Essential oils improve nursery pigs' performance and appetite via modulation of intestinal health and microbiota

Abstract

Optimal intestinal health and functionality are essential for animal health and performance, and simultaneously intestinal nutrient transporters and intestinal peptides are also involved in appetite and feed intake control mechanisms. Given the potential of essential oil (EO) in improving animal performance and improving feed palatability, we hypothesized that dietary supplementation of cinnamaldehyde and carvacrol could improve performance and appetite of nursery pigs by modulating intestinal health and microbiota. Cinnamaldehyde (100 mg/kg), carvacrol (100 mg/kg), and their mixtures (including 50 mg/kg cinnamaldehyde and 50 mg/kg carvacrol) were supplemented into the diets of 240 nursery pigs for 42 d, and data related to performance were measured. Thereafter, the influence of EO on intestinal health, appetite and gut microbiota and their correlations were explored. EO supplementation increased (P < 0.05) the body weight, average daily gain (ADG) and average daily feed intake (ADFI) of piglets, and reduced (P < 0.05) diarrhea rates in nursery pigs. Furthermore, EO increased (P < 0.05) the intestinal absorption area and the abundance of tight junction proteins, and decreased (P < 0.05) intestinal permeability and local inflammation. In terms of intestinal development and the mucus barrier, EO promoted intestinal development and increased (P < 0.05) the number of goblet cells. Additionally, we found that piglets in the EO-supplemented group had upregulated (P < 0.05) levels of transporters and digestive enzymes in the intestine, which were significantly associated with daily gain and feed utilization. In addition, EO supplementation somewhat improved appetite in nursery pigs, increased the diversity of the gut microbiome and the abundance of beneficial bacteria, and there was a correlation between altered bacterial structure and appetite-related hormones. These findings indicate that EO is effective in promoting growth performance and nutrient absorption as well as in regulating appetite by improving intestinal health and bacterial structure.

Keywords: Appetite; Essential oil; Intestinal health; Microbiota; Nursery pig.

Fig. 1

Effects of cinnamaldehyde and carvacrol supplementation on intestinal morphology in nursery pigs. (A) Nursery pigs were administered Con (basal diet), Cin (basal diet with cinnamaldehyde), Car (basal diet with carvacrol) or Cin + Car (cinnamaldehyde and carvacrol) for 42 d. (B) Linear regression of body weight vs. small intestine length. (C) Small intestine length of nursery pigs and their average daily feed intake throughout the trial. (D) Representative images of the jejunum and ileum stained with H&E (scale bar, 200 μm). (E–G) The CD, VH and VH to CD ratio of jejunum and ileum. (H) Intestinal morphology of jejunum and ileum shown by scanning electronic microscope. (I) Linear regression of average daily gain vs. villus height of jejunum and ileum. CD = crypt depth; VH = villus height. Con means piglets fed basal diet (n = 6); Cin means CON diet with 50 mg/kg cinnamaldehyde (n = 6); Car means CON diet with 50 mg/kg carvacrol (n = 6); Cin + Car means CON diet with 25 mg/kg cinnamaldehyde and 25 mg/kg carvacrol (n = 6). Values for R² and P are indicated in each graph. Data are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01.

Fig. 2

Effects of cinnamaldehyde and carvacrol supplementation on intestinal tight junction, development and mucus barrier in nursery pigs. (A) Immunofluorescence staining for ZO-1 and occludin in jejunum and ileum (scale bar, 100 μm). (B, C) Protein abundance of tight junction proteins (including ZO-1, E-cadherin, occludin, claudin-1 and claudin-5) of jejunum and ileum and their quantification. (D) Serum levels of DAO, ET and D-LA. (E) IL-1β, TNF-α and IL-6 levels in colonic mucosa. (F, G) Protein abundance of Ki67, mucin-2, Lgr5 and Lyz of jejunum and ileum and their quantification. (H) Immunofluorescence staining for Ki67 in jejunum and ileum (scale bar, 100 μm). (I, K) PAS and AB-PAS staining of jejunum and ileum (scale bar, 200 μm). (J, L) Quantitative analysis of glycogen-positive and goblet cell relative densities. ZO-1 = zonula occludens-1; DAO = diamine oxidase; ET = endothelin; D-LA = D-lactic acid; TNF-α = tumor necrosis factor alpha; Lgr5 = leucine-rich repeat containing G protein-coupled receptor 5; Lyz = lysozyme. Con means piglets fed basal diet (n = 6); Cin means CON diet with 50 mg/kg cinnamaldehyde (n = 6); Car means CON diet with 50 mg/kg carvacrol (n = 6); Cin + Car means CON diet with 25 mg/kg cinnamaldehyde and 25 mg/kg carvacrol (n = 6). Data are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01.

Fig. 3

Effects of cinnamaldehyde and carvacrol supplementation on intestinal digestion and absorption in nursery pigs. (A–D) Levels of trypsin, lipase, amylase and chymotrypsin in jejunal and ileal contents. (E, F) The mRNA expression of nutrient transporters of jejunum and ileum. (G, H) Protein abundance of SLC1A1, SLC5A1, GLUT2 and GLUT5 of jejunum and ileum and their quantification. (I) Correlation analysis of digestive enzymes and nutrient transporters of jejunum and ileum with ADG and FCR. SLC1A1 = solute carrier family 1 member 1; SLC5A1 = solute carrier family 5 member 1; GLUT = glucose transporter type; γ⁺LAT = γ⁺ system L-type amino acid transporter; RBAT = B(0,+)-type amino acid transport protein; CAT1 = cationic amino acid transporter 1; SNAT2 = sodium-coupled neutral amino acid transporter 2; ASCT2 = alanine-serine-cysteine transporter 2; PepT1 = peptide transporter 1; ADG = average daily gain; FCR = feed conversion ratio. Con means piglets fed basal diet (n = 6); Cin means CON diet with 50 mg/kg cinnamaldehyde (n = 6); Car means CON diet with 50 mg/kg carvacrol (n = 6); Cin + Car means CON diet with 25 mg/kg cinnamaldehyde and 25 mg/kg carvacrol (n = 6). Data are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01.

Fig. 4

Effects of cinnamaldehyde and carvacrol supplementation on appetite and gut microbiota in nursery pigs. (A) Expression of appetite-related hormones in serum. (B) Chao 1 index. (C) Shannon index. (D) Simpson index. (E) Relative abundance of microbiota at the phylum level. (F) Relative abundance of microbiota at the genus level. (G) Cladogram generated from LEfSe analysis. (H) Correlation analysis of top 20 microbes with appetite-related hormones and feed intake. Correlation analysis of (I) Lactobacillus and (J) Prevotella with occludin and mucin-2 protein expression, serum ET level. GH = growth hormone; GIP = gastric inhibitory polypeptide; CCK = cholecystokinin; GLP-1 = glucagon-like peptide-1; LEP = leptin; PYY = peptide YY; ADFI = average daily feed intake; ET = endothelin. Con means piglets fed basal diet (n = 6); Cin means CON diet with 50 mg/kg cinnamaldehyde (n = 6); Car means CON diet with 50 mg/kg carvacrol (n = 6); Cin + Car means CON diet with 25 mg/kg cinnamaldehyde and 25 mg/kg carvacrol (n = 6). Values for R² and P are indicated in each graph. Data are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01.

Fig. 5

Analysis of correlation and function of differential intestinal microbiota. (A) Correlation network of the microbiota at the genus level. (B) Predicted functionality of the microbiota. (C) The schematic diagram depicting EO driving improvements in intestine health and microbiota structure to promote performance and appetite in nursery pigs. Obviously, dietary supplementation of EO can improve the performance of nursery pigs and improve the digestion and absorption efficiency of feeds by ensuring the integrity, turnover and development of the intestinal barrier and increasing digestive enzyme activity and nutrient transporter abundance. Furthermore, the increased diversity and structure of intestine microbiota will affect appetite-related hormones, thereby promoting appetite.