Effect of inclusion of Myristica fragrans on methane production, rumen fermentation parameters and

Aim: The present study was done to evaluate the effect of Myristica fragrans fruit active compounds addition on methane production in vitro. Materials and Methods: Methanolic extract of Myristica fragrans fruit powder was prepared and checked for its inhibitory action on methane production in diet containing roughage 50 percent and concentrate 50 percent respectively. Methane production was estimated by Gas Chromatography. Results: It has been shown that supplementation of Myristica fragrans reduces the methane production up to 48 percent as compared to control diet without supplementation of Myristica fragrans. Similarly real time quantification of mcr-A gene also shown the significant (P<0.05) reduction in the number of methanogens. Myristica fragrans appeared to reduce methane production by inhibiting methanogens directly. However, digestibility of dry matter also decreased due to myristica fragrans supplementation in total mixed diet, which may affect the production of volatile acid production. Conclusion: The active compounds extracted in methanol of Myristica fragrans emerged out to be a useful natural plant source for the inhibition of methanogenesis and its supplementation in animal feed may proves to be an effective measure to control methane emission from ruminants.


Introduction
the advancement of molecular biological approaches it is easy to quantify the number of methanogens In India, methane emission from Agricultural without practicing the tedious methods of culturing sector, the livestock is the major contributor to the methanogens in different samples.mcr-A gene is global warming.In 2009 India livestock methaneubiquitously present in all methanogens, so it is used emission was 11.75 million metric tons per year higher as a standard to quantify the methanogens under than the 9 million metric tons estimated in 1994 [1].To different treatments and diet conditions [9].mitigate methane emission is considered as an In the present paper an effort has been made to international goal in order to reduce global warming as study the anti-methanogenic potential of Myristica methane is considered to be a potent green house gas.fragrans (Jaiphal) plant extract by in vitro Level of methane emission from the ruminants is fermentation study, using Gas Chromatography and affected by a number of factors such as level of feed quantify the expression of mcr-A gene in treated and intake, addition of lipids and ionophores in their diet, control samples.change in rumen microbial environment and the level Materials and Methods of animal productivity have been identified [2,3,4].Plants having secondary metabolites have been Preparation of plant extracts: The Myristica thought to play an important role in reducing fragrans (Jaiphal) fruits were obtained, crushed into 0 methanogenesis in rumen [5].Saponins or saponinsmall pieces and oven dried at 70 C. 50 % aqueous like substances have been reported to suppress methanol was used as a solvent to prepare plant methane production, reduce rumen protozoa counts, extracts.The plant material was then ground to pass and modulate fermentation pattern [6,7,8].Now with through a 1 mm screen.A known quantity of finely ground sample was weighed into 250 ml conical flask.rumen liquor, 30 ml of incubation medium was The 50% aqueous methanol was added (1:10 dried injected to the syringes using auto dispenser.The plant to solvent) and the flask was tightly sealed and syringes were shaken gently and residual air or air 0 bubbles if any was removed and outlet was closed.The kept in a rotary shaker at 25 C and 120rpm for 24 hour.level of piston was recorded and syringes were placed After shaking the content of the flask, methanol in water bath maintained at 39 ± 0.5°C.The syringes extract of Myristica fragrans is directly filtered were shaken every one hour up to 10 hour of through Whatman no. 1 filter paper and used for incubation.These trials were conducted along with further analysis.
respective blank and control in triplicate.After 24 hour Collection of Rumen Liquor for In-Vitro Gas of incubation, volume of gas was withdrawn from the Production Test: Rumen samples were obtained tip of the incubation syringe using Hamilton gas tight after manual mixing of rumen contents from three syringe and analyzed for methane with the help of gas rumen fistulated adult male buffalo (Bubalus bubalis) chromatograph (Nucon 5700, India) Flame ionizing after taking the permission from the Animal Ethics detector (FID) is used.The temperature of injection Committee of the institute.The buffalo were kept on a port, column and detector was 40°C, 50°C and 50°C standard diet comprising concentrate and roughage in respectively.Volume of gas taken for injecting was a ratio 50:50.Rumen liquor samples from the 200µl.The flow rate of carrier gas (N ) through the 2 buffaloes were collected prior to the first morning column was 30ml/min and H was 30ml/min and air 2 feed.Just after collection of sample in insulated flask 0 was 300 ml/min.The standard gas for methane pre-warmed at 39 C taken to lab, immediately passed estimation (Spantech caliberation gas, Surrey, carbon dioxide and filtered through four layer of England) was composed of 50% methane and 50% muslin cloth and then used for setting up of in vitro gas CO .The peak of methane gas was identified on the 2 production test.basis of retention time of standard methane gas and the Preparation of diet: To evaluate the effect of response factor obtained was used to calculate myristica fragrans diet was prepared by taking methane percentage in the gas sample.The methane roughage concentrate ratio of 50:50.The roughage produced from substrate during 24 hour incubation part composed of wheat straw and the concentrate part was compared for the blank values.The volume of composed of maize (33%), ground nut cake(GNC) methane produced was calculated as follows: (21%), mustard cake (12%), wheat bran (20%), Methane production (ml) = Total gas produced (ml) x % deoiled rice bran (11%), mineral mixture (2%) and salt methane in the sample.(1%) respectively.Chemical composition of diet Estimation of ammonia nitrogen: The supernatant include 87.84 g/kg DM of Organic matter(OM),12.53 of each syringe including that of blank was used for g/kg DM of Crude protein(CP) and 32.95 g/kg DM of NH -N estimation.Supernatant (5 ml) was mixed with 3 Acid Detergent Fibre (ADF).
1 N NaOH (2 ml) and steam passed on this using KEL

Estimation of Methane Production by Gas Chro-
PLUS -N analyzer (Pelican, India) and the NH evolved 3 matography: For estimating the methane production was collected in boric acid solution having mixed incubations were carried out in 100 ml calibrated glass indicator and titrated against N / 100 H SO .to the sides.2 ml of plant extract was injected before At the end of incubation (24h) 1 ml of the supernatant incubation.The syringes were kept in an incubator at was treated with 25% meta-phosphoric acid (4 ml) and 39+0.5°C.The medium mixture solution was prepared kept for 3-4 h at ambient temperature [13].Thereafter, by mixing 500 ml distilled water, 0.125 ml micro it was centrifuged at 3000 rpm for 10 minutes and clear mineral, 250 ml buffer , 250 ml macro mineral , 1.25 0 supernatant was collected and stored at -20 C until ml resazurine and 50 ml reducing solution (prepared analyzed.IVFA estimated using gas chromatograph fresh and added prior to incubation).Once the medium (Nucon 5700, India) equipped with flame ionization mixture solution becomes colorless, the required detector (FID) and stainless steel column (length 4'; amount of filtered rumen liquor was added.The o.d ¼''; i.d 3 mm) packed with Chromosorb 101.proportion of medium mixture solution to rumen Temperature of injection port, column and detector liquor was 2:1.Just after mixing the medium and 0 used [18] and the specific amplified target region was was set at 200, 180 and 210 C, respectively.The flow cloned by using Stratagene Blunt End Cloning kit rate of carrier gas (N ) through the column was 40 ml/ 2 (Stratagene,USA) in order to establish a quantitative min, and the flow rate of hydrogen and air through FID assay.With the use of the Fermentas Plasmid was 30 and 300 ml/ min.respectively.Sample (2µl) Purification Kit (Fermentas,USA), plasmid DNA was was injected through the injection port using Hamilton isolated and the purified plasmids were quantified by syringe (10 µl).Individual VFAs of the samples were spectrophotometry with multiple dilutions.The target identified on the basis of their retention time and their DNA used for these experiments possessed an concentration (mmol) and calculated by comparing the A260/A280 ratio greater than 1.8.The target DNA was retention time as well as the peak area of standards 1 quantified by using serial 10-fold dilutions from 10 to after deducting the corresponding blank values.each primer.All PCRs were performed in triplicate.The proximate analysis of substrate was carried out as per the methods of [16].The cell wall constituents of Statistical analysis: Experimental data of different substrates were determined according to suggested parameters were analyzed in paired T test with three method [15].
DNA extraction: Total genomic DNA was isolated

from rumen liquor sample after methane
Methane measurement by Gas Chromatography measurement using Bacterial genomic DNA isolation and other nutritional parameters: Chemical kit (Chromas Biotech Pvt. Ltd., Bangalore, INDIA).composition of diet presented in table 1.After 24 hours For rumen samples a 1.5 ml aliquot was taken from the of in vitro experimentation methane was measured out rumen sample after methane measurement using a of the total gas produced in the tubes.Methane wide bore pipette so as to ensure a homogenous production in the control tubes was found out to be sample containing fluid and digesta.This was 20.47 ml/gm of diet where as in the tubes in which centrifuged at 12000 g for 5 min and the supernatant Myristica fragrans (Jaiphal) is supplemented methane was removed before DNA extraction.
production was significantly (P<0.05)reduced to 12.70 ml/gm of diet.Myristica fragrans (Jaiphal) also Conventional PCR: PCR amplification was TM decreased the ratio of acetate to propionate from 3.90 conducted with an My Cycler Thermal Cycler (Bioto 3.74.Rad, USA).The reaction mixture was treated 0 according to the following protocol: 95 C for 5 min, according to the decrease in copy number of = Hemi cellulose, ADL = Acid detergent lignin, TA = Total ash methanogens present in treatment combination.The However, IVDMD was also decreased significantly result shows that there is significant (P<0.05)decrease due to addition of methanolic extract of Myristica in the methanogens population in the treated sample as fragrans (Jaiphal) which also affect the overall compare to that of the control which is the untreated production of individual volatile fatty acid production.one after 24 hour incubation.The reduction in the methane gas production was also Table-3 Quantitative Real Time PCR analysis: Total DNA isolated from both the treated as well as control samples were subjected to Real Time PCR analysis to monitor the quantity of methanogens in both the samples with the help of mcr-A gene targeting primers [18].All the samples were taken in triplicate.The starting concentration of DNA for Real Time PCR assay was 50 ng and other conditions for Real Time PCR were optimized.The mean Ct value for the treated sample was 24.08 where as that of control was  hence decreasing the methane gas production [21].

2 4 syringes
by previous developed method [10].The 0 Total volatile fatty acid (TVFA) estimation:syringes were incubated in water bath at 39 ± 0.5 C TVFA concentration (mmol/100 ml) in the supernatant[11].The 200 mg substrate was weighed and placed was estimated according to prescribed method[12].into the bottom of the glass syringes without sticking Individual volatile fatty acid (IVFA) estimation:

Figure- 2 .
Figure-2.Standard curve obtained by plotting the logarithm 22.54 (Figure-1).The copy number of methanogens of mcr-A gene concentration versus threshold cycle (Ct) mean was calculated on the basis of standards in control diet values.The curve was constructed using data from all the six triplicate standards' amplifications and treatment (Table3).The percentage decrease in the 9.Thauer, R.K. (1998).Biochemistry of methanogenesis: a tribute to Marjory Stephenson.Marjory Conclusions Stephenson Prize Lecture.Microbiology.144: 2377-This paper introduces a new plant extract which 2406. 10.Menke, W. (1979).The estimation of the has anti-methanogenic potential without affecting the digestibility and metabolizable energy content major nutritional parameters.However detailed study of ruminant feeding stuffs from the gas production about the active compounds present in the Myristica when they are incubated with rumen liquor in vitro.J. fragrans (Jaiphal), dosages and mechanism of its agric.Sci.93:217-222.inhibitory action on methanogens is required.11.Blümmel, M.; Ørskov, E.R. (1993).Comparison of gas production and nylon bag degradability of