日粮添加尿素对育肥湖羊空肠菌群结构及氨和尿素转运蛋白表达的影响

Effects of urea supplementation on jejunal bacterial communities and the mRNA expression of ammonia and urea transporter proteins in fattening Hu lambs

[目的]本试验旨在探究日粮添加尿素对育肥湖羊空肠发酵、菌群结构以及氨和尿素转运蛋白表达的影响。[方法]将42只3月龄育肥公湖羊(约24.3 kg)随机均分为3个处理组,在饲喂基础日粮的基础上分别添加0 g·kg-1(U0组)、10 g·kg-1(U10组)、30 g·kg^(-1)(U30组)尿素。试验预饲期1周,正试期10周。试验结束每组随机选取6只屠宰,采集空肠内容物,测定发酵参数和定量总菌,并利用Illumina MiSeq测序分析空肠细菌组成;采集空肠上皮组织样品检测氨转运蛋白b(Rhbg)、氨转运蛋白c(Rhcg)和尿素转运蛋白B(UT-B)基因mRNA表达。[结果]与U0组相比,U10和U30组空肠氨态氮含量显著升高(P<0.05)。与U0组相比,U10组总挥发性脂肪酸和乙酸含量显著降低,U30组丁酸和异戊酸含量相较于U10组显著升高(P<0.05)。相较于U0组,U10和U30组的总菌数量显著升高(P<0.05)。Illumina-MiSeq测序结果显示,添加尿素并未影响空肠Chao 1、ACE、Shannon和Simpson指数(P>0.05),PCoA分析结果显示各组间空肠细菌群落组成明显区分。添加尿素对湖羊空肠中细菌门水平相对丰度无显著影响,但细菌属水平相对丰度存在差异。各处理组主要优势菌属为Lachnospiraceae NK3A20 group、Ruminococcus 2、Acetitomaculum和Bifidobacteriaceae unclassified,其中Lachnospiraceae NK3A20 group和Ruminococcus 2的相对丰度均在U10组中最高。此外,U10组Ruminococcus gauvreauii group和Olsenella的相对丰度相较于U30组显著升高(P<0.05)。与U0和U30组相比,U10组空肠上皮Rhbg基因mRNA表达水平显著上调(P<0.05);Rhcg基因和UT-B基因表达水平随尿素添加水平的升高逐渐降低,但差异不显著(P>0.05)。[结论]日粮补充适宜尿素(10 g·kg^(-1))可提高空肠内氨态氮含量,促进氮利用细菌的生长定殖,改善菌群结构。此外,添加尿素会影响空肠上皮Rhbg基因的表达,但不影响Rhcg基因和UT-B基因的表达。

[Objectives]This study was conducted to evaluate the effects of incremental urea supplementation on jejunal bacterial communities and the mRNA expression of ammonia and urea transporter proteins in fattening Hu lambs.[Methods]Forty-two 3-month-old selected fattening male lambs with similar body weight(24.3 kg)were randomly assigned to three experimental diets,and supplemented with 0 g·kg^(-1)(U0),10 g·kg^(-1)(U10)and 30 g·kg^(-1)(U30)urea on the basis of basal diets,respectively.The experiment consisted of 1 week of adaptation followed by 10 weeks of dietary treatments.After an 11-week feeding trial,six animals from each group were harvested.Jejunal contents of each lamb were collected to measure fermentation parameters and total bacterial populations and to analyze jejunal bacterial communities by using Illumina MiSeq.Samples of jejunal epithelial tissue were collected to determine the mRNA expression of ammonia transporter protein b(Rhbg),ammonia transporter protein c(Rhcg)and urea transporter protein B(UT-B).[Results]Compared with U0 group,the content of jejunal ammonia-N was significantly higher in U10 and U30 group(P<0.05).Compared with U0 group,the content of total volatile fatty acids(TVFA)and acetate greatly decreased in U10 group(P<0.05).The content of butyrate and isovalerate in U30 group were significantly higher than U10 group(P<0.05).Compared with U0 group,the populations of total bacteria greatly increased in U10 and U30 groups(P<0.05).The Illumina MiSeq sequencing results showed that no difference were found on Chao 1,ACE,Shannon or Simpson index with urea supplementation.The PCoA results based on unweighted-Unifrac indicated that urea supplementation altered the jejunal bacterial communities.Urea supplementation had no significant effect on the abundance of jejunal bacteria at phylum level,but there was a difference at genus level.The dominant bacteria in different diet treatment groups were Lachnospiraceae NK3A20 group,Ruminococcus 2,Acetitomaculum and Bifidobacteriaceae unclassified,and the relative abundance of Lachnospiraceae NK3A20 group and Ruminococcus 2 were highest in U10 group.Furthermore,the relative abundance of Ruminococcus gauvreauii group and Olsenella in U10 group were higher than U30 group(P<0.05).Compared with U0 or U30 groups,the mRNA expression of Rhbg in the jejunum epithelium was up-regulated in U10 group(P<0.05).The mRNA expression level of Rhcg and UT-B decreased with the increasing urea addition to the diet,but the data was not significant(P>0.05).[Conclusions]A basal diet with appropriate urea addition level(10 g·kg^(-1))can increase the content of ammonia-N in the jejunum.At the same time,it can promote the jejunal nitrogen-using bacteria colonization,and modulate the bacterial communities.In addition,urea supplementation in diets affects the expression of Rhbg in jejunum epithelium but does not affect the expression of Rhcg and UT-B.