Abstract: This study was conducted to evaluate the effect of oak leave tannin (OT, 25 and 45 g kg^-1 DM) on in vitro gas production and fermentative parameters of Soybean Meal (SB). Treatments were SB treated with 25 and 45 g kg^-1 oak leave tannin (OT1 and OT2, respectively). The results of experiment showed that oak leave caused to reduce the fermentable fraction (b) and gas production rate Constant (c) of SB (p<0.05) and the lowest (b) and (c) was for SB treated by 45 g kg^-1 oak leave tannin (104.5 mL, 0.04 mL h^-1, respectively). Soybean meal treated with OT2 had the lowest the Organic Matter Digestibility (OMD) and Metabolizable Energy (ME) (153.3 g kg^-1 OM, 13.6 MJ kg^-1 DM, respectively). Concentration of ammonia-N (NH₃-N) and Short Chain Fatty Acid (SCFA) decreased (p<0.05), when SB treated with OT and the highest NH₃-N and SCFA concentrations was for untreated soybean meal (36.6 mg dL^-1 and 0.88 μmol). Therefore, it may be that in vitro rumen fermentation and nutritive value of soybean meal are influenced by tannin content of oak leave.

INTRODUCTION

Approximately 3 million ha of forest are covered by various oak species, in the north-west of Iran (Fatahi, 1995) that leaves used as important source of forage. Soybean meal is used as supplemental protein in dairy rations and is classified as highly degradable. It is reported that oak is containing the high level of tannin and tannin used as chemical additives for protecting and decreasing ruminal degradation of protein sources such as soybean meal because bind with protein (El-Waziry et al., 2007). At normal pH of the rumen, protein remains bound to the tannin but at the low pH of abomasum, the protein is released, so protein becomes available for digestion in the small intestine (El-Waziry et al., 2005).

The in vitro gas production is more efficient than the in sacco method in evaluating the effects of tannins (El-Waziry et al., 2007). Effects of tannin from different sources in reducing ruminal gas production and ammonia levels have been reported but there is not enough information on the effect of oak tannins on rumen fermentation of soybean meal.

Therefore, the aim of this study was to estimation the effect of oak leave tannins (OT, 25-45 g kg^-1 DM, respectively) on rumen degradation of soybean meal by in vitro gas production.

MATERIALS AND METHODS

In vitro Gas Production (GP) technique was conducted according to the Menke and Steingass (1988). Rumen fluid was supplied from two fistulated sheep were fed a 40:60 concentrate: forage (3 kg concentrate: 2 kg alfalfa hay and 4.5 kg corn silage) in prior to the morning meal, homogenized in a laboratory blender filtered through three layers of cheese-cloth and purged with CO₂. The well mixed and CO₂ flushed rumen fluid was added to the buffered rumen fluid solution (1:2 v/v), which was maintained in a water bath at 39°C. Experimental samples were Untreated SB (USB), SB treated with 25 and 45 g kg^-1 oak leave tannin (SB + OT1 and SB + OT2, respectively). Tannin content of leave were determined by using Folin-Ciocalteu reagent in calorimetric method (Makkar and Singh, 1991). About 200 mg experimental sample (1.0 mm screen) incubated with 35 mL buffered rumen fluid under continuous CO₂ reflux in 100 mL calibrated glass syringes for 2, 4, 6, 8, 10, 12, 16, 24, 48, 72 and 96 h in a water bath maintained at 39°C. Samples were incubated in triplicate together with three syringes containing only incubation medium (blank).

After 96 h of incubation, the medium of each syringe used for determination ammonia-N (NH₃-N) concentration using distillation method (Kjeltec 2300 Autoanalyzer, Foss Tecator AB, Hoganas, Sweden). Cumulative gas production data were fitted to the exponential equation:

Where:

The values of Organic Matter Digestibility (OMD) and Metabolisable Energy (ME) of samples were calculated by the equation of Menke and Steingass (1988), OMD (g kg^-1 OM) = 148.8 + 8.89 GP + 4.5 CP + 0. 651 XA and ME (MJ kg^-1 DM) = 2.20 + 0.136 GP + 0.057 CP + 0.0029 CP². Short Chain Fatty Acids (SCFA) were determined by the equation reported by (Getachew et al., 1999). SCFA (Fmol L^-1) = 0.0239 GP B 0.0601. CP and XA were crude protein and ash and GP was the net gas production after 24 h incubation.

Data of in vitro gas production, ME, OMD, NH₃-N and SCFA were subjected to analysis as a completely randomized design using the General Linear Model (GLM). Duncan’s multiple range test was used to compare treatment means at (p<0.05).

RESULTS AND DISCUSSION

Tannin content of oak leave was 39 g 100^-1 DM, respectively. Oak leave tannin decreased in vitro gas production parameters, ME, OMD, SCFA (Table 1) and NH₃-N (Fig. 1) of soybean meal (p<0.05). Soybean meal treated with oak 45 g kg^-1 DM leave had the lowest b and c fractions. This result reported by El-Waziry et al. (2007) for soybean meal treated by querbracho tannin.

Tabacco et al. (2006) reported that tannins significantly depressed gas production, probably hampering rumen microorganisms. Also the result of Bento et al. (2005) showed reduction in gas production by mimosa tannin may be associated with binding of it to microorganisms or their enzymes but McMahon et al. (2000) concluded that tannins do not simply inhibit cellulose digestion but the inhibitory effects of tannins involved the bacterial cells themselves.

The estimated ME, OMD and SCFA were decreased when SB was treated by oak leave and this decrease was maximum for SB treated by 45 g kg^-1 DM. Reducing these parameters reported by Mohammadabadi et al. (2010) for sunflower meal treated with tannic acid. Also Tabacco et al. (2006) explained that OM digestibility decreased by about 5.1% with tannin. Reduction of volatile fatty acid reported by EL-Waziry et al. (2005). The decrease in OMD and ME was probably due to decreased rumen degradability and formation of complexes between tannins and dietary proteins and carbohydrates, as well as reducing rumen microbial proteolytic and cellulolytic enzyme activities, general fermentative activities (Muhammed et al., 1994).

Table 1:	Gas production parameters of soybean meal treated with tannin of oak leave
USB: Untreated SB; SB + OT1: SB treated with 25 g kg-1 oak leave tannin; SB + OT2: SB treated with 45 g kg-1 oak leave tannin b: gas production from fermentable fraction; c: gas production rate constant; OMD: Organic Matter Digestibility; ME: Metabolizable Energy; SCFA: Short Chain Fatty Acid; SEM: Standard Error of Mean, means within each column with different letters are significantly different (p<0.05)

Fig. 1:	The effect of tannin of oak leave on in vitro NH3-N concentration of soybean meal

Tannin caused to altered microbial profiles in the fermentation, reduced microbial numbers and or enzyme production from the microbes (Apajalahti et al., 2004) and reduced microbial degradation of carbohydrates (Muhammed et al., 1994).

When SB treated with tannin of oak leave, NH₃-N concentration decreased that was in agreement with researches of Sliwinski et al. (2002). The lower ammonia concentrations by tannin were mainly due to reduce proteolysis, degradation of peptides and deamination of amino acids in the rumen (Newbold et al., 1990).

CONCLUSION

In the study, the treatment of soybean meal by 25 and 45 g kg^-1 tannin of oak leave decreased degradation and gas production, OMD, ME, SCFA and NH₃ and the highest decrease was observed for soybean meal treated by 45 g kg^-1 tannin of oak leave.