Effect of supplements of Tithonia diversifolia, and potassium nitrate or urea, on methane production in an in vitro system using sugar cane stalk as energy source

Sangkhom Inthapanya, Siton Kongvongxay and T R Preston*   

Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR
inthapanyasangkhom@yahoo.com
Livestock Research Center, National Agriculture and Forestry Research Institute, Laos
*Finca Ecologica, TOSOLY, UTA (Colombia)
AA#48, Socorro, Santander, Colombia

Abstract

The aim of this in vitro study was to evaluate the effect on methane production of potassium nitrate or urea as NPN sources in substrates containing varying ratios of sugar cane and Tithonia diversifolia. The treatments in a 6*2 factorial arrangements with 4 replications were: proportions of Tithonia leaf meal  of  0, 20, 40, 60, 80 and 100% replacing sugar cane (DM basis) and source of NPN  (Potassium nitrate or urea).

Gas production, methane percentage in the gas, percent of substrate solubilized, and methane produced per unit substrate solubilized, were lower for nitrate than for  urea at all levels of Tithonia leaf meal replacing derinded sugar cane in the substrate. When urea was the NPN source the level of Tithonia leaf meal did not affect gas production, but reduced it when nitrate was the source of NPN. The methane percentage in the gas, percent of substrate solubilized and methane produced per unit substrate solubilized, decreased linearly for both urea and nitrate with increasing levels of Tithonia leaf meal. There were no differences in ammonia concentration but VFA concentration was increased as Tithonia replaced sugar cane in the substrate. Ammonia and VFA concentrations were lower with nitrate than with urea.

Keywords: climate change, fermentation, gas production, potassium nitrate, greenhouse grass, tannins, urea

Introduction

Wild sunflower (Tithonia diversifolia) is a shrub widely spread in northern Laos where it is appreciated as a valuable green manure. In Western Kenya, it is renowned as a component of agro-forestry systems as it is rich in N, P and K which are essential for soil fertility (Jama et al 2000). According to Lijzenga (1998), the biomass from Tithonia has proved to be valuable in improving soil fertility for crop production in areas constrained by soil N, P and K deficiencies. It has been reported that addition of foliage of Tithonia diversifolia to the cropping area leads to double the yield of the crops and that it is more effective than urea when applied at the same N rate (Sanchez 2001). 

The forage of Tithonia is also considered to have a potential use for livestock production (Mahecha and Rosales 1905). According to Pathoummalangsy and Preston (2008) the leaves of Tithonia foliage are readily consumed by goats in the fresh state. However, the protein in Tithonia leaves was shown to be 79% degradable in the rumen in 24h (Vargas 1994),  which according to Preston and Leng (1987) would indicate that it would not be a good source of  "bypass" protein. Thus one of the initiatives to improve the utilization of Tithonia foliage in goats was centered on supplying rapidly fermentable carbohydrate to utilize better the ammonia derived from fermentation of the soluble protein in the rumen. Thus supplementation of Tithonia foliage with cassava root chips gave positive results in terms of improved nitrogen retention in growing goats (Pathoummalangsy and Preston 2008).  A similar response to fermentable carbohydrate in the form of sugar cane was reported by Sitone et al (2012), with improvements in feed intake and growth rate compared with feeding Tithonia foliage as the only feed.  

There are many reports of antimicrobial activity in leaf extracts of Tithopnia: to protect crops from termites (Adoyo et al 1997), to control insects (Carino and Rejestes 1982; Dutta et al 1993), with medicinal value for treatment of hepatitis (Lin et al 1993; Kuo and Chen 1997),  to control amoebic dysentery (Tona et al 1998) and to have broad spectrum activity against a range of  bacteria associated with wound infections (Ogunfolakan et al 2010)]. folakan et al (2010). Leaf extracts of Tithonia were found to be as effective as Ivermectin in treating Scabies in rabbits (Hang et al 2012).

It was therefore hypothesized that such antimicrobial activity in the foliage of Tithonia diversifolia might be effective in reducing methane production in an in vitro rumen incubation.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang province, Lao PDR, from January to February 2012. 

Treatments and experimental design 

The experimental design was a 6*2 factorial arrangement with four replications of the following treatments in a random block design:

Proportion of Tithonia leaf meal (TLM):  

• TLM at 0, 20, 40, 60, 80 and 100 % of the substrate DM

Source of NPN:

• Urea at 1.8% of substrate DM

• Potassium nitrate at 6% of substrate DM

The in vitro system

The equipment and procedure was that used by Sangkhom Inthapanya et al (2011) (Photos 1 and 2).

Experimental procedure

Fresh sugar cane stalk was collected from the farm of Souphanouvong University. The outer rind was removed with a knife and the central part (the sugar-rich pith) chopped into small pieces (about 2mm section) and then ground (1mm sieve). The Tithonia leaves were brought from Parkou district, Luang Prabang province (Photo 3). The sample was chopped into small pieces of around 1-2 cm of length, then ground (1 mm sieve). They were mixed with sugar cane stalk as substrate and either potassium nitrate or urea as NPN sources (Table 1) prior to adding 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid (obtained from a newly slaughtered buffalo in the village abattoir), and put in the incubation bottle which was then gassed with carbon dioxide. The bottles were incubated at 38 ⁰C in a water bath for 24 hours.

Data collection and measurements

The gas volume and the percentage of methane in the gas (Crowcon infra-red analyser; Crowcon Instruments Ltd, UK) were recorded for the separate incubations after 24 hours. At the end of the incubation the residual DM in the incubation bottle was determined by filtering through cloth and drying (100°C for 24 hours). Samples of filtrate (10 ml) were used to determine ammonia (NH₃). Samples of 20 ml were used to determine volatile fatty acid (VFA). Methods for ammonia and VFA were those described by Ly and Nguyen Van Lai (1997). 

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2000) Software. Sources of variation in the model were: NPN source; proportion of Tithonia leaves, interaction NPN*proportion of leaves and error.

Results

Composition of substrate

Effect of proportion of Tithonia leaves and NPN source on methane production

Gas production, methane percentage in the gas, percent of substrate solubilized, and methane produced per unit substrate solubilized, were higher for  urea than for nitrate, for all levels of TLM (Table 1; Figures 1-4). When urea was the NPN source the level of TLM did not affect gas production, but reduced it when nitrate was the source of NPN. For methane percentage in the gas, percent of substrate solubilized and methane produced per unit substrate solubilized, the values decreased linearly for both urea and nitrate as tghe proporion of TLM was increased..    

Rumen ammonia and VFA

Ammonia levels were higher for urea than for nitrate and were not affected by level of TLM (Table 2; Figure 5). VFA concentrations were also higher for urea than for nitrate, but were increased with a curvilinear trend by 35% overall on nitrate and by 54% on urea, as the TLM was increased from zero to 100% of the substrate (Table 2; Figure 6).

Discussion

The effect of the rumen fermentable carbohydrate source (derinded sugar cane) in increasing methane production from TLM was the opposite of the decrease in methane production when cassava root meal was added to sweet potato vines and water spinach (Ho Quang Do et al 2012). Both sweet potato vines and water spinach have high levels of soluble protein, similar to that in TLM. The difference between TLM and sweet potato vines and water spinach is that the latter two forages do not possess anti-nutritional or antimicrobial compounds, in contrast with Tithonia leaves which are apparently rich in a range of such compounds according to the following reports [Extracts from Tithonia plant parts protect crops from termites (Adoyo et al 1997), contain chemicals that inhibit plant growth (Baruah et al 1978; Tongma et al 1997), control insects (Carino and Rejestes 1982; Dutta et al 1993), have medicinal value for treatment of hepatitis (Lin et al 1993; Kuo and Chen 1997),  control amoebic dysentery (Tona et al 1998) and have broad spectrum activity against a range of  bacteria associated with wound infections (Ogunfolakan et al 2010)].

It can be expected therefore that inclusion of Tithonia foliage in the diet of ruminants will lead to a decrease in enteric methane emissions. This supposition needs to be verified by experiments directly comparing Tithonea leaves with water spinach foliage and/or sweet potato vines in substrates in which the energy component is either sugar cane or cassava root meal. 

Conclusions

• In an in vitro system, gas production, methane percentage in the gas, percent of substrate solubilized, and methane produced per unit substrate solubilized, were lower for nitrate than for  urea at all levels of Tithonia leaf meal replacing derinded sugar cane in the substrate.

• When urea was the NPN source the level of Tithonia leaf meal did not affect gas production, but reduced it when nitrate was the source of NPN.

• The methane percentage in the gas, percent of substrate solubilized and methane produced per unit substrate solubilized, decreased linearly for both urea and nitrate with increasing levels of Tithonia leaf meal.

• It is concluded that inclusion of Tithonia foliage in the diet of ruminants will probably  lead to a decrease in enteric methane emissions.

Acknowledgements

The authors express gratitude to the MEKARN project, supported by Sida, for financial support for this research. Special thanks are given to Mr Sengsouly Phongphanith and Mr Aloun Ounalom who provided valuable help in the laboratory. We also acknowledge the Souphanouvong University, Faculty of Agriculture and Forest Resources, Department of Animal Science, for providing infrastructure support.

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