Effect of bamboo vinegar powder on the expression of the immune-related genes MyD88 and CD14 in weaning piglets.

The aim was to explore the feasibility of using bamboo vinegar powder as an antibiotics substitute in weaning piglets. Forty-five healthy Duroc × Landrance × Yorshire piglets (weight 6.74 ± 0.17 kg; age 31 days) were randomly divided into the control group (basic diet), ANT group (basic diet + 0.12% compound antibiotics), BV1 group (basic diet + 0.1% bamboo vinegar powder), BV5 group (basic diet + 0.5% bamboo vinegar powder) and BV10 group (basic diet + 1% bamboo vinegar powder). MyD88 and CD14 expression in immune tissues was examined using real-time PCR. MyD88 expression in the control group were significantly lower than that in other groups in all tissues (p⟨0.05), while CD14 expression showed the opposite trend. MyD88 expression was significantly higher in the BV10 group than in other groups in lung tissue (P⟨0.05), significantly higher in the ANT group than in the BV1 group in the kidneys (P⟨0.05), significantly higher in the BV10 group than in the BV1 group in the thymus (P⟨0.05), and signifi- cantly higher in the BV1 group than in the BV10 group in the lymphatic tissue (P⟨0.05). These differences between experimental groups were not observed for the CD14 gene (P>0.05). Thus, adding bamboo vinegar powder to the basic diet of weaning piglets had immune effects similar to antibiotics and the effect was dose-dependent. Moreover, the MyD88 and CD14 genes appear to play a role in these immune effects.

Nramp1 gene expression in different tissues of Meishan piglets from newborn to weaning.

Natural resistance-associated macrophage protein gene 1 (Nramp1) plays an important role in the innate immune response of swine, and is believed to influence disease resistance. In this study, a real-time quantitative polymerase chain reaction technique was used to investigate Nramp1 expression in 12 different tissues in newborn and 7-, 14-, 21-, 28-, and 35-day-old Meishan piglets. Results indicated that Nramp1 was expressed to varying degrees in all sample tissues, although expression differed among growth stages. For example, Nramp1 was highly expressed in the spleen, but minimally expressed in heart, liver, and muscle tissues among the various piglet age classes. Overall, Nramp1 expression increased with age, reaching significant levels in 21- and 28-day-old animals. Nramp1 was expressed in all 12 tissues tested; however, expression in spleen, lung, kidney, and thymus tissues was highest among newborns, which is consistent with this gene's role in innate immunity improvement. Before and after weaning, Nramp1 was highly expressed in digestive (stomach) and intestinal (duodenum, jejunum, and ileum) tissues, further indicating a genetic role in both immune regulation to compensate for weaning stress and enhanced development of intestinal immunity.

Promoter identification and analysis of key glycosphingolipid biosynthesis-globo series pathway genes in piglets.

Glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1, and NAGA) play an important regulatory role in the defense against Escherichia coli F18 in piglets. In this study, we identified the transcription initiation site and promoter of this gene cluster by mined previous RNA-seq results using bioinformatics tools. The FUT1 transcription initiation region included five alternative splicing sites and two promoter regions, whereas each of the six other genes had one promoter. Dual luciferase reporter results revealed significantly higher transcriptional activity by FUT1 promoter 2, indicating that it played a more important role in transcription. The promoters of glycosphingolipid biosynthesis genes identified contained a CpG island within the first 500 bp, except for the B3GALNT1 promoter which included fewer CpG sites. These results provide a deeper insight into methylation and the regulatory mechanisms of glycosphingolipid biosynthesis-globo series pathway genes in piglets.

LBP gene methylation involved in mRNA expression and resistance to E. coli F18 in weaned piglets.

Lipopolysaccharide binding protein (LBP) plays an important role in recognizing and regulating endotoxin. In this study, we aimed at clarifying the relationship between the methylation of LBP gene and it's expression, to identify mechanisms involved in resistance to E. coli F18 in Sutai weaned piglets. LBP expression was detected by real-time PCR in duodenum and jejunum tissues from E. coli F18-sensitive or -resistant piglets. The LBP methylation status of the regions with many CG sites upstream of the transcription start site was analyzed by Bbisulfite Sequencing PCR (BSP) +Miseq in jejunum and duodenum tissue. The results showed that LBP expression was significantly higher in the sensitive group than the resistant group in duodenum tissue (p<0.05). There was a negative correlation between the methylation of CpG islands upstream of the LBP transcription start site and its expression; the methylation at two CpG sites in particular was significantly correlated with reduced LBP expression (CpG-1 and CpG-2; p<0.05 and p<0.01, respectively). These indicated that the methylation of CpG-1 and CpG-2 sites in the LBP region is involved in the regulation of LBP expression, and may provide key contributions to resisting E. coli F18 in Sutai weaned piglets.

Expression of key glycosphingolipid biosynthesis-globo series pathway genes in Escherichia coli F18-resistant and Escherichia coli F18-sensitive piglets.

A pioneering study showed that the glycosphingolipid biosynthesis-globo series pathway genes (FUT1, FUT2, ST3GAL1, HEXA, HEXB, B3GALNT1 and NAGA) may play an important regulatory role in resistance to Escherichia coli F18 in piglets. Therefore, we analysed differential gene expression in 11 tissues of two populations of piglets sensitive and resistant respectively to E. coli F18 and the correlation of differential gene expression in duodenal and jejunal tissues. We found that the mRNA expression of the seven genes was relatively high in spleen, liver, lung, kidney, stomach and intestinal tract; the levels in thymus and lymph nodes were lower, with the lowest levels in heart and muscle. FUT2 gene expression in the duodenum and jejunum of the resistant population was significantly lower than that in the sensitive group (P < 0.01). ST3GAL1 gene expression was also significantly lower in the duodenum of the resistant population than in the sensitive group (P < 0.05). No significant differences were observed among the remaining genes. The expression level of FUT1 was extremely significantly positively correlated with FUT2 and B3GALNT1 expression (P < 0.01) and also had a significant positive correlation with NAGA expression (P < 0.05). The expression level of FUT2 had extremely significant positive correlations with FUT1, ST3GAL1 and B3GALNT1 (P < 0.01). These results suggest that FUT2 plays an important role in E. coli F18 resistance in piglets. FUT1, ST3GAL1, B3GALNT1 and NAGA may also participate in the mechanism of resistance to E. coli F18.

Differential expression of FUT1 and FUT2 in Large White, Meishan, and Sutai porcine breeds.

To assess the relationship between the expression of a(1,2)-fucosyltransferase (FUT1 and FUT2) genes and resistance to Escherichia coli F18 in weaned pigs, FUT1 and FUT2 expression levels in Large White, Meishan, and Sutai pigs (with resistance to E. coli F18) were determined using real-time PCR. The results revealed that FUT1 and FUT2 expression levels were higher in the liver, lungs, kidneys, stomach, duodenum, and jejunum than in the muscle and heart. Medium FUT2 expression levels were detected in the spleen, thymus, and lymph nodes. Intestinal FUT1 expression levels were higher in Sutai pigs than in Large White and Meishan pigs (P < 0.05). However, intestinal FUT2 expression levels were lower in Sutai pigs than in Large White and Meishan pigs (P < 0.05). FUT1 and FUT2 expression levels did not differ between Large White and Meishan pigs (P > 0.05). The results revealed that high FUT1 expression levels and low FUT2 expression levels in the intestines of Sutai pigs affected FUT1 and FUT2 enzymes, the synthesis of type 2 H and type 1 H antigens, and E. coli F18 adhesion. Moreover, low FUT2 expression levels conferred resistance to E. coli F18.

Developmental expression of LTßR and differential expression in Escherichia coli F18 resistant/sensitive piglets.

We analyzed LTßR mRNA expression in piglets from birth to weaning and compared the differential expression between Escherichia coli F18-resistant and sensitive populations to determine whether this gene could be used as a genetic marker for E. coli F18 resistance. Sutai piglets of different age groups (8, 18, 30, and 35 days; N = 4 each) and piglets demonstrating resistance/sensitivity to E. coli F18 were used. LTßR expression levels were determined by real-time PCR. The LTßR expression levels in the lymph node, duodenum, and jejunum were significantly higher in 8-day-old piglets than in the other age groups (P < 0.01), and the expression levels were significantly higher in the lungs of 8-day-old piglets than in 35-day-old piglets (P < 0.01) and 30 day-old piglets (P < 0.05). In liver tissue, the expression level was significantly higher in the 35-day-old piglets than in other age groups (P < 0.01). In the stomach tissue, the expression level was significantly higher in 35-day-old piglets than in 18-day-old piglets (P < 0.05). LTßR expression in the lymph nodes was significantly higher in the resistant group than in the sensitive group (P < 0.01), but there was no significant difference in the other tissues (P > 0.05). These results indicate that 8 days after birth is a crucial stage in the formation of mesentery lymph nodes and immune barriers in pigs, and increased expression of LTßR may be beneficial for developing resistance to E. coli F18.

Use of Fluorescence Quantitative Polymerase Chain Reaction (PCR) for the Detection of Escherichia coli Adhesion to Pig Intestinal Epithelial Cells.

An efficient and accurate method to test Escherichia coli (E. coli) adhesion to intestinal epithelial cells will contribute to the study of bacterial pathogenesis and the function of genes that encode receptors related to adhesion. This study used the quantitative real-time polymerase chain reaction (qPCR) method. qPCR primers were designed from the PILIN gene of E. coli F18ab, F18ac, and K88ac, and the pig ß-ACTIN gene. Total deoxyribonucleic acid (DNA) from E. coli and intestinal epithelial cells (IPEC-J2 cells) were used as templates for qPCR. The 2-ΔΔCt formula was used to calculate the relative number of bacteria in cultures of different areas. We found that the relative numbers of F18ab, F18ac, and K88ac that adhered to IPEC-J2 cells did not differ significantly in 6-, 12-, and 24-well culture plates. This finding indicated that there was no relationship between the relative adhesion number of E. coli and the area of cells, so the method of qPCR could accurately test the relative number of E. coli. This study provided a convenient and reliable testing method for experiments involving E. coli adhesion, and also provided innovative ideas for similar detection methods.

Differential expression of Toll-like receptor 4 signaling pathway genes in Escherichia coli F18-resistant and - sensitive Meishan piglets.

The Toll-like receptor 4 (TLR4) signaling pathway is an important inflammatory pathways associated with the progression of numerous diseases. The aim of the present study was to investigate the relationship between TLR4 signaling and resistance to Escherichia coli F18 in locally weaned Meishan piglets. Using a real-time PCR approach, expression profiles were determined for key TLR4 signaling pathway genes TLR4, MyD88, CD14, IFN-α, IL-1ß and TNF-α in the spleen, thymus, lymph nodes, duodenum and jejunum of E. coli F18-resistant and -sensitive animals. TLR4 signaling pathway genes were expressed in all the immune organs and intestinal tissues, and the expression was generally higher in the spleen and lymph nodes. TLR4 transcription was higher in the spleen of sensitive piglets (p<0.05), but there was no significant difference in TLR4 mRNA levels in other tissues. Similarly, CD14 transcription was higher in lymph nodes of sensitive animals (p<0.05) but not in other tissues. IL-1ß expression was higher in the spleen and in the duodenum of resistant piglets (p<0.05, p<0.01, respectively), and there were no significant differences in other tissues. There were also no significant differences in the expression of MyD88, TNF-α and IFN-α between sensitive and resistant piglets (p>0.05). These results further confirm the involvement of the TLR4 signaling pathway in resistance to E. coli F18 in Meishan weaned piglets. The resistance appeared to be mediated via downregulation of TLR4 and CD14, and upregulation of MyD88 that may promote the release of cytokines TNF-α, IL-1ß, IFN-α and other inflammatory mediators which help to fight against E. coli F18 infection.

Association between polymorphisms in exons 4 and 10 of the BPI gene and immune indices in Sutai pigs.

The bactericidal/permeability-increasing protein (BPI) gene has been identified as a candidate gene for disease-resistance breeding. We evaluated whether polymorphisms in exons 4 and 10 of the BPI gene are associated with immune indices [interleukin-2 (IL-2), IL-4, IL-6, interferon-b (IFN-b), IL-10, and IL-12]. In this study, we identified one mutation (C522T) in the BPI exon 4 site and two mutations (A1060G and T1151G) in the BPI exon 10 site. Correlation analysis revealed that in the Sutai pig population, the effect of genotypes at the BPI exon 4 site on the level of IL-6 was significant (P < 0.05), with an effective genotype of CD; moreover, the effect of genotypes at the BPI exon 10 site on the level of IL-12 was significant (P < 0.05), and the effective genotype was AB. The optimal combined genotype was CD-AB, which was more effective regarding the IL-6 and IL-12 levels compared to the other combined genotypes (P < 0.05). These results indicate that single nucleotide polymorphisms and the combined genotypes of BPI exons 4 and 10 affect immune indices in Sutai pigs. Therefore, these genotypes should be further examined as effective markers for disease-resistant breeding of pigs.

Identification of BPI protein produced in different expression system and its association with Escherichia coli F18 susceptibility.

The super antibiotic bactericidal/permeability-increasing (BPI) protein is a member of a new generation of proteins that have been implicated as endotoxin-neutralizing agents. In this study, recombinant porcine BPI protein was obtained by generating porcine BPI encoding prokaryotic, eukaryotic, and yeast expression vectors. Recombinant protein expression was detected in yeast GS115, Escherichia coli, and 293-6E cells by gel electrophoresis and Western blotting. Escherichia coli F18 is the primary Gram-negative bacteria in the gut and the main pathogen leading to diarrhea and edema dis-ease in weaning piglets. Therefore, E. coli F18-resistant and -sensitive Sutai piglets were used to test differential expression of BPI protein by Western blotting and to investigate the potential correlation between BPI protein expression and E. coli F18-susceptibility. Recombinant porcine BPI protein expression was not detected in the prokaryotic and yeast expression systems; however, soluble protein was detected in the eukaryotic expression system. These data indicate the strong bacterio-static action of the BPI protein and confirm the feasibility of obtaining large amounts of recombinant porcine BPI recombinant protein using this eukaryotic expression system. In addition, the BPI protein expres-sion levels in the E. coli F18-resistant group were significantly higher than those in the sensitive group, indicating that high BPI protein ex-pression is associated with resistance to E. coli F18. Our findings pro-vide a basis for further investigations into the development of a drug designed to confer resistance to E. coli F18 in weaning piglets.

Differential expression of porcine TAP1 gene in the populations of pigs.

Transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum (ER) for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. TAP is consisted of two subunits: TAP and TAP2. Using Real-time PCR technology, this study detected tissue expression profile and analyzed the differential expression of TAP1 gene in Sutai Escherichia coli-resistant group, Yorkshire and Meishan pigs. Tissue expression profile revealed that TAP1 gene expressed in all tissues we detected, and the expression levels were high in lung, immune tissues and intestines. Through the comparation of gene expression differention in different populations, TAP1 expression level of Sutai E. coli-resistant group was significantly higher than that of Yorkshire and Meishan populations in liver, spleen, lung, kidney, thymus, lymph, duodenum and jejunum (P<0.05). Meanwhile TAP1 gene was more highly expressed in Sutai E. coli-resistant group than that of Meishan population in stomach (P<0.05). In conclusion, the upregulation of TAP1 expression level in E. coli-resistant group could be related to E. coli F18 infection. In addition, Chinese local pigs may have special immune response and genetic mechanism in resisting E. coli F18 infection which is differing from MHC I moleculars.

Dynamic changes in TAP1 expression levels in newborn to weaning piglets, and its association with Escherichia coli F18 resistance.

The transporter associated with antigen processing (TAP) transports peptides from the cytosol into the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. This study showed the dynamic changes in the TAP1 expression level in newborn to weaning piglets. Tissue expression profiles revealed that the TAP1 gene was expressed at low levels in all tissues, and the expression levels were relatively higher in the lung, spleen, lymph, and thymus; further, no significant difference was observed in the expression in each tissue among the 3 unweaned stages (8, 18, and 30 days). Nevertheless, the postweaning (35 days) expression levels in tissues, including the spleen, lung, lymph, duodenum, and jejunum were significantly higher than those in the unweaned stages. Furthermore, gene ontology and pathway analysis showed that TAP1 took part in 38 biological functions and 5 pathway processes, including ABC transporters and antigen processing and presentation. These analyses showed that the TAP1 gene, which was related to MHC I immune regulation, had a stable and low expression level in unweaned stages; however, its expression increased in the postweaning stages. The high expression level of TAP1 indicated that the gene might play an important role in Escherichia coli F18 resistance.

Age-dependent expression of the BPI gene in Sutai piglets.

We compared and analyzed the expression of the BPI gene of Sutai piglets ranging from newborn to post-weaning days 8, 18, 30, and 35 by the real-time PCR method, in order to determine if it is involved in protection against disease caused by ETEC F18. There was a significant difference between 18 and 35-day expression in the jejunum. There were also significant differences between 35-day expression and expression at the other development stages in the duodenum. There were no significant differences in expression at 8, 18, and 30 days in the jejunum. We conclude that the porcine BPI gene may be the direct factor that resisted the ETEC F18 in weaning piglets, and that the resistance to ETEC F18 in weaning piglets is related to up-regulation of mRNA expression of BPI gene to a certain extent.

TLR4 gene expression in pig populations and its association with resistance to Escherichia coli F18.

TLR4 is the main recognition receptor of bacterial lipopolysaccharides, which play an important role in innate and adaptive immunity. We used real-time PCR to analyze the tissue expression profile and differential expression of TLR4 in 4 pig populations (Escherichia coli F18-resistant Sutai, E. coli F18-sensitive Sutai, Large White, Meishan), in order to determine the role that the TLR4 gene plays in resistance to E. coli F18. We found that TLR4 expressed consistently in the 4 populations, with relatively high levels in immune tissues and the highest level in the lung. Generally, the expression of TLR4 in E. coli F18-sensitive individuals was the highest, followed by that in E. coli F18-resistant, Large White and Meishan. In the spleen, lung, kidney, lymph nodes, and thymus gland, TLR4 expression is significantly higher in the E. coli F18-sensitive than in the other 3 populations; there were no significant differences among E. coli F18-resistant Sutai, Large White, and Meishan. In addition, Gene Ontology and pathway analysis showed that TLR4 takes part in the inflammatory response. We found that porcine TLR4 has consistent tissue specificity in each breed, and downregulation of expression of the TLR4 gene is related to resistance to E. coli F18 in weaning piglets.

Novel TiO2/GO/M-MMT nano-heterostructured composites exhibiting high photocatalytic activity.

This study proposed a technique to enhance the photocatalytic properties of TiO2 using graphene oxide (GO) and modified Montmorillonite (M-MMT). TiO2/GO/M-MMT nano-heterostructured composites were prepared via hydrothermal and co-precipitation. The photocatalytic performance was evaluated by investigating the photodegradation rate and absorption behavior of methyl orange (MO) under visible light irradiation. The results showed that TiO2/GO/M-MMT heterojunction exhibited excellent photocatalytic degradation performance, as the degradation rate of MO was observed to be 99.3% within 150 min. The density of adsorbed MO decreased by 62.1% after 210 min of dark adsorption using the TiO2/GO/M-MMT composite, which was significantly higher than that achieved using M-MMT, GO/M-MMT, and TiO2/M-MMT. The nano-heterostructure increased the effective interface between TiO2, GO, and MMT, which increased the charge transfer ability and prolonged the electron-hole separation time. Therefore, the results of this study can be used to design novel photocatalysts to eradicate environmental pollutants.

Microarray analysis of differential gene expression in sensitive and resistant pig to Escherichia coli F18.

In this study, Agilent two-colour microarray-based gene expression profiling was used to detect differential gene expression in duodenal tissues collected from eight full-sib pairs of Sutai pigs differing in adhesion phenotype (sensitivity and resistance to Escherichia coli F18). Using a two-fold change minimum threshold, we found 18 genes that were differentially expressed (10 up-regulated and eight down-regulated) between the sensitive and resistant animal groups. Our gene ontology analysis revealed that these differentially expressed genes are involved in a variety of biological processes, including immune responses, extracellular modification (e.g. glycosylation), cell adhesion and signal transduction, all of which are related to the anabolic metabolism of glycolipids, as well as to inflammation- and immune-related pathways. Based on the genes identified in the screen and the pathway analysis results, real-time PCR was used to test the involvement of ST3GAL1 and A genes (of glycolipid-related pathways), SLA-1 and SLA-3 genes (of inflammation- and immune-related pathways), as well as the differential genes FUT1, TAP1 and SLA-DQA. Subsequently, real-time PCR was performed to validate seven differentially expressed genes screened out by the microarray approach, and sufficient consistency was observed between the two methods. The results support the conclusion that these genes are related to the E. coli F18 receptor and susceptibility to E. coli F18.

Polymorphism of M307 of the FUT1 gene and its relationship with some immune indexes in Sutai pigs (Duroc x Meishan).

The alpha (1,2)fucosyltransferase (FUT1) gene has been identified as a candidate gene for controlling the expression of the enterotoxigenic Escherichia coli (ETEC) F18 receptor. Polymorphisms were detected at the M307 position in FUT1 of a breeding base population of Sutai pigs and their correlations to immune parameters analyzed. After digestion by Hin6I, three genotypes were identified at M307, AA (frequency 0.235), AG (0.609), and GG (0.156), with significant deviation from Hardy-Weinberg equilibrium (P < 0.01). The hemoglobin and white blood cell count of the AA genotype pigs were significantly higher than those of AG and GG pigs (P < 0.05). The results indicated that AA pigs not only are resistant to edema disease and post-weaning diarrhea in piglets but also have relatively strong resistance to disease in general.

Effects of porcine epidemic diarrhea virus infection on Toll-like receptor expression and cytokine levels in porcine intestinal epithelial cells.

To explore the role of Toll-like receptors (TLRs) and interferon (IFN) in the innate immunity against porcine epidemic diarrhea virus (PEDV), we detected the expression of TLR genes in PEDV-infected IPEC-J2 cells by real-time PCR. We also detected the level of interferon α (IFN-α) and interferon γ (IFN-γ) by enzyme-linked immunosorbent assay (ELISA). Results showed that IPEC-J2 cells exhibited a clear pathological change after PEDV infection at 24 h. In addition, TLR7, TLR9 and TLR10 expressions were significantly upregulated in PEDV-infected IPEC-J2 cells at 24 h. Interestingly, the expression patterns of TLR2 and TLR4 were consistent at different stages of PEDV infection. The expression level of TLR3 decreased significantly with the increase of infection time, but the expression levels of TLR5 and TLR8 genes at 6 h and 12 h were significantly lower than those in the control group (p⟨0.01). There were significant correlations among the expression levels of TLR genes (p⟨0.05). Cytokine detection showed that the secretion level of IFN-α in the PEDV-infected group was significantly higher than that in the control group (p⟨0.01), and IFN-γ at 6 h and 12 h after PEDV infection was significantly higher than that in control group (p⟨0.01). Therefore, our results suggest that PEDV infection can induce innate immune responses in intestinal porcine jejunum epithelial cells, leading to changes in the expression of Toll-like receptors, and can regulate the resistance to virus infection by affecting the release levels of downstream cytokines.

Combined effect of mutations in ADSL and GARS-AIRS-GART genes on IMP content in chickens.

1. The objective of this study was to investigate the effect of ADSL gene, GARS-AIRS-GART gene and their combination genotype on inosine monophosphate content (IMP) in chicken. 2. The chicken breeds used for this study were Recessive White chicken (RW, Jiang-13 strain of white Plymouth Rock) and preserved population of 4 Chinese native chicken breeds, including Silkies, Baier, Tibetan and Xiaoshan. 3. The primers for exon 2 in ADSL gene and 5'UTR region in GARS-AIRS-GART gene were designed and the single nucleotide polymorphisms (SNPs) were detected by PCR-SSCP and DNA sequencing. 4. Two SNPs were detected, C/T substitution at position 3484 in exon 2 of ADSL gene, which was a silent mutation, and C/T point mutation at position -179 in 5'UTR region of GARS-AIRS-GART gene. In ADSL gene, individuals with TT genotype had significantly higher IMP content than CT and CC genotype individuals. No significant difference was observed between CT and CC genotypes. Similar results were obtained for GARS-AIRS-GART gene. The combination of genotypes ADSL and GARS-AIRS-GART genes also had a significant effect on IMP content. Individuals with TTTT genotype had the highest muscle IMP content, while individuals with CCCT genotype had the lowest. 4. We putatively drew the conclusion that the SNPs in these two genes, as well as the combination genotypes, could be used as potential molecular markers for meat quality in chicken.