Metabolomics and proteomics insights into hepatic responses of weaned piglets to dietary Spirulina inclusion and lysozyme supplementation

Abstract

Background: Studying the effect of dietary Spirulina and lysozyme supplementation on the metabolome and proteome of liver tissue contributes to understanding potential hepatic adaptations of piglets to these novel diets. This study aimed to understand the influence of including 10% Spirulina on the metabolome and proteome of piglet liver tissue. Three groups of 10 post-weaned piglets, housed in pairs, were fed for 28 days with one of three experimental diets: a cereal and soybean meal-based diet (Control), a base diet with 10% Spirulina (SP), and an SP diet supplemented with 0.01% lysozyme (SP + L). At the end of the trial, animals were sacrificed and liver tissue was collected. Metabolomics analysis (n = 10) was performed using NMR data analysed with PCA and PLS-DA. Proteomics analysis (n = 5) was conducted using a filter aided sample preparation (FASP) protocol and Tandem Mass Tag (TMT)-based quantitative approach with an Orbitrap mass spectrometer.

Results: Growth performance showed an average daily gain reduction of 9.5% and a feed conversion ratio increase of 10.6% in groups fed Spirulina compared to the control group. Metabolomic analysis revealed no significant differences among the groups and identified 60 metabolites in the liver tissue. Proteomics analysis identified 2,560 proteins, with 132, 11, and 52 differentially expressed in the Control vs. SP, Control vs. SP + L and SP vs. SP + L comparisons, respectively. This study demonstrated that Spirulina enhances liver energy conversion efficiency, detoxification and cellular secretion. It improves hepatic metabolic efficiency through alterations in fatty acid oxidation (e.g., upregulation of enzymes like fatty acid synthase and increased acetyl-CoA levels), carbohydrate catabolism (e.g., increased glucose and glucose-6-phosphate), pyruvate metabolism (e.g., higher levels of pyruvate and phosphoenolpyruvate carboxykinase), and cellular defence mechanisms (e.g., upregulation of glutathione and metallothionein). Lysozyme supplementation mitigates some adverse effects of Spirulina, bringing physiological responses closer to control levels. This includes fewer differentially expressed proteins and improved dry matter, organic matter and energy digestibility. Lysozyme also enhances coenzyme availability, skeletal myofibril assembly, actin-mediated cell contraction, tissue regeneration and development through mesenchymal migration and nucleic acid synthesis pathways.

Conclusions: While Spirulina inclusion had some adverse effects on growth performance, it also enhanced hepatic metabolic efficiency by improving fatty acid oxidation, carbohydrate catabolism and cellular defence mechanisms. The addition of lysozyme further improved these benefits by reducing some of the negative impacts on growth and enhancing nutrient digestibility, tissue regeneration, and overall metabolic balance. Together, Spirulina and lysozyme demonstrate potential as functional dietary components, but further optimization is needed to fully realize their benefits without compromising growth performance.

Keywords: Carbohydrase; Liver metabolome; Liver proteome; Lysozyme; Piglets; Spirulina.

Fig. 1

Influence of experimental diets on: Final weight (a), ADFI (b) and FCR (c) of piglets. ADFI—Average daily feed intake; FCR—feed conversion ratio. Control—control diet; SP—10% Spirulina diet; SP + L—10% Spirulina diet supplemented with lysozyme..^a,b Values within a row with different superscripts differ significantly at P < 0.05

Fig. 2

Representative 800 MHz ¹H 1D NOESY spectrum of liver aqueous fraction in piglets. Key: (1) 2-Hydroxyvalerate; (2) 4-Hydroxyphenyllactate; (3) 4-Pyridoxate; (4) acetate; (5) adenine; (6) adenosine; (7) ADP; (8) alanine; (9) allantoin; (10) AMP; (11) anserine; (12) arginine; (13) ascorbate; (14) asparagine; (15) aspartate; (16) ATP; (17) betaine; (18) choline; (19) creatine; (20) creatinine; (21) cytidine; (22) ethanolamine; (23) formate; (24) fumarate; (25) glucose; (26) glucose-6-phosphate; (27) glutamate; (28) glutamine; (29) glutathione; (30) glycerol; (31) glycine; (32) GTP; (33) guanosine; (34) histidine; (35) IMP; (36) inosine; (37) isoleucine; (38) lactate; (39) leucine; (40) lysine; (41) maltose; (42) mannose; (43) methanol; (44) methionine; (46) NAD + ; (47) NADP + ; (48) nicotinurate; (49) o-phosphocholine; (50) o-phosphoethanolamine; (51) phenylalanine; (52) proline; (53) riboflavin; (54) sarcosine; (55) serine; (56) sn-glycerol-3-phosphocholine; (57) succinate; (58) taurine; (59) threonine; (60) trigonelline; (61) tryptophan; (62) tyrosine; (63) UDP-galactose; (64) UDP-glucose; (65) UDP-glucuronate; (66) UDP-N-Acetylglucosamine; (67) UMP; (68) uracil; (69) uridine; (70) valine

Fig. 3

Box plot of identified higher concentration metabolites in aqueous fraction of liver tissue of piglets

Fig. 4

Box plot of lower identified metabolites in aqueous fraction of liver tissue of piglets

Fig. 5

PCA (A) and PLS-DA (B) scatterplot of liver metabolite composition for the three experimental groups. PCA—Principal Components Analysis; PLS-DA—Partial Least Squares Discriminant Analysis. 1—Control; 2—10% Spirulina diet (SP); 3—10% Spirulina diet supplemented with lysozyme (SP + L)

Fig. 6

sPLS-DA scatterplot of liver metabolite composition for the three experimental groups sPLS-DA—Sparce Partial Least Squares Discriminant Analysis. 1—Control; 2—10% Spirulina diet (SP); 3—10% Spirulina diet supplemented with lysozyme (SP + L)

Fig. 7

PCA of quantified proteins from the liver of piglets fed with the three experimental diets. PCA -Principal Component Analysis. Control - control diet; SP - 10% Spirulina diet; SP+L - 10% Spirulina diet supplemented with lysozyme

Fig. 8

Volcano plots for each comparison: SP vs. Control (A), SP + L vs. Control (B) and SP + L vs. SP (C). Significantly different proteins are marked in green, and non-significant are marked in red

Fig. 9

Venn diagram showing the overlap between differentially expressed proteins in the three experimental groups

Fig. 10

Heatmaps of differentially abundant proteins in the piglet liver proteome considering all three experimental comparisons together. Protein accession numbers are displayed in rows and normalized protein abundance per sample is displayed in columns. Blue indicates abundance increases whereas red indicates abundance decreases. Control1, Control2, Control3, Control4, Control5 - Control diet; SP6, SP7, SP8, SP9, SP10 - 10% Spirulina diet; SPL11, SPL12, SPL13, SPL14, SPL15 - 10% Spirulina diet supplemented with lysozyme

Fig. 11

Heatmaps of differentially abundant proteins in the liver proteome of piglets fed different diets. Heatmaps showing protein intensity clusters for the SP+L vs. Control (A), the SP vs. Control (B) and the SP+L vs. SP (C). Protein accession numbers are displayed in rows and normalized protein abundance per sample is displayed in columns. Blue indicates abundance increases whereas red indicates abundance decreases. Control1, Control2, Control3, Control4, Control5 - Control diet; SP6, SP7, SP8, SP9, SP10 - 10% Spirulina diet; SPL11, SPL12, SPL13, SPL14, SPL15 - 10% Spirulina diet supplemented with lysozyme

Fig. 12

Functional classification of differentially abundant proteins according to the molecular function, biological process and cellular component

Fig. 13

Protein interaction network for the differentially expressed proteins in piglet liver in SP and Control groups comparison

Fig. 14

Protein interaction network for the differentially expressed proteins in piglet liver in Control and SP + L groups comparison

Fig. 15

Protein interaction network for the differentially expressed proteins in piglet liver in SP + L and SP groups comparison