Effects of treatment of rice straw with lime and/or urea on its chemical composition, in-vitro gas production and in-sacco degradation characteristics

Nguyen Xuan Trach, Magne Mo* and Cu Xuan Dan

Faculty of Animal Science and Veterinary Medicine, Hanoi Agricultural University
* Department of Animal Science, Agricultural University of Norway

Abstract

Chopped rice straw was treated according to a 3 x 3 factorial arrangement, using quick lime (0, 3 and 6%, w/w) and urea (0, 2 and 4%, w/w). The treated straws (TS) were stored at a 50% moisture level for 3 weeks. The treatments were repeated three times, 2 months apart, within an ambient temperature range from 20 to 35^oC. Effects of these treatments were evaluated based on straw chemical composition, in-vitro gas production, and in-sacco degradation.

It was found that treatment with urea increased nitrogen (N) content, and solubilised NDF and hemicellulose. Lime treatment did not affect N content, but appeared to be more powerful in delignification, reducing not only NDF and hemicellulose , but also ADF and ADL. In general, all the treatments increased in-vitro gas production and in-sacco degradability of rice straw. The effects were increased with increasing levels of lime and/or urea in spite of some negative interactions between the two chemicals. However, a level of 2% urea alone seemed to be too low for effective treatment and a level of 6% lime seemed to be too high for rumen cellulolysis.

The present studies indicate that 3% lime alone, 4% urea alone, and a combination of 3% lime with urea (2 or 4%) are promising for rice straw treatment.

Key words: Rice straw, urea, lime, composition, gas production, rumen degradability

Introduction

Rice straw is potentially an important energy feed for ruminants as it is rich in carbohydrates. However, its potential as an energy source is limited because of the complex lignified cell wall structure, which shields the energy yielding nutrients from microbial degradation in the rumen (Jung et al 1993). The utilization of rice straw can be increased, provided some measures are taken to modify the structure of its cell walls (Chenost and Kayouli 1997; Chaudhry 1998a).

Treatment of straw with sodium hydroxide (NaOH) and ammonia (NH₃) to improve its digestibility and intake has been extensively examined and well documented (Jackson 1977; Sundstøl and Owen 1984). However, both chemicals have potential hazards for animals, humans and the environment in addition to economic and technological limitations (Owen et al 1984). Consequently, their application has been limited, especially in developing countries. Treatment of rice straw with urea as a source of NH₃under warm climates has been given more attention and has proved to be effective (Doyle et al 1986; Schiere and Ibrahim 1989; Dolberg 1992; Chenost and Kayouli 1997). However, the technology has not been widely taken up by farmers (Devendra 1997). Moreover, a standard level of 4-6% urea for biologically effective treatment should be too much as a source of non-protein nitrogen (NPN) in the straw for efficient utilization by the ruminant (Preston 1995). Therefore, it is warranted to find treatment alternatives, which will be not only technically effective, but also cost-effective and convenient for farmers.

Lime (CaO/Ca(OH)₂) has been thought of as a potential alkali for straw treatment since it is cheap and readily available (Owen et al 1984; Sundstøl 1985; Doyle et al 1986). Calcium (Ca) residues, which remain in the treated straw, cause no serious problems to the animal or to the environment (Chaudhry 1998a) and can be a supplement to rice straw, which is deficient in Ca when used to feed cattle (Nath et al 1969). Research has already been carried out to examine effects of treatments with lime on the nutritive quality of cereal straw (Doyle 1982; Owen et al 1984; Garmo 1986; Sirohi and Rai 1997; Zaman and Owen 1990, 1995; Pradhan et al 1997; Chaudhry 1998b,c 2000). However, results have been equivocal and contradicting.

Combinations of lime and urea may be attractive for straw treatment (Owen et al 1984; Sundstøl 1985). Such a mixture would be able to combine treatment effects of both chemicals (Hadjipanayiotou 1984; Sirohi and Rai 1994, 1995, 1996, 1999), together with the supplemental effects of the added Ca and nitrogen in the TS. In addition, mould inhibition is an important effect of ammonia released from urea from this mixture in moist straw (Zaman and Owen 1990, 1995; Zaman et al 1994; Pradhan et al 1997). In such a combination neither the level of urea, and thus residual NH₃, nor Ca would be necessarily as high as if the chemicals were used alone. In addition, since the amount of urea can be reduced at the expense of a cheaper chemical (lime), the mixture would be more economical, as long as the overall treatment effect is maintained or enhanced. Moreover, the quality of animal excreta as fertiliser would be increased due to the residual N and Ca.

Nevertheless, there may be associative effects when the two chemicals are put together for straw treatment. According to Van Soest (1994) calcium hydroxide may enhance the treatment effect of urea because urea normally hydrolyses into ammonium carbonate, a weaker base than ammonia that yields ammonium hydroxide in water as the treatment chemical, when urea is used with lime calcium hydroxide will remove carbonate from ammonium carbonates and generate hydroxide. Calcium hydroxide has been shown to become more effective when combined with ammonia or urea for straw treatment (Males 1987). Nevertheless, one may also reasonably suspect that high alkalinity induced by lime can inhibit the activity of urease. Research works have indicated, on the one hand, the presence of urease activity and, on the other hand, its partial inhibition under lime plus urea treatment conditions (Zaman and Owen 1995; Pradhan et al 1997).

Apparently, the use of lime or lime plus urea for straw treatment needs further research before farm-scale application is possible. A series of studies were therefore undertaken to verify the possibility to improve the feeding value of rice straw by one or more treatment combinations using lime (0, 3, and 6%) and urea (0, 2, and 4%). Results of work on chemical analysis, in-vitro gas production and in-sacco degradation are reported in this paper.

Materials and methods

Straw treatment procedure

To prepare samples for chemical composition, in-vitro gas production and in-sacco degradability determination, rice straw (90% DM) was first chopped into about 4-5 cm in length by a small cutter. The straw was then taken in 500g lots to be treated with 3% or 6% (w/w) of quick lime (87% CaO) and/or 2% or 4% (w/w) of urea (46% N). The chemical(s) were dissolved and sprayed in a required amount of water to ensure 50% moisture content for the treated straw. After being thoroughly mixed manually, the treated material was placed in double-layered polyethylene bags and sealed after being carefully pressed to remove as much air as possible. The bags were then stored in the laboratory for 3 weeks. Three batches of such treatments were made 2 months apart in a range of ambient temperature of 20 to 35^oC. To feed fistulated cattle for rumen ecosystem studies, long form straw in 10kg lots was subjected to the same treatments, but one after another. The straws were stored in sealed double-layered plastic sacks for 3 weeks in a shed in a range of 18.2 to 28.5^oC.

Chemical analysis

At the end of the treatment period, samples of treated straw from the three batches were taken together with untreated straw samples for separate analyses of dry matter (DM), nitrogen (N), total ash, neutral detergent fibre (NDF), acid detergent fibre (ADF), and acid detergent lignin (ADL). For the determination of nitrogen content, wet samples of all types of straw were taken right after opening the bags to be acidified without drying. Samples of untreated straw were also analysed for N without drying. DM, N and total ash were determined following Official Methods of AOAC (Cunniff 1997). Cell wall components (NDF, ADF and ADL) were determined according to Van Soest and Robertson (1985). Hemicellulose and cellulose were calculated as the differences between NDF and ADF and between ADF and ADL, respectively. For the purpose of comparison, all these components were calculated as percentages of the organic matter (OM) to avoid being confounded with the added ash in lime treated straw..

In-vitro gas production determination

The gas production method proposed by Menke and Steingass (1988) was used. Air-dried untreated and treated straw were ground to pass a 1.00mm sieve. The weight of an in-vitro sample was calculated based on 200mg straw OM (to exclude differences in the ash content). These samples in three replicates were put into 100ml calibrated glass syringes (Haberle Labortechnik, D-89173 Lonseeettlenschieu, Oberer Seesteig 7, Germany), fitted with plungers. A mixture containing 475 ml distilled water, 240ml buffer solution, 240ml macro-mineral solution, 0.12ml micro-mineral solution, and 1.22ml reazurin solution was prepared in a 2 litre flask. After warming up to 40^oC, a reducing solution consisting of 47.5ml distilled water, 2.0ml 1N NaOH, and 0.336mg NaS9H₂O was added. Thereafter, the flask with the buffer solution was placed in a small water bath kept at 38.5^oC and gently bubbled with CO₂ until the blue colour turned to pink and then clear. Rumen liquor was taken from two fistulated Yellow oxen fed on a diet consisting of 50% medium hay and 50% direct-cut grass at a maintenance level. The rumen liquor was strained through gauze tissue cloth and mixed into the buffer solution at a ratio of 1:2. An amount of 30 ml of the rumen liquor/buffer medium was then taken to put into each syringe with a dispenser. The syringes were placed vertically in an incubator kept at 38.5^oC. Three blank syringes containing 30 ml of the medium only were included at the beginning, in the middle and at the end of the process. All the syringes were gently shaken 30 min after the start of the incubation and four times daily at later times. Gas production was read at 6, 9, 12, 24, 48, 72, and 96 hours of incubation.

To describe rate and extent of straw fermentation, the readings of gas volume from 9h to 96h of incubation were fitted to the exponential equation P = a + b(1-e^-ct) (Ørskov and McDonald 1979), where P represents gas volume at time t, a the intercept, a+b the potential gas production, and c the rate constant of gas production during incubation. Earlier gas production was not fitted in the equation to exclude the deviation from an exponential course of the cell wall fermentation (Blümmel and Becker 1997). Due to the effect of the lag phase (see below), the equation parameter a (with negative values) was not used in place of the initial gas production, but the accumulative gas volume up to 6h of incubation, denoted as A, was used instead (Blümmel and Ørskov 1993) to reflect the fermentation of the soluble and readily fermentable fraction. Value B = (a+b) – A was called later gas production to describe the fermentation of the insoluble but fermentable fraction. The Neway Excel program (Chen 1997) was used for the computation.

In-sacco studies

The nylon bag technique was used to determine:

(1) degradation characteristics of untreated straw and the 8 types of treated straw incubated together in the rumens of 3 fistulated Yellow oxen fed on a fixed diet consisting of 50% medium hay and 50% green grass given at a maintenance level to evaluate treatment effects on degradability of rice straw as a substrate,

(2) degradation characteristics of pangola hay as a fixed substrate incubated in the rumens of 3 fistulated cattle fed ad libitum in turn on the 9 types of straw to see how the rumen cellulolysis efficiency was affected by the treatments of the straw fed, and

(3) degradation kinetics of the 9 types of straw incubated in the rumens of 3 fistulated Yellow oxen fed ad libitum in turn on the same respective straws to look at the combined effects of the treatments both on straw as a substrate and the rumen ecosystem shaped by the straw fed.

For the 9 diets used for (2) and (3), which were taken together, rice straw was the only OM source, made iso-nitrogenous with urea and supplemented with a mineral-vitamin mixture prior to feeding. The animals were fed the same type of straw in each period, undergoing a preliminary period of 15 days to stabilize the rumen ecosystem, followed by 10 days for the in-sacco incubation of samples. Since there were 9 types of straw under comparison, but only 3 fistulated cattle were available, a Latin square design - which should have been better - was impossible, therefore the present design had to be accepted with a risk of period effects. To minimize possible confounding effects, the periods were randomised with a sufficient transition time. Ambient temperature was considered to be the most important period-related factor, which might have affected both treatment effectiveness and the animals during experimentation, and thus recorded to be treated as a covariate if it was significant.

The nylon bag technique as described by Ørskov et al (1980) was applied for degradability determination. Air-dried substrate samples were ground to pass a 2.5 mm sieve. In-sacco samples of 3 g each were then taken into nylon bags in duplicates. The pore size of the nylon bags was 37 micron and the inner size of the bag was 6 cm x 12 cm. The bags were incubated starting 1h after the cattle were offered the morning meal. The incubation times were 8, 16, 24, 48, 72, 96 and 120 hours. After incubation, the bags with residues were taken out of the rumen, dipped immediately into cold water to stop microbial activity, then rinsed by cold tap water to remove the rumen matter from the outside of the bags. Thereafter, the bags with contents were rinsed with cold water for 30 minutes in a washing machine. Finally, they were dried at 60^oC for 48 hours. To determine the contents of water-soluble fraction, two sample bags of each straw type were soaked in a water bath for 24 hours and then underwent the same washing and drying procedures as the incubated bags. Duplicate bags of each sample were similarly dried for determination of the DM content of the samples for calculation of DM disappearance.

The DM degradation data were fitted to the exponential equation P = a + b(1-e^-ct) originally proposed for protein feed evaluation (Ørskov and McDonald 1979), where P is the percent degradation at time t, a the immediate soluble fraction (the intercept), b the insoluble but rumen degradable fraction, and c the rate constant at which the insoluble rumen degradable fraction is degraded. However, for roughages there is often a lag phase when microbes become attached to the fibrous material during which time there is no net disappearance of substrate. Consequently, the value of a in the above equation could be negative and does not represent the immediate soluble fraction. Therefore, in the present studies degradation kinetics of straw and hay was described by: (i) the washing loss (A) determined in the laboratory as described above, (ii) the insoluble (not washable) but degradable fraction denoted as B, which is now B = (a+b) – A, (iii) the rate constant c is the same, (iv) potential degradability: A+B = a+b, and (v) the lag phase (L) = 1/c log_e[b/(a+b-A)] (Ørskov and Ryle 1990; Ørskov and Shand 1997). The Neway Excel program (Chen 1997) was used for the computation.

Statistical analysis

Data were analysed using the GLM (General Linear Model) procedure of SAS (1996). The basic model for analysis of variance (ANOVA) was a 3x3 factorial one with treatment inputs (0, 3, and 6% lime x 0, 2, and 4% urea) and their interaction as fixed effects. Except for gas production data, treatment batch (for chemical composition data) or animal (for in-sacco data) was additionally included as a random block to make it a blocking factorial model. For cases (2) and (3) of the in-sacco studies, where 9 periods of feeding were applied, at first the average period ambient temperature was also included in the model as a covariate, but since no significant influence was detected it was eventually excluded from the model. When the F-test showed a significant effect after ANOVA, pair-wise comparisons between all 9 treatment combination means were performed, using the LSMEANS statement with the PDIFF option for testing the hypothesis H_o: LSM(i) = LSM(j). The Ryan-Einot-Gabriel-Welsch (REGWQ) multiple range test was used to compare factor level means, using the MEANS statement. Planned comparisons were also applied to contrast linear combinations of factor level means.

Results

Chemical composition of straw

Table 1 shows the chemical composition of rice straw as affected by treatment with lime and/or urea. The N content was increased (P<0.001) due to urea addition, while lime did not show any effect on it. On average, the N content was increased by 9.7 or 20.3 g/kg OM in straw treated with 2% or 4% urea, respectively. Urea was effective in solubilising NDF (P<0.01) and hemicellulose (P<0.05), but did not significantly affect the other cell wall components. Lime seemed to be more powerful, not only reducing NDF (P<0.001) and hemicellulose (P<0.001), but also ADF (P<0.001) and ADL (P<0.001). The more each chemical was applied the lower the cell wall components became. The lime x urea interactions were non-significant for chemical composition. However, when the two chemicals were used in combination the treatment effects were not fully additive.

In-vitro gas production

Gas production from straw during in-vitro incubation was highly increased by treatment with lime and/or urea (Table 2). The higher the levels of lime and/or urea, the greater the values of initial gas production (A), 48 h gas production (48hGP), potential gas production (a+b) and rate constant (c). The effect was most apparent for c and 48hGP. Very little gas was produced within the first 6 h, but most produced before 48 h. The difference between untreated straw and treated straw was more apparent for 48hGP than for potential gas production. There was a tendency to reduce the additive effects of the two chemicals when they were used together, but no statistically significant interactions between them were found.

In-sacco degradation of straws incubated in cattle fed on a fixed diet

The kinetic parameters of DM degradation of the 9 types of straw determined with a fixed independent diet fed to the fistulated cattle are presented in Table 3. All the treatments brought about significant increases in all the values of water solubility (A), insoluble but degradable fraction (B), the potentially degradable proportion (A+B), and the rate constant (c), compared to untreated straw. Comparing among treatments, the effects of the different treatments on these dynamic parameters can also be ranked in the same order as based on 48h dry matter degradability (48hDMD) or 48h gas production (48hGP). The lag phase (L) was effectively reduced by urea (P<0.05) and especially by lime (P<0.001). The dose responses were almost linear with higher responses to increasing levels of lime and/or urea. The interaction between lime and urea was found significant for 48hDMD (P<0.01) and A+B (P<0.05). That is, when the two chemicals were put together the improvements in these parameters were smaller than the sums of effects when they were used separately. It was also noteworthy that there were no significant differences in the effects between 3% lime and 4% urea when the two chemicals were used alone.

In-sacco degradation of hay incubated in cattle fed on the different types of rice straw

Results of the in-sacco degradation of Pangola grass hay as a standard substrate incubated in the rumens of cattle fed the different straws are presented in Table 4. Treatments with lime and/or urea significantly increased the rate constant (c), and 48hDMD, reduced the lag phase (L), but did not affect water-insoluble fraction degradability (B) and potential degradability (A+B). Treatment with 4% urea gave significantly higher values of c and 48hDMD compared with 2% urea, but the figures were significantly lower for 6% compared with 3% lime. Although both lime and urea significantly shortened the lag phase, the differences between the two levels of each chemical were not significant. Combination of 3% lime with 2 or 4% urea, or 4% urea alone, gave the best responses in terms of c and 48hDMD.

In-sacco degradation of straws incubated in cattle fed on the same type of straw

The kinetic parameters of straw DM degradation of the 9 types of straw incubated in turn in fistulated cattle fed on the same straw are given in Table 5. As can be seen, the degradation kinetics of the straws had almost the same tendency as when the straws were incubated in a fixed independent rumen environment (Table 3). All the observed parameters were significantly changed for the better by lime and/or urea treatment. The improvement in each parameter was increased with increasing dosage of lime and/or urea applied. The interaction between lime and urea was significant for all observations, except for the lag phase (L). However, the extents to which the degradation rate was increased by the treatments were generally lower than those found when the cattle were fed on the independent fixed diet.

Nitrogen content of urea-treated straw

It has been noted that around one third of the urea-N applied for straw treatment is left after storage and aeration (Sundstøl and Coxworth 1984; Doyle et al 1986; Djajanegara and Doyle 1989; Chenost and Kayouli 1997). However, if the treated straw is not aerated after treatment the loss of added N is much lower. Wanapat (1985) has reported 11.9 and 17.7% CP in wet samples of rice straw treated with 3% and 5% urea, respectively, compared to 4.2% CP in untreated straw, i.e. only around 10% of the added N was lost. Pradhan et al (1996) have calculated that 79% of the added N was retained in urea treated straw. The present study has demonstrated even higher levels of the added N left (82-85%) in the wet urea treated straw after opening the bags.

However, it would be difficult to know how much of the added N remains in urea treated straw as the loss of nitrogen occurs during treatment, at opening the silo, during exposure to the air after that, and during processing of the sample for analysis (Jayasuryia and Pearce 1983). The time of sampling and the method of sample processing may be the most affecting factors. It may be because ammonia after being released from urea is dissolved in the water in a labile form of ammonium. When the treated straw is exposed to the air, the ammonia and the water evaporate and the more they evaporate the greater is the amount of ammonia converted from ammonium. Therefore, increased time of exposure and/or ambient temperature will reduce the N content in urea treated straw. This loss could be reduced by the trapping of excess ammonia in water in the urea treated wet straw.

Since the N content of urea treated straw is reduced over time when the straw is exposed to air after opening the treatment bag/silo, the feeding practice would affect the amount of non-protein nitrogen (NPN) consumed by the animal. To save labour and time for straw aeration, urea treated straw should be fed wet to cattle. This is possible because cattle are able to tolerate high levels of ammonia and the smell of ammonia seems to attract them (Leng 1986). However, if 4% urea treated wet straw is fed to the animal, the level of NPN in the diet would be well above the requirement of the rumen microbes (Preston 1995). The animal has to excrete the extra NPN in the form of urea in the urine. This is a waste of N and also energy, which is needed for the synthesis of urea from ammonia. Otherwise, if the straw is exposed to air for a certain period of time after treatment before feeding, it should be another way of wasting N, to say nothing of time and labour required for the aeration.

To illustrate, in 4% urea treated straw, which has 30.6 g N/kgOM and an organic matter digestibility (OMD) of, say, 57%, there should be 30.6*100/57 = 53.7 g N/kg DOM, which is too high compared with the N requirement of rumen microbes. According to Durand (1989), the total level of N required is only 26g N/kgdigestible organic matter (DOM). Preston and Leng (1984) have suggested 30 g fermentable N/kgDOM to optimise the activity of rumen microbes. Thus, as far as N is concerned, a level of urea as low as 2% would be more reasonable. However, to ensure treatment effectiveness another cheaper non-nitrogenous alkali should be combined.

Lime as an alkali for straw treatment

Lime has been shown to be effective in solubilising the cell walls of rice straw and thus increasing its degradability. Similar effects of lime have also been reported by Sirohi and Rai (1994, 1995, 1997, 1998). Moreover, the present studies have proved that lime can induce stronger effects on chemical composition, in-sacco degradability and in-vitro gas production as compared with urea. These results may be explained by virtue of the fact that lime is a stronger alkali than ammonia, the treatment agent released from urea. When applied with water CaO quickly changes into Ca(OH)₂. The latter is a much weaker base and thus requires a longer reaction time compared with NaOH for straw treatment (Chaudhry 1998a). Yet, Ca(OH)₂ is a stronger base than ammonium hydroxide (NH₄OH), which is formed from NH₃ in water (pK_b = 2.43 vs. 4.75 for NH₄OH) (Weast 1975). Therefore, excluding effects of supplemental N, urea should induce a lower alkalinity in the treated straw compared with lime.

Since lime has a very low solubility (0.165% at 20^oC) (Weast 1975), one may reflect that not much lime should be needed to attain the highest possible alkalinity in the treated straw. Nevertheless, in the present studies as well as in others (Owen et al 1984; Sirohi and Rai 1995, 1997), increasing the level of lime application (2-6%) increased the effects. At first thought this may be an effect of higher hydration heat produced from a higher level of quick lime dissolved in water. However, it has been demonstrated that above 20^oC, treatment effects of lime were almost unaffected by temperature (Zaman and Owen 1990; Zaman et al 1994, Chaudhry 1998b). Pradhan et al (1997) have shown that the increased IVDMD of straw with increasing level of Ca(OH)₂from 4% to 6% was not because of additional hydration heat. Thus, another possibility, which may be more important, is that lime is weakly soluble and non-volatile, so that a large amount of lime is needed to get it properly distributed and act as a reserve which gradually dissolves and maintains the concentration of hydroxide (OH^-) in the water. As OH^- is taken up by the straw, more Ca(OH)₂comes from the reserve into solution (Zaman et al 1994).

Although 6% lime was more effective than 3% lime in cell wall solubilisation (Table 1), improving gas production (Table 2) and degradability (Table 3) of straw as a substrate, a level of 3% lime seemed to be better for the rumen ecosystem (Table 5). This may be due in part to the greater effect of 6% lime on the lignin molecule to release phenolics, which would have been effective in improving degradability of the delignified cell walls, but, at the same time, toxic to microbes in the rumen ecosystem when the treated straw was actually fed to the animal (Akin et al 1988; Chaudhry 2000).

Combination of lime and urea for straw treatment

High levels of deligninification (as reflected by reduced NDF, ADF and ADL), increased rumen degradability and increased gas production of rice straw were obtained in the present studies due to treatments with lime and urea in combination. The combinations showed, to varying extents, additive effects of the two treatment inputs in improving the nutritive value of rice straw. Similar positive results have also been reported by Zaman and Owen (1995), Sirohi and Rai (1994, 1995, 1996, 1998), Pradhan(1997) and Sahoo et al (2000), based on their studies on chemical analysis, in-vitro gas production, in-sacco degradation and in-vitro digestibility of straw treated with lime plus urea.

However, negative interactions between lime and urea which reduced their additive effects have also been found to be significant in the present in-sacco studies. Hadjipanayiotou (1984) and Zaman and Owen (1995) have also reported negative interaction between lime and urea used for straw treatment. This may be due to the fact that the two chemicals in combination are not actually independent in affecting pH and acting on the same substrate which has only a certain potential for further improvement. Inhibition of urease activity by high alkalinity induced by lime may be another possibility. The formation of ammonia from urea has been reported to be reduced in the presence of lime, although mould growth was prevented (Zaman and Owen 1990, 1995; Pradhan et al 1997).

Although full additive effects were not obtained, the very high increases in cell wall delignification, in-vitro gas production and in-sacco degradability after rice straw was treated with lime combined with urea in the present studies, would confirm the importance of the additive and supplementary effects of urea in such a combination. It seems that urease can tolerate relatively high alkalinity. Clearly, urea extensively hydrolyses in urea-treated (stored) materials, where pH may reach 8.5 to 9.6 (Dias-da-Silva et al 1988). It is also of interest that Pradhan et al (1997) have found the formation of NH₃ in rice straw treated with 4% Ca(OH)₂ plus 2% (pH = 8.97) or 4% urea (pH = 9.11) or even treated with 2% NaOH plus 2% (pH = 9.18) or 4% urea (pH = 9.17), although more NH₃ was produced in 4% urea alone treated straw (pH = 8.98). That is, in straw treated with lime plus urea, urease activity is partially, not totally, inhibited.

A combination of lime and urea could thus be applied under different ambient conditions. For example, when the ambient temperature increases, the solubility of lime will decrease as a fact (Weast 1975), but it should be compensated for by enhanced ureolysis (Sundstøl and Coxworth 1984) producing more ammonia to maintain high pH and inhibit mould growth (Zaman et al 1994; Zaman and Owen 1995), that also means, vice versa, that at low temperatures ureolysis is slowed down when mould is not usually encountered (Zaman and Owen 1990) but the solubility of lime will increase to enhance alkalinity. These complementary properties of the two chemicals would ensure high alkalinity and absence of mould for the treated material under different or changing ambient temperatures without high application levels of each. Moreover, uneven distribution of lime in the treated straw due to its low solubility may be compensated for by more ureolysis where little or no lime is present. To say the worst, if urease activity were totally hindered by lime inducing high alkalinity, which means the primary aim of alkali treatment of straw has been achieved, urea would still have a role to play as a source of supplemental NPN, and lime would then be the alkalinity enhancer.

As discussed above, too high a level of lime (6%) may create unfavourable rumen conditions for microbial activity and limit straw palatability, and two high a level of urea may be uneconomical. Among the presently tried treatments, a combination of 3% lime with 2% urea seems to be most reasonable for effective treatment. Assuming that 3% lime plus 2% urea treated straw, which had 20.5 g N/kgOM, has an OMD of 59%, it would have 20.5*100/59 = 35 g N/kg DOM. This level of N is still somewhat higher than 26g N/kgDOM (Durand, 1989) or 30 g N/kgDOM (Preston and Leng 1984), which is needed for rumen microbes. However, 2% urea would be a safe level to account for N lost in the form of NH₃ during the time when the treated straw is left in the trough before consumed by the animal (Jayasuryia and Perera 1982) and to prevent mould in straw stored with lime (Zaman et al 1994).

Relationships between results of the different measurements

In general, the present observations have appeared to agree with one another. The more the straw cell wall fibres were solubilised, the more the straw DM was degraded in-sacco, and the more gas was produced in-vitro. It has been commonly known that due to alkali treatment straw cell wall fibres are modified, the bonds between lignin and structural carbohydrates are partially cleaved, making it easier for the rumen cellulolytic microbes to colonize and degrade the ingested straw, reducing the lag phase and increasing its degradation/fermentation rate. This is also due to the fact that more readily available fibres are liberated, and consequently rumen microbes can multiply faster and thus degrade straw more quickly (Cheng and Hungate 1976; Silva and Ørskov 1988).

Since the rumen is the primary site for degradation of fibrous feed in ruminants, it is important to monitor degradation kinetics of straw in response to alkali treatment. Therefore, the in-sacco technique should be a powerful tool for the purpose. However, the rumen conditions used for the determination may be a factor to consider. In the present studies, improvements in the straw degradation rates, as found when the standard diet was used (Table 3), were higher than those found when the same types of straw were fed to the fistulated animals (Table 5). The fixed diet containing hay and green grass should have created a more favourable environment for cellulolysis than when straw was fed as the only energy source in the diet. Silva and Ørskov (1988) have clearly demonstrated that degradability of straw was markedly increased when it was incubated in a rumen environment with a more abundant supply of readily digestible cellulose/hemicellulose.

Among other possibilities, the negative effects of phenolic acids released from straw cell walls during treatment on rumen microbes (Chesson et al 1982; Varel and Jung 1986; Akin et al 1988) would offset, at least in part, the positive effect on straw degradability when the straw is actually fed. Consequently, there is a risk of over-evaluating degradability of rice straw when a good independent diet is used as a constant diet to feed the fistulated animals. Using the same type of straw to feed the fistulated animal to test in-sacco degradability of treated straw would give more practical results because, at the end of the day, the treated straw is to feed the ruminant. When the straw is actually fed, especially ad libitum, it would shape the rumen environment, which in turn determines the efficiency of straw degradation (Table 4). However, the use of a fixed standard diet should be more convenient, less expensive, less time consuming and well justified for comparative studies. Since the potential degradability (A+B) of straw was found to be significantly affected by treatment, but relatively independent of the feed given to the fistulated animals diet (Table 4), it may be used as a stable indicator to evaluate effects of treatment on roughage quality.

It can be seen from the present studies that the increased degradability of straw due to treatment was well reflected in greater gas volumes and the two methods ranked the treatments in the same order. However, the rate of gas production (c) was much greater than that of in-sacco degradation. Shen et al (1998) and Tolera et al (1998) have also shown a similar relationship between the two methods. That may be because the in-vitro gas produced consists of not only carbon dioxide and methane arising directly from substrate fermentation, but also carbon dioxide released from the buffer solution. Another explanation for the discrepancy is the differences in the microbial biomass. Blümmel and Ørskov (1993) have indicated that the microbial biomass was reduced due to microbial lysis and part of the lysed microbial debris was further fermented to VFA and gas. In spite of this, in-vitro gas production should be a good compensatory method to in-sacco degradation for comparative evaluation of straw treatment effects.

Acknowledgments

The authors would like to thank the Norwegian Council of Universities' Committee for Development Research and Education (NUFU) for the financial support to the present study. Special thanks are extended to Dr. Frik Sundstøl, Dr. Le Viet Ly and Dr. Nils Petter Kjos for their facilitation and advice during the study and preparation of this manuscript.

References

Akin D E, Rigsby L L, Theodorou M K and Harley R D 1988 Population changes of fibrolytic bacterium in the presence of phenolic acids and plant extracts. Animal Feed Science and Technology. Volume 19: 261-275.

Blümmel M and Ørskov E R 1993 Comparison of in-vitro gas production and nylon bag degradability of roughages in predicting feed intake in cattle. Animal Feed Science and Technology. Volume 40: 109-119.

Blümmel M and Becker K 1997 The degradability characteristics of forty four roughages and roughage neutral-detergent fibres as described by in-vitro gas production and their relationship to voluntary feed intake. British Journal of Nutrition. Volume 77: 757-768.

Chaudhry A S 1998a Chemical and biological procedures to upgrade cereal straws for ruminants. Nutritional Abstracts and Reviews. Volume 68: 319-331.

Chaudhry A S 1998b In-vitro and in-sacco digestibility of wheat straw treated with calcium oxide and sodium hydroxide alone or with hydrogen peroxide. Animal Feed Science and Technology. Volume 74: 301-313.

Chaudhry A S 1998c Nutrient digestion and rumen fermentation in sheep of wheat straw treated with calcium oxide, sodium hydroxide and alkaline hydrogen peroxide. Animal Feed Science and Technology. Volume 74: 315-328.

Chaudhry A S 2000 Rumen degradation in-sacco in sheep of wheat straw treated with calcium oxide, sodium hydroxide and sodium hydroxide plus hydrogen peroxide. Animal Feed Science and Technology. Volume 83: 313-323.

Chen X B 1997 Neway Excel: A utility for processing data of feed degradability and in-vitro gas production (version 5.0). Rowett Research Institute. UK.

Cheng K J and Hungate R E 1976 Effect of alfalfa fiber substrate on culture counts of rumen bacteria. Applied Environmental Microbiology. Volume 32: 649-652.

Chenost M and Kayouli C 1997 Roughage Utilization in Warm Climates. FAO Animal and Health Paper 135. Rome.

Chesson A, Stewart C S and Walace R J 1982 Influence of plant phenolic acids on growth and cellulolytic activity of rumen bacteria. Applied Environmental Microbiology. Volume 44: 597-603.

Cunniff P (ed.) 1997 Official Methods of Analysis of AOAC International. Maryland, USA.

Devendra C 1997 Crop residues for feeding animals in Asia: Technology development and adoption in crop/livestock systems. In Renard C (ed.) Crop Residues in Sustainable Mixed Crop/Livestock Farming Systems. CAB International. pp 241-268.

Dias-da-Silva A A, Ferreira A M, Guedes C V M, and Mascarenhas-Ferriera A 1988 Effects of moisture level, treatment time and soyabeen addition on the nutritive value of urea treated maize stover. Animal Feed Science and Technology. Volume 19: 67-77.

Djajanegara A and Doyle P T 1989 Urea supplementation compared with pre-treatment. 1. Effects on intake, digestion and live-weight change by sheep fed a rice straw. Animal Feed Science and Technology. Volume 27: 17-30.

Dolberg F 1992 Progress in the utilization of urea treated crop residues: biological and socio-economic aspects of animal production and application of the technology on small farms. Livestock Research for Rural Development. Volume 4 No 2
[http://www.fao.org/ag/aga/agap/frg/FEEDback/lrrd/lrrd4/2/dolberg.htm]

Doyle P T 1982 Review of treatment of fibrous roughages in South-East Asia. Proceedings of 3rd Seminar on “Maximum Livestock Production from Minimum Land”, Bangladesh Agricultural Research Institute. Joydebpur, Bangladesh. pp 111-122.

Doyle P T, Devendra C and Pearce G R 1986 Rice straw as a feed for ruminants. International Development Program of Australian Universities and Colleges Limited (IDP), Canberra, Australia.

Durand M 1989 Conditions for optimizing cellylolytic activity in the rumen. In Chenost M and Reiniger P (Eds.) Evaluation of Straws in Ruminant Feeding. Elsevier. Amsterdam. pp 66-74.

Garmo T H 1986 Treatment of straw with sodium, potassium and calcium hydroxide in laboratory scale. Agricultural University of Norway. Deptment of Animal Nutrition. Report No. 239.

Hadjipanayiotou M 1984 Effect of level and type of alkali on the digestibility in-vitro of stored, chopped barley straw. Agricultural Wastes. Volume 10: 187-194.

Jackson M G 1977 Review article: the alkali treatment of straws. Animal Feed Science and Technology. Volume 2: 105-130.

Jayasuryia M C N and Perera H G D 1982 Urea ammonia treatment of rice straw to improve its nutritive value for ruminants. Agricultural Wastes. Volume 4: 143-150.

Jayasuryia M C N and Pearce G R 1983 The effect of urease enzyme on treatment time and the nutritive value of straw with ammonia as urea. Animal Feed Science and Technology. Volume 8: 271-281.

Jung H G, Buxton D R, Hatfield R D and Ralph J 1993 Forages cell wall structure and digestibility. American Society for Agronnomics, Wisconsin, USA.

Leng R A 1986 Drought Feeding Strategies: Theory and Practice. Penambul Books. Armidale. NSW. Australia.

Males J R 1987 Optimizing the utilization of cereal crop residues for beef cattle. Journal of Animal Science. Volume 65: 1124-1130.

Menke K H and Steingass H 1988 Estimation of the energetic feed value obtained from chemical analysis and in-vitro gas production using rumen fluid. Animal Research and Development. Volume 28: 7-25.

Nath K, Sahai K and Kehar N D 1969 Effect of water washing, lime treatment and lime and calcium carbonate supplementation on the nutritive value of paddy straw. Journal of Animal Science. Volume 28: 383-391.

Ørskov E R and McDonald I 1979 The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. Journal of Agricultural Science. Volume 92: 499-503.

Ørskov E R and Ryle M 1990 Energy Nutrition in Ruminants. Elsevier. Amsterdam.

Ørskov E R and Shand W J 1997 Use of the nylon bag technique for protein and energy evaluation and for rumen environment studies in ruminants. Livestock Research for Rural Development. Volume 9 No 1 [http://www.fao.org/ag/aga/agap/frg/FEEDback/lrrd/lrrd9/1/orskov.htm]

Ørskov E R, De b Hovell F D and Mould F 1980 The use of the nylon bag technique for the evaluation of feedstuffs. Tropical Animal Production. Volume 5: 195-213.
[http://www.fao.org/ag/aga/agap/frg/tap53/53_1.pdf]

Owen E, Klopfenstein T, Urio N A 1984 Treatment with other chemicals. In Sundstøl F and Owen E C (Eds.) Straw and Other By-products as Feed. Elsevier. Amsterdam. pp 248-275.

Pradhan R, Tobioka H and Tasaki I 1996 The effect of urea and ammonia treatments of rice straw on the voluntary intake, digestibility and nitrogen retention in sheep. Grassland Science. Volume 42: 227-234.

Pradhan R, Tobioka H and Tasaki I 1997 Effect of moisture content and different levels of additives on chemical composition and in-vitro dry matter digestibility of rice straw. Animal Science and Technology (Japan). Volume 68: 273-284.

Preston T A and Leng R A 1984 Supplementation of diets based on fibrous residues and by-products. In Sundstøl F and E C Owen (Eds.) Straw and Other By-products as Feed. Elsevier. Amsterdam. pp 373-413.

Sahoo B, Saraswat M L, Haque N and Khan MY 2000 Energy balance and methane production in sheep fed chemically treated wheat straw. Small Ruminant. Research. Volume 35: 13-19.

SAS 1996 SAS User’s Guide: statistics (Version 6). SAS Institute Inc. USA.

Schiere J B and Ibrahim M N M 1989 Feeding of Urea-ammonia Treated Rice Straw. Pudoc. Wageningen. Netherlands.

Shen H S, Sundstøl F and Ni D B 1998 Studies on untreated and urea-treated straw from three cultivation seasons 2. Evaluation of straw quality through in-vitro gas production and in-sacco degradation measurements. Animal Feed Science and Technology. Volume 74: 193-212.

Silva A T and Ørskov E R 1988 Fibre digestion in the rumens of animals receiving hay, untreated or ammonia treated straw. Animal Feed Science and Technology. Volume 19: 299-287.

Sirohi S K and Rai S N 1994 Chemical composition and in-sacco degradability of wheat straw as influenced by lime and/or urea treatment under different ruminant environments. International Journal of Animal Science. Volume 9: 59.

Sirohi S K and Rai S N 1995 Associative effect of lime plus urea treatment of paddy straw on chemical composition and in-vitro digestibility. Indian Journal of Animal Science. Volume 65: 1346-1351.

Sirohi S K and Rai S N 1996 Synergistic effect of lime and urea treatment of paddy straw on in-sacco digestibility and in-vitro gas production rate. Indian Journal of Animal Nutrition. Volume 13: 214-219.

Sirohi S K and Rai S N 1997 Effect of different levels of lime, moisture and reaction periods on in-sacco digestibility and in-vitro gas production of paddy straw. Indian Journal of Dairy Science. Volume 50: 367-371.

Sirohi S K and Rai S N 1998 Optimization of treatment conditions of wheat straw with lime: Effect of concentration, moisture content and treatment time on chemical composition and in-vitro digestibility. Animal Feed Science and Technology. Volume 74: 57-62.

Sirohi S K and Rai S N 1999 Synergistic effect of lime and urea treatment of wheat straw on chemical composition, in-sacco and in-vitro digestibility. Asian-Australian Journal of Animal Science. Volume 12: 1049-1053.

Sundstøl F 1985. Recent advances in development and utilization of chemically treated low quality roughages. In Wanapat M and Devendra C (Eds.). Relevance of Crop Residues as Animal Feeds in Developing Countries. Funny Press. Bangkok. Thailand. pp 121-145.

Sundstøl F and Coxworth E M 1984 Ammonia treatment. In Sundstøl F and Owen E C (Eds.) Straw and Other By-products as Feed. Elsevier. Amsterdam. pp 196-247.

Sundstøl F and Owen E C (Eds.) 1984 Straw and Other By-products as Feed. Elsevier. Amsterdam.

Tolera A, Sundstøl F and Said A N 1998 The effect of stage of maturity on yield and quality of maize grains and stover. Animal Feed Science and Technology. Volume 75: 157-168.

Van Soest P J 1994 Nutritional Ecology of the Ruminant, 2nd ed. Cornell University Press. USA.

Van Soest P J and Robertson J B 1985 Analysis of Forages and Fibrous Foods. A Laboratory Manual for Animal Science 613. Cornell University. USA.

Varel V H and Jung H G 1986 Influence of forage phenolics on ruminal fibrolytic bacteria and in-vitro fiber degradation. Applied Environmental Microbiology. Volume 52: 275-280.

Wanapat M 1985 Improving rice straw quality as ruminant feed by urea treatment in Thailand. In Wanapat M and Devendra C (Eds.). Relevance of Crop Residues as Animal Feeds in Developing Countries. Funny Press. Bangkok. Thailand. pp 121-145.

Zaman M S and Owen F 1990 Effect of calcium hydroxide or urea treatment of barley straw on intake and digestibility in sheep. Small Ruminant Research. Volume 3: 237-248.

Zaman M S and Owen F 1995 The effect of calcium hydroxide and urea treatment of barley straw on chemical composition and digestibility in-vitro. Animal Feed Science and Technology. Volume 51: 165-171.

Zaman M S, Owen E and Pike D J 1994 The calculation method used for optimizing conditions of treatment of barley straw with calcium hydroxide and urea, moisture, treatment time and temperature on in-vitro digestibility. Animal Feed Science and Technology. Volume 45: 271-282.

Table 2: Initial gas production after 6h (A), later gas production (B), potential gas production (A+B), rate constant c) and gas production after 48h (48hGP) of rice straw as affected by treatments with lime and/or urea
Treatment		Chemical input (%)				A	B	A+B	c	48hGP
Treatment		Lime			Urea	(ml)	(ml)	(ml)	(fractions/h)	(ml)
Means by treatment
I	0			0		2.8^AB	34.3^A	37.2^A	0.034^A	27.7^A
II	0			2		3.2^B	36.3^BC	39.5^B	0.037^A	30.5^B
III	0			4		3.7^BC	36.6^BC	40.3^BC	0.044^BC	32.7^C
IV	3			0		3.7^BC	35.9^BC	39.5^B	0.042^B	31.3^BC
V	3			2		4.5^C	36.8^BC	41.3^CD	0.047^C	35.5^D
VI	3			4		4.3^C	38.1^D	42.4^D	0.048^C	36.7^DE
VII	6			0		4.3^C	36.8^C	41.1^C	0.049^C	35.5^D
VIII	6			2		4.7^C	38.0^D	42.7^DE	0.051^C	36.7^DE
IX			6	4		4.8^C	39.1^E	43.9^E	0.052^C	38.2^E
SEM						0.3	0.3	0.4	0.001	0.7
Factorial effects and contrasts
Lime						***	***	***	***	***
Urea						*	***	***	***	***
Urea x Lime						Ns	Ns	Ns	Ns	Ns
No lime vs. Lime						***	***	***	***	***
No urea vs. Urea						**	***	***	***	***
Notes: P<0.05, P<0.01, * P<0.001, Ns: non-significant, Means within each column under the same subheading bearing same letter (^{a, b, c, d}) are not different at P<0.05.*

Table 4: Water-insoluble degradability (B), potential degradability (A+B), rate constant (c), lag phase (L), and 48 hour degradability (48hDMD) of hay as a standard substrate incubated in cattle fed on rice straw treated with lime and/or urea
TTreatment		Chemical input (%)			B	A+B	c	L	48hDMD
TTreatment		Lime		Urea	(%)	(%)	(fractions/h)	(h)	(%)
Means by treatment
I	0		0		44.3	60.6	0.030^a	4.0^a	48.4^a
II	0		2		42.6	59.3	0.036^b	3.1^b	50.3^bc
III	0		4		44.6	60.9	0.041^cd	2.7^bc	52.9^d
IV	3		0		43.1	59.3	0.040^c	2.5^c	51.8^cd
V	3		2		43.7	59.8	0.041^cd	2.5^c	52.7^d
VI	3		4		44.2	60.5	0.042^d	2.5^c	53.3^d
VII	6		0		42.3	59.1	0.036^b	2.7^bc	49.9^b
VIII	6		2		43.1	60.0	0.037^b	2.5^c	51.1^c
IX	6		4		43.1	59.8	0.040^c	2.7^bc	51.7^cd
SEM					0.5	0.5	0.001	0.2	0.4
Factorial effects and contrasts
Lime					Ns	Ns	***	***	***
Urea					Ns	Ns	***	*	***
Urea x Lime					Ns	Ns	***	*	**
Animal					*	*	**	**	***
No lime vs. Lime					Ns	Ns	***	***	***
No urea vs. Urea					Ns	Ns	***	**	***
Notes: * P<0.05, P<0.01, * P<0.001, Ns: non-significant, Means within each column under the same subheading bearing same letter (a,b,c,d) are not significantly different at P<0.05.

Table 5: Water-insoluble degradability (B), potential degradability (A+B), rate constant (c), lag phase (L), and 48 hour degradability (48hDMD) of rice straw treated with lime and/or urea and incubated in fistulated cattle fed on the same type of straw
Treatment	Chemical input (%)			B (%)		A+B (%)	c (fractions/h)	L (h)	48hDMD (%)
	Lime	Urea
	Means by treatment
I	0	0	49.8^a		59.7^a		0.024^a	4.9^a	40.8^a
II	0	2	52.9^c		64.5^b		0.026^b	4.5^ab	48.4^b
III	0	4	55.4^d		69.1^c		0.031^d	4.1^bc	55.1^d
IV	3	0	54.7^d		68.9^c		0.029^c	4.3^b	53.5^c
V	3	2	53.2^c		69.5^c		0.035^ef	4.3^b	57.6^e
VI	3	4	54.9^d		72.4^d		0.035^ef	3.7^c	59.6^f
VII	6	0	52.4^bc		71.1^d		0.034^e	4.1^bc	59.8^f
VIII	6	2	51.7^b		74.0^e		0.034^ef	3.8^c	60.9^g
IX	6	4	52.5^bc		75.3^f		0.036^f	3.4^c	62.2^h
SEM			0.4		0.5		0.001	0.2	0.2
Factorial effects and contrasts
Lime			***		***		***	***	***
Urea			***		***		***	***	***
Urea x Lime			***		***		***	Ns	***
Animal			**		**		Ns	**	***
No lime vs. Lime			0.09		***		***	***	***
No urea vs. Urea			**		***		***	***	***
Notes: P<0.05, P<0.01, * P<0.001, NS non-significant, Means within each column under the same subheading bearing same letter (a,b,c,d,e,f,g,h) are not different at P<0.05.*