Effect of a supplement of fermented rice on methane production in an in vitro rumen incubation of ensiled cassava root, urea and cassava leaf meal

Sangkhom Inthapanya, T R Preston¹, Le Duc Ngoan² and Le Dinh Phung²

Animal Science Department, Faculty of Agriculture and Forest Resource Souphanouvong University Lao PDR
inthapanyasangkhom@gmail.com
¹ Centro para la Investigación en Sistemas Sostenibles, de Producción Agropecuaria (CIPAV), Carrera 25 No 6-62 Cali, Colombia
² Faculty of Animal Sciences, Hue University of Agriculture and Forestry, Hue University, Vietnam

Abstract

The aim of the research was to determine the effects on methane production of pre-steaming two varieties of rice – normal rice and “sticky” rice - before fermentation to produce a supplement that would reduce rumen methane production. The experiment was arranged as a 2*2 factorial in a completely randomized design with 4 replications of the treatments which were: sticky rice versus normal rice; and with or without steaming prior to fermentation with yeast in an anaerobic fermentation for 7 days. The products of fermentation were tested as additives (4% DM basis) in an in vitro rumen incubation of ensiled cassava root, urea, minerals and bitter cassava leaf meal.

Gas production and methane produced per unit DM solubilized were increased when the rice was steamed but were not affected by the source of the rice. These results suggest that there are no benefits from using sticky rice as opposed to normal rice, or by pre-steaming, when the objective is to produce a fermented rice product which will reduce methane production in a diet of ensied cassava root, urea and cassava leaf meal.

Key words: incubation, methane production, prebiotic, steaming, sticky rice

Introduction

In previous reports we showed that the byproduct from the fermentation of sticky rice, as in the farmer system of making rice wine, led to improvements in growth rate and feed conversion as well as reducing rumen methane production, when it was added at a low level (4%) to a cattle fattening diet based on ensiled cassava root, urea and cassava foliage (Sangkhom et al 2017; Sengsouly and Preston 2017). In an initial attempt to simulate the rice distillers’ byproduct, without the concomitant production of ethanol (Inthapanya et al 2019), it was found that; (i) methane production was increased when urea and di-ammonium phosphate were added during the initial pre-fermentation stage; and (ii) there were no benefits from boiling the fermented rice or distilling off the ethanol prior to using it as an additive to reduce methane production.

The objective of the present experiment was to determine if the use of sticky rice, instead of normal rice, and steaming it prior to the fermentation (as is standard practice in making rice wine) was necessary when the objective was to reduce methane production in an in vitro rumen incubation of a cattle fattening diet of ensiled cassava root, urea and cassava leaf meal.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Animal Science department, Faculty of Agriculture and Forest Resource, Souphanouvong University, Lao PDR, through September - October 2019.

Treatments and experimental design

The treatments in an in vitro rumen incubation of ensiled cassava root, bitter cassava leaf meal and urea, were two additives made from fermented rice: (i) according to the source of rice (Normal or “sticky” rice); and (ii) steaming or not prior to the fermentation. The design was a 2*2 factorial in a completely randomized arrangement with 4 replications of each treatment. The factors were:

Source of rice:

· SR: sticky rice

· NR: normal rice

Steaming:

· ST: steaming for 30 minutes

· NS: no steaming

In vitro system

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

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

Experimental procedure

The cassava root and cassava leaves (bitter) were collected from the Souphanouvong University farm. The leaves were chopped into small pieces of 1-2 cm, and then dried at 80ºC for 24h before grinding. After chopping into small pieces, the root was ground and then ensiled in closed plastic bags for 7 days.

TThe sticky rice and normal rice (1 kg of each) were wet-milled in a liquidizer, then soaked in 1.5 liters of water for 5h, finally being put in a porous bamboo basket which was suspended in a barrel containing 1.5 liters of water. A wood fire under the barrel converted the water into steam which was absorbed by the rice through the pores in the bamboo basket. After steaming the rice was cooled for 30 minutes then mixed with yeast (Saccharomyces cerevisiae) at 3% DM basis. The mixture was then put in closed plastic bags and allowed to ferment for 7 days before evaluation in the rumen in vitro incubation following the procedure developed by Sangkhom et al (2011).

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

Data collection and measurements

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

Chemical analyses

Samples were analyzed for pH, DM, ash, crude protein and true 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 treatments, treatment interaction and random error. The statistical model used was:

Y_ijk = µ +a_i +b_j + (a*b)_ij + e_ijk

Where: Y_ijk is dependent variable; µ is overall mean; a _i is the effect of rice source; b_j is the effect of steaming; (a*b)_ij is the interaction between source of rice and steaming and e _ijk is random error.

Results

Chemical composition of diet ingredients

The pH, DM, crude protein and true protein values were not differed among the sticky rice and normal rice but were higher for Ash in the sticky rice (Table 3).

Gas production

During all incubation intervals after 6h, and over the full 24h period, the gas production was increased when the rice was steamed, but there were no differences between normal rice and sticky rice (Table 4; Figures 1 and 2).

Figure 1. Total gas production after fermentation	Figure 2. Total gas production after fermentation

Methane production

The percentage of methane in the gas was increased when the rice was steamed prior to the incubation and when normal rice rather than sticky rice was the source of the supplement (Table 4, Figures 3 and 4).

Figure 3. Effect of steaming the rice on the methane content in the gas during the incubation	Figure 4. Effect of the source of the rice on the methane content in the gas during the incubation

Substrate digestibility

The DM solubilized during the incubation was increased when the rice was steamed prior to use in the in vitro incubation (Table 4; Figure 5). The DM solubilized was not affected by the source of the rice (Figure 6).

Figure 5. Effect of steaming on DM digestibility	Figure 6. Effect of source of rice on DM digestibility

Methane production per unit substrate solubilized

Methane produced per unit DM solubilized was increased when the rice was steamed (Table 4; Figure 7) but was not affected by the source of the rice (Figure 8).

Figure 7. Effect of steaming sticky rice or normal rice on methane production per unit substrate solubilized.	Figure 8. Effect of sticky rice and normal rice, steamed or not steamed, on methane production, per unit substrate solubilized

Discussion

The farmer system to make rice wine is based on the use of “sticky” rice which is steamed prior to fermentation with yeast, after which the ethanol is recovered by distillation. The residue from this process (rice distillers’ byproduct) has been shown to reduce rumen methane production both in vitro and in vivo when added at a low concentration (4% as DM) to the diet (Sengsouly and Preston 2017; Sangkhom et al 2019). In an attempt to emulate this process, it was found that boiling or distilling the fermented rice conferred no additional benefit in terms of the capacity of the residue to reduce rumen methane (Inthapanya et al 2019). The experiment described in this paper has shown that there is also no advantage in use of “sticky” as opposed to “normal” rice nor of steaming prior to the fermentation.

It remains to be confirmed that the simple process of fermenting “normal” rice without steaming will support the same positive effects in vitro and in vivo as has been demonstrated with the traditional “rice distillers’ byproduct.

Conclusions

• With the aim of simplifying the process to make a simulated” rice distillers’ byproduct”, two kinds of rice (“sticky” and “normal” rice) were compared with and without steaming prior to fermentation.
• There was no advantage to use of sticky as opposed to normal rice as choice of substrate while the effect of steaming was to reduce the capacity of the fermented product to decrease rumen methane in an vitro fermentation of ensiled cassava root, urea and cassava leaf meal.

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

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

Inthapanya S, Preston T R, Ngoan L D and Phung L D 2019 Effect of a simulated 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 cassava. Livestock Research for Rural Development. Volume 31, Article #164. Retrieved November 24, 2019, from http://www.lrrd.org/lrrd31/10/sang31164.html

Minitab 2010 Minitab Software Release 16.0

Sangkhom Inthapanya, 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

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

Sengsouly P and Preston T R 2016 Effect of rice-wine distillers’ byproduct, biochar and sweet or bitter cassava leaves on gas production in an in vitro incubation using ensiled cassava root as substrate. Livestock Research for Rural Development. Volume 28, Article #190. http://www.lrrd.org/lrrd28/10/seng28190.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 24 November 2019; Accepted 7 December 2019; Published 2 January 2020

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