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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.

In Korea, various insect species such as crickets and grasshoppers, as well as honey bee and silkworm pupae, have been consumed as food and used in oriental medicine. In this study to evaluate useful the bioactivities and potentially adverse effects of edible insects, ethanol extracts of Allomyrina dichotoma (AD), Tenebrio molitor (TM), Protaetia brevitarsis (PB), Gryllus bimaculatus (GB), Teleogryllus emma (TE), and Apis mellifera (AM) were prepared and evaluated with regard to their anti-thrombosis, anti-oxidant and haemolysis activities against human red blood cells. AD and TE extracts showed strong anti-oxidant activities, which were not related to polyphenol content. All ethanol extracts, except AM extract, showed strong platelet aggregation activities. The platelet aggregation ratios of the extracts were 194%&ndash, 246% of those of the solvent controls. The effects of the AD, TM, PB, GM, and AM extracts on thrombin, prothrombin and various coagulation factors were negligible. Only the extract of TM showed concentration-dependent anti-coagulation activities, with a 1.75-fold aPTT (activated Partial Thromboplastin Time) extension at 5 mg/mL. Of the six insect extracts, TM and AM extracts exhibited potent haemolytic activity. Our results on the insect extracts&rsquo, functional properties suggest that edible insects have considerable potential not just as a food source but as a novel bio-resource as well.

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 the present study, we examined the effect of single nucleotide polymorphisms (SNPs) of leptin (LEP), skeletal muscle glycogen synthase (GYS1), myogenic differentiation 1 (MYOD1), and myogenic factor 5 (MYF5) genes on economic trait association in pigs. LEP/HindIII, MYOD1/DdeI, MYF5/FokI, and GYS1/FokI genotypes were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) from 466 pigs comprised of Duroc, Landrace and Yorkshire breeds. The LEP/HindIII polymorphism differed significantly with respect to average daily gain (ADG) in Duroc pigs (P

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.

Bromodomain-containing protein 2 (BRD2) is a nuclear serine/threonine kinase involved in transcriptional regulation. We investigated the expression and association of the BRD2 gene as a candidate gene for meat quality traits in Berkshire pigs. BRD2 mRNA was expressed at relatively high levels in muscle tissue. Statistical analysis revealed that the c.1709G>C polymorphism of the BRD2 gene was significantly associated with carcass weight, meat color (a*, redness), protein content, cooking loss, water-holding capacity, carcass temperatures 4, 12 and 24 h postmortem, and the 24 h postmortem pH in 384 Berkshire pigs. Therefore, this polymorphism in the porcine BRD2 gene may be used as a candidate genetic marker to improve meat quality traits in 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.

Cell death-inducing DFF45-like effector (CIDE) B is a member of the CIDE family of apoptosis-inducing factors. In the present study, we detected a single nucleotide polymorphism (SNP), c.414G>A, which corresponds to the synonymous SNP 414Arg, in CIDE-B in the Berkshire pigs. We also analyzed the relationships between the CIDE-B SNP and various meat quality traits. The SNP was significantly associated with post-mortem pH24h, water-holding capacity (WHC), fat content, protein content, drip loss, post-mortem temperature at 12 h (T12) and 24 h (T24) in a co-dominant model (P < 0.05). A significant association was detected between the SNP and post-mortem pH24h, fat content, protein content, drip loss, shear force, and T24 in gilts; and color parameter b*, WHC, and T24 in barrows (P < 0.05). The SNP was significantly correlated with the fat content, and CIDE-B mRNA expression was significantly upregulated during the early stage of adipogenesis, suggesting that CIDE-B may contribute towards initiation of adipogenesis (P < 0.05). Furthermore, CIDE-B mRNA was strongly expressed in the liver, kidney, large intestine, and small intestine, and weakly expressed in the stomach, lung, spleen, and white adipose tissue. These results indicate that the CIDE-B SNP is closely associated with meat quality traits and may be a useful DNA marker for improving pork quality.

This study was performed to analyze the difference between the meat properties classified into 5 ranges depending on Berkshire meat quality traits. Post-mortem pH24hr was positively correlated with water holding capacity, but negatively correlated with meat color, protein content, drip loss, and cooking loss. Berkshire gilt was discovered by higher population of RFN (reddish pink, firm, non-exudative), a normal meat class when compared to barrow, whereas DFD (dark, firm, and dry) was not detected and PSE (pale, soft and floppy, exudative) and RSE (reddish pink, soft and floppy, exudative) were found by a low number of individuals. It was assumed that Berkshire gilt is less susceptible to stress when compared with barrow. Therefore, from the results of this study, we suggest that Berkshire gilt is high possibility for production of good meat due to a forming ability for better meat quality than that of Berkshire barrow.

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.

This study was carried out to predict the relationship between the colour and physicochemical traits in pork by using canonical correlation analysis. The variables of pork colour traits were lightness (L*), redness (a*) and yellowness (b*), whereas the variables of physicochemical traits were post-mortem pH24 h, water-holding capacity (WHC), collagen content, fat content, moisture content, protein content, drip loss, cooking loss and shear force. The canonical correlation coefficient (0.819) between the first pair of canonical variates, V1 and W1, was significant (P < 0.01). According to cross loadings, drip loss, cooking loss and fat content provided the relatively high positive correlations with the variates of colour traits (V1), while pH24 h, WHC and moisture content displayed negative relationships with the variates. Otherwise, L* and a* strongly contribute to the variates of physicochemical traits (W1). In addition, a redundancy index of 0.256 suggests that 25.6% of the variance in V1 is explained by W1. Therefore, in order to obtain the reddish pink colour in pork that is preferred by consumers depending on the relationships between the pork quality traits, we suggest that producer leads to the proper maintenance of post-mortem pH24 h, higher WHC, lower drip loss and cooking loss in pork.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and is a leading cause of death. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available human liver tissue maps and studied the differential expression of proteins in normal and cancer tissues. Reference two-dimensional electrophoresis (2-DE) maps of human liver tumor tissue include labeled 2-DE images for total homogenate and soluble fraction separated on pH 3-10 gels, and also images for soluble fraction separated on pH 4-7 and pH 6-9 gels for a more detailed map. Proteins were separated in the first dimension by isoelectric focusing on immobilized pH gradient (IPG) strips, and by 7.5-17.5% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels in the second dimension. Protein identification was done by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-time of flight-mass spectrometry (DE-MALDI-TOF-MS). In total, 212 protein spots (117 spots in pH 4-7 map and 95 spots in pH 6-9) corresponding to 127 different polypeptide chains were identified. In the next step, we analyzed the differential protein expression of liver tumor samples, to find out candidates for liver cancer-associated proteins. Matched pairs of tissues from 11 liver cancer patients were analyzed for their 2-DE profiles. Protein expression was comparatively analyzed by use of image analysis software. Proteins whose expression levels were different by more than three-fold in at least 30% (four) of the patients were further analyzed. Numbers of protein spots overexpressed or underexpressed in tumor tissues as compared with nontumorous regions were 9 and 28, respectively. Among these 37 spots, 1 overexpressed and 15 underexpressed spots, corresponding to 11 proteins, were identified. The physiological significance of the differential expressions is discussed.

Two-dimensional gel electrophoresis (2-DE) maps for human stomach tissue proteins have been prepared by displaying the protein components of the tissue by 2-DE and identifying them using mass spectrometry. This will enable us to present an overview of the proteins expressed in human stomach tissues and lays the basis for subsequent comparative proteome analysis studies with gastric diseases such as gastric cancer. In this study, 2-DE maps of soluble fraction proteins were prepared on two gel images with partially overlapping pH ranges of 4-7 and 6-9. On the gels covering pH 4-7 and pH 6-9, about 900 and 600 protein spots were detected by silver staining, respectively. For protein identification, proteins spots on micropreparative gels stained with colloidal Coomassie Brilliant Blue G-250 were excised, digested in-gel with trypsin, and analyzed by peptide mass fingerprinting with delayed extraction-matrix assisted laser desorption/ionization-mass spectrometry (DE-MALDI-MS). In all, 243 protein spots (168 spots in acidic map and 75 spots in basic map) corresponding to 136 different proteins were identified. Besides these principal maps, overview maps (displayed on pH 3-10 gels) for total homogenate and soluble fraction, are also presented with some identifications mapped on them. Based on the 2-DE maps presented in this study, a 2-DE database for human stomach tissue proteome has been constructed and is available at http://proteome.gsnu.ac.kr/DB/2DPAGE/Stomach/. The 2-DE maps and the database resulting from this study will serve important resources for subsequent proteomic studies for analyzing the normal protein variability in healthy tissues and specific protein variations in diseased tissues.