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The aim of this research was to screen polymorphism and to perform association study of porcine AMBP (alpha-1-microglobulin/bikunin precursor), GC (group-specific component protein) and PPP1R3B (protein phosphatase 1, regulatory (inhibitor) subunit 3B) genes with meat quality traits as well as to unravel the transcriptional regulation of these genes by expression QTL (eQTL) study. For this purpose, Duroc × Pietrain F2 resource population (DuPi; n = 313) and a commercial breed Pietrain (Pi; n = 110) were used for association and only DuPi for expression and eQTL study. A SNP was identified in the genes AMBP (g.22229C>T), GC (g.398C>T) and PPP1R3B (c.479A>G), respectively. In DuPi SNP of AMBP was associated (P < 0.05) with meat colour, pH1L, pH24L, pH24H and conductivity24L; SNP of GC showed tendency to association (P < 0.10) with pH24H, conductivity1L and thawing loss, and SNP of PPP1R3B was associated (P < 0.05) with meat colour, pH1L, pH24L, pH24H and shear force. In Pi SNPs of AMBP and GC was associated with pH24H and PPP1R3B SNP was associated with pH24L. The mRNA levels in Longissimus dorsi muscle tissue of these three genes were evaluated by using qRT-PCR to identify association between gene expression and meat quality traits as well as to analyse eQTL. The mRNA expression of PPP1R3B associated with pH24L (P < 0.05). Expression of these three genes was higher in animals with low pH of muscle. Linkage analysis using QTL Express revealed ten trans-regulated eQTL on seven porcine autosomes. Suggestive eQTL [P < 0.05, CW (chromosome-wide)] were found for PPP1R3B on SSC3 and 13. These results revealed that genetic variation and gene expression of these genes are associated with the meat quality traits. These three genes could influence meat quality and could be potential positional, physiological and functional candidate gene for meat quality traits in pigs. However, the analysis of eQTL also suggested that we need to consider additional genes encoding for transcrip

The aim of the present study was to detect quantitative trait loci (QTL) for the serum levels of cytokines and Toll-like receptors as traits related to innate immunity in pig. For this purpose, serum concentration of interleukin 2 (IL2), interleukin 10 (IL10), interferon-gamma (IFNG), Toll-like receptor 2 (TLR2) and Toll-like receptor 9 (TLR9) were measured in blood samples obtained from F2 piglets (n = 334) of a Duroc × Piétrain resource population (DUPI) after Mycoplasma hypopneumoniae (Mh), tetanus toxoid (TT) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) vaccination at 6, 9 and 15 weeks of age. Animals were genotyped at 82 genetic markers covering all autosomes. QTL analysis was performed under the line cross F2 model using QTL Express and 33 single QTL were detected on almost all porcine autosomes. Among the single QTL, eight, twelve and thirteen QTL were identified for innate immune traits in response to Mh, TT and PRRSV vaccine, respectively. Besides single QTL, six QTL were identified by a two-QTL model, of which two for TLR9_TT were in coupling phase and one for IL10_PRRSV was in repulsion phase. All QTL were significant at 5% chromosome-wide level including one and seven at 5% genome- and 1% chromosome-wide level significance. All innate immune traits are influenced by multiple chromosomal regions implying multiple gene action. Some of the identified QTL coincided with previously reported QTL for immune response and disease resistance, and the newly identified QTL are potentially involved in the immune function. The immune traits were also influenced by environmental factors like year of birth, age, parity and litter size. The results of this work shed new light on the genetic background of innate immune response and these findings will be helpful to identify candidate genes in these QTL regions related to immune competence and disease resistance in pigs.

Male fertility is impaired through the lack of ESR1 (Estrogen Receptor 1) but little is known about the ESR1 roles in boar spermatogenesis and fertility. Therefore, this research was aimed at investigating the association with sperm quality and boar fertility traits in a total of 300 boars both from purebred Pietrain and Pietrain × Hampshire crosses. A SNP in coding region of ESR1 g.672C > T in exon 1 was associated with sperm motility (P < 0.05) and plasma droplet rate (P < 0.01) while the polymorphism in non-coding region of ESR1 g.35756T > C in inton 1 was associated with non-return rate (P < 0.05). Furthermore, to analyse the mRNA and protein expression of ESR1 in boar reproductive tissues, a total of six boars were divided into two groups [Group I (G-I) and Group II (G-II)], where G-I had relatively better sperm quality. ESR1 expression was higher in tissues collected from G-I boars than those of collected from G-II boars, and the difference in mRNA expression was significant (P < 0.01) in head of epididymis. The ESR1 protein expression results from western blot coincided with the results of qRT-PCR. The ESR1 protein localization observed a strong staining in the cytoplasm of Sertoli cell in the testis, in the epithelial cells in head and tail of epididymis, in smooth muscle in tail of epididymis, and in the post acrosomal region and tail of the spermatozoa. These results will improve the understanding of the functions of the ESR1 in spermatogenesis within the reproductive tract and will shed light on ESR1 as a candidate in the selection of boar with good sperm quality and fertility.

The research aimed to screen for polymorphism, expression of Tenascin C (TNC) and association with meat and carcass quality traits. Three single nucleotide polymorphisms were detected. In a Duroc × Pietrain F2 cross (DuPi) population, g.44488 C > T was associated with meat color and ham weight; g.68794A > G was associated with pH at 24 h post mortem in ham (pH24H) and muscle area but g.68841 C > T was not statistically associated. Genotyping in a commercial Pietrain (Pi) population showed that g.44488 C > T was associated with pH24H, whereas g.68794A > G was associated with conductivity at 45 min post mortem in loin and backfat thickness. Diplotypes showed significant effects on pH24H in both populations. The expression was associated with pH at 45 min post mortem in loin and cooking loss. TNC was significantly higher in animals with higher muscle pH. Linkage analysis revealed four trans-regulated eQTL on four autosomes. These results suggest that TNC could be a potential candidate gene for meat quality traits in pigs.

Cluster-of-differentiation antigen 9 (CD9) gene expressed in the male germ line stem cells is crucial for sperm–egg fusion, and was therefore selected as candidate gene for boar semen quality. The association of CD9 with boar sperm quality and fertility trait was analyzed using a total of 340 boars both from purebred Pietrain and Pietrain × Hampshire crosses. A single nucleotide polymorphism (g.358A>T) in intron 6 was significantly associated with sperm motility (MOT) (P < 0.001), plasma droplet rate (PDR) (P < 0.001) and abnormal spermatozoa rate (ASR) (P < 0.01). Boars were divided into two groups with group 1 (G-I) boars having a higher SCON and SMOT, lower SVOL (sperm volume) and group 2 (G-II) having a lower SCON and SMOT, higher SVOL. The mRNA and protein expression levels were evaluated in reproductive, non-reproductive tissues and spermatozoa from G-I and G-II animals by using quantitative real-time PCR and western blotting. When both reproductive and non-reproductive tissues were examined, highest mRNA was expressed in prostate gland, then in the body of the epididymis, vas deferens and tail of the epididymis. In case of reproductive tissues, CD9 expression was higher in tissues and spermatozoa collected from G-I boars than those collected from G-II boars. The mRNA expression was significantly different (P < 0.05) in body of epididymis from G-I and G-II boars. The CD9 protein expression results from western blot were coincided with the results of qRT-PCR. Moreover, CD9 protein localization in Leydig cells, Sertoli cells, epithelial cells and spermatozoa was remarkable which indicated the important role of CD9 in spermatogenesis process. By using mRNA and protein expression profiles, it could be shown that CD9 plays a crucial role during sperm development, especially within the epididymis where the maturation of the sperm, a key process for the sperm quality and motility takes place. These results will improve the understanding of the functions of the CD9 in

Background Leg weakness issues are a great concern for the pig breeding industry, especially with regard to animal welfare. Traits associated with leg weakness are partly influenced by the genetic background of the animals but the genetic basis of these traits is not yet fully understood. The aim of this study was to identify quantitative trait loci (QTL) affecting leg weakness in pigs. Methods Three hundred and ten F2 pigs from a Duroc × Pietrain resource population were genotyped using 82 genetic markers. Front and rear legs and feet scores were based on the standard scoring system. Osteochondrosis lesions were examined histologically at the head and the condylus medialis of the left femur and humerus. Bone mineral density, bone mineral content and bone mineral area were measured in the whole ulna and radius bones using dual energy X-ray absorptiometry. A line-cross model was applied to determine QTL regions associated with leg weakness using the QTL Express software. Results Eleven QTL affecting leg weakness were identified on eight autosomes. All QTL reached the 5% chromosome-wide significance level. Three QTL were associated with osteochondrosis on the humerus end, two with the fore feet score and two with the rear leg score. QTL on SSC2 and SSC3 influencing bone mineral content and bone mineral density, respectively, reached the 5% genome-wide significance level. Conclusions Our results confirm previous studies and provide information on new QTL associated with leg weakness in pigs. These results contribute towards a better understanding of the genetic background of leg weakness in pigs.

Phospholipase C zeta (PLCz) and cyclooxygenase isoenzyme type 2 (COX-2) are important in spermatogenesis, but their effect has not yet confirmed in pigs. Therefore, this study was aimed to analyse their association with sperm quality and fertility and to identify the mRNA and protein expression in boars reproductive tissues. DNA samples from 231 Pietrain (PI) and 109 Pietrain × Hampshire (PIHA) pigs with records of sperm quality [sperm concentration (SCON), motility, semen volume, plasma droplet and abnormal spermatozoa rate] and fertility (non-return rate and number of piglet born alive) traits were available. A SNP in non-coding region of PLCz g.158 A > C was associated with SCON (p < 0.05) in PIHA population while the polymorphism of COX-2 g.68 G > A in 3′ UTR was not associated with any traits. For mRNA and protein expression study, a total of six boars were divided into two groups with G-I and G-II, where G-I was characterized for relatively better sperm quality. Both genes expressed higher in reproductive tissues compared with non-reproductive tissues. Phospholipase C zeta mRNA expressed higher in testis (p < 0.01), all parts of epididymis and spermatozoa from G-I, while COX-2 expressed higher in testis (p < 0.05), head and body of epididymis (p < 0.01), and spermatozoa from G-II boar. Both proteins were localized in Leydig cells and spermatozoa. These results might shed light on roles of these genes in spermatogenesis as candidate for boar sperm quality and fertility, but still the lack of association across populations should be considered.

ESR2 is involved in oestrogen-related apoptosis in cell cycle spermatogenesis but their effects have not yet confirmed in pig. Therefore, this study was aimed to investigate the association of ESR2 polymorphism with sperm quality and boar fertility traits and to analyse the ESR2 mRNA and protein expressions in boar reproductive tissues. DNA samples from 203 Pietrain (PI) and 100 Pietrain × Hampshire (PIHA) pigs with records of sperm quality [sperm concentration (SCON), motility (MOT), semen volume (VOL), plasma droplet rate (PDR) and abnormal spermatozoa rate (ASR)] and fertility [non-return rate (NRR) and number of piglet born alive (NBA)] traits were available. A SNP in coding region of ESR2 g.35547A>G in exon 5 was associated with MOT and PDR in the PI and with SCON, VOL, MOT and PDR in PIHA population. For mRNA and protein expression study, a total of six boars were divided into two groups with group I (G-I) and group II (G-II) where G-I characterized for relatively a better sperm quality according to the mean of two groups. mRNA expression was higher in brain and testis than that in all parts of epididymis. Both qRT-PCR and western blot analysis revealed that the ESR2 gene expression and protein expression were significantly higher in testis collected from G-II compared with that of G-I boars. Moreover, ESR2 protein localization in germ cell, Leydig and Sertoli cells, epithelial cells and spermatozoa was remarkable, which indicated the important role of ESR2 in spermatogenesis process. These results might shed new light on the roles of ESR2 in spermatogenesis as candidate for boar fertility, but still the lack of association across populations should be considered.

The aim of this research was to screen for polymorphism and to perform an association study of IFI6 with meat and carcass quality traits. A SNP (g.370A>G) was detected which was associated (P < 0.05) with meat colour, pH 24 h post mortem (p.m.) in ham, conductivity 45 min p.m. in loin and conductivity 24 h p.m. in ham, drip loss and carcass length in Duroc × Pietrain and with meat colour, muscle area and ham percentage in the Pietrain population. Highest expression of IFI6 mRNA was detected in skeletal muscle (longissimus dorsi) by qRT-PCR comparing different tissues. Both qRT-PCR and western blot revealed that the IFI6 gene and protein expressions were significantly (P < 0.05) higher in skeletal muscle with low drip loss compared to that of high drip loss. IFI6 protein was localized in the myocytes membrane. Results suggested that IFI6 might play roles in meat and carcass quality and is a potential positional, physiological and functional candidate gene for improving meat quality traits in pigs.

The aim of the present study was to detect quantitative trait loci (QTL) for innate and adaptive immunity in pigs. For this purpose, a Duroc × Pietrain F2 resource population (DUPI) with 319 offspring was used to map QTL for the immune traits blood antibodies and interferon-gamma using 122 microsatellites covering all autosomes. Antibodies response to Mycoplasma hyopneumoniae and tetanus toxoid vaccine and the interferon-gamma (IFNG) serum concentration were measured at three different time points and were used as phenotypes. The differences of antibodies and interferon concentration between different time points were also used for the linkage mapping. Line-cross and imprinting QTL analysis, including two-QTL, were performed using QTL Express. A total of 30 QTL (12, 6, and 12 for mycoplasma, tetanus antibody, and IFNG, respectively) were identified at the 5% chromosome-wide-level significant, of which 28 were detected by line-cross and 2 by imprinting model. In addition, two QTL were identified on chromosome 5 using the two-QTL approach where both loci were in repulsion phase. Most QTL were detected on pig chromosomes 2, 5, 11, and 18. Antibodies were increased over time and immune traits were found to be affected by sex, litter size, parity, and month of birth. The results demonstrated that antibody and IFNG concentration are influenced by multiple chromosomal areas. The flanking markers of the QTL identified for IFNG on SSC5 did incorporate the position of the porcine IFNG gene. The detected QTL will allow further research in these QTL regions for candidate genes and their utilization in selection to improve the immune response and disease resistance in pig.

Osteochondrosis (OC) or leg weakness is an economically important disease of young fast growing pigs and is a concern of animal welfare. The etiology and pathogenesis of osteochondrosis is not fully understood yet, but any abnormalities in the formation of hypertrophic chondrocytes and disrupted blood supply to the growth cartilage are very important predisposing factors. Matrix gla protein (MGP) as a potential calcification inhibitor of extracellular matrix might contribute to the development of OC. Molecular characterization, polymorphisms analysis, methylation at promoter region and expression of MGP gene and protein were performed in both healthy and OC cartilage collected from a Duroc × Pietrain resource population. The porcine MGP gene consists of 4 exons and 3 introns. The full-length MGP cDNA isolated from articular cartilage consists of 606 bp with a 69-bp 5′ UTR, a 312-bp open reading frame with a start codon, a 225-bp 3′ UTR. Three single-nucleotide polymorphisms (SNP) were detected in the intron 1 (A-115G, C-1073T and C-1135A) and one in the 3′UTR (C-3767T). The relative abundance of MGP mRNA was lower (P < 0.05) in OC compared with healthy cartilage. Moreover, the intensity of MGP band was lower (P < 0.05) in OC group when quantified by western blot. Furthermore, one CpG region was identified in MGP promoter and DNA methylation of three CG sites were higher in OC compared with normal cartilage. This suggested that the high DNA methylation at specific CG sites in the MGP promoter might be involved in the down regulation of MGP in OC. Immunofluorescence of normal cartilage collected from pigs of different ages revealed that MGP signals were higher in younger pigs and decreased in the older pigs. The MGP protein was expressed more near to the cartilage canals. These results suggest that the MGP gene might be a potential candidate gene for the development of OC in pigs.