In pre-weaning calves, both leucine and threonine play important roles in growth and muscle metabolism. In this study, metabolomics, proteomics and clinical chemistry were used to assess the effects of leucine and threonine supplementation added to milk replacer on 14 newborn Holstein male calves: 7 were fed a control diet (Ctrl) and 7 were fed the Ctrl diet supplemented with 0.3% leucine and 0.3% threonine (LT) from 5.6 days of age to 53.6 days. At this time, blood and semitendinosus muscle biopsies were collected for analysis. Integrated metabolomics and proteomics showed that branched-chain amino acids (BCAA) degradation and mitochondrial oxidative metabolism (citrate cycle and respiratory chain) were the main activated pathways in muscle because of the supplementation. BCAA derivatives and metabolites related to lipid mobilization showed the major changes. The deleterious effects of activated oxidative phosphorylation were balanced by the upregulation of antioxidant proteins. An increase in protein synthesis was indicated by elevated aminoacyl-tRNA biosynthesis and increased S6 ribosomal protein phosphorylation in skeletal muscle. In conclusion, LT group showed greater BCAA availability and mitochondrial oxidative activity; as the muscle cells undergo greater aerobic metabolism, antioxidant defenses were activated to compensate for possible cell damage. Data are available via ProteomeXchange (PXD016098). SIGNIFICANCE: Leucine and threonine are essential amino acids for the pre-weaning calf, being of high importance for growth. In this study, we found that leucine and threonine supplementation of milk replacer to feed pre-weaning calves led to differences in the proteome, metabolome and clinical chemistry analytes in skeletal muscle and plasma, albeit no differences in productive performance were recorded. This study extends our understanding on the metabolism in dairy calves and helps optimizing their nutritional status.

Having sensitive serum biomarkers able to determine the structural changes of the small intestine suffering from bacterial digestive diseases could be a valuable tool particularly in piglets at weaning, when intestinal infections are highly prevalent. We evaluated the usefulness of three inflammatory and gut-wall-integrity biomarkers to assess the degree of intestinal histo-morphological damage in piglets. Piglets were orally challenged with Salmonella Typhimurium or enterotoxigenic Escherichia coli (ETEC) to get a variable range of response according to individual variability. Forty-eight piglets were challenged with Salmonella Typhimurium and seventy-two with enterotoxigenic Escherichia coli K88. Clinical signs and faecal score were recorded. At Days 4 and 8 post-inoculation, blood was sampled, animals euthanised and distal ileum dissected. Morphological measures were obtained from the gut tissue, and serum tumour necrosis factor-alpha (TNF-α), pig major acute-phase protein (Pig-MAP) and intestinal fatty acid-binding protein (I-FABP) were determined. Animals developed mild-to-severe diarrhoea after the challenge. When analysing the complete set of analytical results, a high correlation was found among the three serum biomarkers. The most representative morphological indicator was the villus:crypt ratio (V:C), which showed a strong negative correlation with all three biomarkers. Regression analyses between faecal score and the previous variable showed linear relations. When the range of V:C was analysed, based on the quartile distribution of each serum variable, a marked increase in their concentration was observed with greater villus damage. Summarising, the combination of I-FABP, Pig-MAP and TNF-α may be useful for determining the intestinal injury degree and barrier integrity in recently weaned pigs.

The purpose of the present study is to establish the influence of age on serum biochemistry reference intervals (RIs) for unweaned calves and recently-weaned piglets using large number of animals sampled at different ages from populations under different season trials. Specifically, milk replacer (MR)-fed calves from April–July 2017 (n = 60); from December 2016–March 2017 (n = 76) and from April–August 2018 (n = 57) and one group of healthy weaned piglets (n = 72) were subjected to the study. Serum enzymes and metabolites of calves at age of 24 h (24 h after colostrum intake), 2, 5, and 7 weeks from merged trials and piglets at 0, 7, and 14 days post-weaning (at 21, 28, and 35 days of age) were studied. The main variable is age whereas no major trial- or sex-biased differences were noticed. In calves, ALT, AST, GGT, GPx, SOD, NEFAs, triglycerides, glucose, creatinine, total protein, and urea were greatly elevated (p < 0.001) at 24 h compared with other ages; glucose, creatinine, total protein, and urea constantly decreased through the age; cholesterol's lowest level (p < 0.001) was found in 24 h compared with other ages and the levels of haptoglobin remained unchanged (p > 0.1) during the study. In comparison with the adult RIs, creatinine from 24 h, NEFAs from 2 w, GGT from 5 w, and urea from 7 w are fully comparable with RIs or lie within RIs determined for adult. In piglets, no changes were noticed on glucose (p > 0.1) and haptoglobin (p > 0.1) and there were no major changes on hepatic enzymes (ALT, AST, and GGT), total protein, creatinine and urea even though several statistical differences were noticed on 7 days post-weaning. Cholesterol, triglycerides, NEFAs, cortisol and PigMAP were found increased (p < 0.05) while TNF-alpha was found less concentrated (p < 0.001) at 0 days post-weaning compared with other times. Moreover, the RIs of creatinine and GGT are fully comparable with RIs or lie within RIs determined for adult. In conclusion, clinical biochemistry analytes RIs were established for unweaned calves and recently-weaned piglets and among them some can vary at different ages.

The effects of different amino acid (AA) supplementations of milk protein-based milk replacers in pre-ruminant calves from 3 days to 7 weeks of age were studied. Animals were divided into 4 groups: Ctrl) Control group fed with milk protein-based milk replacer without supplementation; GP) supplementation with 0.1% glycine and 0.3% proline; FY) supplementation with 0.2% phenylalanine and 0.2% tyrosine; MKT) supplementation with 0.62% lysine, 0.22% methionine and 0.61% threonine. For statistical analysis, t-test was used to compare AA-supplemented animals to the Ctrl group. At week 7, body weight and average daily gain (ADG) were measured and blood samples and skeletal muscle biopsies were taken. Blood biochemistry analytes related to energy metabolism were determined and it was shown that MKT group had higher serum creatinine and higher plasma concentration of three supplemented AAs as well as arginine compared with the Ctrl group. GP group had similar glycine/proline plasma concentration compared with the other groups while in FY group only plasma phenylalanine concentration was higher compared with Control. Although the AA supplementations in the GP and FY groups did not affect average daily gain and metabolic health profile from serum, the metabolome analysis from skeletal muscle biopsy revealed several differences between the GP-FY groups and the Ctrl-MKT groups, suggesting a metabolic adaptation especially in GP and FY groups.

Peripheral blood mononuclear cells (PBMC) are an interesting sample for searching for biomarkers with proteomic techniques because they are easy to obtain and do not contain highly abundant, potentially masking proteins. Two groups of pigs (n = 56) were subjected to mixing under farm conditions and afterwards subjected to different management treatments: negative handling (NH) and positive handling (PH). Serum and PBMC samples were collected at the beginning of the experiment one week after mixing (t0) and after two months of different handling (t2). Brain areas were collected after slaughter and neurotransmitters quantified by HPLC. Hair cortisol and serum acute phase proteins decreased and serum glutathione peroxidase increased at t2, indicating a lower degree of stress at t2 after adaptation to the farm. Differential gel electrophoresis (DIGE) was applied to study the effects of time and treatment on the PBMC proteome. A total of 54 differentially expressed proteins were identified, which were involved in immune system modulation, cell adhesion and motility, gene expression, splicing and translation, protein degradation and folding, oxidative stress and metabolism. Thirty-seven protein spots were up-regulated at t2 versus t0 whereas 27 were down-regulated. Many of the identified proteins share the characteristic of being potentially up or down-regulated by cortisol, indicating that changes in protein abundance between t0 and t2 are, at least in part, consequence of lower stress upon adaptation to the farm conditions after group mixing. Only slight changes in brain neurotransmitters and PBMC oxidative stress markers were observed. In conclusion, the variation in hair cortisol and serum APPs as well as the careful analysis of the identified proteins indicate that changes in protein composition in PBMC throughout time is mainly due to a decrease in the stress status of the individuals, following accommodation to the farm and the new group.