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Effect of Radix Bupleuri herbal supplementation on diversity of the bacterial community and cellulolytic bacteria in the rumen of lactating dairy cows analyzed by DGGE and RT-PCR
Numerous studies have been completed on use of herbal medicine in substitute for chemical feed additives to modify rumen fermentation. This experiment was conducted to investigate effects of Radix Bupleuri herbal supplementation (RBH) on diversity of the bacterial community and cellulolytic bacteria including Fibrobacter Succinogenes, Ruminococcus albus and Ruminococcus flavefaciens in the rumen of lactating dairy cows analyzed by DGGE and RT-PCR. Forty Holstein cows were assigned to one of 4 groups (n = 10) according to milk yield (37.5 ± 1.8 kg/d), day in milk (75 ± 15) and parity (1.7 ± 0.4) in a completely randomized block design. Four treatment diets consisted of supplemental RBH at 0, 0.25, 0.5 or 1.0 g/kg based on dry matter, which were randomly assigned to one of 4 groups. Cows were individually fed the treatment diets, and the experiment lasted for 10 wk in Shanghai. Rumen fluid samples were collected at 2 h post-feeding using stomach-tube on wk 6 of the trial. Rumen samples were strained through 4 layers of cheesecloth and frozen at -20 ℃ until being analyzed. Rumen microbial DNA was extracted with CTAB plus bead beating and analyzed by DGGE with subsequent cluster and optimal density of the bands analysis. The copy number of rumen cellulolytic bacteria was detected by quantitative RT-PCR with species-specific PCR primers amplifying partial 16S rDNA regions. Diversity index values and data of copy numbers of 16s rDNA were analyzed using GLM procedure of SAS 9.2. DGGE profiles showed that the quantity of bands was similar, while the optimal density was different between control and treatment. The dendrogram of the DGGE fingerprint showed that they were assigned to different clusters by different supplemental levels with DGGE fingerprint similarity less than 0.54 overall. Shannon-Weiner index decreased (P < 0.05) especially with 1.0 g/kg RBH, while the dominance index increased (P< 0.05) especially at 0.25 and 0.5 g/kg levels compared with the control. RT-PCR revealed no difference in cellulolytic bacteria among groups. In total, RBH supplementation proved little effects on diversity of the bacterial community and cellulolytic bacteria in the rumen of lactating dairy cows potentially owing to gradual adaptation of rumen bacteria to the RBH supplementation.
Keywords: bacterial community, cellulolytic bacteria, Radix Bupleuri