Maternal Malic Acid May Ameliorate Oxidative Stress and Inflammation in Sows through Modulating Gut Microbiota and Host Metabolic Profiles during Late Pregnancy

Abstract

Sows suffer oxidative stress and inflammation induced by metabolic burden during late pregnancy, which negatively regulates reproductive and lactating performances. We previously found that L-malic acid (MA) alleviated oxidative stress and inflammation and improved reproductive performances in sows. However, the mechanism underlying the MA's positive effects remains unexplored. Here, twenty Large White × Landrace sows with similar parity were randomly divided into two groups and fed with a basal diet or a diet supplemented with 2% L-malic acid complex from day 85 of gestation to delivery. The gut microbiome, fecal short-chain fatty acids, and untargeted serum metabolome were determined. Results showed that Firmicutes, Bacteroidota, and Spirochaetota were the top abundant phyla identified in late pregnancy for sows. Maternal MA supplementation modulated the composition but not the richness and diversity of gut microbiota during late pregnancy. Correlation analysis between gut microbiota and antioxidant capacity (or inflammation indicators) revealed that unclassified_f_Ruminococcaceae, unclassified_f_Lachnospiraceae, UCG-002, norank_f_norank_o_RF3, and Lactobacillus might play a role in anti-oxidation, and Lachnospiraceae_XPB1014_group, Lachnospiraceae_NK4A136_group, UCG-002, unclassified_f_Ruminococcaceae, Candidatus_Soleaferrea, norank_f_UCG-010, norank_f_norank_o_RF39, and unclassified_f_Lachnospiraceae might be involved in the anti-inflammatory effect. The improved antioxidant and inflammation status induced by MA might be independent of short chain fatty acid changes. In addition, untargeted metabolomics analysis exhibited different metabolic landscapes of sows in the MA group from in the control group and revealed the contribution of modified amino acid and lipid metabolism to the improved antioxidant capacity and inflammation status. Notably, correlation results of gut microbiota and serum metabolites, as well as serum metabolites and antioxidant capacity (or inflammation indicators), demonstrated that differential metabolism was highly related to the fecal microorganisms and antioxidant or inflammation indicators. Collectively, these data demonstrated that a maternal dietary supply of MA can ameliorate oxidative stress and inflammation in sows through modulating gut microbiota and host metabolic profiles during late pregnancy.

Keywords: L-malic acid; gut microbiota; late pregnancy; metabolic; sow.

Figure 1

Influence of L–malic acid on gut microbiota diversity in sows during late pregnancy. (A–D) The α–diversity and (E) β–diversity of the gut microbiota in sows by 16S rRNA sequencing. (A–D) Comparison of Shannon index (A), Simpson index (B), Ace index (C), and Chao 1 index (D) between CON and MA groups. (E) Unweighted principal coordinate analysis (PCoA) at the OTU level. CON, basal diet; MA, basal diet containing 2% malic acid complex. Data were shown as means ± SEM (n = 6).

Figure 2

Modulation of L–malic acid on the constitution of gut microbiota in sows during late pregnancy by 16S rRNA sequencing: (A,B) shows the relative abundance of the bacteria community at the phylum and genus level, respectively; (B) displays the top 20 abundant genera; and (C) shows the communities with significant differences in relative abundance at genus level. CON, basal diet; MA, basal diet containing 2% L–malic acid complex. Data are presented as mean ± SEM (n = 6). Statistics were performed with Student’s t-test. * p < 0.05.

Figure 3

Correlation between top 50 abundant gut microbiota and antioxidant ability and inflammatory factors in the sows based on Spearman coefficient. Red indicates a positive correlation, and blue indicates a negative correlation. The star symbol in the small square presents the correlation results. * p < 0.05, ** p < 0.01, *** p < 0.001; ** on the right of the picture represents significantly different microorganisms between control and malic acid group.

Figure 4

Functional prediction of fecal microbiota and the impact of L-malic acid on short-chain fatty acid content in feces. (A) Functional prediction of differential microbiota based on the MetaCyc database. (B) Differential functional analysis of metabolic pathways based on the KEGG Orthology database. (C) Levels of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, and isohexanoic acid in feces in the CON and MA groups. CON, basal diet; MA, basal diet containing 2% L-malic acid complex. Data are presented as mean ± SEM (n = 6). Statistics were performed with Student’s t-test. * p < 0.05, ** p < 0.01.

Figure 5

Effects of L-malic acid supplementation on the serum metabolic profiles of sows during late pregnancy. (A,B) PLS-DA plots of the metabolites in serum in positive ion and negative ion modes. (C) Volcano plots of the identified metabolites. The red ones represent upregulated metabolites and green ones represent downregulated metabolites. (D) VIP scores and the relevant abundance of the top 30 metabolites. The length of the bar represents the contribution of the metabolite to the difference between the two groups. * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Enrichment of KEGG pathways based on the differential metabolites between the CON and MA groups. CON, basal diet; MA, basal diet containing 2% L-malic acid complex. OS, HD, M, EIP, and CP were the first categories of the KEGG pathway; they represent Organismal Systems, Human Diseases, Metabolism, Environmental Information Processing, and Cellular Processes, respectively.

Figure 6

Correlation analysis between the top 20 abundant metabolites and differential microorganisms (A), and between the top 20 differential metabolite and antioxidant or inflammation indices (B) based on Spearman correlation coefficients. Red indicates a positive correlation, and blue indicates a negative correlation. * p < 0.05, ** p < 0.01, *** p < 0.001.