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.

Transcriptome signature of liver tissue with divergent mutton odour and flavour using RNA deep sequencing.

Mutton consumption is less popular in many Asian countries including Indonesia, whose consumers often complain about the unpleasant flavour and odour of the meat. The main causes of mutton odour are the two compounds of branched chain fatty acid (BCFA): methylnonanoic (MNA), phenol, 3-methyl (MP), 4-methylnonanoic (MNA) and 4-ethyloctanoic (EOA) present in all the adipose tissue; and the 3-methylindole (MI) or skatole and indole, which are originated from pastoral diets. It is crucial to understand the genetic mechanism of mutton odour and flavour (MOF) to select sheep for lower BCFA and indole thus reduce the unpleasant flavour of meat. The aim of the present study was to investigate transcriptome profiling in liver tissue with divergent MOF using RNA deep sequencing. Liver tissues from higher (n = 3) and lower (n = 3) MOF sheep were analysed using Illumina HiSeq 2500. The total number of reads produced for each liver sample ranged from 21.37 to 25.37 million. Approximately 103 genes were differentially expressed (DEGs) with significance level of p-adjusted value <0.05. Among them, 60 genes were up-regulated, and 43 were down-regulated (p < 0.01, FC > 1.5) in higher MOF group. Differentially regulated genes in high MOF liver samples were enriched in biological processes such as cellular response to chemical stimulus and endogenous stimulus; cellular components such as such as basement membrane and extracellular matrix; and molecular functions such as haeme binding and oxidoreductase activity. Among the DEGs, metabolic phase I related genes belonging to the cytochrome P450 CYP2A6 were dominantly expressed. Additionally, phase II conjugation genes including UDP glucuronosyltransferases UGT2B18, sulfotransferase SULT1C1, and glutathione S-transferase GSTM1 were identified. The dominant candidate genes for SOF could be cytochrome P450, sodium-channel protein, transmembrane protein, glutathione transferase, UDP glucuronosyltransferases and sulfotransferase. Pathway analysis identified steroid hormone biosynthesis and chemical carcinogenesis by cytochrome P450 pathways which may play important roles in MOF-related molecules metabolism. This work highlighted potential genes and gene-networks that may affect meat off flavour and odour in sheep.

Preliminary study of FMO1, FMO5, CYP21, ESR1, PLIN2 and SULT2A1 as candidate gene for compounds related to boar taint.

An association study between polymorphisms of six genes and boar taint related compounds androstenone, skatole and indole was performed in a boar population (n=370). Significant association (P<0.05) was detected for SNP of FMO5 (g.494A>G) with all boar taint compounds, SNP of CYP21 (g.3911T>C) with skatole and indole, and SNP of ESR1 (g.672C>T) with androstenone and indole. mRNA expression of CYP21 and ESR1 was higher in CAB (castrated boar) compared to non-castrated boars; whereas, the expression of FMO5 and ESR1 was higher in LBT (low boar taint) compared to HBT (high boar taint) in liver tissue. FMO5, CYP21 and ESR1 proteins were less detectable in HBT compared with LBT and CAB in liver tissues. These findings suggest that FMO5, CYP21 and ESR1 gene variants might have effects on the boar taint compounds.

Age-associated differential production of IFN-γ, IL-10 and GM-CSF by porcine alveolar macrophages in response to lipopolysaccharide.

The aim of the present study was to investigate the age-related production variation of T helper (Th)-type cytokines (IL-2, IL-4, IFN-γ and IL-10), granulocyte macrophage-colony stimulating factor (GM-CSF) and nitric oxide (NO) by lipopolysaccharide (LPS)-stimulated porcine alveolar macrophages (AMs) in a time-dependent manner. For this purpose, AMs were isolated from 5-days (newborn), 40-days (post-weaned) and 120-days (young) old pigs. Cells were incubated for 24h in the absence or presence of increasing concentrations of LPS (0.0, 0.01, 1.0, 5.0 and 10.0 µg/mL). IL-10, IFN-γ and GM-CSF mRNA expression was upregulated in a dose-dependent manner for all age groups (P<0.05). Age-related differences included a significantly increased IL-10 mRNA and protein production in newborn piglets compared to post-weaned and young pigs. IL-10 production pattern was similar with a higher peak between 12 and 36 h post-induction in all age groups. In contrast, IFN-γ mRNA and protein level was significantly elevated in young pigs 12h and 24h post-induction, respectively, while the time course production of IFN-γ was mostly consistent in newborn and post-weaned piglets. GM-CSF mRNA expression was significantly lower in newborn piglets than in post-weaned and young pigs. The kinetic of GM-CSF expression peaked at 12h in young and post-weaned pigs and at 24h in newborn piglets. IL-4 mRNA levels were very low and no apparent change of IL-2 expression was observed following LPS stimulation in all age groups. Only very low levels of NO were detected in the cell supernatants of young pigs. Collectively, these studies suggest age-related differences in time-dependent production of IL-10, IFN-γ and GM-CSF by porcine AMs with potential immunoregulatory consequences to be explored further.

Expression of Toll-like receptors and downstream genes in lipopolysaccharide-induced porcine alveolar macrophages.

The aim of the present study was to determine the age-related kinetic changes of Toll-like receptors (TLRs) and downstream genes expression, and secretion of cytokine in lipopolysaccharide (LPS) stimulated porcine alveolar macrophages (AM). For this purpose, AMs were isolated from 5-day-old newborn piglets and 120-day-old young pigs. mRNA expression and cytokine measurement was determined by quantitative real-time PCR and ELISA, respectively. First, AMs were incubated for 24 h in the absence or presence of increasing concentrations of LPS. Results showed the up-regulation of TLRs 2, 4, 5 and 9 mRNA from all concentrations of LPS used, as compared to non-stimulated cells, and TLR4 was the highest expression in both ages (P<0.05). Furthermore, quantitative analysis demonstrated increased expression of mRNAs encoding TLRs 2, 4, 5 and 9, LBP, CD14, MD2, MyD88, IRAK4 and TRAF6 in both ages in a time-dependant manner (P<0.05). Overall, LPS inducible mRNA for TLR4, LBP, CD14 and MyD88 had higher expression in newborn piglets compared with those of young pigs (P<0.05). The level of cytokine protein IL6 and TNFα in supernatant fluid significantly varied with time of incubation and age of animals. Their concentration increased immediately at 1 h after LPS stimulation and remained significantly higher up to 48 h in both ages. Production of pro-inflammatory cytokine protein IL6 and TNFα in supernatant was significantly higher in young pigs than those of piglets. This study suggests that differential age-related changes in the expression of TLRs and downstream genes, and pro-inflammatory cytokine could contribute to a different age-related innate immune response during pulmonary infection. Further investigation is warranted to determine the precise effects of LPS on porcine AMs by means of a functional study across a wider age range.

Association study and expression analysis of porcine ESR1 as a candidate gene for boar fertility and sperm quality.

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 ESR1g.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 ESR1g.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.

Association study and expression analysis of CD9 as candidate gene for boar sperm quality and fertility traits.

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 spermatogenesis within the reproductive tracts and will shed light on CD9 as a candidate gene in the selection of good sperm quality boars.