Application of Real Time PCR assay for quantifying bacterial density in the rumen of Goats fed tannin rich diets
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Date
2009
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AAU, Anand
Abstract
India possess second largest population of Goats. Grazing goats are the backbone of most of the world's marginal land enterprises. They are capable of utilizing effectively a vast variety of plant species and vegetation types including unconventional feeds. Goats are normally habituated to eat vast variety of tree leaves which usually contain anti-nutritional factors like tannins. Their tannin tolerance is higher than other livestock. This may be due to speciaUzed microbial ecosystem. Present study was aimed to explore the effect of tannins on ruminal microbes using real time PCR approach. Tannins are most effective against the fiber-degrading (cellulolytic) bacteria like Fibrobacter succinogens, Riiminococcus species, Butyrivibrio fibrisolvens, Ruminobacter amylophilus. The ruminants which continuously feed upon diets rich in tannins usually develop a microflora which is tolerant to high tannins such bacteria includes Streptococcus capriniis, Streptococcus bovis, Selenomonas ruminantium, Clostridium species and class proteobacteria. The experiment was conducted on eight adult goats divided into 4 groups viz. Tl: control (0% acacia nilotica pods in TMR, 0% tannin), T2: 25% acacia nilotica pods in TMR (3.5% tannin), T3: 43% acacia nilotica pods in TMR (6% tannin) and T4: 59% acacia nilotica pods in TMR (8.5%) tannin). The Total Mixed rations with different levels of Acacia pods were produced and fed to respective goats ad lib. Rumen liquor (200 ml) was collected on 0, 15th and 30th day of experiment at 0, 3 and 6 hrs post feeding from each animal to study the effect of tannins on bacterial population. The bacterial DNA was extracted from pooled samples of both animals in each group by enzyme-chemical lysis method from rumen fluid. The DNA stock samples were quantified using Nano-drop spectrophotometer at 260 and 280 nm. Purity of DNA was judged on the basis of optical density ratio at 260:280 nm which was between 1.8 to 2.0 for all the samples indicating desirable purity. Species specific primers were used to amplify the bacteria (Selenomonas niminantium, Streptococcus bovis, Fibrobacter succinogenes, Treponema bryantii, Anaerovibrio lipolytica, Total Bacteria, Prevotella ruminicola, Ruminococcus albus, Methanobacteriales and Methanomicrobiales targeting 16S rRNA gene were used for amplification of DNA. The amplified products were visualized as a single compact band of expected size under UV light. The PCR products were purified by eluting the PCR product from the agarose using QIAquick Gel Extraction Kit - Qiagen and were ligated in pTZ57R/T vector of InsT/Aclone TM kit (Fermentas). This was followed by transformation into competent cells (DH5-α strain) of E.coli. Recombinant colonies were picked up by Blue white screening. White colonies were confirmed for presence of insert by colony PCR using M13 primers. Recombinant colonies were inoculated in Luria Broth for 16-18 hrs. Plasmid extraction from overnight culture was carried out by using QIAprep plasmid extraction kit. The plasmids contain species specific amplified DNA fragment so these plasmids were used as standards while running the real time PCR. Their copy number was calculated using optical density and molecular weight of plasmids. The plasmids were serially diluted and standard plot was prepared and according to the plot, the concentration of amplified DNA and ultimately the bacterial population was measured. All samples along with standard plasmids were amplified with species specific primers using real time PCR. The population of bacteria Selenomonas ruminantiwn increased with increase in level of tannins in different group (1314% increase in group IV followed by 747% and 210%) in group III and II respectively at 30th day of experiment) of animals and also with period of experimentation (Increased with 844%) and 1314% at 15th and 30th day respectively in group IV). Similarly the population of Streptococcus bovis also increased with increase in level of tannins and with period of feeding (555%o increase in group IV at 30th day). The lipolytic bacteria Anaerovibryo lipolytica increased with increase in level of tannins in feed (3645% increase in group IV). The results revealed Selenomonas ruminantium and Streptococcus bovis as tannin tolerant bacteria. Tarmins have inhibitory activity against fibrolytic bacteria Fibrobacter succinigenes and reduction in population was more prominent at 30th day of experiment (decreased by 73%, 67%) and 57% in group FV, III and II respectively). Similar inhibitory effect (78% decrease in group IV) was also seen in Treponema bryantii which is saccharolytic spirochete and has been shown to be associated with the fibrolytic bacteria of the rumen. The population of proteolytic bacteria Prevotella niminicola was not affected at any level of tannins throughout the experiment. At low level (3.5%), tannins have beneficial effect on microbial growth for total bacterial population but no effect was seen at other levels. The population of methanogens of the order Methanobacteriales and Methanomicrobiales also remain unchanged even at the highest level of tannins. The population of Ruminococcus albus increased at 15th day with the highest in group FV (496%) followed by group III (416%) and group II (308%). At 30th day the population decreased compared to 15th day but remained at higher level to that of 0 day population. The study revealed that Selenomonas ruminantium, Streptococcus bovis and Anaerovibrio lipolytica are major tannin tolerant bacteria in goats. Tannins exert detrimental effect on fibrolytic bacteria like Fibrobacter succinigenes and Treponema bryantii. However no effect of dietary tannins was observed on Prevotella ruminicola, Methanogenes (Methanobacteriales and Methanomicrobiales) and total bacterial population in goats.
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ANIMAL BIOTECHNOLOGY, A STUDY