Energy value of ruminant feeds and its bio-availability is of great importance for animal feed manufactures and end users. The amount of available energy in feeds for ruminants is described either by its metabolisable energy or by organic matter digestibility (Barber et al., 1990), since the value of organic matter digestibility is very close to the corresponding digestibility of energy (Thomas, 1990). The most accurate way of obtaining information on digestibility of organic matter of feeds for ruminants is by conducting in vivo digestibility experiments. Since this method is expensive, time-consuming and therefore is not suited for routine analysis, there has been a constant search for laboratory methods for routine prediction of the in vivo organic matter digestibility of ruminant feeds, in order to implement an adequate system of quality control in the feed industry.

In vitro methods used to predict the in vivo digestibility of organic matter of feeds for ruminants are generally based on: chemical analysis, fermentation with rumen micro-organisms and hydrolysis with enzymatic preparations.

Prediction of organic matter digestibility of forages using chemical analysis by Weende method or Acid detergent fibre method resulted in variable correlations (Aufrère & Michalet-Doreau, 1988).

Methods for predicting in vivo organic matter digestibility using rumen liquor fermentation techniques have become well established, but there are limitations on the use of rumen liquor for digestibility studies. There must be fistulated animals (which are not available to all laboratories) for the collection of fresh rumen liquor. There is also a concern over the use of surgically modified animals in experimentation (Jones & Theodorou, 2000).

The use of incubation of feeds with exogenous enzymes to predict the in vivo organic matter digestibility has the aim to mimic the digestive process in the animal. Most enzymatic methods for organic matter digestibility estimation have been developed for forage feedstuffs, with a few used for other feedstuffs, e.g. grains and compound feeds (Aufrère & Michalet-Doreau, 1988). Weisbjerg and Hvelplund (1993) developed a multi-enzymatic incubation method for estimation of enzymatic digestibility of organic matter for use on compound feeds. This procedure also showed an ability to estimate the organic matter digestibility of straws (Hvelplund, Weisbjerg & Søegaard, 1999), which demonstrated the potential of this method to predict the in vivo organic matter digestibility of grains and forages. Apart from the above-mentioned two references, more citations of this method the literature have not been found.

The aim of this study was to introduce this under-exploited in vitro enzymatic method for the determination of organic matter digestibility (OMD) of feed in the laboratory practise and to determine some of the method characteristics (accuracy and precision), in order to evaluate the possibility of its use for the development of near infrared spectroscopy calibration model for prediction of the in vivo OMD.

The in vitro method of Weisbjerg and Hvelplund (1993) used in this study consists of several successive steps: treating of dried and grounded sample with pepsin-hydrochloric acid to dissolve the protein content, hydrolysis of starch and inactivation of pepsin at 80 °C, incubation the sample with the mixture of enzymes to dissolve digestible carbohydrates from cell walls, extraction of fat with boiling water and acetone and determination of dry matter content and ash of the undissolved residue.

The set of five reference samples with the known values of organic matter digestibility (OMD) were used. They comprises of different types of feed such as concentrate mix, palm kernel cake, sunflower meal, barley and straw, with the range of OMD values from 35,3 to 91,7%. The reference values were determined by the Department of Animal Health, Welfare and Nutrition (Faculty of Agricultural Sciences, University of Aarhus), Tjele, Denmark.

0.25 g of grounded sample with 1 mg accuracy was measured in a 30-ml filter crucible porosity 2 and 15 ml of pepsinhydrochloric acid solution was added. The crucibles were closed with rubber plugs and incubated for 24 hours at 40 °C. Upon the expiration of this period the crucibles were incubated at 80°C for 45 minutes. The plugs from the crucibles were removed and the liquid was sucked away. The content of the crucibles were washed with 100 ml of water. Fifteen ml of buffered enzyme solution, consisting of Amyloglukosidase (activity 3260 Uml-1) and commercial enzymes: Celluclast 1.5 L FG (activity 798 EGU g-1), Novozym 51054 (activity 1000 KVHCU(m) g-1), Vyscozyme L (activity 120 FBG ml-1, was added to the content of the crucibles. They were firstly incubated at 40°C for 24 hours, and then at 60 °C for 19 hours. Upon the expiration of this period the liquid from the crucibles were sucked away, and the content of the crucibles were firstly washed with 100 ml of boiling water, and then with 20 ml of acetone. The crucibles with the content were dryed at 103°C until a constant weight was reached. After drying the crucibles were cooled down in the desiccator and immediately weighted. The content of the crucibles were then incinerated at 475-500 oC until a constant weight is achieved. A blank correction without matter was included. In order to calculate the OMD, the crude ash content of the samples was determined by incineration at 500 oC.

It is to be pointed out that the original in vitro OMD method (Weisbjerg and Hvelplund, 1993) used 100-ml filter crucible, 0.5 g of sample and 30 ml of enzyme buffer solution, whereas in this study 30-ml crucibles were used, which required decreasing of sample mass to 0.25 g and enzyme buffer volume to 15 ml.

OM= 100 - Crude ash (%)
Insoluble OM= (b − c) ∗100
a
OMD = (OM- Insoluble OM)* 100
OM

Statistical evaluation

RESULTS AND DISCUSSION

Accuracy

Precision

CONCLUSIONS

ACKNOWLEDGEMENTS