Livestock Research for Rural Development 31 (7) 2019 Guide for preparation of papers LRRD Newsletter

Citation of this paper

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

T R Preston1, P Silivong and R A Leng2

Souphanouvong University, Luang Prabang, Lao PDR
1 Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
reg.preston@gmail.com
2 University of New England, Armidale, NSW, Australia

Abstract

In an in vitro rumen incubation of ensiled cassava root-urea supplemented with protein and fiber from leaves of grasses, legume trees, prostrate legumes or a shrub (sweet and bitter varieties of cassava), there were close positive relationships between: (i) the percentage of methane in the gas and the rate of gas production; and ii) between methane production and the solubility of the leaf protein.

It is proposed that these relationships substantiate the hypothesis that “the presence of cyanogenic glucosides, in the cassava component of the diet, alters rumen fermentation, enhancing rumen escape of substrate (both glucogenic materials and potentially readily fermentable substrate) for digestion in the intestines followed by further fermentation in the cecum , thereby reducing the overall production of methane with concomitant improved effiency of feed utilization by the host animal. )reduces the overall rate of rumen fermentation, thus enhancing rumen escape of substrate for post-rumen digestion in the intestine and cecum, thereby reducing the overall production of methane with concomitant improved feed utilization effficiency of the host animal”.

Key words: bitter, bypass nutrients, cecum, fermentation, glucogenic, metabolism, nutrient balance, ruminants, solubility, sweet


Introduction

The hypothesis that rumen methane production is directly related to the solubility of the dietary protein was advanced by Phonethep et al (2016) to explain why supplementing foliage of Tithonia diversifolia with fresh water spinach (Ipomea aquatica) (protein solubility 67%) increased rumen methane production while the opposite effect was observed when the supplement was cassava leaves (protein solubility 21%). The proposed explanation was that for feeds in which the protein is of low solubility (ie: are rich in “bypass” or “escape” protein) the balance of feed fermented in the intestine and cecum/large intestine, relative to the rumen is increased, and that production of methane is reduced because disposal of hydrogen in fermentative degradation in the cecum-colon is dominated by acetogenesis rather than methanogenesis (Demeyer 1991; Immig 1996; Popova et al 2013; Leng 2018).

The objectives of the following experiment were to provide further evidence for the relationship between the solubility of dietary protein and methane production in ruminant feeding systems.


Materials and methods

The experiment was carried out from July to September 2018 at the Animal Science Laboratory of the Faculty of Agriculture and Forest Resources, Souphanouvong University, Luang Prabang Province, Lao PDR. Leaves from a range of grasses, prostrate and tree legumes, shrubs and a vegetable (Table 1) were compared as protein supplements in a rumen in vitro incubation of ensiled cassava root and urea simulating the feeding systems described by Phanthavong et al (2016) and Sangkhom et al (2017). Separate incubations were made for each protein source over time periods of 12, 18 and 24h, each of which was replicated three times. The in vitro system (Inthapanya et al 2011; Phanthavong et al 2018) used recycled plastic bottles as flasks for the incubation and gas collection (Photo 1). The leaves were chopped into small pieces (3-5mm) and dried at 65°C for 48h then ground with a coffee grinder prior to being mixed with the ground fresh cassava root and urea. The proportions of the leaves, of urea and of cassava root meal, in the substrates were adjusted to provide a crude protein content in the substrate DM of 14-15% (Table 1).

Photo 1. The in vitro system using recycled plastic water bottlesDiagram 1. Arrangement of the plastic bottles for the incubation
and collection of the gas by water displacement


Table 1. Ingredients in the substrates, content of crude protein in the mixtures and DM mineralized after 24h

Ingredients in the substrate, % as DM

% in substrate

Source of leaves

Leaves

Cassava
root

Urea

Crude
protein

DM
mineralized

P. pupureum

79

20

1

14.5

59.0

P. maximum

70

28.5

1.5

14.5

55.2

B. humidicula

73

26

1

14.5

57.5

S. guiensis

64

35.5

0.5

14.6

62.7

C. ternatea

58

41.5

0.5

14.8

64.8

A. pinzoi

60

39.5

0.5

14.6

64.0

S. grandiflora

58

41.5

0.5

14.8

61.0

G. sepium

57.5

42

0.5

14.7

52.7

L. leucocephala

61.5

38

0.5

14.8

61.0

M. esculenta S

57.5

42

0.5

14.7

59.1

M. esculenta B

60

39.5

0.5

14.8

58.7

I. aquatica

67.5

32

0.5

14.9

67.5

Amounts of the mixtures (Table 1) equivalent to 12g DM were put in the incubation bottles with 960ml of buffer solution (Table 2) and 240ml of rumen fluid. The rumen fluid was taken at 3.00-4.00am from a buffalo immediately after the animal was killed in the Luang Prabang abattoir. A representative sample of the rumen contents (including feed residues) was put in a vacuum flask and taken to the laboratory, and stored until 5.00am, when the contents were filtered through a layer of cloth before being added to the incubation bottles. The remaining air in the fermentation bottle was flushed out with carbon dioxide. The bottles were incubated at 38°C in a water bath for intervals of 12, 18 and 24h, with separate incubations in triplicate for each interval.

Table 2. Ingredients of the buffer solution (g/liter)

CaCl2

NaHPO4.12H2O

NaCl

Cl

MgSO4.7H2O

NaHCO3

Cysteine

0.04

9.30

0.47

0.57

0.12

9.80

0.25

Source: Tilly and Terry (1963)

At the end of each incubation, measurements were made of total gas production (by water displacement), content of methane (by passing samples of gas through an infra-red detection meter [Crowcom Ltd., UK] and DM solubilized (by filtering the residual substrate through cloth and drying the residue residue (65°C) for 72 h. Solubility of the protein in the leaves was determined by shaking 3 g of dry leaf meal in 100 ml of M NaCl for 3h and filtering through Whatman No. 4 filter paper (Whitelaw et al 1963) prior to determining the N content of the filtrate by kjeldahl digestion (AOAC 2010).

Data were analyzed using the General Linear option of the ANOVA program in the Minitab (2014) software. Sources of variation were: source of leaves and residual error. Linear regressions were derived using the Excel software in the Microsoft Office program.


Results and discussion

The solubility of the protein in the leaves varied widely (Table 3) . The highest value was recorded for the water spinach vegetable (68.7%) followed by leaves from prostrate legumes (45-57%), then the tree legumes (35.1-46.3%), and the grasses (33.4 to 41.5%) with lowest values for the leaves of cassava (31.0 to 32.0%). The trends for gas production and the methane content in the gas were similar to those for protein solubility with the highest values for water spinach and the lowest values for the leaves of cassava.

Table 3. Mean values for production of gas, content of methane (CH4) in the gas, and DM solubilized (sol), in rumen in vitro incubations of substrates based on ensiled cassava root-urea with
protein from leaves taken from a range of grasses, prostrate and tree legumes and a shrub (cassava) (in all cases the total weight of substrate DM was 12 g; fermentation times were 12, 18 and 24h)

Vegetable

Grasses

Prostrate legumes

Tree legumes

Shrub

p

SEM

IA

PP

PM

BH

AP

CT

SG

SGR

GS

LL

ME-SW

ME-BI

0-12h

Gas, ml

663

553

447

593

770

817

610

510

487

343

350

313

<0.001

11.0

CH4, %

13.0

17.3

14.0

11.7

15.7

19.7

21.0

16.0

13.7

12.7

15.3

13.7

<0.001

0.36

DM sol., %

54.5

52.4

49.3

51.9

55.7

56.7

54.3

52.9

51.3

47.1

46.2

49.1

<0.001

0.45

CH4, ml/g DM sol.

13.2

15.3

10.6

11.1

18.1

23.6

19.7

12.9

10.8

7.7

9.7

7.3

<0.001

0.59

0-18h

Gas, ml

967

633

567

483

857

917

763

777

667

563

493

473

<0.001

23.0

CH4, %

23.0

22.7

25.3

20.3

21.0

22.0

20.3

18.7

17.7

16.0

17.0

15.7

<0.001

0.35

DM sol., %

59.1

54.7

51.7

53.1

59.5

60.9

57.3

54.3

52.5

50.1

49.8

51.2

<0.001

0.42

CH4,.ml/g DM sol.

31.4

21.9

23.1

15.5

25.3

27.6

22.6

22.2

18.7

15.0

14.0

12.1

<0.001

1.01

0-24h

Gas, ml

1,367

833

733

683

1,057

1,117

963

940

827

727

663

627

<0.001

27.9

CH4, %

29.3

27.3

30.7

23.0

26.0

26.7

25.3

23.7

21.7

20.7

22.0

20.7

<0.001

0.33

DM sol., %

67.5

59.0

55.2

57.5

64.0

64.8

62.7

61.0

59.1

58.7

53.2

55.2

<0.001

0.50

CH4,.ml/g DM sol.

49.6

32.2

34.0

22.8

35.8

38.3

32.4

30.4

25.2

21.3

22.9

19.6

<0.001

1.29

#Protein solubility, %

68.7

41.5

36.3

33.4

52.1

55.0

47.0

46.3

40.5

35.1

32.7

31.0

IA Ipomoea aquatica; PP Pennisetum purpureum; PM Panicum maximum; BH Brachiaria humidicola; AP Arachis pintoi; CT Clitoria ternatea; SG Stylosanthes guiense; SGR Sesbania
grandiflora
; GS Gliricidia sepium; LL Leucaena leucocephala; ME SW Manihot esculenta (Sweet var.); ME BI Manihot esculenta (Bitter var.)   # Of the leaves


Figure 1. Volumes of gas produced in 24h in vitro rumen incubations of substrates based on cassava root pulp-urea with protein from leaves
from a range of grasses, prostrate and tree legumes and a shrub (sweet and bitter varieties of cassava) (see Table 3 for details)


Figure 2. Methane content of the gas produced in 24h in rumen in vitro incubations of substrates based on ensiled cassava root-urea with protein from leaves
from a vegetable, and a range of grasses, prostrate and tree legumes and a shrub (sweet and bitter varieties of cassava) (see Table 3 for details)


Figure 3. Methane produced per unit substrate DM solubilized in 24h in vitro rumen incubations of substrates based on cassava root pulp-urea with protein from leaves
from a vegetable, and a range of grasses, prostrate and tree legumes and a shrub (sweet and bitter varieties of cassava) (see Table 3 for details)
Relationships between methane content in the gas, the rate of gas production and the solubility of the leaf protein

There were positive linear relationships between: (i) the percentage of methane in the gas (and per unit substrate mineralized) and the rate of gas production (Figures 4 and 5); and (ii) between methane production and the solubility of the leaf protein (Figures 6 and 7).

Figure 4. In a 24h rumen incubation of ensiled cassava root-urea with
protein from a range of leaves the methane content of the
gas was positively related with the gas production
Figure 5. In a 24h rumen incubation of ensiled cassava root-urea with protein
from a range of leaves the methane produced per unit DM mineralized
was positively related with the gas production


Figure 6. In a 24h rumen incubation of ensiled cassava root-urea with protein
from a range of leaves the methane content of the gas was
positively related with the solubility of the leaf protein
Figure 7. In a 24h rumen incubation of ensiled cassava root-urea with protein
from a range of leaves the methane produced per unit DM mineralized
was positively related with the solubility of the leaf protein

These relationships are taken as strong supporting evidence for the hypothesis projected in this experiment, that dietary manipulations that result in reduced rates of rumen fermentation (of a diet of high fermentative potential) are associated with reduced partitioning of hydrogen to methane, and therefore an improved balance of nutrients arriving at sites of metabolism. The outcome of this dietary manipulation will then be an improvement in host animal productivity as demonstrated by Phonethep et al (2016) and Houda et al (2019). In the experiment of Phonethep et al 2016) goats fed a basal diet of foliage of Tithonian diversifolia produced less methane and retained more nitrogen (a proxy for better growth rate) when the Tithonia was supplemented with cassava foliage (protein solubility 22%) than when the supplement was water spinach (protein solubility 67%). In the experiment of Houda et al (2019), rumen fermentation was depressed by feeding an additive containing thymol (an active bactericide), gas production in vitro was depressed, and percent of methane in eructed rumen gas of dairy cows was reduced with concomitant increase in milk production.


Conclusions


Acknowledgements

The authors acknowledge support for this research from the MEKARN II project financed by Sida. Special thanks to Mr Aloun who provided valuable help in the laboratory. I also thank the staff of Department of Animal Science laboratory, Faculty of Agriculture and Forest Resource, Souphanouvong University for providing the facilities to carry out this research.


References

AOAC 2010 Official methods of analysis.15th ed. AOAC, Washington, D.C (935-955)

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

Houda Hamdani, Najat Chami, Mounia Oukhouia, Imane Jabeur, Chaimae Sennouni and Adnane Remmal 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

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

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

Phanthavong V, Khamla S and Preston T R 2016 Fattening cattle in Lao PDR with cassava pulp. Livestock Research for Rural Development. Volume 28, Article #10. http://www.lrrd.org/lrrd28/1/phan28010.html

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

Phonethep P, Preston T R and Leng R A 2016 Effect on feed intake, digestibility, N retention and methane emissions in goats of supplementing foliages of cassava (Manihot esculenta Crantz) and Tithonia diversifolia with water spinach (Ipomoea aquatica). Livestock Research for Rural Development. Volume 28, Article #72. http://www.lrrd.org/lrrd28/5/phon28072.html

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

Sangkhom I, Preston T R, Leng R A, Ngoan L D and Phung L D 2017 Rice distillers’ byproduct improved growth performance and reduced enteric methane from “Yellow” cattle fed a fattening diet based on cassava root and foliage (Manihot esculenta Cranz). Livestock Research for Rural Development. Volume 29, Article #131. http://www.lrrd.org/lrrd29/7/sang29131.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

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 1 January 2019; Accepted 25 June 2019; Published 2 July 2019

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