Effect of various chemical treated-rice straws on rumen fermentation characteristic using in vitro gas production technique

Introduction 

Feed resources for ruminant production in the many tropical regions are becoming increasingly important because of rising costs and limited feed resources. This is especially critical during the time of feed shortage in the dry season. Rice straw is an agricultural by-product which farmers typically store for use as a ruminant feed in many tropical countries. However, rice straw is low in nutritive value with high level of lignifications, low level of nitrogen and mineral contents (Wanapat et al 1985). Numerous methods of rice straw treatments have been extensively researched and developed in order to improve its’ utilization by ruminants practiced (Wanapat et al 1985, 1986).  Improvement of rice straw by treatment with urea (5%) has been successfully and widely used in many counties (Preston and Leng 1984, Preston 1995, Hart and Wanapat 1992, Wanapat et al 1997, Wanapat 1999). However, in recent years, the price of urea is rapidly increasing and affecting on practical use by farmers. Trach et al (2001) suggested that when amount of urea was reduced and then combined with calcium hydroxide [Ca(OH)₂], it improved rumen fiber degradability. However, the alternative alkali source for treatment of RS needs further investigation before recommending for use by farmers. Therefore, the objective of the present study was to determine the impact of chemical treatments of RS on rumen fermentation characteristic using in vitro gas fermentation technique.

Materials and methods 

Rice straw treatments and chemical composition analysis

Rice straw was chopped to 3-5 cm and treated with 3% urea (3UTS), 5% urea (5UTS), 2% urea + 2% lime (2ULTS), 3.5% urea + 3.5% lime (3.5ULTS), 2% sodium hydroxide (NaOH) (2STS), and 3% lime (3LTS). The chemical in respective treatments were dissolved in 100 ml/g straw DM and ensiled at room temperature for 14 days before the samples were collected. Untreated rice straw was used as a control and all sample treatments were performed in duplicates.

The straws were ground to pass a 1 mm sieve screen for subsequent chemical analyses. Determination of Kjeldahl-N content was performed according AOAC (1990), while neutral-detergent fiber (NDF) and acid-detergent fiber (ADF) were determined by the method of Van Soest et al (1991). Both ADF and NDF were expressed exclusively of residual ash. Lignin was determined by solubilization of cellulose with sulphuric acid on the ADF residue (Robertson and Van Soest 1981).

In vitro gas production

In vitro gas production (GP) were measured in triplicates at 2, 4, 6, 8, 12, 24, 36, 48, 72, 84 and 96 h post incubation using cumulative gas technique (Menke and Steingass 1988). Rumen fluids were collected from two donor swamp buffaloes which had been fed twice daily on rice straw (ad libitum) and concentrate (0.5% BW) before morning feeding. Rumen fluid was strained through four layers of gauze into a pre-warmed and insulated bottle at 39°C. Inoculation were done in triplicates with 10 ml rumen fluid injected into 60 ml bottle containing 30 ml of buffered medium and 0.2 g dry substrate at 39 °C. In each incubation run, three blanks were run to be used to correct the gas production values for gas release from endogenous substrates. Other bottles for each treatment were determined for dynamic fermentation variables. To describe the dynamics of GP over time, the following equation (Ørskov and McDonald 1979) was used: GP = a + b(1-e^−ct), where GP = cumulative GP (ml), (a + b) = potential GP (ml/g), c = rate of GP (ml/h), and a, b and c are constants. If the ‘a’ value was negative, the lag time before rapid degradation began was estimated as (1/c)ln[b/(a + b)] (McDonald 1981).

Collection of samples and chemical analysis

The three bottles incubated for each treatment were withdrawn from the incubator at 6 and 24 h of incubation, respectively. The fermentation was stopped and freezen at -20 °C. About 30 ml of mixed fermentation medium was used for analysis of NH₃-N and volatile fatty acids (VFA).

Fermentation variables such as NH₃-N and VFA were determined, respectively by the micro Kjeldahl methods (AOAC 1990) and Samuel et al (1997) using HPLC (instruments: controller water model 600 E; water model 484 UV detector; Novapak C₁₈ column; column size 4mm x 150mm; mobile phase 10 mmol/L H₂PO₄ (pH 2.5)).

Statistical analysis

The effects of chemical treatments on rumen GP and fermentation parameters were analyzed by the General linear model (GLM) procedure of SAS (1998). The differences of means for respective treatments were tested by using Duncan’s New Multiple Range Test.

Results and discussion 

Chemical composition of rice straw

Chemical composition of feeds used for an in vitro trial is presented in Table 1.

The CP was increased due to increased urea level, from 2 to 5% and urea plus lime, while lime alone and sodium hydroxide did not show any changes. Particularly, CP was increased from 5.8 to 11.2 % in the straw treated with 2 to 5% urea and urea plus lime. However, according to Wanapat et al (1985) who reported that 3 to 5% urea treatments increased CP from 11.9 to 17.7% in the wet samples of straw. Treatments also resulted in reduced NDF and ADF concentrations of rice straw especially the hemicellulose content. Sodium hydroxide reduced the hemicelluloses content of straw more than other treatments. The combination of chemicals showed an improvement of nutritive value of rice straw especially in lowering level of fibrous fractions (NDF, ADF and ADL). Similar and positive results have also been reported by Zaman and Owen (1985) in in vitro digestibility of rice straw treated with lime plus urea. Moreover, the chemical treatment of rice straw could improve quality and prevented from mold growth as reported by Zaman and Owen (1995). The formation of ammonia from urea has also been reported to be reduced in the present of lime, although mould growth was prevented (Ørskov et al 1979).

Gas production and fermentation characteristics

Figure 1 shows the cumulative gas production for each substrate treatment. All gas volumes were increased as fermentation time interval proceeded from 0 to 96 hours after incubation.

The cumulative GP at all incubation times was higher from treated RS than in the untreated one. Potential gas production was highest in 5% urea-treatment and followed by 3.5ULTS treatment (Table 2).  

Before 24 hours of incubation, gas were highest produced in 2STS, however, after 24 hours of incubation it was reduced as compared with 5UTS and 3.5ULTS. Sodium hydroxide treatment showed the highest in the rate of gas production (0.07ml/h). Under this study, 2ULTS and 3UTS treatments resulted in lower gas production as compared with other treatments. As reported previously by Wanapat (1989), Trach et al (2001), Liu et al (2002) and Wanapat et al (2009) that chemical treatments increased digestibility of low-quality roughages and sodium hydroxide treatment had stronger effect on cell wall dissolve than urea and calcium hydroxide. As a consequence, sodium hydroxide resulted in higher gas rate. However after 24 hour of incubation, treatment with sodium hydroxide was reduced when compared with 5URS and 3.5ULTS treated-rice straw. This could be explained by the amount of supplemental nitrogen which is generally required for rumen microbes. Higher nitrogen content from 5UTS treatment and 3.5ULTS treatment contributed for an increased NH₃-N concentration in the culture media. Under this study, it is in agreement with Wanapat (1989) and Trach et al (2001) who reported that 5UTS and 3.5ULTS treatments could increase in rumen microbe population and nutrient digestibility in in vivo trials.

Ruminal NH₃-N of treated rice straw was increased as a result of urea level. They were higher in 5URS (29.0 mg/dl), 3.5ULTS (20.6 mg/dl), 3URS (19.8 mg/dl) and 2ULTS (18.3 mg/dl) treatments as compared with others. Sodium hydroxide and lime alone did not increase ammonia-nitrogen of rice straw. In an in vitro study, Satter and Styler (1974) suggested that NH₃-N in excess of 5 mg/dl would not be expected to influence on microbial N yield. Therefore, under this study urea or urea plus lime treatments could support nitrogen for rumen microbial biomass. Total VFA and individual VFAs (acetate, propionate and butyrate) concentrations were responded with the chemical treated straw. Particularly, total VFA and acetate concentrations were significantly increased for 5UTS and 3UTS as compared with untreated rice straw (P<0.05) while propionate concentrations were increased by 5UTS, 3UTS and 2STS.  Butyrate concentration was highest for 2STS (P<0.05). An increase in total VFA of all treated RS was obtained due to the increased fermentation rate in in vitro as reflected by the increased in gas production.

In vitro dry matter digestibility (IVDMD) and microbial biomasses of the treated RS were higher than those in untreated RS and were consistent with the increase in gas production and VFA concentration. IVDMD and microbial mass was highest by 5UTS, and followed by other treatments (3UTS, 3.5ULTS, 2ULTS, and 2STS) as compared with the untreated rice straw. When N is concerned, the level of urea as low as 2% would be therefore required for the treatment of rice straw. However, to ensure treatment effectiveness, cheaper non-nitrogenous alkali sources should be additionally combined for use.

Conclusion and recommendations  

Based on this experiment, it could be concluded that treatment of rice straw with combination of urea and lime resulted in high gas production and increased in total VFA, acetate and propionate concentrations. Combination of 2% of lime with urea (2ULTS) for straw treatments should be considered for further use to improve rumen fermentation, nutrient digestibility and with low-cost as compared with 5% urea-treatment.

Acknowledgements 

The first author would like to express his most sincere gratitude and appreciation to the Commission on Higher Education, Thailand under the Strategic Scholarships for Frontier Research Network Program and the Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand for their financial support of research and the use of research facilities.

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Received 10 October 2010; Accepted 10 November 2010; Published 5 January 2011

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