Metabolic disturbances in synovial fluid are involved in the onset of synovitis in heifers with acute ruminal acidosis

Abstract

Acute ruminal acidosis (ARA) is the result of increased intake of highly fermentable carbohydrates, which frequently occurs in dairy cattle and is associated with aseptic polysynovitis. To characterise the metabolic changes in the joints of animals with ARA, we performed an untargeted gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis of synovial fluid. Seven heifers were challenged with an intraruminal oligofructose overload (13 g/kg of body weight [BW]) dissolved in water. Synovial fluid samples were collected at 0, 9 and 24 h post-overload. Metabolome analysis revealed the presence of 67 metabolites. At 9 h post-overload, glyceric acid, cellobiose, fructose and lactic acid were all increased, whereas at 24 h, sorbitol, lactic acid and fructose levels were all increased >10-fold. At 24 h, citric acid and threonine levels were significantly reduced. We detected increased L- and D-lactate, and the presence of interleukin-6 (IL-6) in synovial fluid. Furthermore, using bovine fibroblast-like synoviocytes, we observed that D-lactate induces IL-6 synthesis. Our results suggest that ARA produces severe metabolomic changes in synovial fluid, including disturbances in starch and sucrose metabolism, and increased lactate levels. These changes were observed prior to the appearance of synovitis, suggesting a potential role in the onset of polysynovitis.

Figure 1

The presence of neutrophils in the joint at 24 h after oligofructose overload. In the upper panel, a representative synovial fluid smear from the tarsocrural joint is shown. A box and whisker plot shows the differential count of leukocytes in the synovial fluid of heifers with acute ruminal acidosis (ARA) at 24 h.

Figure 2

Metabolomic profile of synovial fluid before induction of acute ruminal acidosis (ARA). In (A) a representative chromatogram is shown. (B) Metabolic entities detected by gas chromatography–mass spectrometry (GC-MS). (C) Nine more abundant metabolites in synovial fluid. (D) Six less abundant metabolites in synovial fluid. Each bar represents the mean ± S.E.M.

Figure 3

Heatmap and principal component analysis (PCA) of synovial fluid during the induction of acute ruminal acidosis (ARA). In (A) a heatmap of 25 metabolites with the lowest p-values, obtained by ANOVA, are depicted. In (B) PCA of synovial fluid at 0, 9 and 24 h after the induction of ARA.

Figure 4

Changes in the relative abundance of metabolites in synovial fluid 9 h after the induction of acute ruminal acidosis (ARA). Box and whisker plot showing the median relative abundance. Each point represents an experimental animal. *p < 0.05; **p < 0.01 compared to 0 h; Dunn’s multiple comparison test.

Figure 5

Changes in the relative abundance of metabolites in synovial fluid 24 h after the induction of acute ruminal acidosis (ARA). Box and whisker plot showing the median relative abundance. Each point represents an experimental animal. *p < 0.05; **p < 0.01 compared to 0 h; Dunn’s multiple comparison test.

Figure 6

Metabolic pathway altered in synovial fluid after the induction of acute ruminal acidosis (ARA). All matched pathways according to the p-values from the pathway enrichment analysis and pathway impact values from the pathway topology analysis are shown. (A) 9 h and (B) 24 h after oligofructose overload.

Figure 7

Presence of L- and D-lactate in synovial fluid from heifers with acute ruminal acidosis (ARA). (A) L-lactate and (B) D-lactate concentrations are shown. Each bar represents the mean ± S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001 compared to 0 h; Dunn’s multiple comparison test.

Figure 8

Interleukin-6 (IL-6) is increased in the synovial fluid, but not in the plasma of animals with acute ruminal acidosis (ARA). IL-6 concentrations in (A) the synovial fluid and (B) plasma of heifers challenged with oligofructose overload. Each bar represents the mean ± S.E.M. *p < 0.05; ***p < 0.001 compared to 0 h; Dunn’s multiple comparison test.

Figure 9

D-lactate stimulates the synthesis of interleukin-6 (IL-6) in bovine fibroblast-like synoviocytes (FLS). An increase in (A) the expression and (B) release of IL-6 induced by D-lactate are shown. Each bar represents the mean ± S.E.M. *p < 0.05; **p < 0.01; ***p < 0.001 compared to 0 h; Dunn’s multiple comparison test.