_______________________________________________________________________________________________
The effect of supplemental organic acid on methane reduction to decrease the global warming from dairy cattle
1A.Bharathidhasan, 2R.Karunakaran, 3T.R.Pugazhenthi, 4S.Ezhilvalavan
1,2 Dept. of Animal Nutrition, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai-7
3College of Food and Dairy Technology, Koduvalli, TANUVAS, Chennai-52
4Poultry Research Station, Madhavaram milk colony, TANUVAS, Chennai-51 Email : 1[email protected]
[Received: 11th June 2016; Revised: 15th July 2016;
Accepted: 20th July 2016; Available online from: 2nd August 2016]
Abstract – A study was conducted to evaluate the effect of supplemental organic acid viz. malic acid and fumaric acid either individually or in combination on methane reduction to reduce the global warming from dairy cattle in paddy straw based complete diet by in vitro gas production technique (IVGPT). The IVGPT was carried out by incubating the complete diet and rumen liquor with malic acid and fumaric acid either individually or in combination at varying levels in shaking water bath for a period of 24 hours. After 24 hours the total gas production was measured and methane was estimated in Gas Chromatography. Individually, the minimum level of the malic acid at 0.39% and fumaric acid at 0.21% was able to decrease the methane ml per 100 mg of truly digested substrate significantly by 15.28% (P<0.01) and 9.12%
(P<0.05) respectively than control. The malic acid was more effective than fumaric acid on reduction of methane production. The methane (P<0.05) and methane (ml) per 100 mg of truly digested substrate (P<0.01) were significantly (P<0.01) decreased at 0.26 % malic acid + 0.14
% fumaric acid added group than other treatment groups.
This combination of malic acid (0.26%) and fumaric acid (0.14%) decreased the methane and methane (ml) per 100 mg of truly digested substrate by 14.42 and 14.75%
respectively than control. There was no significant difference on total gas production, in vitro true dry matter digestibility (IVTDMD), CO2 and pH. It was concluded that the minimum combination at 0.26% of malic acid and 0.14% fumaric acid significantly decreased the methane (ml) per 100 mg of truly digested substrate at the maximum extent when compared to individual effect of malic acid and fumaric acid for ruminants by IVGPT.
Key word: malic acid, fumaric acid, methane, ruminants
I. INTRODUCTION
Clean Air and Water are the vital part of healthy life.
These must be protected from bad effects of pollution at any cost. But the emission of air pollutants depletes the air quality which may lead to serious health hazards and environmental consequences [1]. Methane (CH4) is one
The estimated CH4 emission from ruminants was 18–20
% of global methane production [2] and it increases the global temperature. Ruminants produce methane under anaerobic fermentation as a path way for the disposal of metabolic carbon and hydrogen ion produced during microbial fermentation. Methane production also represents a loss of feed energy by 8-12 % which leads to lower animal production. Therefore decreasing methane production is desirable for reducing green house gas emission and ultimately decreasing the global warming. There are various feeding strategies used to reduce the methane emission. Diet modification can help to mitigate methane emissions. By the use of antibiotics like ionophore compounds such as monensin, lasolocid and many other chemical feed additives have been shown to decrease methane production in ruminants.
Recently, plant metabolites [3] modulate the rumen microbial populations and reduce the methane production. In this way, the supplemental organic acids like malic acid and fumaric acid are the ones which needed as precursors to propionate by utilizing hydrogen ions and if the rumen concentrations of these acids could be increased, propionate production would increase and methane production would fall. Hence the present study was carried out to study the effects of supplemental malic acid and fumaric acid either individually or in combination on methane reduction by IVGPT in paddy straw based complete diet from dairy cattle.
II.MATERIALS AND METHODS
The IVGPT was carried out [4] to study the effect of supplemental organic acid like malic acid and fumaric acid either individually or in combination on reduction of methanogenesis in paddy straw (46.67 %), CO4 grass (23.33 %) and concentrate mixture (30%) based complete diet. The digestible crude protein and total digestible nutrients content of the diet was 6.28 and 52
%. The supplemental organic acid like malic acid at 0, 0.13, 0.26, 0.39 and 0.52% of substrate and fumaric acid at 0, 0.07, 0.14, 0.21 and 0.28 % of substrate and in combination of malic acid + fumaric acid at 0, 0.13 +
substrate was added along with rumen inoculum to study the rumen methanogenesis and rumen fermentation characteristics by in vitro.
The rumen fluid collected from three dairy cattle was maintained on grazing and it was squeezed through four layers of gauze in to an Erlenmeyer flask under continuous flushing with CO2 and it was maintained at the temperature of 39 oC. Then rumen fluid was mixed with media [4]. The complete diet or substrate of 200 mg was taken in 100 ml calibrated syringes and weighed quantity of malic acid and fumaric acid were added either individually or in combination to the syringes in triplicate. Then 30 ml of rumen inoculum was anaerobically transferred to glass syringe and it was incubated in a shaking water bath at 39 oC for 24 hrs.
At the end of the incubation period the total gas was measured and pH also was determined in the fermentation fluid. The gas samples were collected in vacuotainer for estimation of methane and fermented fluid was collected for the estimation of true in vitro dry matter digestibility.
Methane concentration was estimated using Gas Chromatography (Perkin Elmer, Claurus 500 model) fitted with Flame Ionization Detector (FID) and capillary column (30 meter length and 250 micrometer diameter). Helium was used as carrier gas with oven temperature at 60o C, injector temperature at 100oC and detector temperature at 110oC. Methane concentration in
samples (%) was calculated using the following formula.
Methane concentration (%) =
(Peak area of sample gas / Peak area of standard gas) x
Methane concentration in standard (%)
Methane emission (ml) = Methane concentration (%) / 100 x
Net gas production (ml)
Estimation of in vitro true dry matter digestibility (IVTDMD)
After incubation, the fermented fluid was centrifuged and the residue or pellet was transferred into the glass crucible and fitted in Fibretec and 100 ml of Neutral Detergent Solution (NDS) was added. Then it was refluxed for one hour and residue was recovered. The true digestibility was calculated as the weight of substrate incubated minus the weight of the residue after NDS treatment [5]. The methane emission per 100 mg of true digestible substrate was also worked out.
III.RESULTS AND DISCUSSION
The effect of supplemental malic acid and fumaric acid either individually or in combination on total gas (ml), methane (ml), percentage of methane on total gas production, methane (ml) per 100 mg of truly digested substrate and pH are presented in Table1, Table 2 and Table 3 respectively.
Effect of malic acid on methane reduction
Methane production and percentage of methane on total gas production were significantly (P<0.05) decreased in 0.39 and 0.52 % malic acid added groups compared to control. Similarly, the methane (ml) per100 mg of truly digested substrate was also significantly (P<0.05) decreased in 0.39 and 0.52 % of malic acid added groups compared to control group.
Table.1.Effect of supplemental malic acid on total gas (ml), methane (ml), percentage of methane on total gas production, IVTDMD( %), methane (ml) per 100 mg of truly digested substrate CO2 (ml)and pH (Mean# ± S.E)
Treat ment
Inclusion level of supplemental malic acid (%)
Total gas (ml) NS
Methane (ml)
Percentage of methane on Total gas production
IVTDMD
% NS
Methane (ml) per 100 mg of truly digested substrate
CO2 (ml) NS
pH NS
1 0 14.17 ±
0.18
3.12 ± 0.01 c
22.06 ± 0.33 d
41.87 ± 0.87
3.73 ± 0.08 b
11.04 ± 0.18
7.07 ± 0.09
2 0.13 14.40 ±
0.36
2.96 ± 0.03 b
20.59 ± 0.33 c
41.72 ± 1.11
3.56 ± 0.12 b
11.44 ± 0.33
7.10 ± 0.10
3 0.26 13.97 ±
0.12
2.84 ± 0.03 b
20.32 ± 0.38 bc
41.44 ± 1.45
3.43 ± 0.15 ab
11.13 ± 0.15
7.07 ± 0.09
4 0.39 14.07 ±
0.09
2.67 ± 0.06 a
19.00 ± 0.38 ab
41.46 ± 0.43
3.16 ± 0.12 a
11.39 ± 0.06
7.07 ± 0.09
5 0.52 14.13 ±
0.12
2.66 ± 0.05 a
18.80 ± 0.49 a
41.50 ± 0.63
3.20 ± 0.10 a
11.48 ± 0.16
7.03 ± 0.09
#Mean of three observations with triplicates; NS Not significant, Means bearing different superscripts in the same column differ significantly (p<0.01)
A significant (P<0.05) reduction in methane (ml) per100 mg of truly digested substrate by 15.28 % was observed at the minimum level of 0.39 % of malic acid added group compared to control. Similarly, an earlier report also stated that the addition of malic acid decreased the methane emission more by 1.9 % than control by in vitro [6]. Foley et al. [7] reported that the supplementation of DL malic acid at 3.75 % and 7.5 % decreased (P<0.001) methane production more by 6.30 and 15.77 % respectively than control. There was no
significant difference was observed in total gas production, in vitro true dry matter digestibility, CO2 and pH. Foley et al. [7] also reported that the supplementation of DL malic acid at 0, 2.5, 5.0 and 7.5
% did not alter the pH of ruminal fluid.
Effect of fumaric acid on methane reduction
The methane emission was significantly (P<0.05) decreased more at 0.21 and 0.28 % fumaric acid added groups than control.
Table.2.Effect of supplemental fumaric acid on total gas (ml), methane (ml), percentage of methane on total gas production, IVTDMD( %), methane (ml) per 100 mg of truly digested substrate CO2 (ml)and pH (Mean# ± S.E) Treat
ment
Inclusion level of supplemental malic acid (%)
Total gas (ml) NS
Methane (ml)
Percentage of methane on Total gas productionNS
IVTDMD%
NS
Methane (ml) per 100 mg of truly digested substrate
CO2 (ml) NS
pH NS
1 0 14.17 ±
0.18
3.12 ± 0.01b
22.06 ± 0.33
41.87 ± 0.87
3.73 ± 0.08 b
11.04 ± 0.18
7.07 ± 0.09
2 0.07 14.35 ±
0.10
2.97 ± 0.07 ab
20.72 ± 0.59
41.49 ± 0.24
3.58 ± 0.10 ab
11.38 ± 0.15
7.10 ± 0.06
3 0.14 14.27 ±
0.96
2.93 ± 0.14ab
20.76 ± 1.73
41.49 ± 0.96
3.53 ± 0.08 ab
11.34 ± 1.01
7.03 ± 0.07
4 0.21 14.62 ±
0.68
2.82 ± 0.09 a
19.36 ± 1.22
41.53 ± 1.01
3.39 ± 0.04 a
11.81 ± 0.71
7.07 ± 0.09
5 0.28 14.36 ±
0.61
2.78 ± 0.08 a
19.48 ± 1.25
41.61 ± 0.75
3.34 ± 0.06 a
11.58 ± 0.67
7.10 ± 0.12
#Mean of three observations; NS Not significant, Means bearing different superscripts in the same column differ significantly (p<0.05)
The methane (ml) per100 mg of truly digested substrate was also significantly (P<0.05) decreased by 9.12% and 10.46 in 0.21 and 0.28 % of fumaric acid added groups respectively more than the control.
A reduction of methane by 12 % [8] and 23 % [9] was also observed in sheep and steers respectively when the animals were fed with fumarate in forage as sole feed.
There was no significant difference was observed in total gas production, percentage of methane on total gas
production, in vitro true dry matter digestibility, CO2
and pH.
Effect of malic acid and fumaric acid on methane reduction
The methane and percentage of methane on total gas production were significantly (P<0.01) decreased in combination of all malic acid and fumaric acid added groups more than control.
Table.3.Effect of supplemental malic acid and fumaric acid either individually or in combination on total gas (ml), methane (ml), percentage of methane on total gas production, IVTDMD( %), methane (ml) per 100 mg of truly digested substrate, CO2 (ml)and pH (Mean# ± S.E)
Treatm ent
Inclusion level of standard fumaric acid (%)
Total gas (ml) NS
Methane (ml)*
Percentage of methane on Total gas production*
IVTDMD%NS Methane (ml) per 100 mg of truly digested substrate*
*
CO2 (ml)NS
pH NS
1 0 14.17 ±
0.18
3.12 ± 0.01 b
22.06 ± 0.33 b
41.87 ± 0.87
3.73 ± 0.08 c
11.04 ± 0.18
7.07 ± 0.09 2 0.13 + 0.07 14.55 ±
0.05
2.83 ± 0.18 ab
19.48 ± 1.27 a
41.59 ± 3.18
3.42 ± 0.08 b
11.72 ± 0.22
7.03 ± 0.12
3 0.26 + 0.14 14.23 ± 2.67 ± 18.79 ± 41.86 ± 3.18 ± 11.57 ± 7.07 ±
0.82 0.10 a 0.59 a 0.86 0.06 a 0.73 0.09
4 0.39 +
0.21
14.47 ± 0.94
2.53 ± 0.13 a
17.53 ± 0.75 a
41.69 ± 0.30
3.03 ± 0.18 a
11.94 ± 0.85
7.10 ± 0.06
5 0.52 +
0.28
14.13 ± 1.01
2.52 ± 0.07 a
17.98 ± 1.27 a
41.99 ± 0.39
3.00 ± 0.06 a
11.62 ± 0.99
7.13 ± 0.03
#Mean of three observations; NS Not significant
Means bearing different superscripts in the same column differ significantly *(p<0.05), **(p<0.01) The methane (ml) per100 mg of truly digested
substrate was significantly (P<0.01) decreased by 14.75 % at the minimum concentration of 0.26 % malic acid + 0.14 % fumaric acid added group compared to control. Earlier studies also revealed that there is a decrease in methane emission by in vitro [10] and in vivo [9]. Lopez et al. [11] also observed that the addition of fumarate and malate decreased the methane emission. Bharathidhasan et al. [12] also observed that the addition of malic acid and fumaric acid in combination decreased the methane ml per 100 mg of truly digested substrate. There was no significant difference was observed in total gas production, IVDMD, CO2 and pH. Gomez et al. [13] also observed that the supplementation of disodium malate at 15.1 g/kg DM in grass hay and concentrate (60:40) and 4.87 g/kg DM in barley straw and concentrate (10:90) based diet did not affect the pH of the fermented medium.
Malic acid and Fumaric acid are the direct metabolic precursor of propionic acid and it has potential to decrease the methane emission by directing hydrogen into succinate rather than in to methane production [11]. There by methane production was decreased.
IV. CONCLUSION
It is concluded that the decrease in methane emission in the present study was due to the addition of fumaric acid and malic acid which were more effective alternate hydrogen sink competing with methanogenesis for ruminants [8].
V. ACKNOWLEDGEMENT
The budget outlay for this research programme was sponsored by the ICAR-NIANP coordinated Methane Scheme is gratefully acknowledged.
REFERENCES
[1] H.C. Sahu. Air Pollution Index of some selected Evergreen Plants across NH60 Road Side of Balasore District, Odisha, India, International Journal of Advanced Chemical Science and Applications, 1(1), 62-65, 2013.
[2] A.R,Moss, J.P0 Jouany, C.J. Newbold, Methane production by ruminants: its contribution to global warming, Ann. Zootech, 49, 231–235, 2000.
[3] A.Bharathidhasan, K.Viswanathan, V.
Balakrishnan, C. Valli, S. Ramesh and SMA.
Senthilkumar, Effects of purified saponin on rumen methanogenesis and rumen fermentation characteristics studied using in vitro gas production technique, Inter. J. Vet. Sci., 2(2), 44-49. 2013.
[4] K.H.Menke, H.Steingass, Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid, Animal Research and Development, 28, 7–55,1988.
[5] P.J.Van Soest and Robertson,A Laboratory manual for animal science, 612, Ithaca Ny:
Cornell University,1988
[6] D.Jalc, S. Kisidayova and F.Nerud, Effects of plant Oils and Organic Acid On Rumen Fermentation in vitro, Folia Microbial., 47(2), 171-177, 2002.
[7] P.A.Foley, D.A.Kenny, J.J.Callan, T.M. Boland and F.P.O, Mara, Effects of di-malic acid supplementation on feed intake, methane emission and rumen fermentation in beef cattle.
J. Animal Sci., 87,1048-1057, 2009.
[8] C.J.Newbold, J.O.Ouda, S.Lopez, N.Nelson, H.Omed, R.J.Wallace and A.R.Moss, Propionate precursors as possible alternative electron acceptors to methane in ruminal fermentation In:J. Takahashi, B.A.Young, C.R.Soliva, M.Kreuzer (Eds.), The 1st International Conference on Green House Gases animal Agriculture GGAA2001, Elsvier Health Sciences. Tokachi Plaza, Japan, pp: 272-279, 2002.
[9] E.Bayaru, S.Kanda, T.Kamada, H.Itabashi, S.Andoh, T.Nishida, M.Ishida, T.Itoh, K.Nagara and Y.Isobe, Effect of fumaric acid on methane production, rumen fermentation and digestibility of cattle fed roughage alone, Anim.Sci. J., 72, 139-146, 2001.
[10] N,Asanuma, M.Iwamoto and T.Hino, Effect of addition of fumarate on methane production by ruminal microorganisms in vitro, J. Dairy Sci., 82, 780-787, 1999.
[11] S.Lopez, C.Valdes, C.J.Newbold and R.J.Wallace, Influence of sodium fumarate addition on rumen fermentation in vitro, Bri. J.
Nutr., 81, 59-64, 1999.
[12] A. Bharathidhasan, R. Balamurugan, R.
Karuakaran, V. Balakrishnan, TR. Pugazhenthi and S. Ezhilvalavan, Influence of malic acid and fumaric acid on methane reduction in paddy straw based diet for ruminants, Proceedings of XVI Biennial Animal Nutrition Conference on Inovative Approaches for Animal Feeding &
Nutritional Research held at NDRI, Karnal, India on 06-08, February 2016, Pp:86, 2016
[13] J.A.Gomez, M.L. Tejido and M.D. Carro, Influence of disodium malate on microbial growth and fermentation in rumen -simulation technique fermenters receiving medium and high concentrate diets, Bri.J Nutri., 93,479-484, 2005.
