Methane production from urea-treated rice straw is reduced when the protein supplement is cassava leaf meal or fish meal compared with water spinach meal in a rumen in vitro fermentation

Sangkhom Inthapanya and T R Preston¹

Faculty of Agriculture and Forest Resources, Souphanouvong University,
Luang Prabang, Lao PDR
inthapanyasangkhom@gmail.com
¹Center for Research and Technology Transfer, Nong Lam University
Thu Duc District, Ho Chi Minh City, Vietnam

Abstract

The aim of this study was to evaluate the effect of protein-rich feeds resistant to fermentation in the rumen in reducing methane production in an in vitro incubation system. The treatments in a completely randomized block design (RCBD) with 4 treatments and 4 replications of each treatment were UTR: Urea-treated rice straw with additional urea to provide approximately 15% crude protein in the substrate DM; WS: same as UTR with water spinach meal at level of 6 g crude protein per100 g substrate DM; FM: same as UTR with fish meal at level of 6g crude protein per 100 g substrate DM; CLM: same as UTR with cassava leaf meal at level of 6 g crude protein per 100 g substrate DM. The quantity of substrate DM in each fermentation bottle was 12g to which were added 240 ml rumen fluid (from slaughtered buffalo) and 960 ml of buffer solution. The incubation was for 24h with measurements of total gas production and methane percentage at intervals of 6, 12 and 24 hours and determination of residual DM unfermented substrate at the end.

On all the treatments, the concentration of methane in the gas increased exponentially with the duration of the incubation, highest values being recorded for the water spinach treatment and lowest values for the cassava leaf meal. Methane production was reduced when the crude protein source was fish meal or cassava leaf meal as compared with water spinach. The implications are that methane production is reduced when the dietary crude protein component is less rumen degradable.

Key words: HCN, rumen degradable protein, solubility

Introduction

terrPrevious reports have shown decreases in methane production in rumen in vitro fermentations systems fish meal rather than groundnut meal was the protein source (Ho Quang Do et al 2013) and when leaf meal was derived from “bitter” varieties of cassava as opposed to “sweet” varieties (Le Thuy Binh Phuong et al 2012). It was concluded that in the former case the determining factor was the lower rumen solubility of the protein in fish meal compared with the protein in groundnut meal (as reported earlier by Whitelaw and Preston 1963); while in the case of cassava it was inferred that it was the higher concentration of cyanogenic precursors (known to be toxic to methanogenic bacteria; Smith et al 1985) in the bitter as opposed to the sweet cassava (Le Thuy Binh Phuong et al 2012).

The present study was designed to produce further evidence to support the hypothesis that supplements rich in proteins resistant to fermentation in the rumen, because of low solubility or through linkages to toxic compounds, would produce less methane in an in vitro rumen fermentation than supplements not possessing these properties.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Department of Animal Science, Faculty of Agriculture and Forest Resource, Souphanouvong University, Luang Prabang province, Lao PDR, from June to July 2014.

Treatments and experimental design

The experiment was arranged in a completely randomized block design (RCBD) with 4 treatments and 4 replications of each treatment. The treatments (Table 1).were:

• UTR: Urea-treated rice straw with additional urea to provide approximately 15% crude protein in the substrate DM
• WS: Same as UTR with water spinach meal at level of 6 g crude protein per100 g substrate DM
• FM: Same as UTR with fish meal at level of 6g crude protein per 100 g substrate DM
• CLM: Same as UTR with cassava leaf meal at level of 6 g crude protein per 100 g substrate DM

The basal substrate was urea–treated rice straw (3g urea, 100 g rice straw DM, 100g water)

 The in vitro system

Gas production  was determined using recycled water bottles (Inthapanya et al 2011). A hole was made in the lid of every bottle and two bottles were connected by a plastic tube in order to transfer gas during incubation. The first bottle served as the incubator; the second bottle had the bottom removed and was suspended in water after being calibrated in steps of 50 ml indicating the amount of gas production by displacement. The gas collection bottle was then suspended in a larger bottle that was filled with water. Finally a buffer solution and rumen fluid were mixed with samples of the substrate and added to the fermentation bottle followed by gassing with carbon dioxide. Clay was  used to cover the lids of all the bottles to prevent gas leakages. 

Experimental procedure

Preparation of urea-treated rice straw (UTR)

The rice straw was chopped into small pieces around 1-2 cm of length, then ground (1mm sieve), then mixed with a solution of urea (3% of straw DM) in water (100% of straw DM). The treated straw was stored in plastic bags at ambient temperature for 14 days.

Fish meal, cassava leaf meal and water spinach meal

Fish waste was bought from sellers in Luang Prabang market. The waste was dried by sunlight for 2-3days before being ground through a 1 mm sieve. Cassava leaves and water spinach (leaves and petioles) were collected in the Souphanouvong University area, chopped into small pieces around 1-2 cm of length, then ground (1mm sieve).

Amounts of the substrates equivalent to 12 g DM were put in the incubation bottle, followed by 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid obtained from a buffalo immediately after being slaughtered at the municpal abattoir in LuangPrabang city). The bottles were then filled with carbon dioxide and incubated at 38 ⁰C in a water bath for 24 h.  

 Data collection and measurements

 During the incubation the gas volume were recorded at 6, 12 and 24h. After each time interval, the methane concentration in the gas was measured with a Crowcon infra-red analyser (Crowcon Instruments Ltd, UK).  At the end of the incubation, the residual DM and N in the incubation bottle were analysed to determine mineralization of the DM and of the nitrogenous compounds. The ammonia concentration in the filtrate was measured to determine the proportion of the N source converted to ammonium salts.

 Chemical analyses 

Samples of urea-treated rice straw, cassava leaf meal, fish meal and water spinach meal were analyzed for DM, ash, crude protein and crude fiber according to AOAC (1990) methods. Protein-N soluble in M NaCl was determined by the method outlined in Whitelaw aand Preston (1963).

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2010) Software. Sources of variation in the model were: treatments, incubation times and error.

Results and discussion

On all the treatments, the concentration of methane in the gas increased exponentially with the duration of the incubation (Table 4; Figure 1), highest values being recorded for the water spinach treatment and lowest values for the cassava leaf meal. Increases in methane concentration with incubation time is a consistent feature of in vitro rumen fermentations (Inthapanya et al 2011; Binh Phuong et al 2011; Thanh et al 2011; Outhen et al 2011; Marin et al 2014).

Methane concentration was directly related with the degree of mineralization of the nitrogen in the substrate during the fermentation (Figure 2).

Figure 1. Trends in methane content of the gas according to duration of incubation	Figure 2. Relationship between the substrate nitrogen fermented and the production of methane per unit DM mineralized (CTR treatment excluded)

These results support those of Ho Quang Do et al (2013) and Le Thi Binh Phuong et al 2012) showing a direct relationship between the degree of fermentation of the protein in the substrate and the production of methane. The implication of these findings is that both  performance and the need to reduce enteric methane emissions will benefit when the crude protein supply in ruminant diets  is less rumen degradable.

Conclusions

• In an in vitro rumen fermentation, with urea-treated rice straw, as the basal substrate, methane production was reduced when the crude protein source was fish meal or cassava leaf meal as compared with water spinach.
  
  
• The implications are that methane production is reduced when the dietary crude protein component is less rumen degradable.

Acknowledgements

This research is part of the requirement for the PhD of the senior author. Financial support from the Sida-financed project, MEKARN II, is gratefuly acknowledged.

References

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Received 25 August 2014; Accepted 30 August 2014; Published 5 September 2014

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