New Rumen Ciliate Primers Targeting Full-length 18S RRNA Genes And Its Application In Evaluating Dietary Effect On Ciliate Diversity In The Rumen Of Dairy Cows

Rumen protozoa have caused extensive attention, because they play an important role in the degradation of diets, maintain rumian environment in ruminants, and ruminant methane emission. Uncultured methods were increasingly being used in the rumen protozoal diversity, such as 18 S r RNA gene clone libraries phylogenetic analysis and quantitative PCR. Therefore, this study was designed to optimize and validate best primers targeted to protozoal full-length 18 S r RNA genes, and use the optimal primer to evaluate the effect of different dietary fiber and protein source on rumen protozoal diversity and abundance. This study is divided into three parts:1, 137 Ciliophora gene sequences of were download from Silva database, after aligment in ARB software the conservation regions were used to design new primers. Totoal fiver primer pairs（two from literatures） were detected for their base coverage and specific. As a result, primer pair P. 324 f and P. 1747r₂ had a good base coverage（>98%） which can distinguish rumen protozoa and other eukaryotic microorganisms, and longer amplicon（1,423 bp） suitable for subsequent rumen protozoal community diversity and phylogenetic analysis. Cutoff value for operational taxonomic units（OTU） analysis were calculated from 0.010 to 0.041 within the 137 downloaded sequences, and in 0.015 level the rates of all strains in a species were in the same OTU and one OTU contained only one species were lowest, which indicate this level can be used in later research.2, Three different dietary fiber and protein sources were designed, namely MF group（alfalfa hay, corn silage and soybean meal, miscellaneous meal）, CSA group（corn stover and soybean meal, mixed meal） and CSB group（corn stover and mixed meal）. The health 48 Holstein cows with similar body condition were randomly assigned into three groups, and cows were fed and managed uniformly. In the first 91 days, rumen fluid were sampled before and after the morning feeding（2 h）, and microbial DNA were extracted. The optimal primers were used for the rumen protozoa 18 S r RNA gene clone library, community diversity and phylogenetic analysis. The results showed that the clone libraries between different fiber source were significant different（Libshuff, P<0.005）, but clone libraries between different protein source were similar（Libshuff, P=0.250）. After analysis the abundance and diversity of these libraries, different dietary treatments had no significant effect on the rumen protozoal abundance, but compared to corn straw treatment, alfalfa hay and corn silage could increase the protozoal diversity, whereas different dietary protein source had no consist effect on protozoal diversity before and after morning feeding. Phylogenetic analysis showed that Dasytricha（54%） and Entodinium（35%） were main protozoal genera in this study.3, The numbers of rumen protozoa were explored under the treatments of different dietary fiber and protein sources. Materials and methods were same with the second part, except that all the treatments were changed to MF after 91 day and last for 16 days. Rumen fluid were sampled on the day 31, 61, 91 and 107, and the microbal DNA were extracted from 91 day samples. There were no significant differences of protozoal abundance between treatments by microscopic counting, except 31 day. The similar results were producted by q PCR which indicated different dietary fiber and protein sources had no significant effect on rumen protozoal abundance. In order to confirm the results from clone libraries, the main rumen protozoa Dasytricha and Entodinium were conducted relative quantitive PCR, which showed that different treatments did not affect the relative abundance of Entodinium, but the relative abundance of Dasytricha in corn straw treatments were higher（P<0.05） than alfalfa hay and corn silage treatment.In summary, a primer pair with better base coverage, higher specificity and longer amplicon and a better cutoff value for protozoa OTU assignment were obtained and used to evaluate the rumen protozoal diversity in cows fed different dietary fiber and protein sources. Alfalfa hay and corn silage group had higher protozoal diversity than corn straw group. After blasting all the sequences in clone libraries, most of the sequences were contributed to Dasytricha and Entodinium. However, compared with alfalfa hay and corn silage as fiber source, corn and straw treated had higher（P<0.05） relative abundance of Dasytricha.