牦牛瘤胃碳水化合物降解酶与微生物宏基因组对饲粮添加混合异位酸的响应

Responses of ruminal carbohydrate-degrading enzymes and microbial metagenome in yaks to dietary supplementation with mixed isoacids

摘要:

【背景】异位酸属于支链挥发性脂肪酸，具有为瘤胃微生物提供营养物质、促进瘤胃纤维降解菌生长的作用。【目的】研究饲粮添加混合异位酸(mixed isoacids,MI)对牦牛瘤胃碳水化合物降解酶活性、物种组成及功能基因的影响，并分析瘤胃碳水化合物活性与瘤胃微生物及功能基因相对丰度的相关性，为合理利用MI以改善牦牛瘤胃发酵性能提供参考。【方法】采用单因素试验设计，选取16头200 kg左右的健康麦洼公牦牛，随机分为4组，每组4个重复，每个重复1头牛。对照组(CON组)饲喂不添加MI的基础饲粮，试验组MI添加量分别为基础饲粮的0.1%(T1组)、0.2%(T2组)和0.3%(T3组)。试验预饲期10 d，正饲期85 d，试验结束时采集瘤胃液，通过宏基因组测序分析瘤胃微生物菌群及紫外分光光度法分析瘤胃碳水化合物降解酶活性。【结果】与对照组相比，饲粮添加MI能显著提高牦牛瘤胃羧甲基纤维素酶和淀粉酶的活性。物种α多样性指数分析结果表明，添加0.3% MI显著降低了瘤胃物种丰富度，提高了组内物种组成相似度和功能基因组成相似度；物种与功能差异分析表明，厚壁菌(Firmicutes)、丹毒丝菌(Erysipelotrichia)、糖细菌(Saccharibacteria)及气单胞菌科(Aeromonadaceae)等差异细菌在T2组显著富集；磷转移酶(2.7.1.68)、泛素水解酶(3.4.19.12)、氨基酰转移酶(2.3.2.27)和蛋白丝氨酸/苏氨酸激酶(2.7.11.11)等KEGG酶差异基因在T1组显著富集。瘤胃酶活性与瘤胃宏基因组相关分析结果显示，瘤胃淀粉酶活性与丙酸菌(Profundibacterium)、气单胞菌属(Aeromonas)、糖细菌(Saccharibacteria)和气单胞菌科(Aeromonadaceae)显著正相关，与GH13_20家族、GH77家族和CBM72家族基因显著正相关；瘤胃羧甲基纤维素酶活性与GT90家族基因显著正相关。【结论】在牦牛饲粮中添加MI能显著提高牦牛瘤胃淀粉酶和羧甲基纤维素酶活性，提高瘤胃微生物物种和功能基因的相似度，提高瘤胃细菌及功能基因的相对丰度，且瘤胃细菌及其功能基因与瘤胃酶活性存在一定相关性，表明添加MI能一定程度改善牦牛瘤胃发酵特性。

关键词:混合异位酸;牦牛;碳水化合物降解酶;宏基因组学

Abstract:

[Background] Isoacids are branched-chain volatile fatty acids, providing nutrients to the microorganisms and promoting the growth of fiber-degrading bacteria in the rumen. [Objective] This study aimed to investigate the effects of dietary supplementation with mixed isoacids (MI) on the activities of carbohydrate-degrading enzymes, microbiota composition, and functional genes in the yak rumen. In addition, the correlations of the enzyme activities with the relative abundance of rumen microorganisms and functional genes were analyzed. The results are expected to provide a reference for the rational use of MI to improve the fermentation performance in the yak rumen. [Methods] A single factorial experimental design was used and 16 male yaks weighing approximately 200 kg were randomized into four groups with four replicates per group and one yak per replicate. Yaks in the control group were fed with a basal diet, while those in the other groups were fed a basal diet supplemented with 0.1% (T1 group), 0.2% (T2 group), and 0.3% (T3 group) of MI, respectively. The pre-experiment lasted for 10 days, and the formal feeding lasted for 85 days. At the end of the experiment, rumen fluid samples were collected and the rumen microbiome was analyzed by metagenomic sequencing. Activities of carbohydrate-degrading enzymes were measured by and UV spectrophotometry. [Results] Compared with the control group, dietary MI supplementation significantly increased the activities of carboxymethyl cellulase and amylase in the yak rumen. Supplementation with 0.3% MI reduced the microbial richness and increased the similarity of microbiota composition and functional gene composition in the yak rumen. The differential bacteria such as Firmicutes, Erysipelotrichia, Saccharibacteria, and Aeromonadaceae had high relative abundance in the T2 group. The KEGG pathway enrichment analysis showed that the differentially expressed genes encoding phosphotransferase (2.7.1.68), ubiquitin hydrolase (3.4.19.12), aminoacyltransferase (2.3.2.27), and protein serine/threonine kinases (2.7.11.11) had high relative abundance in the T1 group. Rumen amylase activity was positively correlated with Profundibacterium, Aeromonas, Saccharibacteria, and Aeromonadaceae, as well as members of the GH13_20, GH77, and CBM72 gene families. The carboxymethyl cellulase activity in the rumen was positively correlated with members of the GT90 gene family. [Conclusion] Supplementation of MI in yak diets increased the activities of amylase and carboxymethyl cellulase, improved the similarity of rumen microbiota composition and functional gene composition, and increased the relative abundance of bacteria and functional genes in the yak rumen. In addition, rumen microbiome and their functional genes were correlated with the activities of rumen enzymes. The results suggest that MI can improve the rumen fermentation in yaks to a certain extent.

Key words:mixed isoacids;yak;carbohydrate-degrading enzymes;metagenomics