Ⅰ. INTRODUCTION

Although the crude protein (CP) system in ruminants feed has been used as a standard for evaluating the digestible protein requirement, however some studies have found no response to an increase in the dietary CP level in dairy cattle (Chiou et al., 1995; Van Straalen et al., 1997). In addition, feeding excess CP can lead to decreased nitrogen (N) efficiency causing increased manure N content (St-Pierre and Thraen, 1999). Therefore, current recommendations for feeding protein to ruminants are based on the concept of rumen degradable protein (RDP), rumen undegradable protein (RUP), and intestine absorbable individual amino acids (AA) (Van Straalen et al., 1997; NRC, 2001).

The food industry (both agriculture and food processing sectors) has been growing continuously and the production of by-products has also increased (Ishida et al., 2012). As a result, disposal of these by-products has increases additional processing costs as well as environmental pollution such as nitrate in soil and surface water eutrophication (Van Dyk et al., 2013; San Martin et al., 2016). Therefore, there has been increasing interest in the appropriate use of relatively inexpensive by-products feedstuffs (BPF) for ruminants feed (Oh et al., 2010; Chang et al., 2013). However, ruminant protein feeding systems are constrained by limited knowledge of the metabolism of protein and AA in the intestinal and total gastrointestinal tract of ruminants (O’Mara et al., 1997; Taghizadeh et al., 2005). Therefore, to improve usefulness of BPF for ruminants feed, precise value of CP, RDP, RUP, and AA profile should be precede.

This study was conducted to evaluate the dry matter (DM), CP, and AA degradation and digestion of two types of six BPF (rice bran, RB; wheat bran, WB; corn gluten feed, CGF; tofu residue, TR; spent mushroom substrate from Pleurotus ostreatus, SMSP; brewers grain, BG) in the rumen, small intestine, and total tract using the In situ nylon bag and mobile bag techniques.

Ⅱ. MATERIALS AND METHODS

All experimental procedures were reviewed and approved by the Animal Care and Use Committee of the National Institute of Animal Science (NIAS), Korea (No. NIAS 2016-881).

1 Animals and diets

Three early fattening stages of Hanwoo steers (40 months of age, 520 ± 20.20 kg of body weight) were fitted with permanent rumen cannula (Bar Diamond Inc., Parma ID, USA) and a T-shaped duodenal cannula (Bar Diamond). The steers were housed in separate individual tie-stalls (127 × 250 × 200 cm) consisted of the rubber mat floors, individual feed bunk, and automatic water bowl. They were fed a diet consisting of 1 kg of rice straw and 3.5 kg concentrate mixture (47.9% ground corn grain, 40.9% WB, 5.0% soybean meal, 2.0% rapeseed meal, 2.0% beet molasses, 1.5% dicalcium phosphate, 0.4% salt, and 0.2% vitamin-mineral premix) on a DM basis. The vitamin-mineral premix contained 2,650,000 IU vitamin A, 530,000 IU vitamin D3, 1,050 IU vitamin E, 10,000 mg/kg niacin, 4,400 mg/kg Mn, 13,200 mg/kg Fe, 440 mg/kg I, 2,200 mg/kg Cu, and 440 mg/kg Co. The basal diet was formulated to meet NRC requirements (NRC, 2001). The animals were fed twice daily (at 0900 and 1700 h) with water available ad libitum and free-choice minerals in equal portions, to maintain a relatively stable rumen environment. Each experimental period consisted of a 14-day adaptation period and a 3-day data collection period.

2 Feed samples collection, chemical composition and amino acid analysis

Feed samples of RB, WB, CGF, TR and BG were collected from feed factory, NIAS, Wanju, Korea. The SMSP was obtained from Mushroom Research Institute (GARES), Gwangju, Korea. All feed samples were dried in a forced-air oven at 60°C for 48 h and milled to pass through a 2-mm mesh screen of a Wiley mill (Model 4; Thomas Scientific, Swedesboro, NJ, USA) for the chemical composition analysis and In situ nylon bag and mobile bag studies.

The Association of Official Analytical Chemists (AOAC, 2000) methods were used to determine dry matter (DM), CP and ether extract (EE) contents in the feed samples as described by procedures 930.15, 948.13 and 920.39, respectively. The acid detergent fiber (ADF) and neutral detergent fiber (NDF) were evaluated were performed by the sequential procedure of Van Soest et al. (1991) using a fiber analyzer (ANKOM²⁰⁰⁰, ANKOM Technology Corporation, Macedon, NY, USA). Calcium (Ca) and phosphorus (P) were analyzed using inductively coupled plasma atomic emission spectroscopy (Optima 8300; Perkin Elmer, Inc., Waltham, MA, USA).

The AA analysis was performed after the feed samples were hydrolyzed in 6 M HCl at 110°C for 24 h. The AA analysis was conducted using the ninhydrin method in a Hitachi 8900 (Hitachi, Tokyo, Japan).

3 In situ rumen incubation of tested feeds

Rumen degradation was measured with three steer, applying incubation times of 2, 4, 8, 16, 24, 48, and 72 h. Intestinal digestibility was measured using the rumen undegraded feed residues after 16 h of rumen incubation.

We measured degradability of DM and CP in the rumen by using the nylon bag technique (Ørskov and McDonald, 1979). Nylon bags (made of dacron cloth, approximately 8 × 15 cm; 45-μm pore size; sample size: surface area = 16.7 mg/cm2) were filled with 5g of dry ground feed samples and were tightly sealed with a heat-sealed. The nylon bags were placed in larger polyester mesh bags (25 × 40 cm; 3 mm pore size) and tied with a rubber band. The nylon bags were presoaked in warm water (40°C) for 15 min before rumen incubation. Each feed sample containing nylon bag was incubated in the rumen of a steer for 0, 2, 4, 8, 16, 24, 48, and 72 h; there were nine replicates (three replicates per each steer). Time zero represented bags that were not incubated, but treated in the same manner as the other bags. The nylon bags were removed at their assigned incubation time, then immediately gently rinsed with cold tap water to stop microbial activation, washed in a washing machine for 30 min, and followed by dried in a forced-air oven at 60°C for 48 h until constant weight was achieved before determination of residual DM. Each bag was weighed and the feed sample residues were analyzed for CP using standard methods (AOAC, 2000). The loss of nutrients at each incubation time was calculated relative to the original nutrient content of the feed samples that were not incubated and washed. The feed samples were mild ground to pass through a 1-mm screen before the AA analysis.

4 In situ intestinal incubation of tested feeds

The small intestinal digestibility of AA and CP was determined using the mobile bag technique as described by De Boer et al. (1987). Mobile bags with an approximately 3 × 4.5 cm (made of dacron cloth, 35-μm pore size; sample size: surface area = 37.04 mg/cm2) were filled with 0.5 g of dry ground feed samples and heat-sealed. The mobile bags were placed in a larger polyester mesh bag (25 × 40 cm; 3 mm pore size) and tied with a rubber band. The mobile bags were presoaked in warm water (40°C) for 15 min before rumen incubation. Each feed sample containing mobile bag was pre-incubated in the rumen of a steer for 16h; there were nine replicates (three replicates per each steer). Following rumen incubation, all mobile bags are remove from the rumen, and were hand washed until the rinse water remained clear and then mobile bags were combined with in a 0.004 M HCl solution containing 1 g of pepsin/L (pH 2.4) for 3 h in a 39°C water bath stirring to simulate abomasum digestion. Following the pepsin-HCl incubation, mobile bags were rinsed with distilled water to wash out the pepsin-HCl solution. Subsequently, mobile bags were inserted every 10 min into the duodenum through the T-shaped cannula (Varvikko and Vanhatalo, 1993). After 36 h, Mobile bags were collected from the manure and hand washed until the rinse water remained clear and then dried in a forced-air oven at 60°C for 48 h until a constant weight was achieved before determination of residual DM. Each bags were weighed, digestibility of DM was determined and expressed on DM basis, and feed sample residues were analyzed for CP. Disappearance of CP was expressed relative to the original CP content of the feed samples. The feed samples were mild ground to pass through a 1-mm screen before the AA analysis.

5 Calculations and statistical analysis

The percentages of DM, CP, EE, and AA after a 16 h-incubation in the rumen were calculated as the difference between the feed samples and the portion remaining after incubation in the rumen, intestine, and total tract. CP degradation values in the rumen were calculated according to Ørskov and McDonald (1979), with a passage rate of 5%/h (Van Straalen and Tmminga, 1990). The measured percent CP after the 16 h incubation in the rumen was calculated as the difference between the samples. The rumen degraded protein (RDP) and rumen undegraded protein (RUP) were determined according to the following two equations (NRC, 2001):

where:

where:

Differences in DM, CP, EE, and individual AA digestion rates between the feed samples in the rumen and intestine were calculated according to Ørskov and McDonald (1979) using the PROC GLM procedure of SAS, version 9.1 (SAS Institute Inc, Cary, NC, USA) with one-way ANOVA. Duncan’s multiple range test was used to comparison of means. Experimental results presented are expressed as mea ± standard error from independent measurements. A p-value less than 0.05 was considered statistically significant.

Ⅲ. RESULTS AND DISCUSSION

1 Chemical and AA composition of tested feeds

The chemical and AA compositions of the tested feeds are presented in Table 1. All tested feeds had a CP content was less than 20%. Compared with the other feeds, the bran CGF and food-processing residue BG had the higher CP contents, while the bran RB and food-processing residue SMSP had the lower CP contents. The percentage of NDF in the feeds was highest for the bran CGF and food-processing residue SMSP. The CP, EE, NDF, and ADF compositions of all of the tested feeds were similar to those tabulated by NRC (2001) (Table 1). Interestingly, the percentages of NDF was higher CGF in brans and SMSP in food-processing residues than in the other feeds.

Compared with the other feeds, the bran CGF and food- rocessing residue TR had the highest levels of the Lys, while the Met levels were highest in the bran RB and CGF and food-processing residue BG. The percentage of AA of all tested feeds were similar to those reported by the NRC (2001). Overall, these results suggest that CGF, TR, and BG have a high potential bioavailability in the rumen digestive system.

2 In situ rumen degradation

The CP residues at each incubation times are presented in Table 2. The bran RB and food-processing residue SMSP had the highest CP content, except for 0 and 2 h. In the last 72 h incubation time, the CP residue content was relatively low in the bran WB and CGF than in RB, and in the food-processing residues TR and BG than in SMSP (Table 2). Interestingly the CP content of SMSP did not change with the length of rumen incubation. Because the main component of SMSP is sawdust (lignocellulosic materials) (Terashita and Kono, 1984). Previously study showed that CGF and BG are good protein sources for growing and finishing cattle (Preston et al., 1973; Firkins et al., 2001). These results suggest that the low residual CP content of the bran CGF and food-processing residue TR has higher potential bioavailability than the other feeds.

RDP provides a mixture of peptides, free AA, and ammonia for microbial growth and the synthesis of microbial proteins, and RUP is important source of absorbable AA for ruminant animals (NRC, 2001). Therefore, sufficient RUP should pass through the rumen unchanged and be absorbed in the small intestine of the host animal (NRC, 2001). Table 3 shows variations in the CP fraction, RDP, and RUP of the tested feeds. For the brans, WB had a low soluble A fraction and high degradable B fraction of CP, RB had an intermediate soluble A fraction and high degradable B fraction, and CGF had a high soluble A fraction and low degradable B fraction. For the food-processing residues, SMSP and BG had relatively low soluble A fractions (17.7% and 16.8%, respectively) and high degradable B fractions (138.3% and 73.7%, respectively), while TR had an intermediate soluble A fraction and high degradable B fraction. The degradation rate of the degradable B fraction (Kd) did not differ among the feeds. The bran RB had the lowest RDP and highest RUP content, but was no observed significant different (p>0.05). The SMSP had the lowest RDP and highest RUP content but was no observed significant different (p>0.05), which might have resulted from the NDF content (Table 1). The variation in rumen degradation could arise from differences in the physical properties (solubility, structure, etc.) of the feed, and from limitations of the experimental apparatus, species studied and rumen environment (Van Straalen et al., 1997; Clark et al., 2010).

3 In situ mobile bag digestion in the rumen

In particular, the bran RB and food-processing residue SMSP were resistant to degradation in the rumen (Table 4). For the brans, ruminal degradation of CP had highest in CGF and lowest in RB (p<0.05). For the food-processing residues, ruminal degradation of CP had highest in TR and lowest in SMSP (p<0.05). For the brans, the ruminal degradation of Lys was higher in CGF than in RB and WB (p<0.05). For the food-processing residues, the ruminal degradation of Lys was higher in BG and TR than in SMSP (p<0.05). For the brans, the ruminal degradation of Met was highest in CGF, intermediate in WB, and lowest in RB (p<0.05). For the food-processing residues, the ruminal Met degradation was higher in TR compared than in SMSP (p<0.05). In the tested feeds, His and Met were degraded the most, Arg, Lys, Val, and Thr were degraded by intermediate amounts, and Ile, Leu, Phe, and Thr were degraded the least in the rumen. Many studies agree with these findings and suggest that differences in the ruminal degradation of AA among feeds are influence by the protein class (albumin, globulin, etc.) and various physical properties (Messman and Weiss, 1994; van Straalen et al., 1997; Maxin et al., 2013).

4 In situ mobile bag digestion in the intestine

The intestinal digestion of CP and AA contents are presented in Table 5. For the brans, the intestinal digestion of CP was highest in RB and lowest in CGF (p<0.05). For the foodrocessing residues, the intestinal digestion of CP did not differ significantly among the feeds. For the brans, the intestinal digestion of Lys was highest in RB and in WB, and lowest in CGF (p<0.05). For the food-processing residues, the intestinal digestion of Lys was higher in SMSP than in TR and BG (p<0.05). For the brans, the intestinal digestion of Met was highest in RB and lowest in CGF (p<0.05). For the foodrocessing residues, the intestinal digestion of Met did not differ significantly (p>0.05). Lys and Met have been recognized as limiting AA (NRC, 2001). Mjoun et al. (2010) suggested that the AA composition of the RUP fraction is important in formulating feeds. Our results showed that intestinal digestion of AA in the tested feeds followed the intestinal degradation of CP. These variations might affect the CP and AA contents in RUP after rumen exposure (Gao et al., 2015).

5 In situ mobile bag digestion in the total tract

Table 6 summarizes digestion of CP and AA for the total tract. For the brans, the total tract digestion of CP did not differ significantly among the feeds. For the food-processing residues, the total tract digestion of CP was significantly higher in TR and BG than in SMSP (p<0.05). The total tract digestion of Lys was significantly higher in the bran WB, and in the food-processing residues BG and TR, compared with the other feeds (p<0.05). The total tract digestion of Met was significantly higher in the bran WB and CGF, and in the food-processing residues TR and BG, compared with the other feeds (p<0.05). The mobile nylon bag technique gives an estimate of true digestibility, and the in vivo digestibility is probably slightly higher than our results (Von Keyserlingk and Mathison, 1989). In addition, our results showed that the total tract degradation of AA for all feeds was slightly different from the sum of the ruminal and intestinal degradation results (Tables 4 and 5). Microbial contamination of the RUP might result in a different AA profile of feeds after ruminal incubation and processing (Yoon et al., 1996; Boucher et al., 2009). Notably, the estimated intestinal degradation rate of the bran RB and food-processing residue SMSP were relatively higher that the estimate ruminal degradation rate.

6 In situ mobile bag digestion in the rumen, intestine, and total tract

The Lys and Met (g/kg of CP) absorbance of the tested feeds in the rumen, intestine, and total tract are shown in Table 7. The ruminally absorbable Lys was significantly higher in the bran CGF and food-processing residues TR and BG compared with the other feeds (p<0.05). The intestinally absorbable Lys did not differ significantly among the brans, while for the food-processing residues it was significantly higher in TR and BG compared than in SMSP (p<0.05). For the brans, the total tract absorbable Lys was highest in CGF and equal in RB and WB, while for the food-processing residues was higher in BG and TR than in SMSP (p<0.05). For the brans, the ruminally absorbable Met was highest in CGF and lowest in RB and WB (p<0.05). The intestinally absorbable Met was highest in RB, followed by WB and CGF in brans, and for the food-processing residues, BG was significantly higher than in TR and SMSP (p<0.05). For brans, the total tract absorbable Met was highest in CGF and lowest in RB and WB. For food-processing residues, the total tract absorbable Met was highest in BG, followed by TR and lowest in SMSP (p<0.05). The formulation of diets for suitable RUP requires low digestibility of AA in the rumen and high digestibility of AA in the intestine and total tract (NRC, 2001). Although we observed some irregular ruminal, intestinal and total tract absorbability of AA in the tested feeds, the bran RB and food-processing residue BG have high proportions of Lys and Met for ruminant feeds.

Our study showed that the ruminal degradation and intestinal digestion of CP, RDP, RUP, and AA in the tested feeds (RB, WB, CGF, TR, SMSP, and BG) varied substantially. For the brans, RB had low CP degradability in the rumen and high intestinal digestibility of Lys and Met. Similarly, the foodrocessing residue BG had high intestinal digestibility of Lys and Met. Present studies suggested that based on the total tract absorbability of Lys and Met, the bran RB and food-processing residue BG are suitable supplemental feedstuffs for RUP for Hanwoo cattle. Concluded that these results can be used as baseline data for ruminant ration formulation, thereby achieving a better balance of AA in ruminant feed, and reduce feed costs as well as environmental pollution.