Cecal Microbial Diversity and Metabolome Reveal a Reduction in Growth Due to Oxidative Stress Caused by a Low-Energy Diet in Donkeys

Abstract

Dietary energy level plays an important role in animal growth and development. The present study investigated the effect of dietary energy on the growth performance, antioxidative state, and nutrient digestion of meat donkeys. It simultaneously explored the probable reason for cecal microbiota and metabolome. Twelve meat donkeys (male) aged 1 year with a similar weight (150 ± 25 kg) were divided into two treatment groups: low-energy group (E1) and high-energy group (E2). The experiment was divided into a 10-day pre-trial period and a 135-day trial period. Donkeys in the trial periods were fed diets with digestible energy values (in dry matter) of 12.08 and 13.38 MJ/kg (pre-fattening, 1-45 d), 13.01 and 14.27 MJ/kg (mid-fattening, 46-90 d), and 13.54 and 14.93 MJ/kg (late-fattening, 91-135 d). The results show that E1 decreases body weight, average daily gain (ADG), and the digestibility of crude protein, ether extract, neutral detergent fiber, and acid detergent fiber (p < 0.05), but increases cecal acetate and butyrate concentrations, non-esterified fatty acids (NEFAs) in serum, and the ratio of dry matter intake to ADG(F/G). E1 diminished the activities of catalase and glutathione peroxidase, while it increased the content of interleukin, tumor necrosis factor-alpha, and reactive oxygen species (ROS) (p < 0.05). Cecal microbiome showed that the abundance of Firmicutes and Actinobacteria in E1 was significantly lower than in E2 (p = 0.029, p = 0.002), whereas Bacteroidetes was higher (p = 0.005). E1 increased the genera Ruminococcaceae-UCG-004, Acinetobacter, and Rikenellaceae_RC9_gut_group. Meanwhile, cecal metabolome showed that formyl-5-hydroxykynurenamine, chorismate, 3-sulfinoalanine, and 3-isopropylmalate were upregulated in E1, while brassinolide was downregulated. The altered metabolites were mainly enriched in metabolic pathways related to energy metabolism and metabolism to mitigate oxidative stress in the meat donkeys, such as tryptophan metabolism, brassinosteroid biosynthesis metabolism, etc. In conclusion, low-energy diets resulted in a negative energy balance in meat donkeys, resulting in more nutrients being oxidized to provide energy, inducing oxidative stress, and thereby leading to decreasing growth. The reduction in meat donkey growth from low-energy diets was related to changes in cecum microbiota and metabolites.

Keywords: cecal microbiome and metabolome; dietary energy; growth performance; meat donkey; oxidative stress.

Figure 1

Effects of dietary energy level on the composition of microflora at the genus level. Each color represents one treatment: The red curves represent donkeys fed a low-energy (E1) diet and the blue curves represent donkeys fed a high-energy (E2) diet.

Figure 2

(A) Differential metabolites in E2 vs. E1 in Pos. (B) Differential metabolites in E2 vs. E1 in Neg. The red curves represent upregulated metabolites, the blue represents downregulated metabolites, and the gray represents no change. E1 = low-energy group; E2 = high-energy group.

Figure 3

Pathway enrichment analysis was performed using the significantly different metabolites for E2 vs. E1. E1 = low-energy group; E2 = high-energy group. The red bubbles represent metabolic pathways.

Figure 4

Correlation analysis of cecal content bacteria and growth performance, nutrient digestibility, NEFA (non-esterified fatty acid) concentration, cecal volatile fatty acids. Red color: positive correlation; blue color: negative correlation. * p < 0.05; ** p < 0.01; *** p < 0.001. Abbreviations: BW = body weight; ADG = average daily gain; F/G = the ratio of dry matter intake to ADG. CP% = the digestibility of crude protein; EE% = the digestibility of ether extract; ADF% = the digestibility of acid detergent fiber; NDF% = the digestibility of acid detergent fiber. TVFAs = total volatile fatty acids; A/P, the ratio of acetate to propionate.

Figure 5

Correlation analysis of cecal differential metabolites and growth performance, nutrient digestibility, NEFA (non-esterified fatty acid) concentration, and cecal volatile fatty acids. Red color: positive correlation; blue color: negative correlation. * p < 0.05; ** p < 0.01; *** p < 0.001. Abbreviations: BW = body weight; ADG = average daily gain; F/G = the ratio of dry matter intake to ADG. CP% = the digestibility of crude protein; EE% = the digestibility of ether extract; ADF% = the digestibility of acid detergent fiber; NDF% = the digestibility of acid detergent fiber. TVFAs = total volatile fatty acids; A/P, the ratio of acetate to propionate.

Figure 6

Correlation analysis of cecal content bacteria and (A) serum antioxidant and immune indicators; (B) cecum antioxidant and immune indicators. Red color: positive correlation; blue color: negative correlation. * p <0.05; ** p < 0.01; *** p < 0.001. Abbreviations: CAT = catalase; GPx = glutathione peroxidase; T-SOD = total superoxide dismutase; MDA = malondialdehyde; IL = interleukin; TNF-α = tumor necrosis factor-alpha; NO = nitric oxide; and ROS = reactive oxygen species.

Figure 7

Correlation analysis of cecal content bacteria and (A) serum antioxidant and immune indicators; (B) cecum antioxidant and immune indicators. Red color: positive correlation; blue color: negative correlation. * p < 0.05; ** p < 0.01; *** p < 0.001. Abbreviations: CAT = catalase; GPx = glutathione peroxidase; T-SOD = total superoxide dismutase; MDA = malondialdehyde; IL = interleukin; TNF-α = tumor necrosis factor-alpha; NO = nitric oxide; and ROS = reactive oxygen species.

Figure 8

Correlation analysis of cecal differential metabolites and bacteria. Red color: positive correlation; * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 9

A mechanism map for linking the microbiota and the metabolites involved in the metabolic pathway of enrichment. Abbreviations: BW = body weight; NEFAs = non-esterified fatty acids; ROS = reactive oxygen species; G = glucose; CAT = catalase; GPx = glutathione peroxidase; IL-1β = interleukin-1β; IL-6 = interleukin-6; TNF-α = tumor necrosis factor-alpha; 5-HT = 5-hydroxytryptamine; f5-HK = formyl-5-hydroxykynurenine; Butyryl-CoA = butyryl coenzyme A; Acetyl-CoA = acetyl coenzyme A; Trp = tryptophan; Val = valine; Leu = leucine; Ile = isoleucine; Phe = phenylalanine; Tyr = tyrosine; TCA cycle = tricarboxylic acid cycle. “+” indicates a positive correlation between microorganisms and metabolites or other substances, and “−” indicates a negative correlation. The blue boxes represent enriched differential metabolic pathways in E2 (high dietary energy) vs. E1 (low dietary energy). “ ” represents a positive increase, “ ” represents a negative increase, and “ ” represents a negative decrease.