Methane production in an in vitro rumen incubation is lower for leaves with low compared with high protein solubility

Ho Quang Do, Tran Duy Khoa, Trinh Phuc Hao and T R Preston*

Cantho University, Cantho, Vietnam
hqdo@ctu.edu.vn
* Nong lam University, Ho Chi Minh City, Vietnam

Abstract

The objectives of this study were to determine: the protein solubility of leaves of cassava and Sesbania grandiflora and of foliage of water spinach and sweet potato vines;  and the effect of these feed resources on methane production in an in vitro incubation system with or without cassava root meal as substrate.

Crude protein solubility was lower for leaves of cassava  and Sesbania grandiflora  than for water spinach foliage and sweet potato vines.   The gas production and methane production per unit DM solubilized were lower on the leaves with low solubility of the protein, and were lower still when cassava root meal was included in the substrate.

Key words: cassava root meal, cassava leaves, gas production, Sesbania grandiflora, water spinach

Introduction

There is a need to develop feeding systems for ruminants that will result in reduced emissions of methane gas from the enteric fermentation in these animals. Previous research showed that methane production was less when  fish meal rather than groundnut meal was the substrate (Preston et al 2013). The differences in methane production between the two protein meals appeared to be related to the solubility of the protein which was 16% in fish meal compared with 76% in groundnut meal.

The aim of this study was to determine if similar relationships existed in leaves/foliages of different protein solubility.  Whitelaw and Preston (1963) showed that meals of low protein solubility (heat-treated fish and groundnut meals) supported greater nitrogen retention in early-weaned calves than similar meals of high  protein solubility (enzyme-hydrolyzed fish meal and non-heat treated groundnut meal). These authors hypothesized that these results were indicative of the escape (bypass) of protein from the rumen fermentation for subsequent enzyme digestion in the small intestine, a process that results in a higher protein-energy (P:E) ratio in absorbed nutrients and therefore improved animal production (Preston and Leng 1987). Feeds which supply bypass protein and are also associated with reduced methane production would increase their relative importance as components of ruminant feeding systems. The report by  Ffoulkes and Preston (1978) showed that cattle growth rates were greater when a protein-free basal diet (molasses with 3% urea) was supplemented with fresh cassava foliage than sweet potato vines, feeds which in this present study have been shown to have, respectively,  low and high levels of soluble protein.

The hypothesis that underlined the present study was that plants having leaves/foliage with low protein solubility would produce less methane than plants with higher levels of soluble protein, when incubated with rumen fluid in an in vitro system.

Materials and methods

Location and experiment design

The experiment was conducted in the Laboratory of the Department of Animal Science, College of Agriculture and Applied Biology, Can Tho University. A completely randomized design with 3 repetitions was used to determine the  methane production from leaves of cassava (CL) and Sesbania grandiflora (SG), and the foliage of  Water spinach (WS) and  vines of Sweet potato (SP) in an in vitro incubation with rumen fluid.

Each incubation was carried out on the ground dried leaves/foliages as the only substrate or with additional cassava root meal supplying 50% of the substrate (Table 1).

Experimental procedure

The leaves/foliages were dried at 55°C for 48 hours then ground through a 1mm screen. Solubility of the protein was determined by shaking 3 g of dry leaf meal in 100 ml of M NaCl for 3h then filtering through Whatman No. 4 filter paper, and determining the N content of the filtrate (Whitelaw et al 1962). The samples of dried leaves were analyzed for DM, ash, NDF, ADF and N according to procedures in AOAC (1990).

The quantity of substrate used in the in vitro incubation was 2 g to which were added 40 ml rumen fluid (from rumen-fistulated cattle) and 160 ml buffer solution (Tilley and Terry 1963). The incubation was for 24h with measurements of total gas production recorded by water displacement (Inthapanya et al 2011; Photo 1). Samples of gas were analyzed for the proportions of methane with a Triple plus +IR meter (Crowcon Instruments Ltd, UK;  Photo 2).

Photo 1: The in vitro incubation system	Photo 2: The gas meter for measuring methane system

Statistical analysis

The data were analyzed by the General Linear Model option of the ANOVA program in the Minitab software (Minitab 2000). Sources of variation were: treatments and error.

Results and discussion

The contents of crude protein were similar in the leaves and foliages (Table 2). NDF and ADF values were lowest in the Sesbania leaves. The mineral content was relatively high in all the leaves/foliages but very low in the cassava root.

The solubility of the crude protein was twice as high in sweet potato vines and water spinach compared with Sesbania and cassava leaves (Table 3). 

Values for gas production, the methane content of the gas and the methane produced per unit substrate solubilized in most cases were lower for the leaves of Sesbania and cassava compared with the foliages (leaves plus stems) of water spinach and sweet potato vines (Table 4). The lower methane production in the leaves of Sesbania  and cassava compared with the foliages of water spinach and sweet potato, coincides with the much reduced solubility of the protein in the leaves of the Sesbania and cassava compared with the foliages of water spinach and sweet potato. This association between methane production and protein solubility was also observed in in vitro incubations with fish meal and groundnut meal (Ho Quang Do et al  2013) and in a wide range of leaves from fodder trees (Silivong et al 2013).  Further research is needed to elucidate the mechanism explaining these relationships.

Figure 1. Methane production per unit substrate DM solubilized from leaves of Sesbania and cassava and foliages of water spinach and sweet potato when incubated alone or with cassava root meal	Figure 2. Relationship between methane production per unit substrate DM solubilized and the solubility of the protein in M NaCl (leaves of Sesbania and cassava and foliages of water spinach and sweet potato  incubated alone or with cassava root meal)

A strict comparison cannot be made of the incubation with and without cassava root meal, as the incubations were done on different days. However, it is fairly obvious that the addition of cassava root meal to the substrate reduced gas production, methane content of the gas and methane produced per unit substrate DM solubilized (Table 4; Figure 1). It is to be expected that the effect of adding cassava root meal to the substrate (50% of the DM) would be to enhance propionate production, which in turn would mean less hydrogen available for conversion to methane. A similar result (lower methane and more rumen propionate) was reported by Sabri Yurtseven et al (2009) for milking sheep that consumed a higher concentrate: roughage ratio (80:20) when they had free access to concentrates and roughage, compared with being given  a complete mixed diet having a 60: 40 ratio of concentrate to roughage.

Methane production appeared to be associated with the solubility of the protein in the substrates (Figure 2). It was higher for water spinach and sweet potato vines (protein solubility 70.7 and 70.9%)  compared with the leaves  of Sesbania and cassava (protein solubility 33.0 and 35.0%). A similar relationship was observed with by-product meals of different degrees of solubility (Preston et al 2013)with lower methane production from fish meal (protein solubility 16%) than from groundnut meal (protein solubility 76%).

Conclusions

• Crude protein solubility was lower for leaves of Sesbania grandiflora and cassava than for foliages of water spinach and sweet potato.

  
  

• The gas production and methane production per unit DM solubilizied were lower for the leaves with lower solubility of the protein, compared with the foliages of high solubility, and were lower still when cassava root meal was included in the substrate. 

References

AOAC 1990 Official methods of analysis. 15th edition. AOAC, Washington, D.C.

Ffoulkes D and Preston T R 1978 Cassava or sweet potato as roughage in molases – urea based diet; effect of supplementation with soybean. Tropical Animal Production. pages: 186-192 http://www.utafoundation.org/UTAINFO1/TAP/TAP33/3_3_1.pdf
 

Preston T R, Do H Q, Khoa T D, Hao T P and Leng R A 2013 Protein solubility of fish meal and groundnut meal and methane production in an in vitro incubation. Livestock Research for Rural Development. Volume 25, Article #16. http://www.lrrd.org/lrrd25/1/hqdo25016.htm

Inthapanya S, Preston T R and Leng R A 2011 Mitigating methane production from ruminants; effect of calcium nitrate as modifier of the fermentation in an in vitro incubation using cassava root as the energy source and leaves of cassava or Mimosa pigra as source of protein. Livestock Research for Rural Development. Volume 23, Article #21. http://www.lrrd.org/lrrd23/2/sang23021.htm

Minitab 2000 Minitab reference manual release 13.31for Window. Minitab Inc., State College, USA. http://www.minitab.com

Sabri Yurtseven, Mehmet Cetin, Irfan Ozturk, Abdullah Can, Mustafa Boga, Tekin Sahin and Huseyin Turkoglu 2009 Effect of Different Feeding Method on Methane and Carbon Dioxide Emissions, Milk Yield and Composition of Lactating Awassi Sheep. Asian Journal of Animal and Veterinary Advances, 4: 278-287.

Tilley J M A and Terry R A 1963 A two stage technique for the in vitro digestion of forage crops. Journal of the British Grassland Society 18 : 104.

Whitelaw F G and Preston T R 1963 The nutrition of the early-weaned calf.  III. Protein solubility and amino acid composition as factors affecting protein utilisation. Animal Production. Volume 5 pp 131-145. http://www.utafoundation.org/publications/whitelaw&preston 1963.PDF

Received 5 August 2012; Accepted 24 June 2013; Published 1 July 2013

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