Effect of Alfalfa Hay and Starter Feeding Intervention on Gastrointestinal Microbial Community, Growth and Immune Performance of Yak Calves

Abstract

The present study aims to evaluate the effects of different early weaning paradigms, which supplied with extra alfalfa hay, or starter feeding, or both alfalfa hay and starter feeding, along with the milk replacer, on the gastrointestinal microbial community, growth, and immune performance of yak calves. Twenty 30-day-old male yak calves were randomly assigned to four groups, including the control (CON), alfalfa hay (A), starter feeding (S), and starter plus alfalfa hay (SA) groups. The gastrointestinal microbial colonization, the gastrointestinal development and function, and the growth and immune performance of all the yak calves were separately measured. Supplementation with alfalfa and starter feeding during the pre-weaning period significantly increased body weight, body height, body length, and chest girth. The significantly improved rumen fermentation and promoted intestinal digestion-absorption function in alfalfa and starter feeding groups, including the identified significantly increased concentrations of ruminal total volatile fatty acid (VFA); the significantly increased concentrations and proportions of acetate, butyrate, and isovalerate; the increased α-amylase activities in the duodenum, jejunum, and ileum; the increased papillae length and width of rumen epithelium and rumen wall thickness; and the increased villus height and crypt depth of the duodenum, jejunum, and ileum, could all contribute to promote the growth of calves. These significant improvements on rumen fermentation and intestinal digestion-absorption function could be further attributed to the increased proliferation of starch-decomposing, and cellulose- or hemicellulose-decomposing bacteria identified in the rumen, jejunum, and ileum. Furthermore, based on the expression of intestinal inflammatory cytokines and the rumen epithelial RNA sequencing results, alfalfa supplementation reduced the occurrence of ruminal and intestinal inflammation, whereas starter feeding supplementation was mainly beneficial to the differentiation of immune cells and the improved immune function. Meanwhile, the significantly altered relative abundances of genera in the SA group, including increased relative abundance of Limnobacter, Escherichia/Shigella, and Aquabacterium in the rumen and increased relative abundance of Coprococcus, Pseudobutyrivibrio, Flavonifractor, Synergistes, and Sutterella in jejunum, were able to reduce gastrointestinal inflammation and enhance the immune function, which enhanced the immune function of the yak calves fed with alfalfa and starter feeding. Overall, milk replacer supplemented with alfalfa and starter feeding during the pre-weaning period could alter gastrointestinal microbiota and then benefit the gastrointestinal development, digestion-absorption function, growth, and immune performance of the yak calves.

Keywords: alfalfa; gastrointestinal microbiota; growth performance; immune homeostasis; starter feeding; yak calves.

FIGURE 1

Effects of individual and simultaneous supplementation with alfalfa and starter feeding during pre-weaning period on average dry matter intakes (DMI) of overall experiment (A), daily DMI of each measured day (B), individual daily DMI of alfalfa hay and starter feed (C), and body weight and body size traits (D) of yak calves. ^a–bDifferent superscripts above the error bars for the same index indicated significant differences among different groups (P < 0.05); CON, supplemented with milk replacer only; S, supplemented with milk replacer and starter feed; A, supplemented with milk replacer and alfalfa hay; SA, supplemented with milk replacer, starter feed, and alfalfa hay.

FIGURE 2

Effects of individual and simultaneous supplementation with alfalfa and starter feeding during the pre-weaning period on the ruminal (A,C) and intestinal morphology of yak calves (B,D–F). ^a–dDifferent superscripts above the error bars for the same index indicated significant differences among different groups (P < 0.05); CON, supplemented with milk replacer only; S, supplemented with milk replacer and starter feed; A, supplemented with milk replacer and alfalfa hay; SA, supplemented with milk replacer, starter feed, and alfalfa hay.

FIGURE 3

Effects of individual and simultaneous supplementation with alfalfa and starter feeding during the pre-weaning period on the ruminal (A), abomasum (B), and intestinal enzymic activities (C–E) of yak calves. ^a–cDifferent superscripts above the error bars for the same index indicated significant differences among different groups (P < 0.05); CON, supplemented with milk replacer only; S, supplemented with milk replacer and starter feed; A, supplemented with milk replacer and alfalfa hay; SA, supplemented with milk replacer, starter feed, and alfalfa hay.

FIGURE 4

Effects of individual and simultaneous supplementation with alfalfa and starter feeding during the pre-weaning period on the sIgA and immune cell cytokine content of mucous membrane samples of the duodenum (A), jejunum (B), and ileum (C). ^a–cDifferent superscripts above the error bars for the same index indicated significant differences among different groups (P < 0.05); CON, supplemented with milk replacer only; S, supplemented with milk replacer and starter feed; A, supplemented with milk replacer and alfalfa hay; SA, supplemented with milk replacer, starter feed, and alfalfa hay.

FIGURE 5

Differentially expressed genes (DEGs) of rumen epithelium between the yak calves induced by individual and simultaneous supplementation with alfalfa and starter feeding. (A–C) The top 30 mostly significantly enriched pathways based on the identified DEGs of different compared groups includes SA vs. CON, S vs. CON, and A vs. CON; (D) validation of the accuracy of RNA-seq data by qRT-PCR. CON, supplemented with milk replacer only; S, supplemented with milk replacer and starter feed; A, supplemented with milk replacer and alfalfa hay; SA, supplemented with milk replacer, starter feed, and alfalfa hay. ^a,bwithin a row with different superscripts means significantly difference.

FIGURE 6

The rumen microbial community differences induced by the individual and simultaneous supplementation with alfalfa and starter feeding. (A) α-diversity analyses based on the rumen microbiota; (B) β-diversity analyses based on the rumen microbiota; (C) significantly increased genera in SA group (red bar) and S group (green bar) in compared group of SA vs. S; (D) significantly increased genera in SA group (red bar) and A group (green bar) in compared group of SA vs. A; (E) significantly increased genera in S group (red bar) and A group (green bar) in compared group of S vs. A; (F) significantly Spearman correlation between the identified differential ruminal genera and the significantly altered growth performance, healthy condition, ruminal enzymic activities, and ruminal development of yak calves. Blue block indicated negative correlation and red block indicated positive correlation. CON, supplemented with milk replacer only; S, supplemented with milk replacer and starter feed; A, supplemented with milk replacer and alfalfa hay; SA, supplemented with milk replacer, starter feed, and alfalfa hay.

FIGURE 7

The jejunal microbial community differences induced by the individual and simultaneous supplementation with alfalfa and starter feeding. (A) α-diversity analyses based on the rumen microbiota; (B) β-diversity analyses based on the rumen microbiota; (C) significantly increased genera in SA group (red bar) and S group (green bar) in compared group of SA vs. S; (D) significantly increased genera in SA group (red bar) in compared group of SA vs. A; (E) significantly increased genera in SA group (red bar) and CON group (green bar) in compared group of SA vs. CON; (F) significantly increased genera in S group (red bar) and A group (green bar) in compared group of S vs. A; (G) significantly increased genera in S group (red bar) and CON group (green bar) in compared group of S vs. CON; (H) significantly increased genera in CON group (red bar) in compared group of A vs. CON; (I) Significantly Spearman correlation between the identified jejunal differential genera and the significantly altered growth performance, healthy condition, and jejunal enzymic activities, and development of yak calves. Blue block indicated negative correlation and red block indicated positive correlation. CON, supplemented with milk replacer only; S, supplemented with milk replacer and starter feed; A, supplemented with milk replacer and alfalfa hay; SA, supplemented with milk replacer, starter feed, and alfalfa hay. ^a,bwithin a row with different superscripts means significantly difference.

FIGURE 8

The ileal microbial community differences induced by the individual and simultaneous supplementation with alfalfa and starter feeding. (A) α-diversity analyses based on the rumen microbiota; (B) β-diversity analyses based on the rumen microbiota; (C) significantly increased genera in SA group (red bar) and S group (green bar) in compared group of SA vs. S; (D) significantly increased genera in SA group (red bar) and A group (green bar) in compared group of SA vs. A; (E) significantly increased genera in SA group (red bar) and CON group (green bar) in compared group of SA vs. CON; (F) significantly increased genera in S group (red bar) and A group (green bar) in compared group of S vs. A; (G) significantly increased genera in S group (red bar) and CON group (green bar) in compared group of S vs. CON; (H) significantly increased genera in A group (red bar) and significantly increased genera in CON group (green bar) in compared group of A vs. CON; (I) Significantly Spearman correlation between the identified ileal differential genera and the significantly altered growth performance, healthy condition, and ileal enzymic activities and development of yak calves. Blue block indicated negative correlation and red block indicated positive correlation. CON, supplemented with milk replacer only; S, supplemented with milk replacer and starter feed; A, supplemented with milk replacer and alfalfa hay; SA, supplemented with milk replacer, starter feed, and alfalfa hay. ^a,bwithin a row with different superscripts means significantly difference.