日粮中添加鱼油后肉牛瘤胃细菌区系的变化

目的：研究肉牛日粮中添加2%鱼油后瘤胃细菌区系的变化。方法：分别于添加鱼油前后取瘤胃液提取总DNA,根据细菌通用引物27F/1492R扩增16S rDNA基因序列,分别构建2个16S rDNA基因文库;从每个文库中各随机挑取384个克隆,对阳性克隆用HhaⅠ进行限制性片段长度多态性（RFLP）分析、聚类,取各RFLP类群中的代表克隆进行16S rDNA序列测定,构建系统发育树,研究细菌区系多样性的变化。结果：添加鱼油前、后,文库的RFLP图谱分别可以分成74和41个RFLP类群;添加鱼油前后的优势菌群均为噬纤维菌-屈挠杆菌-拟杆菌（CFB）和低（G＋C）含量的革兰阳性菌（LGCGPB）,但添加鱼油后文库中LGCGPB比例降低,而且未检测到纤维菌门细菌;有50%～60%的测定序列与GenBank数据库中的序列相似性高于97%,90%以上为未培养的细菌;添加鱼油后瘤胃细菌的多样性减少。结论：日粮添加鱼油后CFB比例增高但多样性下降,LGCGPB、纤维杆菌比例下降,这一结果为调控瘤胃发酵提供了依据。

Changes of Rumen Bacteria Flora of Beef Cattle After Fed with Fish Oil

Objective：To investigate the change of rumen bacteria floras after diets of beef cattle supplemented with 2% fish oil.Methods：Total genomic DNA was extracted from rumen fluid obtained before and after fish oil supplementation.16S rDNA was amplified with bacteria universal primer 27F/1492R and two 16S rDNA libraries were constructed.384 clones were picked up randomly from each library and positive clone were evaluated by restriction fragment length polymorphism（RFLP） with HhaⅠ.The represent clones in each RFLP class were sequenced.Phylogenetic tree were constructed to study the structural differences of rumen bacteria floras before and after fish oil supplementation.Results：Cluster analysis identified 74 and 41 unique RFLP patterns in fish oil supplementation library and without fish oil supplementation library separately.It resulted that CFB（Cytophaga-Flexibacter-Bacteroides） and LGCGPB（low G＋C Gram positive bacteria）were major phyla in both libraries.The percent of LGCGPB declined after fish oil supplying and no Fibrobacter was detected.The sequencing results showed that the similarity of 50%～60% sequences obtained were more than 97% to the 16S rRNA gene sequences reported in GenBank.Uncultured bacteria account for over 90%.The diversity of rumen bacteria flora was reduced after fish oil supplied.Conclusion：The percent of CFB in post-fish oil supplementation library was increased but the diversity decreased.Bacterias in LGCGPB and Fibrobacter phyla decreased.This provided evidence for controlling the fermentation of rumen.