Effect of different levels and sources of crude protein on in vitro digestibility and gas production from rice straw and Para grass (Brachiaria mutica)

Doan Huu Luc, Nguyen Van Thu* and T R Preston**

College of Agriculture, Angiang University, 25 Vo Thi Sau, Longxuyen city, Angiang province, Vietnam
dhluc@agu.edu.vn
* College of Agriculture and Applied biology, Cantho University, 3/2 street, Cantho city, Vietnam
* TOSOLY, AA48 Socorro, Santander, Colombia

Abstract

Three experiments were conducted to evaluate effects of different levels and sources of crude protein (CP) on in vitro digestibility in vitro gas production from rice straw and Para grass. The treatments in experiment 1 were CP levels of 5, 7, 9, 11 and 13% in DM derived from soybean with rice straw as substrate. Experiment 2 was similar to Experiment 1, but the treatments were crude protein levels of 11, 12, 13, 14 and 15% and the substrate was Para grass.. Experiment 3 was a 2 x 2 factorial design. The first factor was protein source (fish meal or copra meal) and the second one was source of substrate (rice straw or Para grass). These feeds were combined to give a crude protein level in the DM of 12.5 %.

In vitro OMD and NDFD of rice straw supplemented with soybean meal increased in curvilinear fashion according to the crude protein level in the mixture with maximum values for OMD being reached with 13% crude protein and of NDFD at 11%. Data for gas production showed similar trends to those for OMD for incubation times of 12 and 24h; however, at 48h, there was no increase in gas production with more than 9% crude protein while after 96h there were no differences among all levels of crude protein. It appeared that the beneficial effect of the higher protein level was only apparent in the early stages of incubation. Increase in protein level with Para grass improved OMD at 0h (ie: water soluble OM) but had only a slight positive effect  after 48h of incubation. After 96h of incubation there was no effect of protein level on OMD. NDFD was not affected by protein level at any of the incubation times. OMD and NDFD were higher when the substrates were supplemented with fish meal compared with copra meal, and were higher for Para grass than for rice straw.

In vitro OM digestibility appeared to be a more suitable method than in vitro gas production, as a tool to predict the effects of protein supplementation on the nutritive value of tropical roughages.

Keywords: Copra meal, fish meal, soybean, supplementation

Introduction

Large ruminant production systems in the Mekong delta of Vietnam are generally based on feeding of rice straw and natural grasses without supplementation.  However, these diets result in only moderate rates of productivity due to low protein content and low digestibility. In order to improve animal performance on these feeds it is recommended first to improve rumen function by supplementation with sources of fermentable nitrogen and minerals and then, in order to secure higher rates of performance, to provide a source of “ bypass” protein (Preston and Leng 1987). This strategy was confirmed in the research reported by Nha et al (2008) where increasing the offer level of a combination of urea and Sesbania foliage led to increases in rumen ammonia levels and in N retention buffaloes and cattle fed rice straw.

Procedures for measuring growth rates, in vivo digestibility and N-balance are time-consuming and require the use of animals and facilities to house them. For this reason there has always been interest in the development of in vitro methods. The  in vitro measurement of gas production has been shown to be a low-cost  way to predict the in vivo digestibility of roughages (Menke and Steingass 1988; Robinson et al 2004; Getachew et al 2004).

The objectives of this study were therefore to compare the gas production technique with the more conventional method for determining in vitro digestibility. The two methods wswere used to evaluate: (i) the effects of protein supplementation on the nutritive value of low-protein roughages, and (ii) the comparative value of different protein sources used to supplement different energy substrates.

Materials and methods

Feed samples, chemical analysis and inoculum

The feeds  used in the experiments were whole soybeans meal, fish meal, copra meal, Para grass and rice straw.  Para grass and rice straw samples were first chopped to 1-2 cm in length for drying at 60^°C for 24h. Whole soybeans were roasted for 30 minutes and shelled. Fish meal was made from fresh sea fish from which whole fins and bones were removed. The residual flesh was dried at 60⁰C for 24h. Copra meal was the byproduct after oil extraction of the flesh of coconuts. All the feed samples were ground to pass a 1mm sieve, prior to analysis for proximate constituents (Table 1) and use in the series of experiments.  

DM was determined by drying samples at 105^°C overnight. OM was determined by ashing at 550⁰C for 3h.  Crude protein and ether extract were determined by procedures of AOAC (1990). The analyses of NDF and ADF were done by the method described by Goering and Van Soest (1970).

Rumen fluid was collected from cattle after slaughter in the local abattoir, and put in a thermos flask for bringing to the laboratory, where it was strained through 3 layers of muslin cloth prior to being used as the inoculum in the in vitro studies.

Experimental designs

Experiment 1

This was a completely randomized design with 5 treatments and 3 replications. The basal substrate was rice straw. The treatments were crude protein levels of 5, 7, 9, 11 and 13% in DM, achieved by adding appropriate proportions of soybean (Table 2). 

Experiment 2

This was similar to Experiment 1, but the substrate was Para grass and the crude protein levels were: 11, 12, 13, 14 and 15% in DM (Table 3).

Experiment 3

This was a 2 x 2 factorial design with 3 replications. The first factor was protein source (FM- fish meal and CM- copra meal) and the second one was substrate (RS- rice straw and PG- Para grass). These feeds were combined in proportions to obtain a crude protein level of 12.5 % in DM.

In vitro digestibility and gas production

The in vitro organic matter digestibility (OMD) and neutral detergent fiber digestibility (NDFD) values were determined for 0, 48 and 96h of incubation, according to the method of Goering and Van Soest (1970).

The in vitro gas production (GP) was recorded for 12, 24, 36, 48, 72 and 96h of incubation and done according to the procedure proposed by Menke and Steingass (1988).

Statistical analysis

Variance of data was analyzed by the One-way (Experiments 1 and 2) and Two-way (Experiment 3) models in the Minitab (version 15.1.0.0) software. Regression analysis (Excel program in the Microsoft Office 2003 software) was used to determine trends between the digestibility and gas production data and the CP levels in the samples,

Results and discussion

Experiment 1

In vitro OMD and NDFD of the rice straw supplemented with soybean meal increased according to the crude protein level in the mixture (Table 4).

The responses were curvilinear with reduced increments in digestibility as the crude protein level increased, maximum values being reached with about13% crude protein for 48h OMD and 11% for 48h NDFD (Figures 1, 2 and 3). 

Figure 1.  Effect of crude protein level on in vitro OM digestibility at 0h of mixtures of rice straw and soybean meal	Figure 2.  Effect of crude protein level on in vitro OMD at 48h of mixtures of rice straw and soybean meal

It is interesting to compare the measurements of OM digestibility at 0h (Figure 1) and 48h (Figure 2), the degree of fit of the data to the regression line being much higher for measurements at 0h (ie: prior to incubation), which is effectively the water solubility (or washing loss) of the OM.  Several researchers (Karsli and Russell 2003; Enoh et al  2005; Nguyen Van Thu 2005; Pheng Buntha and Chhay Ty 2006) have shown that washing loss of DM is closely related with in vivo DM digestibility and superior to NDF and in sacco degradability as a predictor of in vivo DM digestibility.

It was to be expected that OM digestibility would increase with crude protein level as the digestibility of soybean meal is obviously considerably higher than that of rice straw. The increase in NDF digestibility with increase in protein in the sample presumably reflects a stimulating effect on cellulolytic micro-organisms due to increase in the supply of fermentable protein. This is in line with results reported by Dryhurst and Wood (1998), Mould et al (2005), Bailey et al (2008) and Perdok and Leng (1989) showing increases in in vivo DM digestibiliity of low-protein forages as sources of rumen fermentable N were increased by adding urea or protein.

Data for gas production (Table 5; Figure 4) showed similar trends to those for in vitro OM digestibility for incubation times of 12 and 24h; however, at 48h, there was no increase in gas production with more than 9% while after 96h there were no differences among all levels of CP. It appeared that the beneficial effect of the higher protein level was only apparent in the early stages of incubation.

Experiment 2

The effects on in vitro OM and NDF digestibility (Table 6) of incubating para grass with increasing levels of soybean were different from what was observed with rice straw.

Increase in protein level improved in vitro OM digestibility at 0h (ie: water soluble OM) but had only a slight positive effect after 48h of incubation when the  protein level was raised from 11 to 12% with no further improvement at higher protein levels. After 96h of incubation there was no effect of protein level on OM digestibility. NDF digestibility was not affected by protein level at any of the incubation times. 

The effects of protein level on measurements of in vitro gas production from Para grass (Table 7) were broadly similar to what was observed with rice straw, except that the trends for increased gas production with increase in protein level were consistent at all times of incubation (Figure 5).

Experiment 3

OM and NDF digestibility coefficients were higher for fish meal than for copra meal after 48 and 96h of incubation; the values for Para grass were higher than those for rice straw (Table 8). There were no interactions between protein source and substrate. 

In vitro gas production was stimulated more by fish meal than by copra meal; and was higher for Para grass than for rice straw (Table 9).  

The greater response in vitro digestibility to supplementation with fish meal compared with copra meal was probably due to the known superior amino acid balance of fish meal protein compared with that in copra meal. Thus, Husseen and Jordan (1991), Broderick (1992), González et al (1998) and Pavan and Santini (2002) all considered that supplements of fish meal were more efficient in enhancing the rumen ecosystem than other protein sources.

The higher values for Para grass compared with rice straw are to be expected in view of the known superior nutritive value of grasses compared with cereal straws.

The major objective in this series of studies was to assess the usefulness of measurements of in vitro OM and NDF digestibility and in vitro gas production as indicators of:  (i) the effects of supplementation of roughages with a source of crude protein (Experiments 1 and 2); and (ii) the relative nutritive values of different protein supplements and different substrates (Experiment 3). In this respect the determination of in vitro OM digestibility appeared to be the more suitable method the results of which were broadly in line with known effects of dietary CP on digestibility of roughages. This conclusion does not entirely support the claims of Dryhurst and Wood (1998), Mould et al (2005), Getachew et al (2004) and Rotger et al (2006) that gas production techniques have a high potential to predict effects of level and source of supplementary protein for ruminants. The fact that these researchers made their studies with forages grown in temperate latitudes, whereas rice straw and Para grass are found only in tropical latitudes, may be one explanation of the discrepancy in view of documented differences in morphology of plant species grown in tropical versus temperate latitudes (Van Soest 1994).

Conclusions

• In the study reported in this paper, the determination of in vitro OM digestibility appeared to be a more suitable method than in vitro gas production, as a tool to predict the effects of protein supplementation on the nutritive value of tropical roughages.

References

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Received 31 May 2009; Accepted 21 June 2009; Published 1 July 2009

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