Expression of toll-like receptor 4 and its associated cytokines from peripheral blood mononuclear cells in Leghorn chickens.

Background and Aim: Immune cells require toll-like receptor 4 (TLR4) to respond to lipopolysaccharides (LPS) by releasing pro-inflammatory cytokines. Peripheral blood mononuclear cells (PBMCs) are used to assess changes in cytokines released in response to diseases or pathogens. This study aimed to assess TLR4 gene expression in PBMCs from Leghorn chicken and the release of related cytokines. Materials and Methods: Peripheral blood mononuclear cells were isolated from blood samples obtained from Leghorn chicks. The PBMC cultures were stimulated with various concentrations of LPS (0.01-1 µg/ml). Polymerase chain reaction was used to detect TLR4 expression. The production of tumor necrosis factor-alpha (TNF-α) and interleukins (IL-1ß and IL-6) was quantified using an enzyme-linked immunosorbent assay. Results: We found that TLR4 was expressed in both non-stimulated and stimulated Leghorn chicken PBMCs. In addition, the release of TNF-α, IL-1ß, and IL-6 levels in Leghorn chicken PBMCs increased significantly with an increase in LPS concentration (0.01-1 µg/mL) (p < 0.05). Conclusion: Although TLR4 was expressed in both non-stimulated and stimulated Leghorn chicken PBMCs, its expression was significantly higher in LPS-stimulated PBMCs Therefore, the chicken's endotoxin response can be assessed by evaluating the pro-inflammatory cytokine production from PBMCs.

Muscle fiber characteristics and expression level of Troponin T3, Toll-like receptor 2, and Toll-like receptor 4 genes in chicken meat with white striping.

Background and Aim: The poultry industry faces an emerging muscular defect in chicken meat called white striping (WS). The biological processes associated with WS myopathy are immune system activation, angiogenesis, hypoxia, cell death, and striated muscle contraction. We examined the Troponin T3 (TNNT3), Toll-like receptor 2 (TLR2), and Toll-like receptor 4 (TLR4) genes based on their functions related to muscle contraction and the innate immune system. This study aimed to determine the muscle fiber characteristics (MFCs) and expression level of TNNT3, TLR2, and TLR4 genes in white striping chicken meat (WSCM). Materials and Methods: A total of 428 breast samples were randomly collected from a commercial poultry processing plant. The samples were classified into four levels: 0 (normal), 1 (moderate WS), 2 (severe WS), and 3 (extreme WS). Five samples per group were selected to evaluate MFCs, including total number of muscle fibers, muscle fiber diameter, cross-sectional area, endomysium thickness, and perimysium thickness. Five samples per group were selected for ribonucleic acid (RNA) isolation to evaluate the messenger RNA (mRNA) expression levels of TNNT3, TLR2, and TLR4 genes related to WS. Results: Statistical analysis revealed that the total number of fibers, endomysium thickness, and perimysium thickness significantly differed between groups (p < 0.05). Muscle fiber diameter and cross-sectional area did not significantly differ (p > 0.05). The expression of the TNNT3 gene did not significantly differ among groups (p > 0.05). Toll-like receptor 2 and TLR4 mRNA expression significantly differed among groups (p < 0.05). Conclusion: These detailed MFCs will provide baseline information to observe WS in chicken meat. Toll-like receptor 2 and TLR4 genes may play a role in the occurrence of WS in chicken meat through non-specific immune reactions.

Pro-inflammatory cytokine release from chicken peripheral blood mononuclear cells stimulated with lipopolysaccharide.

Background and Aim: The principal cytokines released by the host on infection include pro-inflammatory cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF). These cytokines were regarded as regulators of the host's response to infection. This study aimed to determine the release of pro-inflammatory cytokines from chicken peripheral blood mononuclear cells (PBMCs) following lipopolysaccharide (LPS) stimulation. Materials and Methods: Blood samples were collected from six Betong chickens. To isolate PBMCs, density gradient centrifugation was utilized. PBMC culture in RPMI1640 with 10% fetal bovine serum was stimulated with various concentrations of LPS (0, 0.01, 0.1, and 1.0 µg/mL). The production of TNF-α, IL-1ß, and IL-6 was determined using an enzyme-linked immunosorbent assay. Results: When the PBMCs were cultured for 24 h with varying doses of LPS, there was no significant variation in cell viability. TNF-α, IL-1ß, and IL-6 levels were measured in Betong chicken PBMC. The release of these cytokines increased considerably as LPS concentration (0.01-1 µg/mL) increased (p<0.05). Conclusion: In vitro studies of the chicken immune response, notably the release of pro-inflammatory cytokines, can be conducted using PBMCs obtained from chicken blood.

Expression levels of filaggrin-2 in relation to drip loss in pigs.

OBJECTIVE: The aim of this study was to investigate the expression level of filaggrin-2 (FLG2) in correlation with drip loss. METHODS: The muscle samples were randomly taken from a local meat supplier. Samples were taken from Longissimus lumborum muscles to evaluate the drip loss (n = 100). Five muscles per group (low and high drip loss) were selected to evaluate FLG2 mRNA and protein expression levels. RESULTS: mRNA of FLG2 gene was not significantly different in pigs with different levels of drip loss (p>0.05). Statistical analysis revealed that FLG2 protein expression levels were significantly different between the drip loss groups. Western blot revealed that the high drip loss group had higher FLG2 protein expression level than the low drip loss group (p<0.001). Moreover, immunohistochemistry revealed the high signal intensity was on the muscle cell membrane and cytoplasm. CONCLUSION: FLG2 protein might play roles in drip loss of pork and will provide the basis for information to improving meat quality traits in pigs.

Hematology and toll-like receptors 2 and 4 and lipopolysaccharide-induced tumor necrosis factor-α factor gene expression in peripheral blood mononuclear cells of Thai indigenous chickens.

Background and Aim: Toll-like receptors (TLRs) play crucial roles in the early phase of infection in the innate immune response against bacteria, viruses, fungi, and parasites. Lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF) is an essential transcription factor that regulates the immune system, apoptosis, and inflammatory cytokines. This study aimed to determine the hematological profile reflecting the immune response related to TLR2 and TLR4 and LITAF gene expression in Thai indigenous chickens. Materials and Methods: Blood samples (2 mL) were randomly obtained from three chicken breeds (black-boned chicken, Fah Luang chicken, and Pradu Hang Dam chicken) at 16 weeks of age (n = 5 per breed). The hematological profile and mRNA expression within the peripheral blood mononuclear cells (PBMCs) were determined by hematological analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Results: The hematological profile differed significantly in terms of red blood cells (RBCs), hemoglobin, and white blood cells (WBCs) (p < 0.05). Black-boned chicken and Fah Luang chicken had lower RBC levels than Pradu Hang Dam chicken. Fah Luang chicken had lower hemoglobin than Pradu Hang Dam chicken. However, Fah Luang chicken had higher WBC levels than Pradu Hang Dam chicken. Hematocrit, heterophils, basophils, eosinophils, lymphocytes, and monocytes did not differ significantly among the groups (p > 0.05). According to qRT-PCR, the expression of the TLR2 gene did not differ significantly among the groups (p > 0.05), while TLR4 and LITAF gene expression did (p < 0.05). Toll-like receptor 4 and LITAF genes were most highly expressed in Fah Luang chicken. Conclusion: The PBMCs of Thai indigenous chickens showed evidence of TLR4 and LITAF gene expression, with higher expression levels observed in Fah Luang chicken. From this preliminary study, it is concluded that TLR4 and LITAF genes might play roles in the main immune system response in Thai indigenous chickens.

Influence of freeze-thawed cycles on pork quality.

OBJECTIVE: This study was conducted to determine the effect of freeze-thawed cycles (Fresh meat, F-T 1 cycle and F-T 2 cycles) on the quality characteristics of porcine longissimus dorsi muscle. METHODS: A total of 20 three-crossbred pigs (Duroc×[Large White×Landrace]) were randomly obtained from a commercial slaughterhouse in Thailand. Muscle samples were immediately taken from 10 to 11th of the longissimus dorsi for histochemical analysis. The muscles were cut into 2.54 cm-thick chops. A minimum of 20 chops were used for each treatment (fresh meat, freeze-thawed 1 and 2 cycles). Individually chops were packaged in polyethylene bags and frozen at -20°C for 6 months followed by thawing in refrigerator at 4°C for 24 h (the 1st freeze-thawed cycle). The freeze-thawed procedure was repeated for two cycles (the 2nd freeze-thawed cycle). Thawing loss, shear force value, citrate synthase activity and muscle fiber characteristics were determined on the muscles. RESULTS: Results showed that increasing of freeze-thawed cycle increased the thawing loss (p<0.01) and citrate synthase activity (p<0.001). Shear force value of fresh meat was higher than freeze-thawed 1 and 2 cycles (F-T 1 cycle and F-T 2 cycles). Freeze-thawed cycles affected muscle characteristics. Muscle fiber area and muscle fiber diameter decreased with an increasing number of freeze-thawed cycles (p<0.001), while the thickness of endomysium and perimysium were increased (p<0.001). CONCLUSION: Repeated freeze-thawed cycles degraded muscle fiber structure and deteriorated pork quality.

Isolation of peripheral blood mononuclear cells and the expression of toll-like receptors in Betong chickens.

BACKGROUND AND AIM: Toll-like receptors (TLRs) comprise microbial sensing receptors present on cell surfaces that are capable of detecting pathogens. The present study aims to examine the expression of TLRs within the peripheral blood mononuclear cell (PBMC) of the Betong chickens. MATERIALS AND METHODS: Blood samples were harvested from 12 Betong (KU line) chickens. Hematological values were calculated. PBMC was isolated from the blood utilizing a Histopaque solution and stored in a RPMI1640 culture medium. Cell viability was investigated using a Trypan Blue dye exclusion test. DNA was extracted from PBMC and the expression of the DNA's TLRs was examined using a polymerase chain reaction. RESULTS: Hematological values were determined from the blood samples collected in this study obtained from healthy Betong chickens. PBMC that was isolated from the Betong chickens possessed cell viability higher than 95% (95.37±1.06). From the examination of TLRs gene expression, results revealed instances of TLR1.1, TLR1.2, TLR2.1, TLR2.2, TLR3, TLR4, TLR5, TLR 7, TLR15, and TLR21 that were present in the PBMC of Betong chickens. CONCLUSION: PBMC isolated from the blood of healthy Betong chickens possessed excellent cell quality. All chicken TLRs were discovered within the PBMC of Betong chickens. Hence, PBMC stands out as one of the premier sources for in vitro studies of chicken immune response.

Fiber characteristics of pork muscle exhibiting different levels of drip loss.

The present study aimed to investigate the fiber characteristics of pork muscle exhibiting different levels of drip loss during storage. The samples were taken from Longissimus dorsi muscle to evaluate drip loss (n = 100). Fifteen muscles per group (low and high drip loss) were selected to evaluate the histological characteristics at 0 and 72 h of storage time. The statistical analysis revealed that a high drip loss group had greater endomysium thickness than a low drip loss group at 0 h of storage time (P < 0.05). There were no significant differences in total number of fibers, fiber diameter, fiber area and perimysium thickness at 0 h of storage time (P > 0.05). At 72 h of storage time, a high drip loss was evident in higher total number of fibers (P < 0.01), but smaller fiber diameter (P < 0.05), fiber area (P < 0.01) and endomysium thickness (P < 0.01) than a low drip loss group. There was no significant difference in perimysium thickness (P > 0.05). In conclusion, drip loss might be affected by muscle structural characteristics during storage.

Expression level of the cytochrome P450c21 (CYP21) protein correlating to drip loss in pigs.

Drip loss is an important meat quality trait of fresh meat affecting economic losses. The cytochrome P450c21 (CYP21) protein has a role on cortisol production and depends on stress. This might affect meat quality. The present study aimed to investigate the expression of CYP21 protein in correlation with drip loss. The samples were taken from the Longissimus dorsi muscle to evaluate drip loss (n = 300). Five muscles per group (low and high drip loss) were selected to evaluate CYP21 protein expression levels. Statistical analysis revealed that CYP21 protein expression levels were significantly difference between the drip loss groups. The high drip loss group had higher CYP21 protein expression levels than the low drip loss group (P < 0.05). Moreover, the high drip loss group had higher optical density values of the CYP21 protein band than the low drip loss group (P < 0.05). In conclusion, the expression of CYP21 protein will provide the basis for information and better understanding of the mechanisms related to drip loss in pork. Further study is warranted to validate these results in other populations.

Blood haematology, muscle pH and serum cortisol changes in pigs with different levels of drip loss.

OBJECTIVE: An experiment was conducted to study the blood haematology, muscle pH, and serum cortisol changes in pigs with different levels of drip loss. METHODS: Two groups (low and high) of 20 animals were selected from 100 pigs based on drip loss. All [Duroc× (Large White×Landrace)] pigs were slaughtered according to standard slaughtering procedures. At exsanguinations, blood samples were taken for the haematological parameters and serum cortisol analysis. The muscle samples were taken from longissimus dorsi muscle to evaluate the muscle pH and drip loss. RESULTS: Haematological parameters of low drip loss group showed higher content of white blood cells and monocytes than high drip loss group (p<0.05). The low drip loss group had higher muscle pH at 45 min (p<0.05) and 24 h (p<0.001) post-mortem than the high drip loss group. However, there was no significant difference in serum cortisol levels (p>0.05). CONCLUSION: Drip loss is mainly affected by the muscle pH decline after slaughter and also might be affected by white blood cells and monocytes.

In vitro and in vivo assessment of inhibitory effect of stevioside on pro-inflammatory cytokines.

OBJECTIVE: Stevioside is a natural non-caloric sweetener which has been reported to have anti-inflammatory activity. The aim of the present study was to examine in vitro and in vivo effects of stevioside on rats plasma levels of tumor necrosis factor- α (TNF-α), interleukin-1ß (IL-1ß), TNF-α and IL-1ß release from lipopolysaccharide(LPS)-stimulated rat peripheral blood mononuclear cells (PBMCs). MATERIALS AND METHODS: Male wistar rats weighing between 170-220 g were given stevioside (0, 500 and 1000 mg/kg BW/day) for 6 weeks. Mononuclear cells were separated from peripheral blood samples. TNF-α and IL-1ß levels in plasma and the release of TNF-α and IL-1ß from PBMCs were determined using rat enzyme-linked immunosorbent assay (ELISA) kits. RESULTS: Plasma levels of TNF-α and IL-1ß were found to be non-detectable in control and groups treated with 500 and 1000 mg/kg of stevioside. Regarding TNF-α release from LPS-stimulated PBMCs, rats that were orally fed with 500 and 1000 mg/kg of stevioside were significantly different (p<0.05) from those in LPS-treated control group (186.8+18.6 and 151.4 + 15.4 vs 248.6+21.4 pg/ml). Additionally, IL-1ß levels in rats treated with 500 and 1000 mg/kg of stevioside were significantly different (p<0.05) from those in LPS-treated control group (220.0+12.1 and 158.1 + 22.6 vs 294.4+16.1 pg/ml). CONCLUSION: Consumption of stevioside has an inhibitory effect on the release of TNF-α and IL-1ß from LPS-stimulated PBMCs in rats.

Association and expression analysis of porcine HNF1A gene related to meat and carcass quality traits.

The aim of this study was to investigate the association and expression of HNF1A gene as a candidate gene for meat and carcass quality traits in pigs. Statistical analysis revealed that the g.8260 A>G polymorphism significantly associated with pH 24(H), meat percentage and muscle area in the F2 Duroc × Pietrain (DuPi, n=313) and with pH 24(L), fat area and backfat thickness in the Pietrain (Pi, n=110) population. HNF1A mRNA and protein expressions were higher (p<0.05) in animals with the low post-mortem muscle pH 24(L). The promoter methylation profiling suggested that methylation was not involved on HNF1A expression regulation (p>0.05) in animal with divergent muscle pH. In conclusion, polymorphism in porcine HNF1A gene could be used as a candidate marker to improve the meat and carcass quality traits, with the consideration of breed-specific effect.

Association and expression quantitative trait loci (eQTL) analysis of porcine AMBP, GC and PPP1R3B genes with meat quality traits.

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, pH(1L), pH(24L), pH(24H) and conductivity(24L); SNP of GC showed tendency to association (P < 0.10) with pH24H, conductivity(1L) and thawing loss, and SNP of PPP1R3B was associated (P < 0.05) with meat colour, pH(1L), pH(24L), pH(24H) and shear force. In Pi SNPs of AMBP and GC was associated with pH(24H) and PPP1R3B SNP was associated with pH(24L). 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 pH(24L) (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 transcription factors (TF), via fine-mapping underlying the eQTL peaks, in order to understand interaction among these genes.