THE POSSIBILITY OF IN VITRO MULTI-ENZYMATIC METHOD APPLICATION FOR THE ASSESSMENT OF THE INFLUENCE OF THERMAL TREATMENTS ON ORGANIC MATTER DIGESTIBILITY OF FEED FOR RUMINANTS

In vitro multi-enzymatic method is a two-step procedure that uses exogenous enzymes for incubation of feed that aim to imitate digestive processes in the animal. It is used for determination of organic matter digestibility (OMD) of various feedstuffs and complete mixtures used in ruminant nutrition. The aim of the present work was to determine whether this in vitro multi-enzymatic method can be applied for the assessment of the influence of thermal treatments on OMD of feed for ruminants. Samples of corn and complete mixture for cows (based on corn) were subjected to pelleting, steam flaking, micronization and extrusion. Statistical analysis of the results did not show significant increase of corn and complete mixture OMD after various thermal treatments compared to untreated samples, which can be explained by high digestibility of corn itself (exceeds 90%). Based on obtained results it can be concluded that applied in vitro method is not suitable for the assessment of the influence of thermal treatments on OMD of corn and corn based mixture, while future research should be broadened to analysis of samples with lower OMD, such as sorghum, oats and barley.


INTRODUCTION
Animal feed is analyzed in order to obtain information on its value as a source of nutrients.When there is a possibility in vivo measurements are performed, but it requires animals, relatively large amount of feed and time for testing.In vivo measurements are expensive, and in addition, it is necessary to invest intensive work that does not make them suitable for routine analysis.A possible solution to this problem is fast and cheap laboratory methods for routine prediction of in vivo organic matter digestibility (OMD) in ruminant nutrition.There are several laboratory procedures that are used to predict OMD of feed for ruminants, that are based on chemical analysis or biological methods that simulate the digestion process.Getachew et al. (1998) list three major digestion techniques (biological methods) currently available for determining the nutritional value of ruminant feeds: 1) digestion with rumen microorganisms, 2) hydrolysis with enzymatic preparations, 3) in situ incubations of samples in nylon bag in the rumen.
In vitro techniques are based on the incubation with a microbial inoculum from the rumen which is used as a digestive medium.According to Gosselink et al. (2004) most accepted in vitro techniques are Tilley and Terry method (Tilley and Terry, 1963) and gas production technique developed by Menke and Steingass (1988).The disadvantages of the aforementioned methods arise from the problems associated with inoculum use.Rumen liquid must be fresh which implies the existence of animals with fistulas (which are not available to all laboratories) because the activity of the microorganisms is lost or changed during liquid storage.There are many factors that can influence the obtained results such as the variation in the quality of the rumen liquid that can occur due to the species and diet of the animal from which the inoculum is taken, the collection time and to what extent the anaerobic conditions, optimal pH and temperature are maintained (Jones and Theodorou, 2000;Adesogan, 2002;Lopez, 2005).These disadvantages can be avoided by using exogenous enzymes for incubation of feed that aim to imitate digestive processes in the animal.The use of enzymes as an alternative to a rumen inoculum has the advantage of overcoming the need for fistulated animals and anaerobic procedures, simplifying analytical methodology and eliminating variability in inoculum activity (Lopez, 2005).Weisberg and Hvelplund (1993) developed in vitro multi-enzymatic method for determining OMD of concentrated feedstuffs that are used in ruminant nutrition.The principle of OMD determination consists primarily in the incubation of a sample in acidic pepsin solution for 24 hours in order to degrade the proteins.The mixture is then heated to 80 °C in order to inactivate pepsin and create conditions for starch hydrolysis.After rinsing with water, the sample is incubated in a mixture of enzymes, resulting in the degradation of starch and digestible carbohydrates from cell walls.The insoluble residues are washed with warm water and then with acetone for the purpose of extracting the fat.For the calculation of in vitro OMD it is necessary to determine the content of dry matter and ash in the indigestible residue of the sample.Over the years, the use of this method has been confirmed and the prediction of in vivo OMD of straw (Hvelplund et al., 1999), various feedstuffs (Palić and Muller, 2006) and complete mixtures (Palić and Leeuw, 2009), as well as for the calibration of NIR device (Pojić et al., 2008).In aforementioned papers, the multi-enzyme method proved to be equally reliable in predicting in vivo OMD as well as the in vitro method Tilley and Terry (1963).Also, this method is the official method in Denmark for assessing in vivo OMD of concentrated feedstuffs and complete feed (Akerlind et al., 2011) using the formula: To the best of our knowledge no research was found where thermally treated samples of feed were analyzed using this in vitro method.Therefore, the aim of the present work was to determine whether this in vitro multi-enzymatic method can be applied for the assessment of the influence of thermal treatments on OMD of feed for ruminants.

MATERIAL AND METHODS
Corn variety ZP666 grown in Serbia was used in this research.Using computer program "MIXOLINO" complete mixture for dairy cows was formulated with the aforementioned corn as the only grain in the mixture.Composition of complete mixture for dairy cows is presented in Table 1, while Table 2 presents basic chemical composition of corn and complete mixture.
In the first part of the experiment corn was subjected to pelleting, steam flaking, micronization and extrusion.Prior to pelleting and extrusion corn was milled using hammer mill equipped with a 4 mm sieve, while steam flaking and micronization were conducted on whole corn grain.All samples were conditioned prior to selected thermal treatment except for one (sample MC1, see Table 3).In the second part of the experiment same thermal treatments were applied on complete mixture.Prior to pelleting and extrusion all components of complete mixture were milled, then mixed and conditioned.Since the steam flaking and micronization are possible to apply only on whole corn grain, complete mixture was formed of steam flaked or micronized corn and with the addition of the rest of diet components that were previously mixed and pelleted.Applied temperatures during thermal treatments are presented in Table 3.
In vitro OMD of untreated and thermally treated corn and complete mixture was determined using multi-enzymatic method previously described by Kokić et al. (2013).

Statistical analysis
One-way ANOVA and Tukey HSD test were used to analyze variations of the results.Differences between the means with probability p<0.05 were accepted as statistically significant.The level of confidence was set at 95% (STATISTICA 10.0, StatSoft Inc., Tulsa, OK, USA).

RESULTS AND DISCUSSION
In vitro OMD of corn grain and complete mixture for dairy cows subjected to different thermal treatments is presented in

CONCLUSIONS
According to the results obtained in this study it can be concluded that applied in vitro method is not able to detect the change in OMD of corn after thermal treatments, which can be explained by high digestibility of corn itself (exceeds 90%).In future research, samples with lower OMD should be analyzed, such as sorghum, oats and barley, in order to determine whether this in vitro multi-enzymatic method can be used for the assessment of the influence of thermal treatments on OMD of feed for ruminants.

АCKNOWLEDGEMENTS
This paper is a result of the research within the project III46012 financed by the Ministry of Education, Science and Technological Development, Republic of Serbia.

Table 1 .
Composition of complete mixture for dairy cows

Table 2 .
Chemical composition of corn and complete mixture for dairy cows

Table 4
it can be concluded that various in vitro methods are not able to detect the change in OMD after thermal treatments of samples that has high digestibility (such as corn) and that in such ca-ses other methods should be used, preferably in vivo method.