Effect on methane production of supplementing a basal substrate of molasses and cassava leaf meal with mangosteen peel (Garcinia mangostana) and urea or nitrate in an in vitro incubation

Vo Duy Thanh, T R Preston* and R A Leng**

Cantho University, Vietnam
vothanh69@gmail.com
* Finca Ecologica, TOLOGY, UTA (Colombia)
AA#48, Socorro, Santander, Colombia
** University of New England, Armidale NSW, Australia

Abstract

An in vitro incubation was used to evaluate effects of Mangosteen peel extract with calcium nitrate or urea on methane production from a substrate of molasses and cassava leaf meal. The design was a 2*2 factorial arrangement in a completely random block design with four replications.

Calcium nitrate as replacement for urea, and addition of Mangosteen extract,  lowered methane production during the final phase (32 to 48h) of the incubation. Estimates of effects over the entire incubation period, based partially on results from similar experiments in the literature, support  the effect of calcium nitrate in lowering methane production. The apparently beneficial effect of Mangosteen peel extract in reducing methane production needs to be substantiated by further research.  

Key words: fermentation, gas production, tannins, saponins

Introduction

On a worldwide basis, enteric methane from ruminants is estimated to represent 17–30% of total anthropogenic methane (Beauchemin et al 2009). The methane resulting from methanogenesis represents a loss of dietary energy to the animal (from 2 to 12% of the gross energy intake according to Johnson and Johnson (1995) and it is a significant greenhouse gas (Steinfeld et al 2006). According to Hindrichsen et al (2005),  85-90% of methane is produced by enteric fermentation. Murray et al (1976) indicated that 89% of the methane was excreted in the animal’s breath and 11%  from the anus. These factors have led to a global search for nutritional strategies to mitigate methane emission from ruminants.  

The presence of saponins and condensed tannins in forages has been shown to decrease methane production both in vivo and in vitro (Puchala et al 2005; Holtshausen et al 2009; Szumacher-Strabel and Cieślak  2010 ). Sainfoin (Onobrychis viciifolia), Lotus pedunculatus (lotus) and Lotus corniculatus (Birdsfoot trefoil) contain condensed tannins which have been shown to be beneficial for the rumen fermentation when they are present in moderate quantity (4 to 6% of the diet DM) in the diets (Patra 2007).

Mangosteen (Garcinia mangostan) peel is a fruit by-product which has been used in combination with coconut oil to improve rumen ecology and milk production (Suchitra and Wanapat 2008). According to these authors, mangosteen peel is rich in condensed tannins and crude saponins (16 and 10% in DM, respectively), which they considered to be responsible for the observed reduction in rumen protozoa, and the concomitant increase in rumen bacteria in dairy cattle fed 100 g DM/d of mangosteen  peel.

Leng (2007) reported that nitrate can replace carbon dioxide as an electron acceptor with the generation of another reduced product. In this case, ammonia, i.e. nitrate is reduced to nitrite and then to ammonia, resulting in lower methane gas emission. Bozic at al (2009) reported that methane production dropped by 98% in an in vitro test with sodium nitrate. Nolan et al (2010) found that in sheep supplemented with 4% nitrate instead of urea,  the reduction of methane production was 23%. Several other studies have now shown that nitrate effectively inhibits methane production (Klüber et al 1998, Guo et al 2009, Guangming et al 2010, Mohanakrishnan at al 2008).

The present investigation was aimed to determine the effects of mangosteen peel on methane production in an in vitro incubation with calcium nitrate or urea as the source of non-protein nitrogen and molasses and cassava leaf meal as the basal substrate.

Materials and Methods

Location and duration

The experiment was carried out in the laboratory of the Faculty of Agriculture, An Giang University, An Giang province, Vietnam from August to October, 2010.

Treatments and experimental design

Four treatments were compared in a 2*2 factorial arrangement in a completely random block design with four replications. The factors were: with or without mangosteen peel supplement;  and urea or calcium nitrate as source of NPN. Individual treatments were:

• Mangosteen peel plus calcium nitrate (MP-CaN)

• Mangosteen peel plus area (MP-U)

• Calcium nitrate (CaN)

• Urea (U)

The basal substrate was a mixture of molasses and cassava leaf meal (Table 1). 

The in vitro system

The in vitro system using recycled water bottles (Photo 1) has been described in detail by Sangkhom et al (2011).

Preparation of substrate and rumen fluid

The forage component of the diet (cassava leaf and mangosteen peel) was cut into small pieces, about 1 cm of length, dried at 65^°C during 24h and then milled in a coffee grinder, prior to mixing with molasses and the source of NPN (urea or calcium nitrate). Representative samples (Table 1) of the mixtures (12g DM) were put into the incubation bottle to which were added 0.96 liters of buffer solution (Table 2) and 0.24 liters of rumen fluid, prior to filling each bottle with carbon dioxide.  The rumen fluid was taken at 10-11pm in the slaughter-house from a buffalo immediately after the animal was killed. A representative sample of the rumen contents (including feed residues) was put in a vacuum flask and stored until 8-9am  the following morning when the contents were filtered through one layer of cloth before being added to the incubation bottle The flasks were then incubated at 39^°C for 48h.

Measurements

Gas volume and the content of methane was recorded during periods of incubation from 0-8h, 9-21h, 22-31h and 32-48h. The methane content of the gas was measured by pasing the gas sample through an infra-red meter (Crowcon Ltd, UK; Photo 2). At the end of the incubation the total gas volume was calculated and the residual DM in the incubation bottle measured by filtering through cloth and oven-drying at 100°C for 24h.

Statistical analysis

Data were analyzed by the General Linear Model in the ANOVA program of the MINITAB software (Version 13.2; Minitab 2000). Sources of variation in the model were: Mangosteen peel, NPN source, interaction Mangosteen*NPN source and error. 

Results and discussion

Effects of dietary mangosteen peel and NPN sources on gas production and concentration  of methane

Gas production was not affected by Mangosteen peel but was reduced in the incubations 0-8h, 9-21h and overall 90-48h, when calcium nitrate rather than urea was the NPN source (Table 3). Methane concentration in the gas in the incubation 0-8h was reduced by Mangosteen peel (Figure 2) and when calcium nitrate rather than urea was the NPN source (Figure 1). The effect of the Mangosteen peel was seen at all incubation times as was that of calcium nitrate, with the exception of the 22-31h period (Table 3). The proportion of the substrate fermented was reduced when calcium nitrate rather than urea was the NPN source, but was not affected by supplementation with Mangosteen peel. Overall production of methane per unit of substrate fermented was reduced by supplementation with mangosteen peel and by calcium nitrate as opposed to urea.

The beneficial effects in reduction of methane production when calcium nitrate replaced urea as the NPN source in an in vitro incubation are similar to those reported by Phommasack Outhen et al (2011),  Sangkhom et al 2011 and Le Thuy Binh Phuong et al 2011).  There are no reports of the effect of Mangosteen peel on methane production in an in vitro incubation. . Reduced production of methane was reported by Khan and Chaudhry (2009) for a range of spices added to an in vitro incubation, especially for Coriander (Coriandrum sativum) and in the latter case was attributed to the high levels of tannins reducing methanogenisis and/or the uptake of hydrogen for bio-hydrogenation of the unsaturateed fatty acids. Presumably, the high levels of saponins and tannins reported in Mangosteen peel (Suchitra and Wanapat 2008) were the determinant factors in bringing about the reduction in methane in the present study.

In the report of Khan and Chaudhry (2009), Coriander was found to lower methane production from 14ml/g original substrate to 8ml/g substrate after 24h incubation – a drop of 40%.  In our study the lowering of methane with Mangosteen peel after 21h incubation was from 11.4 to 5.5 ml/g substrate (a drop of 51%) while the reduction with calcium nitrate versus urea was from 15.2 to 7.4 ml/g of substrate (also a drop of 51%).

The concentration of methane in the gas increased with curvilinear trends with time of incubation, the pattern being similar for the effects of the NPN source and for the Mangosteen peel additive (Figures 5 and 6). As discussed by Sangkhom et al (2011), these changes almost certainly reflected changes in the nature of the fermentation as carbohydrate was fermented first to VFA, methane and carbon dioxide followed by secondary fermentation of the VFA to methane and carbon dioxide.

Figure 5. Effect of incubation time on methane content of the gas with nitrate or urea as the NPN source	Figure 6. Effect of incubation time on methane content of the gas with and without addition of Mangosteen peel

Conclusions

• Calcium nitrate as replacement for urea, and addition of Mangosteen extract,  lowered methane production an in vitro incubation of molasses and cassava leaf meal.
• The apparently beneficial effects of Mangosteen peel in reducing methane production needs to be substantiated by further research.

Acknowledgements

The authors express their appreciation to the MEKARN project, funded by Sida, for supporting this research  which forms part of the requirements of the senior author for the MSc degree to be submitted to Can Tho University.  The cooperation of the Administration and staff members of An Giang University is gratefully acknowledged.

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Received 20 March 2011; Accepted 24 March 2011; Published 1 April 2011

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