Recombinant Phytase Modulates Blood Amino Acids and Proteomics Profiles in Pigs Fed with Low-Protein, -Calcium, and -Phosphorous Diets

Abstract

A beneficial effect of corn-expressed phytase (CEP) on the growth performance of pigs fed with very low-protein (VLP) diets was previously shown. Little is known whether this improvement is related to alterations in the expression profiles of blood proteins and amino acids (AAs). The objective of this study was to investigate whether supplementation of VLP, low-calcium (Ca), and low-P diets with a CEP would alter the blood AAs and protein expression profiles in pigs. Forty-eight pigs were subjected to one of the following groups (n = 8/group) for 4 weeks: positive control (PC), negative control-reduced protein (NC), NC + low-dose CEP (LD), NC + high-dose CEP (HD), LD with reduced Ca/P (LDR), and HD with reduced Ca/P (HDR). Plasma leucine and phenylalanine concentrations were reduced in NC; however, the LD diet recovered the concentration of these AAs. Serum proteomics analysis revealed that proteins involved with growth regulation, such as selenoprotein P were upregulated while the IGF-binding proteins family proteins were differentially expressed in CEP-supplemented groups. Furthermore, a positive correlation was detected between growth and abundance of proteins involved in bone mineralization and muscle structure development. Taken together, CEP improved the blood profile of some essential AAs and affected the expression of proteins involved in the regulation of growth.

Keywords: corn-expressed phytase; low-protein diet; proteomics.

Figure 1

Venn diagrams of total identified proteins in the serum of pigs fed with low-protein diets with or without reduced calcium and phosphorous supplemented with a corn-expressed phytase. (A) the number of total identified proteins across biological replicates (703 proteins) with 25 proteins identified with significantly differential abundance across dietary groups, (B) NC vs. PC, (C) LD vs. NC, (D) HD vs. NC, (E) LDR vs. LD, (F) HDR vs. HD. PC (positive control): normal protein, adequate calcium (Ca) and available phosphorous (aP), no corn-expressed phytase (CEP) added; NC (negative control): low protein, adequate Ca and aP, no CEP added; LD: NC + CEP added at low dose, i.e., 2000 one phytase unit (FTU)/kg of diet; HD: NC + CEP added at high dose, i.e., 4000 FTU/kg of diet; LDR: LD with reduced Ca and P; HDR: HD with reduced Ca and P. n = 7–8 for each dietary group.

Figure 2

Principal Component Analysis (PCA) score plots of differentially expressed protein in the serum of pigs fed with low-protein diets with or without reduced calcium and phosphorous supplemented with a corn-expressed phytase. (A) Overall, (B) NC vs. PC, (C) LD vs. NC, (D) HD vs. NC, (E) LDR vs. LD, (F) HDR vs. HD. Each circle represents an individual pig and the red, blue, green, orange, yellow and black circles are representative of PC, NC, LD, HD, LDR and HDR groups, respectively. PC (positive control): normal protein, adequate calcium (Ca) and available phosphorous (aP), no corn-expressed phytase (CEP) added; NC (negative control): low protein, adequate Ca and aP, no CEP added; LD: NC + CEP added at low dose, i.e., 2000 one phytase unit (FTU)/kg of diet; HD: NC + CEP added at high dose, i.e., 4000 FTU/kg of diet; LDR: LD with reduced Ca and P; HDR: HD with reduced Ca and P. n = 7–8 for each dietary group.

Figure 3

Hierarchical clustering of differentially expressed protein in serum of pigs fed with low-protein diets with or without reduced calcium and phosphorous supplemented with a corn-expressed phytase. (A) Overall, (B) NC vs. PC, (C) LD vs. NC, (D) HD vs. NC, (E) LDR vs. LD, (F) HDR vs. HD. The red color indicates high abundance, and the green color indicates low abundance. PC (positive control): normal protein, adequate calcium (Ca) and available phosphorous (aP), no corn-expressed phytase (CEP) added; NC (negative control): low protein, adequate Ca and aP, no CEP added; LD: NC + CEP added at low dose, i.e., 2000 one phytase unit (FTU)/kg of diet; HD: NC + CEP added at high dose, i.e., 4000 FTU/kg of diet; LDR: LD with reduced Ca and P; HDR: HD with reduced Ca and P. n = 7–8 for each dietary group.

Figure 4

Network analysis of differentially expressed proteins in serum of nursery pigs fed with low-protein diets with or without reduced calcium and phosphorous supplemented with a corn-expressed phytase. Network of interactions of differentially expressed proteins for (A) NC vs. PC, (B) LD vs. NC, (C) HD vs. NC, (D) LDR vs. LD. No protein interactions were detected for HDR vs. HD. The red and green colors are representative of upregulated and downregulated proteins, respectively, and the grey color represents 40% of additional interactors. PC (positive control): normal protein, adequate calcium (Ca) and available phosphorous (aP), no corn-expressed phytase (CEP) added; NC (negative control): low protein, adequate Ca and aP, no CEP added; LD: NC + CEP added at low dose, i.e., 2000 one phytase unit (FTU)/kg of diet; HD: NC + CEP added at high dose, i.e., 4000 FTU/kg of diet; LDR: LD with reduced Ca and P; HDR: HD with reduced Ca and P. n = 7–8 for each dietary group.

Figure 5

Pearson correlation analysis of overall differentially expressed proteins (protein ID) in serum of pigs fed with low-protein diets with or without reduced calcium and phosphorous supplemented with a corn-expressed phytase identified by proteomics approach and growth performance related measurements. The color of circles is based on the Pearson coefficient distribution: red represents a negative correlation (p < 0.05), blue represents a positive correlation (p < 0.05), and white is indicative of nonsignificant correlation (p > 0.05) with larger size circles having a higher correlation coefficient. Feed intake (FI), final body weight (FBW), average daily gain (ADG), gain:feed ratio (G:F), gain:protein ratio (GP), bone mineral density (BMD), bone mineral content (BMC), triglyceride (TG), glucose (GL), cholesterol (CHOL).