Potential of blood biomarkers to estimate optimum amino acid requirements for pig growth

Current requirements of the branched chain amino acids (BCAA) isoleucine (Ile), leucine (Leu), and valine (Val) have been estimated empirically in dose–response experiments using pig growth as the response criteria. The discriminating metabolites to the optimum dietary BCAA levels may be an alternative to animal growth as the response criteria, and could use fewer animals in short-term studies. Previous dose–response studies in our laboratory demonstrated that 0.52 ± 0.1 standardised ileal digestible (SID) Ile:lysine (Lys), 0.70 ± 0.07 SID Val:Lys, and 0.93 ± 0.1 SID Leu:Lys are the minimum BCAA requirements to support the best growth performance of weaned piglets (Soumeh et al. 2014, 2015a, 2015b). The objectives of the current study were to first identify biomarkers of BCAA intake status that are linked to animal growth and second to develop a method to study BCAA requirements in pigs based on blood metabolites in a short-term trial.

Three dose–response experiments were conducted to study growth performance of pigs (10 to 20k g, n = 96 per study) that were fed with increasing levels of SID Ile:Lys (0.42, 0.46, 0.50, 0.54, 0.58, and 0.62); SID Val:Lys (0.58, 0.62, 0.66, 0.70, 0.74, and 0.78); and SID Leu:Lys (0.70, 0.80, 0.90, 1.00, 1.10, and 1.20). At d 8 and 15 of each experiment, after an overnight fast, pigs were supplied with 25 g/kg BW^0.75 of feed and blood samples were collected 3 h later from eight pigs per treatment. Blood samples were analysed by a HPLC−MS in a non-targeted metabolomics approach to determine the metabolic profile of pigs fed increasing dietary levels of BCAA:Lys. Principle component analyses (PCA) and partial least-squares regression (PLS) were used to identify discriminating metabolites. The identified biomarkers were used as response criteria in the next trial using the diets of the previous studies (stored at −20°C) in a 6 × 6 Latin square design (six BCAA levels × six pigs per BCAA level). The experimental diets were fed for 2 days and then the next diet was fed for a total of 12 days. Blood samples were taken after 2 days and analysed for identified biomarkers. Performance data was analysed using the MIXED procedure of SAS (v9.3, SAS Institute Inc., Cary, NC, USA) and metabolic profiling and biomarker identification analysed using multivariate analysis (LatentiX v2.12, LatentiX Aps, Frederiksberg, Denmark). Of the several identified discriminating metabolites in each study, few showed a significant response to increasing dietary levels of Ile, Leu, and Val in the 2-day trials. Fitting different statistical models to these metabolites (Table 1), however, allowed estimation of a minimum requirement for each BCAA that were close to the values determined using traditional growth performance criteria (Nørgaard et al. 2017).

Table 1.  Optimum SID Ile:Lys, Leu:Lys and Val:Lys values for pig growth estimated by broken-line (BL), curvilinear-plateau (CLP) and quadratic (Q) models fitted to blood metabolites

The results indicate that blood biomarkers have potential as response criteria in short-term dose–response studies to estimate BCAA requirements in pigs.