The effects of dietary supplementation with rumen‐protected amino acids on the expression of several genes involved in the immune system of dairy sheep

Abstract

Amino acids might be a tool to transform animals from a pro- to an anti-inflammatory phenotype through the downregulation of several genes (TLR-4, NF-κB, TNFa, IL-1β, IL-2, IL-6, IL-8, CCL-5 and CXCL-16) whose expression increases during inflammation. To examine this possibility, each of sixty Chios dairy sheep was assigned to one of the following five dietary treatments: A: basal diet (control group); B: basal diet +6.0 g/head rumen-protected methionine (MetaSmart ); C: basal diet +5.0 g/head rumen-protected lysine (LysiGEM ); D: basal diet +6.0 g/head MetaSmart + 5.0 g/head LysiGEM ; and E: basal diet +12.0 g/head MetaSmart + 5.0 g/head LysiGEM. The results showed a significant downregulation in the expression of the TLR-4 gene in both the macrophages and the neutrophils of ewes fed rumen-protected amino acids. Significantly lower mRNA transcript accumulation was also observed for the TNFa, IL-1β and CXCL-16 genes in the macrophages and for the IL-1β gene in the neutrophils of ewes supplemented with amino acids. The ewes that received dietary supplementation with rumen-protected lysine alone (C) had significantly lower CCL-5 transcript levels in their macrophages than the ewes fed the other supplemented diets. Diet D enhanced the mRNA expression of the IL-2 gene in ewe neutrophils. Negative correlations were found between: a. TLR-4, TNFa, IL-1β and CXCL-16 gene expression in macrophages and the milk fat and total solids content; b. CCL-5 gene expression in neutrophils and the milk yield and FCM ; and c. CXCL-16 gene expression and the milk protein content. Moreover, positive correlations were found between the BHBA concentration and the expression of the TLR-4 and CXCL-16 genes in macrophages. In conclusion, the rumen-protected amino acids improved sheep metabolism (as indicated by reduced blood BHBA and urea concentrations), milk chemical composition and immune system function.

The impact of rumen-protected amino acids on the expression of key- genes involved in the innate immunity of dairy sheep

“…Lower mRNA relative expression levels of MyD88 gene in the neutrophils of ewes treated with rumen protected amino acids, in comparison with the normally fed, were found but the results were statistically significant for those consumed the lysine only (Fig 1). These findings are in accordance with the significantly lower transcripts accumulation levels of TLR-4 gene in the neutrophils of ewes fed with the same dietary treatments, as previously reported [10]. Indeed, a significantly positive correlation was found in the neutrophils of ewes between MyD88 and TLR-4 genes (Fig 3).…”

“…The NF-κ B transcription factor can also upregulate the IL-1α gene expression [27]. Thus, the significant reduction in the mRNA transcripts accumulation levels of TLR-4 gene in the neutrophils of treated ewes, which has previously been reported [10], verify the close relationship among TLR-4, NF-κB and IL-1α. This relationship was also confirmed, by the significantly positive correlations, which were found between the expression levels of TLR-4 and NF-κB genes in the neutrophils and TLR-4 and IL-1α genes in monocytes (Fig 3).…”

“…Notwithstanding that the dietary supplementation with rumen protected amino acids did not affect mRNA expression level of MAPK-1 gene, as mentioned earlier evidence confirms the involvement of JunD gene in the IL-1β synthesis in primary macrophages of mouse [35]. Actually, a significant downregulation in the mRNA accumulation level of IL-1β genes has previously been found in both monocytes and neutrophils of treated ewes [10], which may underline the negative regulator role of JunD gene in IL-1β signaling pathway. JunD gene plays also an important role in the defense against oxidative stress [36].…”

“…Cell isolation. Monocytes and neutrophils were isolated from 60 individual blood samples as described by [10]. RNA extraction, removal of gDNA and cDNA synthesis was also performed according to [10].…”

“…Indeed, it has been found that chronically raises concentrations of NEFA in dairy cows, lead to reduced viability of polymorphonuclear neutrophil leukocytes in vitro, which could reduce the immune response to pathogens [9]. However, to the best of our knowledge, scarce information exists on the impact of rumen-protected amino acids on the expression of genes involved in the immune systems of high-producing sheep [10]. More specifically, the dietary supplementation with rumen-protected amino acids such as methionine and/or lysine not only downregulates the expression levels of proinflammatory cytokines (IL-1β, TNF-α), chemokine (CXCL-16) and pathogens recognition receptor-4 (TLR-4) but reduces significantly the BHBA content in ewes [10].…”