Protein solubility of fish meal and groundnut meal and methane production in an in vitro incubation

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

Cantho University, Cantho, Vietnam
preston@lrrd.org
* UTA-TOSOLY, AA 48 Socorro, Colombia
** University of New England, Armidale NSW, Australia

Abstract

The study aimed to measure the effects of two protein meals of widely different solubility on production of methane in a rumen in vitro system with cassava root meal as the energy substrate and sodium nitrate or urea as a source of non-protein-nitrogen (NPN). The protein meals of low and high solubility were fish meal (FM) and groundnut meal (GM); sources of NPN were sodium nitrate (SN) and urea (U). The incubation with rumen fluid from rumen- fistulated cattle was for 24h with measurements of total gas production,  content of methane in the gas and substrate DM solubilized.

The solubility of the crude protein in the fish and groundnut meals was 17 and 76%, respectively. Gas production was lower with nitrate than with urea and lower for the substrate containing fish meal than for that containing groundnut meal. Percentage methane in the gas and methane produced per unit substrate DM solubilized were reduced 26 and 58%, respectively by nitrate compared with urea and by 9.2 and 18%, respectively for fish meal compared with groundnut meal.

Key words: bypass protein, cassava root meal, escape protein, gas production, PE ratio

Introduction

Whitelaw et al (1962) showed that the solubility of the protein was more important than the amino acid composition in determining the rate of retention of nitrogen and live weight gain in early-weaned calves fed fish meal or groundnut meal as the protein supplement in all-concentrate diets. These early findings were substantiated in feeding trials in Cuba which showed that cattle fed molasses-based diets increased their growth rates with a curvilinear response to increasing replacement of urea by fish meal as the dietary N-source (Preston 1971). These and subsequent findings (eg: Preston et al 1976; Ffoulkes and Preston 1978; Zhang Weixian et al 1994) led to the development of the concept of  “bypass” protein to compare protein supplements in terms of their potential to escape the rumen fermentation and be digested in the small intestine. This in turn leads to an increase in the protein: energy (P:E) ratio in absorbed nutrients and hence the likelihood of increased productivity and efficiency of feed utilization in ruminant animals (Preston and Leng 1987 [2009]).

Recent research has emphasized the need to reduce greenhouse gas emissions from ruminant animals, especially the methane arising from enteric fermentation. A major step forward in achieving this aim was the hypothesis (Leng 2008) that nitrate is a preferred electron sink for hydrogen arising from rumen fermentation of carbohydrate, and that this leads to the production of ammonia rather than methane. The efficacy of this approach has now been demonstrated both in in vitro and in vivo experiments (see review by Leng and Preston 2010).  

Dietary proteins that are highly soluble are converted rapidly to ammonia in the rumen and the S amino acids give rise to hydrogen sulphide, which is an effective electron sink. It is thus possible that there might be some negative feedback of this rapid production of ammonia from dietary protein on the pathway of ammonia formation from hydrogen.

The hypothesis underlying the present study was that methane production would be lower in an in vitro rumen incubation with a protein source of low compared with one of high solubility. The study was carried out with protein sources of contrasting solubility in the form of fish meal and groundnut meal.

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. Four treatments were arranged in a 2*2 factorial design, the factors being:

Protein meals of low and high solubility:

• Fish meal (FM) or groundnut meal (GM)

Source of non-protein nitrogen:

• Nitrate (N) or urea (U)

The energy source was cassava root meal; nitrate was in the form of sodium nitrate (Table 1).

 The level of crude protein was 12.8% of the DM in each of the substrates, with the NPN source providing 52% of the total N (Table 2).  Samples of the ingredients were analyzed for DM, ash, NDF, ADF and N according to AOAC (1990).  The quantity of substrate used in the in vitro incubation was 2 g to which were added 40 ml rumen fluid (taken 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 system	Photo 2. The Crowcon gas meter for measuring methane

Statistical analysis

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

Results and discussion

Whitelaw et al (1962) reported crude protein solubilities of  83.8 and 13.6 % for groundnut meal and fish meal, respectively, similar to the values recorded in this study (Table 2).

Gas production was lower with nitrate than with urea and lower for fish meal than groundnut meal in the substrates fed  (Table 3). Percentage methane in the gas and methane produced per unit substrate DM solubilized were lower for nitrate than for urea and were lower for fish meal than for groundnut meal (Figures 1-4). 

Figure 1.  Effect of protein source on methane percentage in the gas	Figure 2.  Effect of nitrate versus urea on methane percentage in the gas
Figure 3.  Effect of  protein source on methane production per unit DM solubilized	Figure 4.  Effect of nitrate versus urea on methane production per unit DM solubilized

There is no obvious explanation for the reduction in methane with decreasing solubility of the protein. For methane production to be decreased either there has to a reduced production of hydrogen in the rumen fermentation or an increase in methanotroph activity resulting in utilization of the methane that would normally be produced.  Less hydrogen would be produced if the rumen fermentation resulted in increased microbial growth (microbial dry materials are more reduced then the substrate they use ( Hungate 1966), or if some of the fermentable diet DM escaped to the lower intestinal tract. In the case of fish and groundnut meals the evidence is that there is much more escape of protein from the rumen with fish meal than with groundnut meal as evidenced by the increased N retention in calves fed a fish meal-based, compared with groundnut meal-based,  diet (Whitelaw and Preston 1962). The amount of fat and its unsaturated content will also affect  methane production particularly per unit of dry matter solubilised as the fat remains with the insoluble components but also is a hydrogen sink. Fish meal contains more sulphur amino acids than groundnut meal, but is less degraded in the rumen,  thus the overall production of hydrogen sulphide from the two proteins may be similar with similar effects of the released hydrogen sulphide acting as a hydrogen sink. Further research is needed to elucidate possible mechanisms for the observed differences.

In terms of practical application, the implication is that dietary protein supplements with characteristics that facilitate protein escape from the rumen (eg:  oilseed or animal byproduct meals given additional heat treatment; presence of appropriate levels of tannins to protect the protein from rumen microbial degradation as in foliage/tree leaves) will both improve animal production and reduce enteric methane emissions.

In a subsequent paper (Silivong et al 2013) we examine methane production from tree leaves and from forages and relationships between methane production and crude protein solubility in these different substrates.

Conclusions

• The solubility of the crude protein in the fish and groundnut meals was 17 and 76%, respectively.
  
  
• Gas production was lower with nitrate than with urea and lower for fish meal than groundnut meal.
  
  
• Percentage methane in the gas and methane produced per unit substrate DM solubilized were reduced 26 and 58%, respectively by nitrate compared with urea and by 9.2 and 18%, respectively for fish meal compared with groundnut meal.  

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 molasses – urea based diet; effect of supplementation with soybean. Trop Animal Prod 1978. page: 186- 192 http://www.utafoundation.org/UTAINFO1/TAP/TAP33/3_3_1.pdf

Hungate R E 1966 The Rumen and Its Microbes, Academic Press, New York, pp. 91-147.

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

Leng R A 2008  The potential of feeding nitrate to reduce enteric methane production in ruminants. A Report to The Departmernt of Climate Change Commonwealth Government of Australia. ACT Canberra Australia For paper and PPT presentation see http://www.penambulbooks.com/

Leng R A and Preston T R 2010: Further considerations of the potential of nitrate as a high affinity electron acceptor to lower enteric methane production in ruminants. Livestock Research for Rural Development. Volume 22, Article #221. http://www.lrrd.org/lrrd22/12/leng22221.htm

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

Preston T R 1971 Quantitative aspects of animal protein production from NPN in ruminants Source:FAO/IAEA. Tracer studies on non-protein nitrogen for ruminants. http://www.utafoundation.org/preston.papers/Preston1971(IAEA).pdf

Preston T R, Carcaño C, Alvarez F J  and Gutíerrez  D G 1976 Rice polishings as a supplement in a sugar cane diet effect of level of rice polishings and of processing the sugar cane by derinding or chopping. http://www.utafoundation.org/UTAINFO1/TAP/TAP13/1_3_1.pdf

Preston T R and Leng R A 1987 Matching ruminant production systems with available resources in the tropics and sub-tropics . Penambul Books Armidale (2009 New online edition) http://www.utafoundation.org/P&L/preston&leng.pdf

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, Preston T R and Dawson G S 1962 The nutrition of the early-weaned calf  a comparison of commercial groundnut meal, heat-treated groundnut meal and fish meal as the major protein source in the diet. Animal Production Volume 4. http://www.utafoundation.org/publications/whitelaw&preston 1962.pdf

Zhang Weixian, Gu Chuan Xue,  Dolberg D and Finlayson P M 1994 Supplementation of ammoniated wheat straw with hulled cottonseed cake. Livestock Research for Rural Development.  6 (1) http://www.lrrd.org/lrrd6/1/china1.htm

Received 6 June 2012; Accepted 21 December 2012; Published 4 January 2013

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