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

Abstract

Rumen protected amino acids inclusion in ewes' diets has been proposed to enhance their innate immunity. The objective of this work was to determine the impact of dietary supplementation with rumen-protected methionine or lysine, as well as with a combination of these amino acids in two different ratios, on the expression of selected key-genes (NLRs, MyD88, TRIF, MAPK-1, IRF-3, JunD, TRAF-3, IRF-5, IL-1α, IL-10, IKK-α, STAT-3 and HO-1). Thus, sixty Chios dairy ewes (Ovis aries) were assigned to one of the following five dietary treatments (12 animals/ treatment): A: basal diet consist of concentrates, wheat straw and alfalfa hay (control group); B: basal diet +6.0 g/head rumen-protected methionine; C: basal diet + 5.0 g/head rumen-protected lysine; D: basal diet +6.0 g/head rumen-protected methionine + 5.0 g/head rumen-protected lysine and E: basal diet +12.0 g/head rumen-protected methionine + 5.0 g/head rumen-protected lysine. The results revealed a significant downregulation of relative transcript level of the IL-1α gene in the neutrophils of C and in monocytes of D ewes compared with the control. Significantly lower mRNA transcript accumulation was also observed for the MyD88 gene in the neutrophils of ewes fed with lysine only (C). The mRNA relative expression levels of JunD gene were highly induced in the monocytes, while those of IL-10 and HO-1 genes were declined in the neutrophils of ewes fed with the C and D diets compared with the control. Lower transcript levels of STAT-3 gene were observed in the neutrophils of ewes fed with either C or with E diets in comparison with the control. In conclusion, our results suggest that the dietary supplementation of ewes with rumen-protected amino acids, down regulate the expression of some genes involved in the pro-inflammatory signalling.

Fig 1. Transcript abundance of several genes in the neutrophils of sheep.

NOD-like receptors (NLRs), Interleukin 1a (IL-1a), Myeloid-Differentiation-primary response gene 88 (MyD88), Mitogen-Activated Protein Kinase-1 (MAPK-1), Transcription factor JunD (JunD), TIR (Toll/Interleukin-1 Receptor) domain-containing adaptor protein inducing interferon beta (TRIF), Interferon Regulatory Factor 3 (IRF-3), TNF Receptor-associated Factor 3 (TRAF-3), Interferon Regulatory Factor 5 (IRF-5), Interleukin 10 (IL-10), Signal Transducer and Activator of Transcription 3 (STAT-3), Heme Oxygenase-1 (HO-1) and Conserved Helix-Loop-Helix-Ubiquitous Kinase (CHUK) or IKK-α relative to the geometrical mean of the references genes (Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) and Tyrosine 3-monoxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Bars show means ± SEM of each (n = 12) of the five dietary treatments; A: control, basal diet; B: basal diet +6.0 g/head rumen- protected methionine; C: basal diet +5.0 g/head rumen- protected lysine; D: basal diet +6.0 g/head rumen- protected methionine +5.0 g/head rumen- protected lysine and E: basal diet +12.0 g/head rumen- protected methionine +5.0 g/head rumen- protected lysine fed in ewes. Superscripts with small letters (a, b, c) between the five dietary treatments (A, B, C, D, E) differ significantly (p ≤ 0.05). The units in diagrams are relative.

Fig 2. Transcript abundance of several genes in monocytes of sheep.

NOD-like receptors (NLRs), Interleukin 1a (IL-1a), Myeloid-Differentiation-primary response gene 88 (MyD88), Mitogen-Activated Protein Kinase-1 (MAPK-1), Transcription factor JunD (JunD), TIR (Toll/Interleukin-1 Receptor) domain-containing adaptor protein inducing interferon beta (TRIF), Interferon Regulatory Factor 3 (IRF-3), TNF Receptor-associated Factor 3 (TRAF-3), Interferon Regulatory Factor 5 (IRF-5), Interleukin 10 (IL-10), Signal Transducer and Activator of Transcription 3 (STAT-3), Heme Oxygenase-1 (HO-1) and Conserved Helix-Loop-Helix-Ubiquitous Kinase (CHUK) or IKK-α relative to the geometrical mean of the references genes (Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) and Tyrosine 3-monoxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Bars show means ± SEM of each (n = 12) of the five dietary treatments; A: control, basal diet; B: basal diet +6.0 g/head rumen- protected methionine; C: basal diet +5.0 g/head rumen- protected lysine; D: basal diet +6.0 g/head rumen- protected methionine +5.0 g/head rumen- protected lysine; and E: basal diet +12.0 g/head rumen- protected methionine +5.0 g/head rumen- protected lysine fed in ewes. Superscripts with small letters (a, b, c) between the five dietary treatments (A, B, C, D, E) differ significantly (p ≤ 0.05). The units in diagrams are relative.

Fig 3. Pearson’s heat map correlations between the mRNA expression of several genes in neutrophils and monocytes of sheep.

Toll-like receptors 4 (TLR-4), nuclear factor kappa B (NF-κB), tumor necrosis factor a (TNFa), interleukin 1β (IL-1β), interleukin 2 (IL-2), interleukin 6 (IL-6), interleukin 8 (IL-8), C-C motif chemokine ligand 5 (CCL-5) and chemokine (C-X-C motif) ligand 16 (CXCL-16), NOD-like receptors (NLRs), Interleukin 1a (IL-1a), Myeloid-Differentiation-primary response gene 88 (MyD88), Mitogen-Activated Protein Kinase-1 (MAPK-1), Transcription factor JunD (JunD), TIR (Toll/Interleukin-1 Receptor) domain-containing adaptor protein inducing interferon beta (TRIF), Interferon Regulatory Factor 3 (IRF-3), TNF Receptor-associated Factor 3 (TRAF-3), Interferon Regulatory Factor 5 (IRF-5), Interleukin 10 (IL-10), Signal Transducer and Activator of Transcription 3 (STAT-3), Heme Oxygenase-1 (HO-1) and Conserved Helix-Loop-Helix-Ubiquitous Kinase (CHUK) or IKK-α relative to the geometrical mean of the references genes (Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) and Tyrosine 3-monoxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)).