects differed by gender: the values were significantly associated with almost all traits in gilts, whereas only cooking loss and shear force were shown significantly in barrows. The variant G allele was associated with decreases in backfat thickness, post-mortem pH24h, CIE a*, fat content, and cooking loss, but with increases in CIE b*, water-holding capacity, moisture content, protein content, and shear force. Because the general correlation between meat-quality traits were not validated in the present study, we suggest that certain SNP might be used in the restrictive application to distinguish meat-quality traits.

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.

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.

Single nucleotide polymorphisms (SNPs) are useful genetic markers that allow correlation of genetic sequences with phenotypic traits. It is shown here that HSD17B4, a bifunctional enzyme mediating dehydrogenation and anhydration during β-oxidation of long-chain fatty acids, contains a non-synonymous SNP (nsSNP) of chr2:128,825,976A>G, c.2137A>G, I690V, within the sterol carrier protein-2 domain of the HSD17B4 gene, by RNA-Seq of liver RNA. The HSD17B4 mRNA was highly expressed in the kidney and liver among various other tissues in four pig breeds, namely, Berkshire, Duroc, Landrace, and Yorkshire. The nsSNP was significantly associated with carcass weight, backfat thickness, and drip loss (P < 0.05). Furthermore, HSD17B4 may play a crucial role during the early stages of myogenesis when expression of its mRNA was significantly high. In conclusion, HSD17B4 may serve as a possible regulator of muscle development, and its identification should help to select for improved economic traits of Berkshire pigs such as carcass weight, backfat thickness, and drip loss.

The paraoxonase (Pon) gene family contains three members: Pon1, Pon2, and Pon3. Pon3 modulates superoxide production and prevents apoptosis. The role of Pon3 has not been fully elucidated in the pig. This study is the first to investigate the association between Pon3 and meat quality in the Berkshire pig. We identified a single nucleotide polymorphism in the Pon3 gene (c.227A > G) that resulted in a change in histidine to arginine at position 76. To elucidate the role of this non-synonymous single nucleotide polymorphism in the Pon3 gene, we analysed the Pon3 genotype and meat quality traits in 434 Berkshire pigs. The results of a codominant model show that carcass weight, meat colour (lightness), cooking loss, and the Warner–Bratzler shear force were significantly associated with the Pon3 genotype. Furthermore, the 24-h post-mortem pH had the strongest relationship with the Pon3 genotype. The G allele decreased cooking loss and fat content, whereas the A allele increased the 24-h post-mortem pH and decreased backfat thickness, which contribute to meat storage life and M. longissimus dorsi depth respectively. In conclusion, the non-synonymous single nucleotide polymorphism in the Pon3 gene showed a close correlation with meat quality traits in the Berkshire pig.