Effect of isolate of ruminal fibrolytic bacterial culture supplementation on fibrolytic bacterial population and survivability of inoculated bacterial strain in lactating Murrah buffaloes

Aim: The present study was conducted to evaluate the effect of bacterial culture supplementation on ruminal fibrolytic bacterial population as well as on survivability of inoculated bacterial strain in lactating Murrah buffaloes kept on high fibre diet. Materials and Methods: Fibrolytic bacterial strains were isolated from rumen liquor of fistulated Murrah buffaloes and live bacterial culture were supplemented orally in treatment group of lactating Murrah buffaloes fed on high fibre diet to see it's effect on ruminal fibrolytic bacterial population as well as to see the effect of survivability of the inoculated bacterial strain at three different time interval in comparison to control group. Results: It has been shown by real time quantification study that supplementation of bacterial culture orally increases the population of major fibre degrading bacteria i.e. Ruminococcus flavefaciens, Ruminococcus albus as well as Fibrobacter succinogenes whereas there was decrease in secondary fibre degrading bacterial population i.e. Butyrivibrio fibrisolvens over the different time periods. However, the inoculated strain of Ruminococcus flavefaciens survived significantly over the period of time, which was shown in stability of increased inoculated bacterial population. Conclusion: The isolates of fibrolytic bacterial strains are found to be useful in increasing the number of major ruminal fibre degrading bacteria in lactating buffaloes and may act as probiotic in large ruminants on fibre-based diets.


Introduction
express the ability to rapidly digest the fibre with other strains present in rumen.As the rumen microbial The primary sources of energy found in forages ecosystem is not fully explored and majority of the rumen are the structural polysaccharides, cellulose, hemimicrobes are unculturable and not fully identified cellulose and pectin.These three components generally therefore, there is a need to generate more information account for about 400-600 g/kg forage dry matter.In on rumen microbes using recent molecular tools.tropical countries of the world, the ruminants are fed on Thus, the aim of present study was to identify lignocellulosic agricultural by-products like cereal potent fibre degrading bacteria from the rumen of straws, stovers, sugarcane bagasse, tree foliages and buffaloes and their use as probiotic supplement for the cakes of oil seeds like groundnut, cotton, mohua, neem manipulation of rumen microbial ecosystem towards and mustard.The efficiency of ruminants to utilize improving utilization of low quality forage feeds.such a wide variety of feeds is due to highly diversified rumen microbial ecosystem consisting of bacteria

Isolation of fibre degrading bacteria:
4 6 ciliate protozoa (10 -10 /ml, from 25 genera), anaerobic Fibre-degrading bacteria were isolated from rumen 3 5 fungi (10 -10 zoospores/ml, representing five genera) liquor of three permanently fistulated buffaloes 8 9 and bacteriophages (10 -10 /ml) [1].These numbers maintained at National Dairy Research Institute herd might even be larger as majority of them are nonand fed on high fibre diet (concentrate: roughage: culturable.Because cellulose is the most abundant 40:60).Freshly collected rumen liquor was used for component of plant cell walls, ruminal cellulolytic isolation of fibre degrading bacteria.Prior permissions for taking rumen liquor from experimental male microorganisms play a central role in the nutrition of fistulated buffalo already taken from Institutional ruminant animals fed diets based on forage.
Animal Ethics Committee (IAEC).Digestibility of fibre in ruminants may be improved by Total forty-two numbers of bacterial isolates were the introduction of highly fibrolytic strains of bacteria isolated by Hungate's anaerobic roll tube technique in the rumen.This approach may be feasible only if the using carboxymethylcellulose as substrate in the broth inoculated bacterial isolates survive in the system and medium.Pure cultures were obtained by repeated roll The representative rumen liquor sample was collected tube preparation on agar medium, picking of single individually from all the animals of treatment as well as bacterial colony and sub culturing into broth medium.
control group with the help of stomach tube before The fibre degrading characteristic of the bacterial feeding once in a month i.e.Pre-dosing (prior to start of isolates were tested by their in-vitro fibre degrading the dosing), during dosing (immediately after potential on pure NDF by using in-vitro gas production completion of dosing) and post-dosing (after one technique and based on in-vitro fibre degradability month of completion of dosing) period for three most potent isolates were selected for molecular quantification of inoculated bacteria as well as ruminal study.Molecular characterization of the isolates was carried out using conventional PCR technique.The cellulolytic bacteria.The samples were squeezed genomic DNA extracted from the isolate was amplified through two layers of muslin cloth to remove any using universal as well as gene specific primers for impurities (plant extracts, undigested matter, and so ruminal fibre degrading bacteria.All the isolates found on) and processed for DNA extraction.to be of genus Ruminococcus.The amplified product of DNA extraction: three most potent fibrolytic isolates based on their in-Total genomic DNA was extracted by using vitro true dry matter digestibility potential of pure NDF CTAB method [2].DNA concentrations were isolated from wheat straw, were sequenced and nearly measured at 260 nm with a Nanodrop.The DNA used the complete sequence data were obtained for all the for these experiments possessed an A260/A280 ratio three isolates.All three isolates have shown similarity higher than 1.8.To minimize animal to animal with the Ruminococcus flavefaciens strains.The isolate variations, the equal amount of extracted DNA of these NB-1 showed 97% similarity with R. flavefaciens animals was mixed together before PCR.strain FD-1 and was most potent fibre degrader used as

Conventional PCR:
supplement for experimental lactating Murrah buffaloes.
PCR amplification was conducted with

Selection, distribution and feeding of animals:
TM MyCycler thermal cycler (Bio-Rad, USA) as Twelve lactating Murrah Buffaloes (Mid to Late described [3].The PCR conditions were 95°C for 5 lactation) were selected from the buffalo herd of min, followed by 35 cycles consisting of 95°C for 30 National Dairy Research Institute, Karnal.Approval of sec, various annealing temperatures (Table 1) for 30 use of animals to conduct nutritional studies was taken sec, 72°C for 1 min, and a final extension period of from Institute Animal Ethics Committee.Animals 72°C for 10 min.were divided into two groups of six each according to their milk yield, live body weights and days in Real-time PCR:

lactation. Animals were fed with experimental diets
To establish a quantitative assay, all the targeted (concentrate:wheat straw:green maize in the ratio of groups (Table -1 Treatment group of animals were fed by using 10-fold serial dilutions from 10 to 10 experimental diet plus live bacterial culture of plasmid copies of the previously quantified plasmid Ruminococcus flavefaciens strain FD-1 (300 ml) orally standards.Real time PCR amplifications and on alternate day for a period of one month whereas, detections were performed in a MJ MiniTM personal control group was fed control diet plus autoclaved Thermal cycler system (Bio-Rad,USA) as described bacterial culture (300ml) on alternate day for a period [4].In brief, SYBR Green Master mix (Fermentas, 12 of one month.The bacterial culture contained 3x10 USA) was used for PCR amplification.Samples were number of cells per ml of diluent so total number of assayed in triplicate in a 25 µl reaction mixture 14 bacterial cells dosed in an animal was 9x10 cells on containing 5 mM MgCl2, 12.5 µl of 10× Mastermix alternate day basis continuously for one month period.
(including Fast start enzyme, Fast start Taq DNA polymerase, reaction buffer, dNTP mixture, MgCl2, x 10 /ml of RL whereas in treatment group the population for different ruminal microbes.The standard curves were 8.01×10 , 5.76×10 and 2.75×10 copies/ml for F. obtained were used to quantify DNA of different succinogenes, R. flavefaciens, and R. albus, ruminal microbes. respectively [5].It was found that R. albus was more The population sizes of targeted ruminal microbes abundant than both F. succinogenes and R. flavefaciens are given in Table-2a and Table- to 2.52x10 /ml after post dosing period in treatment Ruminococcus, Fibrobacter succinogenes and group whereas in control group the increase was eukaryotes measured by 16S rRNA probes increased significantly (P<0.05 ) during the dosing period, but significant (P<0.05)only during post dosing period 7 declined post-dosing in the sheep dosed with 500 ml (2.58x10 /ml of RL) in comparison to pre dosing 7 medium containing strain of Ruminococcus continuously (1.86x10 /ml of RL).The increase in R.albus population for two weeks [8] matches with the result of present was also noticed in treatment and control group and the 3 3 finding as in present finding also population of increase was from 1.73x10 /ml of RL to 1.96x10 /ml 3 3 Ruminococcus flavefaciens, Fibrobacter succinogenes and from 1.72x10 to 1.77x10 /ml of RL, respectively and Ruminococcus albus has shown marked increase during dosing period although the effect was not during dosing period whereas declined post dosing significant.The B.fibrisolvens population increased in 7 except in dosed strain.control group and the increase was from 2.5x10 to 3.04 ) were amplified using specific primers 40:30:30) plus live and autoclaved culture of R. and cloned by using the Stratagene Blunt End cloning flavefaciens strain FD-1 after 21 days adaptation of kit (Stratagene, USA) and purified by QIAprep kit animals.The animals were housed in open shed having (Qiagen, Venlo, The Netherlands).The purified proper arrangement of individual feeding and plasmids were quantified by Nano-drop (ND-1000 dye), 50 ng of template DNA, and 0.5 of B.fibrisolvens decreased from 3.29x10 to 3.19 7 µM of each primer.All PCR reactions were performed x10 /ml of RL, which shows antagonistic relation in in triplicate.terms of population growth with R.flavefaciens.The population of F.succinogenes was increased during Results and Discussion dosing in both group but it decreased after post dosing For absolute quantification of different ruminal and the decrease was significant (P<0.05) in comparison microbes, external standards were prepared from a to during dosing population.The graphical representation simulated rumen matrix.For each standard, linear of real time quantification study of ruminal fibrolytic regressions derived from the threshold cycle [Ct] of bacterial strain in experimental buffaloes at different each DNA dilution versus the threshold cycle were time interval has been shown in Figure-1.calculated.Logarithms of the DNA concentrationsThe present finding was very much identical with were plotted against the threshold cycles with a linear the finding of [5] who found that predominant 2 correlation coefficient (r ) ranging from 0.993 to 0.997 cellulolytic bacteria in the rumen of swamp buffalo 6 5 4

Sampling from experimental animals:
2b respectively.in batch and continuous culture [6] while [7] found R. Results indicated that dosing increases R.flavefaciens flavefaciens was slightly more abundant than F.

Table - 2a
: Real time quantification of ruminal fibrolytic bacterial strain in experimental buffaloes Real time quantification of ruminal fibrolytic bacterial strain in experimental buffaloes Sirohi SK(2009).Dominance of population whereas this increase was significant in Methanomicrobium phylotype in methanogen population inoculated bacterial population even after dosing period present in Murrah buffaloes (Bubalus bubalis).Letters in of one month in treatment group as compare to control Applied Microbiology 49(2):274-277.doi:10.1111/j.1472765X.2009.02654.x. group of animals during dosing and post dosing period 4. Chaudhary PP, Dagar Sumit and Sirohi SK(2012).which may lead to increased fibre digestion in rumen.as affected by level of roughage in swamp buffaloes, Current Microbiology, 58: 294-299. population