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

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1 Vet. World, 212, Vol.5(6): RESEARCH Effect of inclusion of Myristic frgrns on methne production, rumen fermenttion prmeters nd methnogens popultion S K Sirohi, P P Chudhry, Nvneet Goel Nutrition Biotechnology Lb, Animl Biotechnology Centre, Ntionl Diry Reserch Institute, Krnl-1321, Indi Corresponding uthor: S K Sirohi, emil: sirohisk@gmil.com Received: , Accepted: , Published Online: doi: /vetworld Abstrct Aim: The present study ws done to evlute the effect of Myristic frgrns fruit ctive compounds ddition on methne production in vitro. Mterils nd Methods: Methnolic extrct of Myristic frgrns fruit powder ws prepred nd checked for its inhibitory ction on methne production in diet contining roughge 5 percent nd concentrte 5 percent respectively. Methne production ws estimted by Gs Chromtogrphy. Results: It hs been shown tht supplementtion of Myristic frgrns reduces the methne production up to 48 percent s compred to control diet without supplementtion of Myristic frgrns. Similrly rel time quntifiction of mcr-a gene lso shown the significnt (P<.5) reduction in the number of methnogens. Myristic frgrns ppered to reduce methne production by inhibiting methnogens directly. However, digestibility of dry mtter lso decresed due to myristic frgrns supplementtion in totl mixed diet, which my ffect the production of voltile cid production. Conclusion: The ctive compounds extrcted in methnol of Myristic frgrns emerged out to be useful nturl plnt source for the inhibition of methnogenesis nd its supplementtion in niml feed my proves to be n effective mesure to control methne emission from ruminnts. Key words: Methnogen, Methne, Mitigtion, Myristic frgrns, Rel Time PCR To cite this rticle: Sirohi SK, Chudhry PP, Nvneet Goel (212).Effect of inclusion of Myristic frgrns on methne production, rumen fermenttion prmeters nd methnogens popultion, Vet. World, 5(6):335-34, doi: /vetworld Introduction In Indi, methne emission from Agriculturl sector, the livestock is the mjor contributor to the globl wrming. In 29 Indi livestock methne- emission ws million metric tons per yer higher thn the 9 million metric tons estimted in 1994 [1]. To mitigte methne emission is considered s n interntionl gol in order to reduce globl wrming s methne is considered to be potent green house gs. Level of methne emission from the ruminnts is ffected by number of fctors such s level of feed intke, ddition of lipids nd ionophores in their diet, chnge in rumen microbil environment nd the level of niml productivity hve been identified [2,3,4]. Plnts hving secondry metbolites hve been thought to ply n importnt role in reducing methnogenesis in rumen [5]. Sponins or sponinlike substnces hve been reported to suppress methne production, reduce rumen protozo counts, nd modulte fermenttion pttern [6,7,8]. Now with the dvncement of moleculr biologicl pproches it is esy to quntify the number of methnogens without prcticing the tedious methods of culturing methnogens in different smples. mcr-a gene is ubiquitously present in ll methnogens, so it is used s stndrd to quntify the methnogens under different tretments nd diet conditions [9]. In the present pper n effort hs been mde to study the nti-methnogenic potentil of Myristic frgrns (Jiphl) plnt extrct by in vitro fermenttion study, using Gs Chromtogrphy nd quntify the expression of mcr-a gene in treted nd control smples. Mterils nd Methods Preprtion of plnt extrcts: The Myristic frgrns (Jiphl) fruits were obtined, crushed into smll pieces nd oven dried t 7 C. 5 % queous methnol ws used s solvent to prepre plnt extrcts. The plnt mteril ws then ground to pss through 1 mm screen. A known quntity of finely Veterinry World, Vol.5 No.6 June

2 ground smple ws weighed into 25 ml conicl flsk. rumen liquor, 3 ml of incubtion medium ws The 5% queous methnol ws dded (1:1 dried injected to the syringes using uto dispenser. The plnt to solvent) nd the flsk ws tightly seled nd syringes were shken gently nd residul ir or ir kept in rotry shker t 25 C nd 12rpm for 24 hour. bubbles if ny ws removed nd outlet ws closed. The After shking the content of the flsk, methnol level of piston ws recorded nd syringes were plced extrct of Myristic frgrns is directly filtered in wter bth mintined t 39 ±.5 C. The syringes through Whtmn no. 1 filter pper nd used for were shken every one hour up to 1 hour of further nlysis. incubtion. These trils were conducted long with respective blnk nd control in triplicte. After 24 hour Collection of Rumen Liquor for In-Vitro Gs of incubtion, volume of gs ws withdrwn from the Production Test: Rumen smples were obtined fter mnul mixing of rumen contents from three tip of the incubtion syringe using Hmilton gs tight rumen fistulted dult mle bufflo (Bublus bublis) syringe nd nlyzed for methne with the help of gs fter tking the permission from the Animl Ethics chromtogrph (Nucon 57, Indi) Flme ionizing Committee of the institute. The bufflo were kept on detector (FID) is used. The temperture of injection stndrd diet comprising concentrte nd roughge in port, column nd detector ws 4 C, 5 C nd 5 C rtio 5:5. Rumen liquor smples from the respectively. Volume of gs tken for injecting ws 2µl. The flow rte of crrier gs (N 2 buffloes were collected prior to the first morning ) through the feed. Just fter collection of smple in insulted flsk column ws 3ml/min nd H 2 ws 3ml/min nd ir pre-wrmed t 39 C tken to lb, immeditely pssed ws 3 ml/min. The stndrd gs for methne crbon dioxide nd filtered through four lyer of estimtion (Spntech clibertion gs, Surrey, muslin cloth nd then used for setting up of in vitro gs Englnd) ws composed of 5% methne nd 5% production test. CO 2. The pek of methne gs ws identified on the bsis of retention time of stndrd methne gs nd the Preprtion of diet: To evlute the effect of response fctor obtined ws used to clculte myristic frgrns diet ws prepred by tking methne percentge in the gs smple. The methne roughge concentrte rtio of 5:5. The roughge produced from substrte during 24 hour incubtion prt composed of whet strw nd the concentrte prt ws compred for the blnk vlues. The volume of composed of mize (33%), ground nut cke(gnc) methne produced ws clculted s follows: (21%), mustrd cke (12%), whet brn (2%), Methne production (ml) = Totl gs produced (ml) x % deoiled rice brn (11%), minerl mixture (2%) nd slt methne in the smple. (1%) respectively. Chemicl composition of diet Estimtion of mmoni nitrogen: The superntnt include g/kg DM of Orgnic mtter(om),12.53 of ech syringe including tht of blnk ws used for g/kg DM of Crude protein(cp) nd g/kg DM of NH3-N estimtion. Superntnt (5 ml) ws mixed with Acid Detergent Fibre (ADF). 1 N NOH (2 ml) nd stem pssed on this using KEL Estimtion of Methne Production by Gs Chro- PLUS - N nlyzer (Pelicn, Indi) nd the NH 3 evolved mtogrphy: For estimting the methne production ws collected in boric cid solution hving mixed incubtions were crried out in 1 ml clibrted glss indictor nd titrted ginst N / 1 H2SO 4. syringes by previous developed method [1]. The Totl voltile ftty cid (TVFA) estimtion: syringes were incubted in wter bth t 39 ±.5 C TVFA concentrtion (mmol/1 ml) in the superntnt [11]. The 2 mg substrte ws weighed nd plced ws estimted ccording to prescribed method [12]. into the bottom of the glss syringes without sticking to the sides. 2 ml of plnt extrct ws injected before Individul voltile ftty cid (IVFA) estimtion: incubtion. The syringes were kept in n incubtor t At the end of incubtion (24h) 1 ml of the superntnt C. The medium mixture solution ws prepred ws treted with 25% met-phosphoric cid (4 ml) nd by mixing 5 ml distilled wter,.125 ml micro kept for 3-4 h t mbient temperture [13]. Therefter, minerl, 25 ml buffer, 25 ml mcro minerl, 1.25 it ws centrifuged t 3 rpm for 1 minutes nd cler ml reszurine nd 5 ml reducing solution (prepred superntnt ws collected nd stored t -2 C until fresh nd dded prior to incubtion). Once the medium nlyzed. IVFA estimted using gs chromtogrph mixture solution becomes colorless, the required (Nucon 57, Indi) equipped with flme ioniztion mount of filtered rumen liquor ws dded. The detector (FID) nd stinless steel column (length 4'; proportion of medium mixture solution to rumen o.d ¼''; i.d 3 mm) pcked with Chromosorb 11. liquor ws 2:1. Just fter mixing the medium nd Temperture of injection port, column nd detector Veterinry World, Vol.5 No.6 June

3 ws set t 2, 18 nd 21 C, respectively. The flow used [18] nd the specific mplified trget region ws rte of crrier gs (N ) through the column ws 4 ml/ cloned by using Strtgene Blunt End Cloning kit 2 min, nd the flow rte of hydrogen nd ir through FID (Strtgene,USA) in order to estblish quntittive ws 3 nd 3 ml/ min. respectively. Smple (2µl) ssy. With the use of the Ferments Plsmid ws injected through the injection port using Hmilton Purifiction Kit (Ferments,USA), plsmid DNA ws syringe (1 µl). Individul VFAs of the smples were isolted nd the purified plsmids were quntified by identified on the bsis of their retention time nd their spectrophotometry with multiple dilutions. The trget concentrtion (mmol) nd clculted by compring the DNA used for these experiments possessed n retention time s well s the pek re of stndrds A26/A28 rtio greter thn 1.8. The trget DNA ws 1 fter deducting the corresponding blnk vlues. quntified by using seril 1-fold dilutions from 1 to 6 1 plsmid copies of the previously quntified Protozo counting: For protozol count one plsmid stndrds. Rel-time PCR mplifiction nd milliliter of the fermenttion fluid ws diluted with 1 TM detection were performed in MiniOpticon Rel ml of formlin (18.5% formldehyde) nd 3-4 drops Time PCR system (BioRd,USA) under similr of brillint green nd then incubted for 24 hours t conditions s were stndrdized during conventionl room temperture. The stined protozo were diluted PCR. In brief, SYBR Green qpcr Mstermix (2X) (if needed) nd counted by hemocytometer s per (Ferments,USA) ws used for PCR mplifiction. method [14]. Smples were ssyed in triplicte in 25 µl rection In vitro true DM degrdbility: True DM degrdbility mixture contined 12.5µl of 2X Mstermix (including of feed smple of ech syringe contining residues FstStrt enzyme, FstStrt Tq DNA polymerse, fter incubtion ws estimted s per method [15]. rection buffer, dntp mixture, MgCl2, nd SYBR Green dye), 5 ng of templte DNA, nd.5 µm of Proximte nlyses nd cell wll constituents: The proximte nlysis of substrte ws crried out s ech primer. All PCRs were performed in triplicte. per the methods of [16]. The cell wll constituents of Sttisticl nlysis: Experimentl dt of different substrtes were determined ccording to suggested prmeters were nlyzed in pired T test with three method [15]. replictes [19]. DNA extrction: Totl genomic DNA ws isolted Results nd Discussion from rumen liquor smple fter methne Methne mesurement by Gs Chromtogrphy mesurement using Bcteril genomic DNA isoltion nd other nutritionl prmeters: Chemicl kit (Chroms Biotech Pvt. Ltd., Bnglore, INDIA). composition of diet presented in tble 1. After 24 hours For rumen smples 1.5 ml liquot ws tken from the of in vitro experimenttion methne ws mesured out rumen smple fter methne mesurement using of the totl gs produced in the tubes. Methne wide bore pipette so s to ensure homogenous production in the control tubes ws found out to be smple contining fluid nd digest. This ws 2.47 ml/gm of diet where s in the tubes in which centrifuged t 12 g for 5 min nd the superntnt Myristic frgrns (Jiphl) is supplemented methne ws removed before DNA extrction. production ws significntly (P<.5) reduced to Conventionl PCR: PCR mplifiction ws 12.7 ml/gm of diet. Myristic frgrns (Jiphl) lso TM conducted with n My Cycler Therml Cycler (Bioto decresed the rtio of cette to propionte from 3.9 Rd, USA). The rection mixture ws treted ccording to the following protocol: 95 C for 5 min, Tble-1. Chemicl composition of totl mixed rtion followed by 4 cycles consisting of 95 C for 3 sec, (5 % Roughge : 5 % Concentrte) 6 C for 3 sec, 72 C for 1 min, nd finl extension Prmeter Diet (5R:5C) period of 72 C for 1 min. OM Reltive Rel Time PCR: Totl Genomic DNA CP isolted from both the sets i.e Diet+R.L.+Plnt extrct EE 3.4 NDF 6.45 nd Diet+R.L. (Control) ws subjected to rel time ADF nlysis. Both the smples were tken in triplicte. HC 27.5 Rel-time PCR ssys for the quntifiction of Cellulose 21.8 methnogens, ws performed [17,18]. The primer sets ADL 5.6 TA for detection nd enumertion of methnogens ws Veterinry World, Vol.5 No.6 June

4 OM = Orgnic mtter, CP = crude protein, EE = Ether extrct, NDF = Neutrl detergent fibre, ADF = Acid detergent fibre, HC = Hemi cellulose, ADL = Acid detergent lignin, TA = Totl sh methnogens is clculted in comprison to control ccording to the decrese in copy number of methnogens present in tretment combintion. The result shows tht there is significnt (P<.5) decrese in the methnogens popultion in the treted smple s compre to tht of the control which is the untreted one fter 24 hour incubtion. However, IVDMD ws lso decresed significntly due to ddition of methnolic extrct of Myristic frgrns (Jiphl) which lso ffect the overll production of individul voltile ftty cid production. The reduction in the methne gs production ws lso supported by other nutritionl prmeters given in Tble 2. A lrge number of plnt extrcts nd spices were evluted erlier for their nti-methnogenic Prmeters Control Diet Diet supplemented ctivity. Nture, ctivity nd concentrtion of the with Myristic frgrns Tble-3. Copy number of methnogens clculted on the bsis mcr-a gene quntifiction in diet supplemented with Myristic frgrns ctive components reflect wht type of effect Threshold cycle (Ct) men vlues b Copy Number x 1 (per 5ng DNA) prticulr plnt species is on methnogenesis [2]. Current study gives us knowledge bout the nti- Different superscript in row significntly different t P <.5 methnogenic ctivity of new plnt extrct Myristic frgrns (Jiphl). Supplementtion of Myristic frgrns (Jiphl) ws observed to elicit rpid reduction in methne relese from the rumen liquor during in vitro experimenttion with reduction of 62.4 % within 24 h of supplementtion compred with the control t the sme time point. Tble-2. Dry mtter digestibility nd rumen fermenttion prmeters in control nd tretment diet supplemented with Myristic frgrns Prmeters Control Diet Myristic frgrns supplemented diet ph DDM (mg) b Methne(ml/gDM) NH3-N(mg/1ml) b Protozo(x1 /ml).8.5 b b Figure-1. Differentil mplifiction of rumen bcteril DNA TVFA(mM/1ml) b templtes with mcr-a gene specific primers s discussed in Acette(mM/1ml) mteril methods. Propionte(mM/1ml) Butyrte(mM/1ml) A/P Rtio DDM = Digestible Dry Mtter, NH3-N = Ammoni nitrogen, TVFA = Totl voltile ftty cid, A/P = Acette to propionte rtio Quntittive Rel Time PCR nlysis: Totl DNA isolted from both the treted s well s control smples were subjected to Rel Time PCR nlysis to monitor the quntity of methnogens in both the smples with the help of mcr-a gene trgeting primers [18]. All the smples were tken in triplicte. The strting concentrtion of DNA for Rel Time PCR ssy ws 5 ng nd other conditions for Rel Time PCR were optimized. The men Ct vlue for the treted smple ws 24.8 where s tht of control ws Figure-2. Stndrd curve obtined by plotting the logrithm (Figure-1). The copy number of methnogens of mcr-a gene concentrtion versus threshold cycle (Ct) men ws clculted on the bsis of stndrds in control diet nd tretment (Tble3). The percentge decrese in the vlues. The curve ws constructed using dt from ll the six triplicte stndrds' mplifictions Veterinry World, Vol.5 No.6 June

5 Dissocition curve nlysis for the mcr-a gene Cheng, K.J. (1996). Dietry, environmentl nd specific primer set produced dissocition curve with microbiologicl spects of methne production in two peks t 81 C nd 85 C in both the treted s well ruminnts. Cn J Anim Sci. 76: s control smple. Amplifiction plot, Melting curve 5. Kmr, D.N.; Ptr, A.K.; Chtterjee, P.N.; Kumr, nd stndrd curve is presented in figure 1 nd 2 R.; Agrwl, N. nd Chudhry, L.C. (28). Effect of plnt extrcts on methnogenesis nd microbil respectively. Methnogens re responsible for the profile of the rumen of bufflo: brief overview. production of methne in the ruminnts which mjorly Animl Production Science. DOI: 1.171/EA7268. contributes to globl wrming. Gs chromtogrphy 6. Mkkr, H.P.S. nd Becker, K. (1997). Degrdtion nd rel time re rpid moleculr techniques idelly of Quillj sponins by mixed culture of rumen suited for the checking methne produced in vitro nd microbes. Lett Appl Microbiol. 25: quntifiction of methnogens respectively. The 7. Wng, Y.; McAllister, T.A.; Newbold, C.J.; Cheeke, number of protozo is significntly ffected in both the P.R. nd Cheng, K.J. (1997). Effects of Yucc extrct control nd treted diets which re closely ssocited on fermenttion nd degrdtion of sponins in the with methnogens. Myristic frgrns (Jiphl) might Rusitec. Proceedings of Western Section. Americn be reducing the methne emission by inhibiting Society of Animl Science. 48: methnogen popultion directly nd / or lso the 8. Hristov, N.A.; McAllister, T.A.; Vn Herk, F.H.; Cheng, K.J.; Newbold, C.J. nd Cheeke, P.R. (1999). methnogen ssocited with protozo. Most of the Effect of Yucc schidiger on ruminl fermenttion studies till dte give us knowledge bout the plnt nd nutrient digestion in heifers. J Anim Sci. extrcts which will inhibit protozo popultion nd 77: hence decresing the methne gs production [21]. 9. Thuer, R.K. (1998). Biochemistry of methnogenesis: tribute to Mrjory Stephenson. Mrjory Conclusions Stephenson Prize Lecture. Microbiology. 144: This pper introduces new plnt extrct which 246. hs nti-methnogenic potentil without ffecting the 1. Menke, W. (1979). The estimtion of the mjor nutritionl prmeters. However detiled study digestibility nd metbolizble energy content bout the ctive compounds present in the Myristic of ruminnt feeding stuffs from the gs production when they re incubted with rumen liquor in vitro. J. frgrns (Jiphl), dosges nd mechnism of its gric. Sci. 93: inhibitory ction on methnogens is required. 11. Blümmel, M.; Ørskov, E.R. (1993). Comprison of Acknowledgements gs production nd nylon bg degrdbility of roughges in predicting feed intke in cttle. Authors re thnkful to Ntionl Fund for Bsic Anim.Feed Sci. Technol., 4: nd Strtegic Reserch in Agriculture (NFBSRA), 12. Brnet, A.J.G., Reid, R.L., (1957). Studies on Indin Council of Agriculturl Reserch(ICAR),New production of voltile ftty cids from grss by rumen Delhi to provide the finncil support under grnt F. liquor in rtificil rumen IVFA production from fresh No. NFBSRA/PCN/AP-1/26-7 to crry out the grss. Journl of Agriculture Science, 48, 315. reserch work. Senior Reserch Fellowship provided 13. Erwin, E.S., Mcro, G.A., Emery, E.M., Voltile ftty cid nlysis of blood nd rumen fluid by the sme funding gency to PPC nd NG is lso by Gs chromtogrph. Journl of Diry Science 44, fully cknowledged Competing interests 14. Dehority, Burk A., (1984). Applied nd Environmentl Microbiology. Vol. 48, No. 1. The uthors declre tht they hve no competing 15. Vn Soest, P.J., Robertson, J.B., Lewis, B.A., interests. Methods for fiber, neutrl detergent fiber nd non- References strch polyscchrides in reltion to niml nutrition. Journl of Diry Science 74, AOAC, (1995). Officil Methods of Anlysis.16th ed. 1. Singh, M. (29). Cows with Gs: Indi's Globl- Assocition of Officil Anlyticl Chemists, Wrming Problem. Time World. Arlington,VA. 2. Johnson, K.A. nd D.E. Johnson, (1995). Methne 17. Denmn, S.E.; McSweeney, C.S. (26). emissions from cttle. J Anim Sci. 73: Development of rel-time PCR ssy for monitoring 3. Hironk, R.; Mthison, G.W.; Kerrign, B.K. nd nerobic fungl nd cellulolytic bcteril Vlch, I. (1996). The effect of pelleting of lflf hy popultions within the rumen. FEMS Microbiol Ecol., on methne production nd digestibility by steers. Sci 58: Totl Environ. 18, Denmn, S.E.; Tomkins, N.W. nd McSweeney C.S. 4. McAllister, T.A.; Okine, E.K.; Mthison, G.W. nd (27). Quntifiction nd diversity nlysis of Veterinry World, Vol.5 No.6 June

6 ruminl Methnogenic popultions in response to production in vitro. Anim.Feed Sci. Technol. 147: the ntimethnogenic compound bromochlorome thne. FEMS Microbiol Ecol., 62: Guo, Y.Q.; Liu, J.X.; Lu, Y.; Zhu, W.Y.; Denmn, S.E. 19. Snedecor, G. W., W. G. Cochrn, (1968). Sttisticl nd McSweeney, C.S. (28). Effect of te sponin on Methods, 5th ed. Iow Stte Univ. Press, Ames., I.A. methnogenesis, microbil community structure nd 2. Gonz lez, R.G.; L opez, S.; Fern ndez, M.; Bods, expression of mcra gene, in cultures of rumen micro- R. nd. Gonz lez, J.S. (28). Screening the ctivity orgnisms. Lett. Appl. Microbiol. doi:1.1111/j of plnts nd spices for decresing ruminl methne 765X x ******** Veterinry World, Vol.5 No.6 June