The effect of different sources of xylanase on in vitro dry matter digestibility measured by the filter bag method *

The effect of including two sources of xylanase (produced by Trichoderma viride or rumen microorganisms, Megazyme, Ireland)) into the incubation solution on in vitro DM digestibility (IVDMD) determined by the cellulase-pepsin method using filter bags and a Daisy" Incubator (Dl; Ankom Co, Fairport, NY) was studied. The source of xylanase did not significantly affect IVDMD. Moreover, irrespective of the concentration used, none of the xylanases improved IVDMD determination.


1NTRODUCT10N
Every year advances in biotechnology make new enzymes available for animal nutrition, including enzymes that can be used effectively in determining in vitro DM digestibility (IVDMD) of feedstuffs.Compared to the traditional Tilley-Terry method (1963), the enzymatic ones do not reąuire cannulated animals as rumen fluid donors.They are also rapid and economical (Adesogan et al., 2000).Cellulase is the most widely and successfully used enzyme in in vitro studies (De Boever et al., 1988;Aufrere and Guerin, 1996).However, sińce the enzymatic activity of the rumen ecosystem is very complex, it seems probable that the introduction of other fibrolytic enzymes, such as xylanase, could improve the precision of the in vitro method (better prediction and reproducibility).Xylanases belong to the glucanase enzyme family that breaks down various xylans to produce short-chain xylooligosaccharides.The aim of this study was to determine the effect of including xylanase from two sources in the incubation solution on IVDMD determined by the cellulase-pepsin method using filter bags and a Daisy" Incubator (DI; Ankom Co, Fairport, NY).

MATERIAŁ AND METHODS
In vitro DM digestibility (IVDMD) was determined on 5 forage samples (3 grass hays, 1 grass silage and 1 maize silage) ground to pass through a 1.5 mm screen.The silages were dried in a forced-air oven for 48 h at 50°C.Their chemical composition was determined by standard methods (AOAC, 1995).About 0.5 g of a feed sample was placed in a filter bag (Ankom F57; 50 * 55 mm) made from polyester-polyethylene extruded filaments of pore size about 25 pm.There were 4 bags for each feed.IVDMD was determined by three methods: cellulase-pepsin (C-P), cellulase+xylanaseA-pepsin (CXA-P), and cellulase+xylanaseB-pepsin (CXB-P).In each method the samples were first incubated for 48 h in cellulase (Onozuka RIO, Trichoderma viride; Yakult Honskha LTD, Tokyo, Japan) solution and then for 24 h in pepsin-HCl (Merck 2000 FIP U/g art.7190; 0.2% solution (P/V) in 0.1 N HC1) solution.Both incubations were performed in DI jars at 39.5°C.The solutions were prepared according to Aufrere and Graviou (1996).In the CXA-P and CXB-P methods the cellulase solution was supplemented with either Ml (Endo-Beta Xylanase, Trichoderma sp., Megazyme, Ireland) or M6 (Beta Xylanase from rumen microorganisms, Megazyme, Ireland) at a rate of 700, 1400 and 450, 900, 1350, 5400 U/L, for Ml and M6, respectively.According to the producer, the specific activity of xylanase Ml and M6 was 157 and 230 U/mg, respectively.After the pepsin stage the bags were rinsed with tap water and dried at 80°C in an oven to a constant weight.The results of IVDMD were subjected to two-way (feed x xylanase concentration) analysis of variance and the Scheffe rangę test (SAS, 1996), separately for each source of xylanase.The results of the C-P method were used as the xylanase 0 level.

RESULTS AND DISCUSSION
The chemical composition of the feeds is presented in Table 1.The wide rangę of crude protein (7.58-16.50% in DM) and NDF (36.71-70.04%)concentrations makes the comparison reliable.None of the xylanases under study, irrespective of the concentration used, increased IVDMD over the values obtained for cellulase alone (Tables 2 and 3).When used in the highest concentration (5400 U/L), the xylanase of rumen microorganisms (M6) even decreased IVDMD (PO.001).Neither of the xylanase sources showed a significant interaction between feeds and the concentration of the xylanase in the incubation medium.The reason for the lack of effect of xylanase on IVDMD is not elear sińce both xylanases are supposed to break down the hemicellulose fraction of the feeds.The enzymes cellulase and xylanase do not compete for the substrate, and addition of the latter should have increased IYDMD.It is possible that the pH rangę of the buffer used for cellulase is not suitable for the xylanases studied here.A destructive effect of cellulase on xylanase seems improbable.
The IVDMD values obtained in the study are lower than in vivo dry matter digestibility coefficients (data not presented).However, when the same feed samples where incubated in water only, dry matter disappearance was in the rangę of about 20%, indicating the presence of cellulase and pepsin activities inside the bags.

CONCLUSIONS
It is concluded that the source and concentration of xylanase did not significantly affect in vitro DM digestibility (IVDMD).Moreover, using a mixture of enzymes (cellulase + xylanase) has no advantage over the use of cellulase alone in IVDMD determination.

TABLE 1
Chemical composition of the feedstuffs, % DM