Ruminal degradation and intestinal digestion of crude protein of tropical protein resources using nylon bag technique and three-step in vitro procedure in dairy cattle

Abstract

The experiment was carried out on four fistulated multiparous Holstein Friesian crossbred (dry period ) dairy cows to evaluate nutritive value of local protein feed resources using the in sacco method and in vitro pepsin -pancreatin digestion. Measurements were made of ruminal degradability, intestinal digestibility of DM and crude protein, and effects of the rumen environment in cattle fed on untreated and urea-treated rice straw. Feeds used were cottonseed meal (CSM),  soybean meal (SBM), dried brewer's grain (DBG),  palm seed meal (PSM),  cassava hay (CH) and leucaena leaf meal  (LLM). Each feedstuff was weighed into duplicate nylon bags and incubated in each of the four rumen fistulated cows for 0, 2, 4, 8, 16, 24, and 48 h post feeding. Rumen feed residues from bags of 16 h of incubation time were used for estimation of lower gut digestibility by  in vitro pepsin-pancreatin digestion.

Ruminal ammonia-nitrogen (NH3-N) concentrations were 4.5 and 12.4 mg% for untreated rice straw and urea-treated rice straw, respectively. Effective degradability of DM of CSM, SBM, DBG, PSM, CH, LLM in cows fed with untreated rice straw and urea-treated rice straw were 41.9,46.7; 56.1,63.3; 30.8,36.1; 47.0,48.3; 41.1,44.1 and 47.5,49.5 %, respectively. Effective degradability of the crude protein in cows fed with untreated rice straw and urea-treated rice straw were 49.6,54.6; 59.2,66.3; 40.9,48.8; 33.5,39.9; 47.3,54.6 and 65.0,70.6 % for the respective feeds. Both DM and crude protein in vitro pepsin-pancreatin digestibility of the same feed sources in cows fed with urea-treated rice straw were higher than in untreated rice straw . Results of crude protein in vitro pepsin-pancreatin digestibility, as ranked from the highest to the lowest, were SBM, CSM, LLM, CH, DBG and PSM for both environments.

It is concluded that the nutritive value of protein-rich supplements,  in terms of rumen degradable, intestinal and total tract digestibility, was improved when urea-treated rice straw was offered as a roughage as compared with untreated rice straw; and that the lower ruminal ammonia concentration inhibited the degradation rate of the supplements when the cows were fed with low quality roughage (untreated rice straw). SBM and LLM were highly degraded in the rumen, while CH, CSM and DBG were less degraded and hence resulted in higher levels of rumen undegradable protein. These protein sources can be used to improve rumen ecology and rumen by-pass protein supply to ruminants.

Key words: Digestibility, in sacco, pepsin-pancreatin, protein sources, rice straw, rumen degradability, urea-treated rice straw.

 
Introduction

The rate and extent of protein degradation in the rumen is very crucial, as it determines the availability of nitrogen to microorganisms in the rumen and the supply of amino acids to the small intestine of the host animals. The protein consumed by the animal should be partly degradable in the rumen, as peptides, amino acids and NH₃-N derived from proteolysis can be used in microbial protein synthesis and to improve rumen ecology. It is, therefore, very important to determine the degradability and digestion of different feed ingredients which are grown and used in different locations. Incubation of feeds in nylon bags in the rumen of fistulated ruminants has been used to determine the extent to which the protein fraction of feeds is degraded in the rumen (Ørskov and McDonald 1979; Rao and Prasad 1989 ). The feed N which escapes rumen degradation and is digested in the intestine can be measured by a three-step in vitro procedure (Calsamiglia and Stern  1995). Ørskov et al (1980) observed  that the nylon bag technique was not only a powerful tool for indexing the relative degradabilities of feedstuffs, but that it may also be used to study rumen processes, as it is possible to vary the factors within the bag, or within the rumen.

Mehrez et al (1977) studied rate of rumen fermentation in relation to ammonia concentration by varying the amount of urea added to a whole barley diet (from 0 to 10 g/kg diet), and then incubated these same diets in the nylon bag in the rumen of the animals. They found that the disappearance of dry matter from the bag was positively correlated with increasing level of ruminal ammonia nitrogen from 10 to 24 mg%. Many reports have been published on the protein degradability of feeds for ruminants (Stern et al 1983; Santos et al 1984; Armentano et al 1986; Voigt and Piatkowski 1987; Arieli et al 1989; Cros et al 1991a,b; Maiga et al 1996; Gralak et al  1997; Wanapat et al 2000; Islam et al  2002; ) and intestinal digestibility (de Boer et al 1987; Cros et al 1991a,b;Maiga et al 1996). However, there is limited information available on characteristics of DM and crude protein degradation in the rumen and digestibility in the lower digestive tract of protein sources locally used for livestock in the tropics with special reference to Thailand. Therefore, the present study was undertaken to assess the degradation characteristics and lower-gut digestibility of different protein sources in cattle fed on two types of roughages, untreated and urea-treated rice straw.

Materials and methods

Experimental animals and design

Four, ruminally fistulated, multiparous, non lactating Holstein-Fresian crossbred dairy cows were used in a completely randomized design (CRD) to evaluate six protein sources (cottonseed meal, CSM; soybean meal,SBM; dried brewery's grain, DBG; palm seed meal, PSM; cassava leaf hay,CH; leucaena leaf meal, LLM). All feeds were ground to pass a 1 mm screen.

The cows were individually penned and clean fresh water and mineral blocks were offered free choice.  Roughage was fed on ad libitum basis and concentrate (13% crude protein, 65% TDN) (Table 1) was fed at 0.5% body weight  in two equal portions, at 0830 am and at 1630 pm.  The animals were given the diets for 14 days before the nylon bag procedure was imposed.

Urea-treated rice straw preparation

Urea (5 kg) was dissolved in fresh water (100 kg). Rice straw (100 kg) was placed on a concrete floor and the urea solution was poured over it. The stack was covered with polyethylene sheet for 10 days before the straw was fed.

Sampling procedure, data collection and analysis

Ruminal disappearance study

DM and CP disappearances in the rumen were estimated for each feed source using the nylon bag technique (Ørskov and McDonald 1979). The bags (7 x 14cm) were made from dacron cloth with a pore size of 38 μm. Five grams of each feed (DM basis) were placed in each bag, which was anchored in the rumen with a 30-cm length of braided fishing line. All samples were prepared in duplicate and incubated in the rumen of each animal for 0, 2, 4, 6, 8, 16, 24 and 48 h before the bags were removed. At each time, ruminal NH₃-N was determined by the hypochlorite-phenol procedure (Beecher and Whitton 1978) on the supernatant of the centrifuged sample. pH was determined with a portable pH and temperature meter (Orion Research portable meter 200 series, USA). After the specific incubation periods, the bags were removed from the rumen and were immediately washed with cold tap water until clear, and dried in a forced air oven at 60 ^oC for 72 h. For the control, bags without incubation (0 h) were washed and dried in a similar condition. DM and CP of feeds and residues in the bag were estimated by AOAC (1990) procedures. Degradation rates of DM and CP were calculated using the equation of Ørskov and McDonald (1979):

 (p = a + b (1- e^-ct )

where p = disappearance rate at time t, a = the  intercept representing the portion of DM or CP solubilized at initiation of incubation (time 0), b = the fraction of DM or CP potentially degradable in the rumen, c = a rate constant of disappearance of fraction b, and t = time of incubation. The nonlinear parameters a, b, and c were estimated by an iterative least squares procedure. The effective degradabilities of DM (EDDM) or of CP (EDCP) were calculated using the following equation:

   EDDM or EDCP = a + (bc)/(c+k)

where k is the estimated rate of out flow from the rumen, and a, b, and c are the same parameters as described earlier. Effective degradability of DM or CP was estimated for each ingredient assuming rumen solid outflow rate from the previous experiment (Ørskov and McDonald 1979). Undegradable protein digestion  of each sample after 16h incubation was used to measure in vitro protein digestion in the lower gut using a three-step in vitro procedure of Calsamiglia and Stern (1995).

In vitro pepsin-pancreatin  digestion procedure

Samples of the feed residue from the bags after 16 h of incubation, after determining N content,  were put into a 50 ml centrifugation tube in quantities equivalent to 15 mg of N. 10 ml of a  0.1 N HCl solution (pH 1.9), containing 1 g/litre of pepsin (sigma P-7012, Sigma) were added and the samples incubated for 1 h in a 38 ^ºC shaker water bath. After incubation,  0.5 ml of a 1 N  NaOH solution and 13.5 ml of a pancreatin solution  (0.5 M KH₂PO₄ buffer standardized at pH 7.8 containing 50 ppm of thymol and 3 g/litre of pancreatin [Sigma P-7545, Sigma]) were added. The samples were incubated at 38^oC for 24 h in a shaker water bath, and mixed (magnetic stirrer) every 8 h. After  incubation, 3 ml of a 100% (wt/vol) solution of TCA were added to the tubes to stop enzymatic action and to precipitate undigested proteins. All tubes were mixed and allowed to stand for 15 min. The samples were centrifuged at 10,000 x g  for 15 min and the supernatant analyzed for soluble N by the Kjeldahl method (AOAC 1990). Pepsin-pancreatin digestion of protein was calculated as TCA-soluble N divided by amount of sample N (dacron bag residue) used in the assay (Calsamiglia and Stern 1995).

 Statistical analyses

Data for  a, b, and c values and for EDDM and EDCP and results from the three-step in vitro procedure were analyzed using the GLM option of the software programme of SAS (1987) according to the  following model :

Y_ij = m + d_ij + e_ij

where Y_ij= the criteria under study, m = overall mean, d_i = feed source effect (or treatment of roughage diet), and e_ij = residual error.

Results

With the exception of soybean meal and palm seed meal , all the six feeds had similar dry matter (DM), ash, neutral detergent fiber (NDF), and acid detergent fiber (ADF) values (Table 2). Palm-seed meal contained the highest NDF and ADF, while  soybean meal had the lowest. CP content was highest in soybean meal and cottonseed meal and lowest in palm-seed meal.

Ruminal ammonia-nitrogen concentrations were almost three times higher in cows fed with urea-treated-rice straw than in cows fed with untreated rice straw (Table 3). The ruminal pH was similar on both diets.

It was found that rate of DM and CP disappearance in the rumen of all feeds was higher in cows fed with urea-treated rice straw as compared with cows fed with untreated rice straw (Tables 4, 5 and 6; Figures 1 and 2). The effective rates of rumen degradability of DM, in both rumen environments, decreased according to the ranking order : SBM, LLM, CSM, DBG, CH and PSM. The corresponding rates for disappearance of CP decreased in the following ranking order; LLM, SBM, CH, CSM, DBG, and PSM. Levels of rumen undegradable protein, as percentage of total protein, were in the range of 55 to 65% for palmseed meal and dried brewer's grains and between 30 and 40% for leucaena  leaf meal and soybean meal (Figure 1). Values for cassava hay and cottonseed meal were intermediate.

59.1

51.2

Intestinal digestibility of CP  (expressed as % of rumen residual CP) was higher in cows fed with urea-treated than untreated rice straw (Table 6; Figure 2). For total tract digestibility of both CP and DM, the values were higher in all feeds in cows fed with urea-treated rice straw. Ranking order from highest to lowest of total tract digestibility of DM was SBM, CSM, LLM, CH, DBG, and PSM for both rumen environments.

Figure 1. Mean values for "effective" rumen undegradable protein (RUP) for six protein sources in cows fed
with untreated rice straw and urea-treated rice straw using the nylon bag technique

Figure 2. Intestinal digestibility of the rumen undegraded protein in six protein sources in cows fed
with untreated rice straw  and urea-treated rice straw using the in vitro pepsin-pancreatin technique

Discussion

Ruminal ecology

The average ruminal pH (Table 3) did not differ between cows fed with untreated and urea-treated rice straw ( 6.5 and 6.6 , respectively). This level was similar to reports by Chanjula et al (2003) and Wanapat et al (2003) that in cattle fed on untreated and urea-treated rice straw, rumen pH was maintained around 6.5 to 6.7. A ruminal pH range of from 6.49 to 6.66 is considered to be optimal for microbial digestion of fiber (Hoover 1986; Firkins 1996). The increase in NH₃-N  from 4.5 to 12.4 mg% in cows fed with untreated and urea-treated rice straw, respectively, is similar to the findings of Chanthai et al (1989)  that in cattle fed untreated rice straw NH₃-N was less than 2 mg% and  increased to 9 mg% when the straw was treated with urea.

Rumen DM and CP degradability of the the protein supplements

The low rumen degradability of Palm seed meal  agrees with the report of Gralak et al (1997) who found slow rates of degradation of both  DM and CP in palm kernel meal. Highest rates for DM and CP degradability were found for SBM which could be due to the structure and solubility characteristics of the protein in SBM which facilitate attack by microorganisms in the rumen (Mahadevan et al 1980). As stated by NRC (2001), there are numerous factors which affect the amount of CP in feeds that will be degraded in the rumen, the two most important considerations being the proportional concentrations of NPN and true protein, and the physical and chemical characteristics of the proteins that comprise the true protein fraction of the feedstuff.  The effective CP degradability of SBM was lower than was reported by NRC (2001), Gralak et al (1997) and Armentano et al (1986). The SBM reported by NRC (2001) was prepared by solvent extraction, while  in Thailand it is prepared by mechanical extraction which results in a high degree of heating.  The effect of heat treatment in decreasing ruminal protein degradability was also reported by Mahadevan et al (1980) and  NRC (2001).

Rate of CP and DM degradability of leucaena was similar to that for SBM.  It is clear from a consideration of the rumen bag technique that the absolute value of the results depends on the way in which the forage is prepared and the pore size of the material from which the bag is made.  In this experiment the leucaena leaf meal had a high proportion of fine dusty particles, which would easily escape from the bag in the rumen.

The low rumen CP degradability in BDG, CSM and CH agrees with reports by several workers (Wohlt et al 1973; Armentano 1986; Gohl 1998; Wanapat et al 2000). BDG has both low protein solubility and degradability (Wohlt et al 1973; Armentano 1986). According to Gohl (1998), cottonseed meal has a relatively low rumen degradability and is therefore a good source of by-pass protein. The formation of tannin-protein complexes in CH is considered to be the reason for it having a high by-pass protein content (Wanapat et al 2000).

Lower gut and total digestibility of feeds

The order of crude protein intestinal digestibility as ranked from the highest to the lowest was; SBM, CSM, DBG, CH, LLM,and PSM.  The values were slightly lower than those given by NRC (2001) (93, 92, 80, 75 and 75%  for SBM, CSM, DBG, PSM and legume forage hay,  respectively). A low CP intestinal digestibility in PSM may be due to the high content of NDF.

Effect of host diet on DM and CP rumen degradability, lower gut and total digestibility

For all protein sources the values of  rumen degradable DM and CP were higher in cows fed with urea-treated than untreated rice straw. This result is in agreement with the report by Mehrez et al (1977) who found that the disappearance of dry matter from the bag was positively correlated with increasing level of ruminal ammonia nitrogen in the range of  10 to 24 mg%. Perdok and Leng (1989) showed that higher levels of rumen NH₃-N (15 to 30 mg%) improved digestibility and feed intakes. Higher bacteria counts (cellulolytic ,proteolytic and amylolytic bacteria) were found by Wanapat et al (2003) in cows fed with urea-treated rice straw than in cows fed untreated rice straw;  Erdman et al (1987) showed that increasing the ruminal NH₃-N from 4.3 to 17.2 mg% by urea infusion could increase DM and CP degradabilty in SBM and CSM.

For all protein sources intestinal digestion of crude protein  (expressed in % of rumen residual protein) was higher in cows fed with urea-treated straw than in untreated rice straw.

Conclusions

• The ruminal degradability of feeds in cows fed with urea-treated rice straw was higher than in those given untreated rice straw, and was associated with much higher levels of rumen ammonia.
• The crude protein in SBM and LLM was  highly degraded in the rumen, while that in  CH, CSM and DBG was less degraded and hence resulted in a higher proportion of the feed protein in the form of rumen undegradable (bypass) protein.

Acknowledgements

The senior author would like to express his most sincere gratitude and appreciation to the Swedish Agency for Research Co-operation with Developing Countries (SAREC) for providing scholarship and research support; and to Skon-Nakhon Agricultural Research and Training center, Rajamangala Institute of Technology, Skon-Nakhon, Thailand for providing animals and laboratory facilities. The help of graduate students in the Ruminant Nutrition Laboratory, Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Thailand is greatly appreciated. An earlier version of this paper was submitted to the Swedish University of Agricultural Sciences in partial fulfillment of the degree of Master of Science.