不同蛋白源日粮添加乳酸链球菌素对育肥湖羊瘤胃发酵及微生物菌群结构的影响

本试验旨在探究不同蛋白源日粮条件下添加乳酸链球菌素（nisin）对育肥湖羊瘤胃发酵及微生物菌群结构的影响。试验采用2×2因子设计，两因子分别为蛋白源豆粕（SBM）、干玉米酒精糟及其可溶物（DDGS）]和nisin （添加水平为0或30.5 mg·kg^-1 DM），两两组合，配制4种等氮等能日粮。选取32只体重（23±2） kg的断奶湖羊公羔，按照随机区组设计，根据体重分为2个区组（低体重组，16只；高体重组，16只），每个区组的湖羊随机分配到4个组并分别饲喂对应日粮，单栏饲喂。试验预饲期1周，正式期9周，试验期结束时每组从高、低体重区组中各随机选取3只湖羊（共24只）进行屠宰，采集瘤胃内容物，提取微生物基因组DNA，利用Illumina MiSeq测序和Real-time qPCR方法分析瘤胃微生物菌群结构。研究结果表明，除Ruminococcaceae NK4A214 group和Unclassified Bacteroidetes相对丰度外，日粮蛋白源与nisin对其他所有测定指标（瘤胃发酵参数、瘤胃菌群数量、瘤胃细菌多样性及相对丰度等）均不存在交互作用（P>0.05）。日粮添加nisin对所有测定指标均无显著影响（P>0.05）。与饲喂豆粕湖羊相比，饲喂DDGS湖羊瘤胃乙酸、氨态氮浓度及总支链脂肪酸（BCVFA）浓度显著降低（P<0.05）。qPCR结果显示，饲喂不同蛋白源日粮湖羊瘤胃总菌、真菌及甲烷菌数量均无显著差异（P≥0.053），但相对于饲喂豆粕湖羊而言，饲喂DDGS湖羊瘤胃内原虫和嗜氨梭菌（C.aminophilum）数量显著降低（P<0.05）。Illumina-MiSeq测序结果表明，饲喂DDGS湖羊瘤胃内细菌Chao1指数和ACE指数显著升高（P<0.05）。不同日粮处理组在门水平上的优势菌门均为Bacteroidetes及Firmicutes；在属水平各处理组的主要优势菌属为Prevotella 1、Christensenellaceae R-7 group和Ruminococcaceae NK4A214 group。饲喂不同蛋白源湖羊瘤胃细菌在门水平上并未产生显著影响（P≥0.14）；在属水平上，饲喂DDGS湖羊瘤胃Pseudobutyrivibrio和Roseburia丰度均显著高于饲喂豆粕湖羊（P<0.05），而Butyrivibrio 2以及Ruminococcaceae UCG-005的相对丰度显著低于饲喂豆粕湖羊（P<0.05）。综上所述，使用DDGS作为日粮蛋白源改变了湖羊瘤胃发酵参数及微生物菌群结构，原虫和C.aminophilum数量的降低可能是导致瘤胃氨态氮浓度显著降低的主要原因；日粮添加30.5 mg·kg^-1 DM的nisin对育肥期湖羊瘤胃发酵参数及微生物菌群结构均无显著影响。

Effects of Different Dietary Protein Sources Supplemented with Nisin on Rumen Fermentation and Rumen Microbiota of Fattening Hu Sheep

This study investigated the effects of different dietary protein sources and nisin on rumen fermentation and rumen microbiota in fattening Hu sheep. Thirty-two male Hu lambs ((23±2) kg initial BW) were assigned to 4 dietary treatments in a randomized block design with a 2×2 factorial arrangement. Two blocks were designed according to body weight with 16 lambs each. Two protein sources (soybean meal (SBM) and dried distillers grains with solubles (DDGS)), and two levels of nisin (0 and 30.5 mg·kg^-1 DM) were used to formulate 4 isonitrogenous and isoenergetic diets. The feeding trial was conducted for 10 weeks, with the first 1 week for adaptation followed by 9 weeks of dietary treatment. At the end of the experimental period, 6 lambs (3 lambs per block) were randomly selected from each group, and slaughtered for rumen contents collection. Metagenomic DNA of rumen content was extracted, and rumen microbiota were analyzed by Illumina MiSeq sequencing and quantitative PCR. No interaction (P>0.05) of protein×nisin was found on all measured indicators (rumen fermentation characteristics, ruminal microbial populations, rumen bacterial diversity and relative abundance, etc.) except for the relative abundance of Ruminococcaceae NK4A214 group and unclassified Bacteroidetes. Nisin supplementation had no impact on all measured indicators (P>0.05). Lambs receiving DDGS had lower concentrations of ruminal acetate, ammonia and branched-chain VFA (BCVFA) compared with lambs fed SBM (P<0.05). The DDGS-fed lambs had a smaller (P<0.05) population of protozoa and C. aminophilum than those fed SBM, but the population of total bacteria, fungi, and methanogens were similar (P ≥ 0.053). For alpha diversity, ACE and Chao1 index were higher (P<0.05) in the DDGS-fed lambs than in those fed SBM. At phylum level, the dominant bacteria in different diet treatment groups were Bacteroidetes and Firmicutes. At genus level, the dominant bacteria in each treatment group were Prevotella 1, Christensenellaceae R-7 group and Ruminococcaceae NK4A214 group. None of the relative abundance of bacteria at phylum level was affected (P ≥ 0.14) by protein sources. Compared with those fed SBM, the relative abundance of Pseudobutyrivibrio and Roseburia were higher (P<0.05), while the relative abundance of Butyrivibrio 2 and Ruminococcaceae UCG-005 were lower (P<0.05) in DDGS-fed lambs. Replacing SBM in an isonitrogenous lambs diet with DDGS changed the rumen fermentation characteristics and the microbial community structure, and the reduction of the population of protozoa and C. aminophilum may be the main reason for the reduction of rumen ammonia concentration. Nisin supplementation at 30.5 mg·kg^-1 DM had no effects on rumen fermentation and rumen microbiota in fattening lambs.