Effect of contrasting sources of carbohydrate (sugar or starch) and bypass protein (leaf meal from sweet or bitter cassava) on methane production in a rumen in vitro incubation

Phuong Le Thuy Binh, T R Preston¹ and R A Leng²

Nong Lam University, Thuc Duc, Ho Chi Minh City, Vietnam
binhphuonglt@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² Emeritus Professor, University of New England, Armidale NSW2351, Australia

Abstract

In a rumen in vitro incubation over 24h, the methane content of the gas and the methane produced per unit substrate solubilized were: (i) increased when the substrate carbohydrate was sugar as compared with cassava root (supplemented in both cases with urea); and (ii) decreased when the bypass protein source was leaf from bitter cassava replacing that from sweet cassava.There were no interactions between carbohydrate source and the relative proportion of leaf meal from bitter and sweet cassava for any of the above criteria.

It is hypothesized that dietary elements (eg: cyanogenic glucosides), or anti-microbial additives (such as tannins, thymol…), which reduce the fermentability of an otherwise highly fermentable substrate, will produce changes in the end products of the rumen fermentation characterized by a lower proportion of the metabolizable energy in the form of methane and more in the rumen VFA, especially the proportion in the form of propionate. This in turn should lead to improvements in ruminant productivity in terms of growth and milk production.

Key words: bypass nutrients, cyanogenic glucosides, fermentation, glucose precursors, tannin, thymol

Introduction

In recent rumen in vitro studies (Maselema and Chigwa 2017; Preston et al 2019)) it was found that the methane content of the gas was higher when the substrates were grasses (Pennisetum spp) or a vegetable (Ipomoe aquatica) compared with leaves from forage trees (eg: Leucaena leucocephala) and shrubs (eg: Manihot esculenta). It was also reported that feeds rich in soluble protein (eg: groundnut meal) produced more methane than feeds in which the protein was of low solubility (eg: fish meal) (Preston et al 2013). Equally it has been shown that incubations in which the protein was derived from leaves from bitter cassava varieties produced less methane than when the leaves were from sweet cassava varieties (Phuong et al 2012; Phanthavong et al 2018).

The objective of the present experiment was to provide further evidence that feeds with high rumen fermentability also produce more methane. For this purpose, contrasting sources of carbohydrate (starch versus sugar) and protein (leaves from sweet versus bitter cassava) were compared in an in vitro rumen incubation).

Materials and methods

Treatments and design

The in vitro incubation was designed as a 2*3 factorial with three replications:

Carbohydrate source

Sucrose: cane sugar

Starch: fresh root of cassava

Cassava leaf meal as source of bypass protein (proportions on DM basis)

100SW: 100% from sweet cassava variety (Gon)

50SW:50B: 50% from sweet cassava; 50% from bitter cassava (KM94)

25SWL75B: 25% from sweet cassava; 75% from bitter cassava

In vitro incubation

The in vitro system was the same as that described by Inthapanya et al (2011).

Diagram 1. A schematic view of the rumen in vitro incubation system (Inthapanya et al 2011)

Composition of substrates

The carbohydrate component, which provided 72% of the substrate, was supplemented with urea as a source of rumen ammonia and fresh cassava leaves to provide bypass protein and fiber (Table 1).

Roots of cassava were ground by dedicated mill prior to incubation.. The sugar was pure sucrose. Fresh cassava leaf (Gon and KM94 varieties) were chopped into small pieces and incubated immediately after collection from the cassava plants. A vitamin mixture (A, D and E) and a mineral mixture (40% Ca₂HPO₄, 52.5% NaCl and 7.5% sulphur) were included in each incubation. Rumen fluid was from a newly slaughtered cow at the local abattoir. The substrates (12 g DM) were incubated at 38⁰C with 0.24 L rumen fluid and 0.96 L of buffer solution (adapted from Tilly and Terry 1964). The total incubation time was 24h.

Gas production was measured by water displacement. The methane percentage in the gas was measured with an infra-red meter (Crowcon Instruments Ltd, UK).

The dry matter and crude protein contents of the substrates were determined according to AOAC (1990) methods. HCN was determined by titration with AgNO ₃ after boiling the sample in KOH to concentrate the HCN.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab (2014) software. In the model the sources of variation were: source of carbohydrate, source of cassava leaf, carbohydrate * cassava leaf interaction and error.

Results

Gas production over 24h, the methane content of the gas and the methane produced per unit substrate solubilized were increased when the substrate carbohydrate was sugar as compared with cassava root (Table 2; Figures 1-4). Increasing the proportion of leaf from bitter cassava replacing that from sweet cassava, reduced the rate of gas production, the methane content of the gas and the production of methane per unit substrate DM solubilized.

The proportion of substrate DM solubilized was increased by 10% when the carbohydrate source was sugar compared with starch (cassava root) and by 13% when the source of protein was 100% sweet cassava leaf compared with 25% from sweet and 75% from bitter leaf.

There were no interactions between carbohydrate source and the relative proportion of leaf meal from bitter and sweet cassava for any of the measurements (Figures 1-4).

Figure 1. Effect of substrate (cassava root or sugar) and source of cassava leaf (sweet or bitter) on gas production in 24h	Figure 2. Effect of substrate (cassava root or sugar) and source of cassava leaf (sweet or bitter) on %methane in the gas after 24h
Figure 3. Effect of substrate (cassava root or sugar) and source of cassava leaf (sweet or bitter) on DM solubilized in 24h	Figure 4. Effect of substrate (cassava root or sugar) and source of cassava leaf (sweet or bitter) on methane produced per unit substrate DM solubilized in 24h

Discussion

The effect of the carbohydrate source (sugar versus starch) and source of protein (leaf from sweet versus bitter cassava) on methane production can be generalized by plotting the methane content of the gas versus: (i) the total gas production in 24h (Figure 5) ; or (ii) the degree of solubilization of the substrate DM in 24h (Figure 6). There were positive relationships between the proportion of the gas in the form of methane and the fermentability/digestibility of the substrate (R² =0.30); and the degree of solubilization (digestibility) of the substrate (R ² =0.80).

Figure 5. Relationship between methane content of the gas and the gas production over 24h	Figure 6. Relationship between methane content of the gas and the proportion of the substrate solubilized in 24h

A positive relationship between fermentability/digestibility of the substrate and the proportion of the gas in the form of methane can also be seen in the data of: Masalema and Chigwa (2017), who studied in vitro methane production from grasses and tree legumes; Preston et al (2019) in an in vitro study of grasses versus leaves from trees and shrubs; Hamdani et al (2019) in studies in vitro and in vivo with dairy cows in which rate of rumen fermentation was depressed as was the methane content of eructed gas by a thymol-based anti-microbial additive; and Inthapanya et al (2020) in an in vitro study in which methane content of the gas was reduced by dietary treatments which also reduced the relative degree of rumen fermentability of the substrate.

It is hypothesized that:

(i) dietary elements (eg: cyanogenic glucosides), or anti-microbial additives, which reduce the fermentability of an otherwise highly fermentable substrate, will produce changes in the end products of the rumen fermentation characterized by a lower proportion of the metabolizable energy in the form of methane and more in the rumen VFA, especially the proportion in the form of propionate. The corollary to a reduced rumen fermentability of an otherwise highly fermentable substrate is that more of the substrate will escape the rumen fermentation for more efficient enzymic digestion in the intestine of diet components (starch and true protein) that are digested by mammalian enzymes, while fibrous elements of the escaping substrate will be captured by acetogenic rather than methanogenic fermentation in the cecum-colon (Demeyer et al 1991; Immig 1996; Popova et al 2013; Leng 2018).

(ii) The result of these changes will be an improved balance of nutrients at sites of metabolism which will be reflected in improved rates and efficiency of growth and milk production in the intact ruminant. This hypothesis has yet to be proved in feeding trials

Conclusions

• Gas production over 24h, the methane content of the gas and the methane produced per unit substrate solubilized were increased when the substrate carbohydrate was sugar as compared with cassava root.
  
  
• Increasing the proportion of leaf from bitter cassava replacing that from sweet cassava, reduced the rate of gas production, the methane content of the gas and the production of methane per unit substrate DM solubilized.
  
  
• It is hypothesized that dietary elements (eg: cyanogenic glucosides), or anti-microbial additives (such as tannins, thymol…), which reduce the fermentability of an otherwise highly fermentable substrate, will produce changes in the end products of the rumen fermentation characterized by a lower proportion of the metabolizable energy in the form of methane and more in the rumen VFA, especially the proportion in the form of propionate.
  
  
• This in turn should improve ruminant productivity in terms of growth and milk production.

References

AOAC 1990 Official methods of analysis.15th ed. AOAC, Washington, D.C (935-955)

Binh P L T, Preston T R, Duong K N and Leng R A 2017 A low concentration (4% in diet dry matter) of brewers’ grains improves the growth rate and reduces thiocyanate excretion of cattle fed cassava pulp-urea and “bitter” cassava foliage. Livestock Research for Rural Development. Volume 29, Article #104. http://www.lrrd.org/lrrd29/5/phuo29104.html

Demeyer D 1991 Differences in stoichiometry between rumen and hindgut fermentation. Adv. 1023 Animal Physiology and Animal Nutrition 22:50-66

Hamdani H, Chami N, Oukhouia M, Jabeur I, Sennouni C and Remmal A 2019 Effect of a thymol-based additive on rumen fermentation, on methane emissions in eructed gas and on milk production in Holstein cows. Livestock Research for Rural Development. Volume 31, Article #107. http://www.lrrd.org/lrrd31/7/houdh31107.html

Immig I 1996 The rumen and hindgut as source of ruminant methanogenesis. Environmental Monitoring and Assessment Volume 42, Issue 1-2, pp 57-72

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

Inthapanya S, Preston T R, Le Duc Ngoan and Le Dinh Phung 2020 Effect of yeast-fermented rice and rice distillers’ byproduct on methane production in an in vitro rumen incubation of ensiled cassava root, supplemented with urea and leaf meal from sweet or bitter varieties of cassava; Livestock Research for Rural Development. Volume 32, Article #52. http://www.lrrd.org/lrrd32/3/intha32052.html

Leng R A 2018 Unravelling methanogenesis in ruminants, horses and kangaroos: the links between gut anatomy, microbial biofilms and host immunity. Animal Production Science, 58, 1175-1191 https://doi.org/10.1071/AN15710

Maselema D and Chigwa F C 2017 The potential of Richardia scabra and fodder tree leaf meals in reducing enteric methane from dairy cows during dry season. Livestock Research for Rural Development. Volume 29, Article #51. http://www.lrrd.org/lrrd29/3/mase29051.html

Minitab 2014 Statistical Software. Minitab Inc. Company. State College (Pennsylvania). http://www.minitab.com

Phanthavong V, Sangkhom I, Preston T R, Dung D V and Ba N X 2018 Effect of leaves from sweet or bitter cassava and brewers’ grains on methane production in an in vitro rumen incubation of cassava root pulp-urea. Livestock Research for Rural Development. Volume 30, Article #167. http://www.lrrd.org/lrrd30/9/phant30167.html

Phuong L T B, Preston T R and Leng R A 2012 Effect of foliage from “sweet” and “bitter” cassava varieties on methane production in in vitro incubation with molasses supplemented with potassium nitrate or urea. Livestock Research for Rural Development. Volume 24, Article #189. http://www.lrrd.org/lrrd24/10/phuo24189.htm

Popova M, Morgavi D P and Martin C 2013 Methanogens and methanogenesis in the rumen and cecum of lambs fed two https://www.researchgate.net/publication/233930442

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

Preston T R, Silivong P and Leng R A 2019 Methane production in rumen in vitro incubations of ensiled cassava (Manihot esculenta Cranz) root supplemented with urea and protein-rich leaves from grasses, legumes and shrubs. Livestock Research for Rural Development. Volume 31, Article #112. http://www.lrrd.org/lrrd31/7/silv31112.html

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

Received 13 February 2020; Accepted 28 March 2020; Published 1 May 2020

Go to top