Effect of potassium nitrate or urea as NPN source and levels of Mangosteen peel on in vitro gas and methane production using molasses, Operculina turpethum and Brachiaria mutica as substrate

Vo Duy Thanh, Nguyen Van Thu* and T R Preston**

Can Tho University, Vietnam
vothanh69@gmail.com
* Department of Animal Sciences, Cantho University, Cantho city, Vietnam
** Finca Ecologica, TOSOLY, UTA (Colombia)
AA#48, Socorro, Santander, Colombia

Abstract

The objective of this study was to evaluate the effect of the level of Mangosteen peel and potassium nitrate or urea as non-protein nitrogen source on methane production in an in vitro incubation. The design wasa 3*4 factorial with 3 replicates. The factors were source of non protein nitrogen: urea (1.83% of substrate, DM basis) and potassium nitrate (4 or 6% of substrate, DM basis); and levels of Mangosteen peel (0, 0.5, 1 and 1.5% of substrate DM basis). The quantity of substrate was 2.5g to which were added 200ml of buffer solution and 50ml of buffalo rumen fluid taken immediately after the animal was killed in the slaughter-house. The incubation was for 48 h with measurements of gas and methane production at 6, 12, 24, 36 and 48 h. The proportion of substrate solubilized at 48h was determined by filtration, followed by measurement of ammonia-nitrogen concentration in the filtrate. 

After 48h incubation, gas and methane production, per cent substrate DM digested and methane produced per unit DM digested, were lower when potassium nitrate was the NPN source compared with urea. The 6% level of potassium nitrate was more effective in reducing methane production than the 4% level. Gas and methane production increased with time of incubation. Similar reductions in the above parameters were observed with increasing level of Mangosteen peel in the substrate. The ammonia concentration in the filtrate after 48h of incubation was lower when potassium nitrate was the NPN source compared with urea.

Keywords: Climate change, greenhouse gases, incubation, rumen

Introduction

Due to the World challenging problems of energy and food crisis, and negative effects of global climate changes which livestock production is facing, particularly the animal nutrition must consider not only nutrient requirements and production but also friendly environment, sustainable productivity and human and animal welfare. Preston (2009) concluded that instead of conflict in the use of biomass for food and fuel, there can be synergism, and that, instead of contributing to climate change, farming systems can have a negative carbon footprint. Hindrichsen et al (2005) indicated that 85-90% of methane is produced by enteric fermentation and Murray et al (1976) also concluded that 89% of the methane is excreted in the animal’s breath and 11% from the anus. There is a great incentive to reduce methane emissions from livestock.

Mangosteen (Garcinia mangostana) is a tropical plant which produces an edible fruit; it is very familiar with people in Southeast Asia. The fruit when ripe has a thick outer skin, of dark purple color. The Mangosteen peel contains both condensed tannins and crude saponins, which exert a specific effect against rumen protozoa, while the rest of the rumen biomass remains unaltered (Ngamsaeng and Wanapat 2005). Ngamsaeng and Wanapat (2005) suggested that supplementation of mangosteen peel (100g DM/d) in cattle can increase the population of rumen bacteria and decrease the protozoal population, and maintain the fungal zoospore population.

Tannins have been found to inhibit methane in the rumen fermentation, which is beneficial for sparing of energy loss by this route. Many types of forage are known to contain condensed tannins and have been shown to decrease methane production both in vivo and invitro such as sainfoin (Onobrychisviciifolia), Lotus pedunculatus (lotus) and Lotus corniculatus (Birdsfoot trefoil) (Patra 2007).  Condensed tannins are beneficial for the rumen fermentation when they are present in moderate quantity (4 to 6% of the total DM) in the diets (Patra 2007).  Woodward et al (2001) investigated the feeding of sulla (Hedysariumcoronarium) on methane emission and milk yield in Friesian and Jersey dairy cows. Cows grazing on sulla had higher daily dry matter intake (13.1 vs. 10.7 kg DM) and daily milk solid production (1.07 vs. 0.81 kg) than when grazing on perennial ryegrass pasture. Total daily methane emission was similar (254 vs 260 g).

Thepresentinvestigation aimed to determine the effects of potassium nitrate or urea as the source of non-protein nitrogenonmethane production in an in vitro incubation in which there were various levels of mangosteen peel.

Hypothesis

The hypothesis to be tested was:

Potassium nitrate and mangosteen peel will reduce methane production in an in vitro incubation using molasses and Operculina turpethum as the basal substrate.

Materials and methods

Location and duration

The in vitro fermentation was conducted in the laboratory at the Department of Animal Sciences, Faculty of Agriculture and Applied Biology, Cantho University, Cantho city, Vietnam.

The experiment was donefrom May to July, 2011.

Experimental design

Thiswas a 3*4 factorial design with 3 replicates. The factors were

Sources of non protein nitrogen: 1.83% urea, 4% and 6% potassium nitrate

Mangostgeen peel: 0%, 0.5%, 1.0% and 1.5% (DM) of the substrate.

The basal substrate was a mixture of 35% (DM) para grass, 35% (DM) Operculina turpethum and22.5% to 28.2% (DM) molasses.

The in vitro system

The in vitro system was based onthe same procedure (Photos 1 and 2) that has been described in an earlier paper (Thanh et al 2011).

Preparation of substrate and rumen fluid

The Operculina turpethum, para grass and mangosteen peel were cut into small pieces, about 1 cm of length and then dried at 65^°C during 24h. Representative samples of the mixtures (2.5g DM) were put into the incubation bottle to which were added 0.2 liters of buffer solution (Table 1) and 0.05 liters of rumen fluid, prior to filling each bottle with carbon dioxide. The rumen fluid was taken at 9-10am in the slaughter-house from a buffalo immediately after the animal was killed. Representative samples of the rumen were filtered through two layers of cloth (for keeping feed residues in the rumen fluid) before being added to the incubation bottle.The flasks were then incubated at 38^°C for 48h.

Measurements

The incubation was for 48 h with measurements of gas and methane production being recorded at 6 h, 12 h, 24 h, 36 h and 48 h of incubation. The methane percentage of the gas was measured by passing the gas sample through an infra-red meter (Crowcon Ltd, UK; Photo 2).  Unfermented solids at 48 h were determined by filtering through two layers of cloth and drying at 100°C for 24h. Ammonia-nitrogen (NH₃-N) concentration was measured in the filtrate.

Chemical analyses

The ingredients in the substrate were analysed for DM, OM, CP and ash according to the standard methods of AOAC (1990).

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: Levels of Mangosteen peel, NPN sources, interaction levels of Mangosteen peel*NPN source and error. 

Results

Chemical composition

Both Mangosteen peel and Operculina turpethum had higher crude protein than the para grass (Table 2).

In vitro gas and methane production

After 48h incubation, gas and methane production, per cent substrate DM digestedand methane produced per unit DM digested, were lower when potassium nitrate was the NPN source compared with urea (Table 3). The 6% level of potassium nitrate was more effective in reducing methane production than the 4% level. Gas and methane production increased with time of incubation (Figures 1 and 2). Similar reductions in the above parameters were observed with increasing level of Mangosteen peel in the substrate (Figures 4 to 6).

Figure 1. Effect of potassium nitrate or urea as source of non protein nitrogen on gas production at 48 h	Figure 2. Effect of potassium nitrate or urea as source of non protein nitrogen on methane production at 48 h

Figure 3. The percentage of methane in the gas with potassium nitrate or urea as source of non-protein nitrogen

Figure 4. Effect of increasing concentrations of Mangosteen peel on gas production at 48 h fermentation	Figure 5. Effect of increasing concentrations of Mangosteen peel on methane production at 48 h fermentation
Figure 6. Effect of incubation time and increasing concentrations of mangosteen peel on methane per cent in the gas

There were close negative relationships between the level of Mangosteen peel in the substrate and methane production (Figures 7-8) and the per cent of substrate solubilized (Figure 9).

Figure 7. Relationship between level of mangosteen peel and methane production	Figure 8. Relationship between level of mangosteen peel and methane production per unit substrate DM fermented

The ammonia concentration in the filtrate after 48h of incubation was lower when potassium nitrate was the NPN source compared with urea (Table 4). There were no differences in pH, the values of which were normal for fermentation by rumen micro-organisms.

Discussion

The beneficial effects of mangosteen peel in reducing methane production n an in vitro incubation were similar to those reported by Thanh et al (2011). In the earlier study, the level of Mangosteen peel was 0.67%. The results of the present experiment indicate that higher levels of Mangosteen peel, up to 1.5%,  can be used with greater reduction in methane and without apparent negative effects on the fermentation. Similar positive effects on methane reduction were reported by Khan and Chaudhry (2009) for a range of spices added to an in vitro incubation, especially for coriander (Coriandrum sativum).  These authors attributed the reduction in methane to the high levels of tannins reducing methanogenisis and/or the uptake of hydrogen for bio-hydrogenation of the unsaturated fatty acids.

The positive effect of potassium nitrate in reducing methane production in an in vitro incubation is in line with the results of many similar studies with similar or different substrates (Outhen et al 2011; Sangkhom Inthapanya et al 2011; Phuong et al 2011) when nitrate salts replaced urea as the source of NPN.  

Conclusion

• Methane production in an in vitro incubation was reduced by potassium nitrate compared with urea and by increasing levels of Mangosteen peel up to 1.5% of the substrate DM..

Acknowledgements

The authors would like to thank the MEKARN project for supporting this research which forms part of the requirement of the senior author for the MSc degree in Animal Production "Specialized in Response to Climate Change and Depletion of Non-renewable Resources" of Cantho University.

References

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Received 3 March 2012; Accepted 12 March 2012; Published 2 April 2012

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