# Multi-Omics Analysis Reveals Concentrate Supplementation Alleviates Body Weight Loss by Regulating Rumen Function in Lactating Tibetan Sheep During the Cold Season

## TL;DR

Adding more concentrate feed helps lactating Tibetan sheep maintain body weight during cold seasons by improving their rumen function and microbial balance.

## Contribution

This study is the first to use multi-omics to show how concentrate supplementation affects rumen function and gene expression in lactating Tibetan sheep.

## Key findings

- Higher concentrate levels reduced body weight loss and improved rumen histomorphology in Tibetan sheep.
- Concentrate supplementation altered rumen microbiota, with distinct microbial signatures observed across different supplement levels.
- Key genes involved in digestion and immunity were differentially expressed with varying concentrate levels.

## Abstract

Systematic studies on the effects of concentrate supplementation on the rumen function and microbiology alterations in Tibetan ewes during their lactation period are limited. Our multi-omics analysis showed that higher concentrate levels significantly alleviated body weight loss by regulating rumen histomorphology, enhancing cellulase activity, and altering the abundance of key bacteria (Lachnospiraceae_XPB1014_group, Anaerovibrio, Ruminococcus, and Pseudobutyrivibrio). Furthermore, concentrate supplementation levels altered the expression of genes (TRPA1, EPHB1, GATA3, C4, ABCG2, THBS4, and TNFRSF11B) involved in immune regulation, signal transduction, and nutrient digestion. Correlation analysis confirmed that these changes are interconnected, as indicated by significant associations between rumen bacteria, fermentation parameters, and digestive enzyme activities. Our findings clarify that concentrate supplementation mitigates weight loss by orchestrating functional improvements across the rumen ecosystem.

The parturition season of grazing Tibetan ewes spans from October to March, a period that exacerbates the adverse impacts of nutrient-deficient herbage on milk yield, body condition, and postpartum recovery. To alleviate the weight loss of ewes during the cold seasons, we provided concentrate supplements at four levels (dry matter (DM) basis), 260 g (C1), 440 g (C2), 520 g (C3), and 610 g (C4), alongside a basal diet of grazed pasture. A total of 96 multiparous Tibetan ewes (third parity, body weight: 45.17 ± 3.69 kg (body weight (BW) were enrolled within 12–18 h postpartum and randomly allocated to four dietary groups (n = 24 ewes per group). We measured growth performance, ruminal histomorphology, fermentation parameters, and digestive enzymes. A multi-omics technique (16S rRNA gene sequencing and RNA-seq) was employed to investigate the mechanisms underlying alterations in ruminal function. The results showed that increasing the concentrate level decreased body weight loss and increased average dry matter intake (p < 0.05). Rumen morphology was significantly altered: papilla width and muscle layer thickness were greatest in the C4 group, whereas submucosal thickness was highest in the C1 group (p < 0.05). Cellulase activity was lowest in the C1 group (p < 0.05). Papilla width of lactating Tibetan ewes in the C4 group was higher (p < 0.05) than that in the C1 and C3 groups. Concentrate supplementation altered ruminal microbiota composition and diversity. Each group exhibited a distinct microbial signature: the C1 group was characterized by Lachnospiraceae_XPB1014_group, Candidatus_Omnitrophus, Paenibacillus, and unclassified_Oligoflexaceae; the C2 group was enriched in Papillibacter, Anaerovibrio, V9D2013_group, and unclassified_Peptococcaceae; the C3 group was characterized by unclassified_Bacteroidales_RF16_group; and the C4 group was characterized by Ruminococcus, Pseudobutyrivibrio, and Mitsuokella (p < 0.05). Transcriptomic analysis identified differentially expressed genes (TRPA1, EPHB1, GATA3, C4, ABCG2, THBS4, and TNFRSF11B) that are predominantly involved in immune regulation, signal transduction, and nutrient digestion. The results of Spearman correlation analysis showed that Anaerovibrio was negatively correlated with propionate (r = −0.565, p < 0.05). However, it was positively correlated with the ratio of acetate and propionate (r = 0.579, p < 0.05). Moreover, Lachnospiraceae_XPB1014_group was negatively correlated with cellulase (r = −0.699, p < 0.05) and α-amylase (r = −0.514, p < 0.05). These findings suggest that the increasing concentrate supplementation alleviates body weight loss in lactating Tibetan sheep by orchestrating improvements in rumen histomorphology, digestive function, altering bacteria composition, and ruminal immune and modulating host epithelial gene expression.