Methane production in vitro rumen incubation of ensiled cassava root and urea is reduced by supplementation with low levels of Brewers’ spent grains and by supplementation with cassava foliage rather than water spinach

Vor Sina and T R Preston¹

Center for livestock and Agriculture Development (CelAgrid). P O Box 2423, Phnom Penh Capital, Cambodia
sina_vor@yahoo.com
¹ Centro para la Investigación en Sistemas Sostenibles de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia

Abstract

The objective of this in vitro rumen incubation was to determine the optimum level of Brewers’ grains as a source of a prebiotic in diets for ruminants, based on ensiled cassava root, urea and cassava leaves or water spinach as sources of protein. The experiment was arranged as a 4*2 factorial in a completely randomized design (CRD) with 4 replications of each treatment. The factors were: level of brewers’ grains; BG: 0, 2, 4 and 6%; source of protein: cassava leaf CL or water spinach WS. Ensiled cassava root, urea, and sulphur-rich minerals were added to all the substrates.

At all stages of the 24h in vitro rumen incubation, the gas production and the methane content of the gas were reduced with linear trends as the level of brewers’ grains was increased and were higher when the protein source was water spinach rather than cassava leaves. The methane content of the gas increased with a linear trend as the gas production increased. There was a similar relationship between substrate digested and methane content in the gas. It is suggested that these findings support the hypothesis that depressing the rate of fermentation (ie: rate of gas production) would facilitate the escape from the rumen of valuable sources of nutrients (eg: starch and protein) for more efficient enzymic digestion in the small intestine while escaping ferrous particles would be digested (fermented) to acetic acid (not methane) in the cecum-colon.

Key words: incubation bottles, method to measure methane, polynomial equations, tropic

Introduction

In a previous experiment it was shown that the addition of 4% of Brewers’ spent grain to a diet of ensiled cassava root pulp, urea and cassava foliage, (bitter variety) was effective in reducing enteric methane production and HCN toxicity and improving growth rates in cattle (Binh et al 2015)). In a feeding trial with goats the growth rate was increased 100% from 60 to 120 g/day when the diet of fresh cassava foliage (sweet variety) was supplemented with 4% of Brewers’ spent grain (Vor Sina et al (2017).

The aim of the present experiment was to test different levels of Brewers’ spent grains as a source of prebiotic in an in vitro rumen incubation of c ensiled cassava root , urea with leaves from cassava or water spinach as sources of protein. The treatments were four levels of Brewers’ spent grain (0, 2, 4 and 6% on DM basis) in a 4*2 factorial arrangement with the leaves of cassava or water spinach as sources of protein.

Materials and methods

Location

The experiment was carried out in Nong Lam University, Ho Chi Minh city.

Treatments and experimental design

The experiment was arranged as a 4*2 factorial in a completely randomized design (CRD) with 4 replications of each treatment. The factors were:

Level of brewers’ grains BG:

• 0, 2, 4 and 6%

Source of protein:

• Cassava leaf CL

• Water spinach WS

Ensiled cassava root, urea, and sulphur-rich minerals were added to all the substrates (Table 1) to provide a total amount of 12 g DM.

In vitro system

The in vitro incubation procedure (Diagram 1) was the same as that developed by Sangkhom et al (2011).

Diagram 1. A schematic view of the rumen in vitro incubation system

Experimental procedure

Feed ingredients

The cassava root and leaves of cassava (sweet variety) and water spinach were collected from the farmer area near the university. The leaves were chopped into small pieces of 1-2 cm, and then dried at 80ºC for 24hours before grinding. The cassava root was chopped into small pieces, ground and then ensiled for 7 days in closed plastic bags.

Amounts of the substrates (Table 1), equivalent to 12g DM, were put in the incubation bottles, followed by 0.96 liters of buffer solution (Table 2) and 240 ml of rumen fluid obtained from a cow immediately after being slaughtered in the local ababttoir . The bottles were then filled with carbon dioxide and incubated at 38 0C in a water bath for 24hours.

Data collection and measurements

During the incubation the gas volume was recorded at 6h intervals (0-6, 6-12, 12-18 and 18-24hours). After each interval, the gas volume was recorded and the methane concentration was measured with a Crowcon infra-red analyzer (Crowcon Instruments Ltd, UK). At the end of the incubation, the remaining substrate was filtered through cloth and the solid residue dried at 100^oC to determine the DM mineralized during the incubation.

Chemical analyses

Samples were analyzed for DM, ash and crude protein according to AOAC (1990) methods.

Statistical analysis

The data were analyzed by the General Linear Model (GLM) option in the ANOVA program of the Minitab software (version 16.0). In the model the sources of variation were level of Brewers’ grans, source of protein, interaction brewes’ grains*protein source and error. Trends in in responses to treatments were determined by fitting polynomial equations to the data using MICROSOFT Excel software.

Results and discussion

Gas production and methane content

Gas production decreased with a linear trend as the level of Brewers’ grains increased (Table 3; Figure 1) 1)and was higher when the protein source was water spinach rather than cassava leaf (Figure 2) The methane content of the gas increased with a linear trend as the gas production increased (Figure 3).There was a similar relationship between substrate digested and methane content in the gas (Figure 4).

Figure 1. Effect of brewers’ grains and protein source on gas production	Figure 2. Effect of brewers’ grains and protein source on methane content of the gas.

Figure 3. Relationship between gas production and percent of methane in the gas	Figure 4. Relationship between mineralization of the substrate and percent of methane in the gas

Similar effects of water spinach compared with cassava leaves of increased rumen gas production and higher methane content of the gas with water spinach were reported by Silivong et al (20015). Researchers in Laos also reported a positive relationship between the rate of gas production and the content of methane in the gas in an in vitro rumen incubation (Sangkhom et al 2020).

It is suggested that the above findings support the hypothesis proposed by Sangkhom et al (2020) that depressing the rate of fermentation (ie: rate of gas production) would facilitate the escape from the rumen of valuable sources of nutrients (eg: starch and protein) for more efficient enzymic digestion in the small intestine while escaping fibrous particles would be (fermented) to acetic acid (not methane) in the cecum-colon as suggested by Phonethiep et al (2019).

The concept that depressing rumen gas (and methane) production can lead to improved ruminant was shown by Hamdani et al (2019) when they used an additive containing the anti-bacterial compound: “thymol” that reduced rumen gas and methane production in vitro. Feeding the thymol-based additine to Holstein cattle increased milk production and decreased enteric methane production.

Conclusions

• At all stages of the 24h in vitro rumen incubation, the gas production and the methane content of the gas were reduced with linear trends as the level of brewers’ grains was increased and were higher when the protein source was water spinach rather than cassava leaves.
  
  
• The methane content of the gas increased with a linear trend as the gas production increased. There was a similar relationship between substrate digested and methane content in the gas.
  
  
• It is suggested that these findings support the hypothesis that depressing the rate of fermentation (ie: rate of gas production) would facilitate the escape from the rumen of valuable sources of nutrients (eg: starch and protein) for more efficient enzymic digestion in the small intestine while escaping fibrous particles would be fermented to acetic acid (not methane) in the cecum-colon.

Acknowledgements

The support from the MEKARN II project, financed by Sida, is gratefully acknowledged, as is the help from the Animal Science Department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR.

References

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