Association between alterations in plasma metabolome profiles and laminitis in intensively finished Holstein bulls in a randomized controlled study

Abstract

Metabolic consequences of an energy and protein rich diet can compromise metabolic health of cattle by promoting a pro-inflammatory phenotype. Laminitis is a common clinical sign, but affected metabolic pathways, underlying pathophysiology and causative relationships of a systemic pro-inflammatory phenotype are unclear. Therefore, the aim of this study was to elucidate changes in metabolome profiles of 20 months old Holstein bulls fed a high energy and protein diet and to identify novel metabolites and affected pathways, associated with diet-related laminitis. In a randomized controlled feeding trial using bulls fed a high energy and protein diet (HEP; metabolizable energy [ME] intake 169.0 ± 1.4 MJ/day; crude protein [CP] intake 2.3 ± 0.02 kg/day; calculated means ± SEM; n = 15) versus a low energy and protein diet (LEP; ME intake 92.9 ± 1.3 MJ/day; CP intake 1.0 ± 0.01 kg/day; n = 15), wide ranging effects of HEP diet on metabolism were demonstrated with a targeted metabolomics approach using the AbsoluteIDQ p180 kit (Biocrates Life Sciences). Multivariate statistics revealed that lower concentrations of phosphatidylcholines and sphingomyelins and higher concentrations of lyso-phosphatidylcholines, branched chain amino acids and aromatic amino acids were associated with an inflammatory state of diet-related laminitis in Holstein bulls fed a HEP diet. The latter two metabolites share similarities with changes in metabolism of obese humans, indicating a conserved pathophysiological role. The observed alterations in the metabolome provide further explanation on the underlying metabolic consequences of excessive dietary nutrient intake.

Figure 1

Exploratory data analysis. (a) Principal component analyses (PCA) of the combined data set of blood metabolome, body weight, insulin concentration, laminitis score, heel horn erosion and interdigital dermatitis score. Group fed low energy and protein diet (LEP): baseline n = 11, slaughter n = 15. Group fed high energy and protein diet (HEP): baseline n = 13, slaughter n = 15. Each dot represents the data of one bull at one sampling day. (b) Scores plot of sparse partial least squares-discriminant analysis (sPLS-DA) between components 1 and 2 applied to the dataset only at slaughter, n = 15 (blood metabolome, blood biochemistry, insulin concentration, body weight, laminitis score, heel horn erosion and interdigital dermatitis score). (c) Loadings of component 1 calculated by sPLS-DA of the dataset at slaughter. The variables listed on the left were ranked by the absolute values of their loadings, the color scheme on the right shows relative differences of the compounds between the feeding groups at slaughter. PC x:y = Phosphatidylcholine with total length of x carbon atoms and y unsaturated bonds in its acyl chains, aa = diacyl, ae = acyl-alkyl.

Figure 2

Claw disease. (a) Score points of laminitis and (b) score points of heel horn erosion at baseline and slaughter. Data shown as means ± SEM, n = 15, different letters indicate significant difference (P < 0.05). HEP = group fed high energy and protein diet, LEP = group fed low energy and protein diet.

Figure 3

Carcass performance. (a) Fat class and (b) conformation class at slaughter. Data shown as means ± SEM, n = 15, different letters indicate significant difference (P < 0.05). HEP = group fed high energy and protein diet, LEP = group fed low energy and protein diet.

Figure 4

Insulin and glucose profile. (a) Insulin concentration in serum at baseline and slaughter. (b) Glucose concentration in plasma at slaughter. Data shown as means ± SEM, n = 15, different letters indicate significant difference (P < 0.05). HEP = group fed high energy and protein diet, LEP = group fed low energy and protein diet.

Figure 5

Heatmap visualizing all differentiating metabolites of interest at slaughter of bulls fed high energy and protein (HEP) and low energy and protein (LEP) diet, including (a) claw disease and growth performance, (b) insulin and blood biochemistry, (c) amino acids, (d) amino acids related, (e) acylcarnitines, (f) sphingomyelins (SM), (g) lyso-phosphatidylcholines (PC), PC aa 34:2 and 6 representative diacyl (aa) and acyl-alkyl (ae) PC. Each column represents one animal’s metabolite profile. All significantly different results are shown in Supplementary Table S1, n = 15, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Proposed pathways from intensive finishing towards laminitis. (a) Possible alterations related to rumen acidosis, (b) possible alterations due to insulin resistance; both potentially causing metabolic inflammation and impaired vascularization which could provoke laminitis. Highlighted factors were observed in Holstein bulls fed high energy and protein diet. Arrows indicate direction of significantly different concentrations in comparison to the group fed low levels of energy and protein. Obesity refers to the documented fat class at slaughter. ROS reactive oxygen species, GLDH glutamate dehydrogenase, PC phosphatidylcholines, SM sphingomyelins, AA amino acids.