Serum metabolomic characterization in pigs in relation to birth weight category and neonatal nutrition

Abstract

The objective of this study was to characterize developmental differences in low birth weight (LBW) and normal birth weight (NBW) piglets with or without pre-weaning nutrient restriction using serum metabolomic profile analysis. At farrowing, 112 piglets were identified as LBW (1.22 ± 0.28 kg) or NBW (1.70 ± 0.27 kg) and were randomly assigned to receive normal nutrition (NN) or restricted nutrition (RN) (6 h/day no suckling) from days 2 to 28 post farrow (n = 8 pigs/group). On day 28, piglets were weaned onto a common diet. Fasted blood samples were obtained on days 28 and 56 (n = 8 pigs/group) and were analyzed using quantitative metabolomics via a combination of direct injection mass spectrometry with a reverse-phase LC-MS/MS custom assay. Data were normalized using logarithmic transformation and auto-scaling. Partial least squares discriminant analysis (PLS-DA) was carried out to further explore the differential metabolites among the groups (metaboanalyst.ca) with an integrated enrichment and pathway topography analysis. On day 28, LBW piglets had lower levels of essential amino acids as well as reduced metabolites associated with fatty acid oxidation, glycolysis, and the tri-carboxylic acid (TCA) cycle compared to the NBW group. The overall reduction of metabolites associated with energy production and regulation suggests that LBW vs. NBW are in an energy-survival state. On day 56, LBW pigs had increased utilization of fatty acids and resultant ketone production, evident by increased carnitines, acetoacetate, β-hydroxybutyrate, and glycerol compared to NBW pigs. In addition, compared to the NBW pigs LBW pigs had a consistent decrease in serum glucose and lactate as well as reduced TCA cycle metabolites: pyruvate, succinate, citrate, and α-ketoglutaric acid similar to day 28. Low reliance on glycolysis and the TCA cycle and higher glycerol production in the LBW pigs may indicate impairments in glucose tolerance at 56 d. In summary, LBW piglets appear to have more metabolic alterations in early life, which is not resolved with adequate nutrition or refeeding and may elucidate physiological and metabolic mechanisms of poor growth and life performance compared to NBW pigs later in life.

Keywords: low birth weight; metabolomics; nutrition; piglets.

Figure 1.

PLS-DA 2D score plot of the metabolites in serum samples showed separated clusters between the age groups (28 d and 56 d). Within age groups, at 28 d, PLS-DA showed separation in clusters between normal birth weight and restricted nutrition (NBW-RN) and the other groups, and, at 56 d, between low birth weight with normal nutrition (LBW-NN) and NBW-RN. Shaded areas in different colors represent the 95% confidence interval.

Figure 2.

PLS-DA 2D score plot of the metabolites in serum samples of low (LBW) or normal birth weight pigs (NBW) (a) with restricted (RN) or normal nutrient allowance (NN) (c) at 28 d (pre-weaning). The top 15 metabolites with VIP > 1 scores are shown in (b) and (d).

Figure 3.

PLS-DA 2D score plot of the metabolites in serum samples of low (LBW) or normal birth weight pigs (NBW) (a) with restricted (RN) or normal nutrient allowance (NN) (c) at 56 d (post-weaning). The top 15 metabolites with VIP > 1 scores are shown in (b) and (d).

Figure 4.

Topology analysis of metabolic pathways identified among the birth weight categories, neonatal nutrient allowance, and age comparisons. The X-axis represents the pathway impact, and Y-axis represents the pathway enrichment. Larger sizes and darker colors represent greater pathway enrichment and higher pathway impact values, respectively. The black and green dotted lines represent the cut-off values for pathway impact (i.e., higher than 0.1) and −log P-value (i.e., higher than 2), respectively. All pathways identified showed a false discovery rate (FDR) lower than 0.05. I: Glycine, serine, and threonine metabolism; II: alanine, aspartate, and glutamate metabolism; III: D-glutamine and D-glutamate metabolism; IV: arginine biosynthesis; V: arginine and proline metabolism; VI: citrate cycle (TCA cycle); VII: glyoxylate and dicarboxylate metabolism; VIII: aminoacyl-tRNA biosynthesis; IX: butanoate metabolism.

Figure 5.

Pathway network view. Metabolic pathways are represented by nodes colored based on significance, with larger sizes and darker colors representing greater pathway enrichment and higher pathway impact values, respectively.

Figure 6.

Differential metabolites between low birth weight (LBW) and normal birth weight (NBW) piglets pre-weaning (28 d) affects primary metabolic pathways which are critical in normal physiological functioning.

Figure 7.

Differential metabolites between low birth weight (LBW) and normal birth weight (NBW) piglets pre-weaning (56 d) affects primary metabolic pathways which are critical in normal physiological functioning.