Effects Of Different Forage And Protein Sources Of Diet On The Quantity Of Functional Bacteria In The Rumen Of Dairy Cows

The ruminants harbor complex symbiotic microbiota which is responsible for the breakdown ofvarious dietary nutritive components. The microbiota is highly responsive to the changes of diet or theraito of dietary concentrate to forage. The present study was conducted to quantify the population ofrumen functional bacteria of Chinese Hostein cows fed with diffrernt forage and protein type diets byquantitative real-time PCR. And we also verified the influence of different diets on the numbers ofRuminococcus albus, concentration of NH3-N and volatile fatty acids by anaerobic culture in vitro. Thisstudy was aimed to reveal the internal changing mechanism of rumen microbes, and providing strategiesfor the completing feeding structure, promoting utilization efficiency of feed and improving animalperformance. The study consisted three parts:1. The roughage of the diets was alfalfa plus corn silage and corn straw respectively, and theprotein supplement was soybean meal, which was named MF (alfalfa plus corn silage, soybean meal)and CSA (straw, soybean meal). Thirty-two Chinese Hostein cows were blocked based on DIM andmilk yield and randomly assigned to two treatments to evaluate the effect of the different diets on thequantity of rumen microorganism. The experiment was conducted for107days and consisted2periods.Period1lasted91days, during which cows were fed with MF and CSA respectively. Period2lasted16days and all animals were fed MF diet. Rumen fluid was collected by inserting a flexible plastic tubeinto the rumen before and after feeding on day91and day107. Then the rumen microbial DNA wasextracted by CTAB plus bead beating DNA extraction method. Species-specific PCR primers werechosen from the literature to amplify partial16S rDNA regions. The copy number of rumen cellulolyticbacteria, hemi-cellulolytic bacteria, proteolytic bacteria, amylolytic bacteria, fat and acid utilizationbacteria and lactic acid producing bacteria was detected by quantitative real-time PCR. The resultsshowed that when we used alfalfa plus corn silage as the dietary roughage source, the copy number ofFibrobacter fuccinogene and Ruminococcus albus was signicantly greater (P<0.05) than that of cornstraw group, while Eubacterium ruminantium was decreased (P<0.05) in alfalfa plus corn silage group.2. The protein supplement of the diets was soybean meal and cottonseed plus rapeseed mealrespectively, and the roughage supplement was corn straw, which was named CSA (straw, soybean meal)and CSB (straw, cottonseed plus rapeseed meal). Thirty-two Chinese Hostein cows were blocked basedon DIM and milk yield and randomly assigned to two treatments to evaluate the effect of the differentdiets on the quantity of rumen microorganism. The experiment was conducted for107days andconsisted2periods. Period1lasted91days, during which cows were fed with MF and CSArespectively. Period2lasted16days and all animals were fed MF diet. Rumen fluid was collected byinserting a flexible plastic tube into the rumen before and after feeding on day91and day107. Then therumen microbial DNA was extracted by CTAB plus bead beating DNA extraction method.Species-specific PCR primers were chosen from the literature to amplify partial16S rDNA regions. Thecopy number of rumen cellulolytic bacteria, hemi-cellulolytic bacteria, proteolytic bacteria, amylolytic bacteria, fat and acid utilization bacteria and lactic acid producing bacteria was detected by quantitativereal-time PCR. The results showed that when we used soybean meal as the dietary protein source, thecopy number of Prevotella brevis, Streptococcus bovis, Bacteroides and Ruminobacter amylophilu wassignicantly greater (P<0.05) than that of cottonseed plus rapeseed meal group, while Fibrobacterfuccinogene and Ruminococcus albus was decreased (P<0.05) in soybean meal group.3. The MF, CSA and CSB meal were used as the part of the substrates. Ruminococcus albus wasincubated for0h,24h,48h and72h at39℃by anaerobic culture in vitro, and then detected the copynumber of Ruminococcus albus by quantitative real-time PCR, and also the concentration of NH3-N andvolatile fatty acids. The results showed that the copy number order of Ruminococcus albus was MF>CSA> CSB (P<0.05). Acetic acid/propionic acid ratio in group MF was higher than CSA (P<0.05),while the concentration of acetic acid and propionic acid in group MF was lower than CSA (P<0.05).The concentration of isobutyrate acid, butyrate acid, isopentoic acid and pentoic acid in group CSB wassignificantly higher than group CSA (P<0.05).In conclusion, when fed the diets with different roughage source, alfalfa plus corn silage couldsignificantly promote the amounts of cellulolytic bacteria; when fed the diets with different proteinsupplements, soybean meal can promote the amounts of proteolytic bacteria, while cottonseed plusrapeseed meal might increase the copy number of cellulolytic and hemicellulolytic bacteria; differentroughage and protein source did produce an significant influence of the amounts of Ruminococcus albusand concentration of volatile fatty acids in vitro.