Effects of Enzyme Application Method and Levels and Pre-treatment Times on Rumen Fermentation , Nutrient Degradation and Digestion in Goats and Steers

Present study investigate the effect of enzyme supplementation, methods (applied to rumen or enzyme treated diet) compared with no enzyme diet, on rumen fermentation and apparent nutrient digestibility in a 3×3 Latin square design with three rumen cannulated Korean Native goats. In situ rumen degradation kinetics was studied in three rumen cannulated Holstein steers. Three diets were, no enzyme, 1% enzyme in rumen and 1% enzyme in diet. The enzyme was sprayed onto forage, and the forage: concentrate ratio was 30:70. Degradation kinetics was studied with three enzyme levels (0, 1 and 2%, w/w) and four pre-treatment times (0, 1, 12 and 24 h). Results suggested that enzyme application method did not affect rumen fermentation, ruminal enzyme activity and total tract apparent digestibility. Nutrient degradation rate and effective degradability of DM, NDF and ADF increased with increasing enzyme level and pre-treatment times. Degradation of nutrients was affected by enzymes levels or pre-treatment times. Therefore, it is probable that the improved degradation may be due to the supplemented exogenous hydrolytic enzymes under a certain condition. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 3 : 389-393)


INTRODUCTION
Enzymes supplements are intensively used to improve the nutritive value of feeds and to remove anti-nutritional factors from feeds for non-ruminants, particularly in broiler diets.However, the use of enzymes in ruminant diets is not common and a technology yet to develop.Beauchemin and Rode (1996) reviewed that studies supplementing dietary enzymes for cattle and sheep improved feed digestibility and animal performance in 1960s, but many of these early preparations were poorly defined and results were often inconsistent.Recently the use of feed enzymes for ruminants showed variable animal responses; the use of feed enzymes improved cattle performance (Beauchemin et al., 1995;Feng et al., 1996;Stokes and Zheng, 1995;Treacher et al., 1997), but no improvement was observed (Chen et al., 1995).Therefore, apparently, the inconsistent results from those studies can be attributed to several factors, including diet composition (Yang et al., 2000), type of enzyme preparation (Morgavi et al., 2001), complement of enzyme activities (Wallace et al., 2001), amount of enzyme (Beauchemin et al., 1997), enzyme stability (Hristov et al., 1998), and method of application (Yang et al., 2000).Several researches demonstrated that enzymes are less effective if infused directly into the rumen than applied directly to feed (Lewis et al., 1996;McAllister et al., 1999;Wang et al., 2001).Beauchemin et al. (2001) recently reviewed that increasing the level of enzyme supplementation is usually not beneficial.Applying enzymes to feed prior to feeding enhances ruminal fibre digestion by altering the structure of the feed thereby making it more susceptible to degradation.In addition, cell wall degrading enzymes are added to the silage to provide more fermentable sugar for silage fermentation and to improve silage DM digestibility (Kung, Jr. et al., 1991;Ridla and Uchida, 1999).In commercial feeding, processed forage and grains are stored prior to feeding, providing an ideal opportunity for the use of enzyme products (Beauchemin et al., 1995).
This study aims to investigate the effects of enzyme supplementation on ruminal fermentation and nutrients digestibilities in ruminants by different enzyme application methods, levels and pre-treatment times.

Rumen fermentation and nutrient digestibility studies
Animal, experimental design and treatments : Six Korean native goats (live weight 25 kg±3.5 kg) were fitted with a rumen cannula were housed in individual pens and had free access to fresh water.The experiment was conducted in a 3×3 Latin square design.Each experimental period consisted of 10 d adaptation to diets and 5 d experimental measurements of feed intake (5 d), feces collection (3 d) and ruminal fermentation characteristics (2 d).Each period, animals received one of three diets, such that each animal received all diets by completion of the experiment.The amount of feed per day was 2% of body weight of experimental animals.The three diets consisted of 70% concentrate, 30% forage as tall fescue (DM base).The contents of crude protein, ether extract and NDF in the concentrate were 12.7%, 3.1% and 48.4% (DM basis), respectively.In addition, forage contained crude protein, ether extract and NDF were 5.6%, 0.9% and 69.7% (DM basis), respectively.The enzyme mixture consisting of mainly cellulase and xylanse was applied in different methods of application : no enzyme, 1% enzyme in rumen-The enzyme was infused into rumen via cannula in a liquid form twice on feeding time; and 1% enzyme treated diet-Enzyme was sprayed onto forage.The enzyme was dissolved in distilled water, made concentration 1% (w/w), and the actual supplemented enzyme level was 0.01% of diet.The guaranteed enzyme activity was 1,400,000 BXU (=Enzyme development corporation in house activity unit).The available xylanase activity in a feed sample is determined by the ratio method.The delta absorbances are calculated and a ratio was set up against the known activity of the control sample.Commercial information observed that 1 unit of activity is defined as the amount of enzyme that is required to release one micromole of xylose reducing sugar equivalents from wheat arabinoxylan (1% w/v) in one minute at 400°C and pH 4.6.It has been determined experimentally that 1 unit=100 BXU.
Experiment procedures and analytical methods : Ruminal fluid was collected for two days (d 4 and d 5) from multiple sites in the rumen.Samples were immediately measured for pH using a pH meter and then squeezed through four layers of cheesecloth with a mesh size of 250 µm.Nine ml of filtrate was preserved with 1 ml of 1% sulfuric acid to determine VFA and NH 3 .The samples were stored frozen at -20°C until analyses.Estimation of VFA contents was performed by Hewlett Packard ® 6890 GC System (Erwin et al., 1961).NH 3 concentration was analyzed by using Milton Roy ® Spectronic 21D spectrophotometer at 630 nm (Chancy and Marbach, 1962).The enzyme activity assay was based on the method of Groleau and Forsberg (1981).An aliquot of 0.5 ml supernatant of cultures were added to the tubes containing 0.5 ml of 1% carboxymethylcellulose (CMC), which was suspended with 0.5 M citrate buffer (pH 5.5), and 0.5 ml of 1% xylan solution, and then, fixed for 30 min at 40°C.After adding 0.6 ml of dinitrosalicyclic acid (DNS), samples were boiled for 5 min.CMCase and xylanse activity were calculated by converting the quantity of separated glucose, which were determined by Milton Ray Spectronic 21D at 550 nm with µmol/min unit.Apparent total tract digestibilities of nutrients were determined using collected fecal samples.Fecal samples were collected for three days (d 1, d 2 and d 3).The fecal samples were dried in a forcedair oven at 60°C till constant weight and ground in a Willey mill (1mm screen).Chemical composition of diet and feces were determined according to AOAC (1990).Additionally, the amounts of NDF and ADF in diet and feces were determined (Van Soest et al., 1991) and the expressions were not including residual ash.

Degradation study
To determine the in situ degradation characteristics of nutrients, 2 g of dry sample milled through a 2.5 mm screen were placed in nylon bags (140×75 mm, pore size 40 to 60 µm).The bags were incubated in the rumen of three ruminally-cannulated Holstein steers.The animals were offered a diet consisted of forage and concentrate with 60:40 ratio.Animals had free excess to water and mineral/vitamin licks.Nylon bags were incubated at 0, 6, 12, 24, 48 and 72 h.The measurement of 0 h bags was obtained by soaking the two bags of each sample in warm water (37°C) for 1 h.The 0 h measurement and incubated bags were then washed with running cold water until the rinse out was clear, and dried till constant weight.The DM, NDF and ADF degradation data were fitted to the exponential equation y=a+b (1-e -ct ) (Ørskov and McDonald, 1979) to determine the degradation constants (a, b and c).Effective degradability of DM (ED) was determined using equation ED=a+[bc/(c+k)].The constant k value was assumed to be 0.05/h in equation for ED value.

Statistical analysis
Data obtained from an in vivo experiment were analyzed using the General Linear Model (GLM) procedure of SAS package (1990).Individual in situ nutrient degradation from incubation was analyzed using marquardt method of proc NLIN (nonlinear regression) to determine a constants (a, b and c) by Ørskov and McDonald (1979)'s exponential model {y=a+b (1-e -ct )}.In situ data were analyzed by the same statistical procedure as those of in vivo experiment.

Nutrient digestibility, rumen fermenttion and enzyme activity
Ruminal parameters such as pH, NH 3 and volatile fatty acids (VFA) contents were not significantly different among three diets (Table 1).No effect of dietary enzyme supplementation on ruminal fermentation parameters was also noted in other studies (Beauchemin et al., 1999;Krause et al., 1998).In fact, it was hypothesized that method of application has an impact on ruminal fermentation.In addition, some studies have shown that enzymes are less effective if infused directly into the rumen than when they are applied to feed (Lewis et al., 1996;McAllister et al., 1999;Wang et al., 2001).However, the present results are not accordance with those results.This is probably because ruminal pH in present study was not in the range between 4.5 and 5.5 (Gashe, 1992).Under these conditions, possibly, exogenous enzymes could make a contribution to rumianl fibre digestion.Ruminal pH in the present study was in the range of 6.65-6.80.It is also possible that animal types affect enzyme efficacy in ruminants.Yang et al. (2000) reported that supplementing the diet with an enzyme product improved digestion in dairy cows, but not in sheep.
The activities of CMCase and xylanase were not affected by exogenous hydrolytic enzymes.This may be due to sub-optimal ruminal pH for enzyme activity as discussed previously.Unchanged enzyme activity in the rumen by enzyme supplementation may also be explained by the fact that exogenous enzyme was not destroyed by ruminal proteases or enzyme activities in the rumen was high enough, and therefore exogenous enzyme did not add any additional activity.
The digestibilities of nutrients in the total tract of goats were not affected by methods of application of enzyme (Table 2).This result is in accordance with other results (Beauchemin et al., 1995;Chen et al., 1995), but in some studies (Beauchemin et al., 1999;Feng et al., 1996;Rust et al., 1965;Rode et al., 1999;Yang et al., 1999Yang et al., , 2000) ) nutrient digestibilities were improved by enzyme supplementation.The improved digestibilities of nutrients in total tract indicates that the effects of enzyme supplementation were mostly intestinal.This hypothesis was raised by Hristov et al. (1996) who demonstrated that a significant proportion of exogenous enzymes escaped ruminal digestion and remained active in the small intestine.However, the present results did not support it.This is because adaptation period might be the cause for non significant effect on digestibilities.Trend of NDF digestibility shows that it could have increased if enzyme treatment given for a longer duration.
The present in vivo experiment showed that ruminal fermentation, enzyme activities and total tract apparent digestibility of nutrients in the total tract were not affected by supplemented enzymes.Therefore, it seems that exogenous hydrolytic enzymes did not influence ruminal and post-ruminal digestion.

Nutrient degradation
The degradation constants "a", "b", "c" and "a+b" fractions, and effective degradability (ED) of DM and NDF were partially affected by the levels of enzyme was increased over the incubation time (Table 3).While, the "a+b" fraction and ED of ADF were increased as levels of enzyme was increased, the constants "a", "b" and "c" of ADF degradation were not affected by levels of enzyme over the incubation time.The increased degradation could be due to the increased damage of feed surface by exogenous enzyme treatment of feed before feeding (Nsereko et al., 2000).
The EDs of DM, NDF and ADF were increased with increasing pre-treatment time of feed with enzyme (p<0.001).Also, the degradation constants "b", "c" and "a+b" fractions of DM were increased as pre-treatment time of enzyme was increased.While, the degradation constants "a", "b", and "a+b" fractions of NDF and ADF were not affected by pre-treatment times of enzyme.Recent research indicates that adding the enzyme mixture just prior to feeding is as effective as treating the forage for 2 wk (Yang et al., 1999) or 1 to 3 d (Lewis et al., 1996;Nussio et al., 1997) before feeding.Enzyme applied to forages immediately before in vitro incubation also improved digestion of DM and NDF (Feng et al., 1996), suggesting that fibrolytic enzymes applied at feeding (direct fed) may enhance digestion of forages by cattle.Beauchemin et al. (2001) reported that applying fibrolytic enzymes to feed prior to feeding enhances ruminal fibre digestion by altering the structure of the feed thereby making it more susceptible to degradation.Exogenous enzymes treatment, prior to  ingestion, increase bacterial colonization and thereby improves DM disappearance of forage (Yang et al., 1999) because exogenous enzymes increased microbial attachment of ruminal microbes to feed and increased activity of enzymes associated feed particles (Wang et al., 2001) There was no interaction effect between level and pretreatment time of enzyme on "a" fraction of DM, but "b", "c", "a+b" and ED was increased as level and pre-treatment time of enzyme were increased.The constant "c" of NDF and ADF were increased as level and pre-treatment time of enzyme were increased.Also, ED of NDF was highest for 24 h pre-treatment time and 2% enzyme treatment (p<0.001).Degradation of nutrients was affected by enzymes levels or pre-treatment times.Therefore, it is probable that the improved degradation may be due to the supplemented exogenous hydrolytic enzymes under a certain condition.

IMPLICATION
Presumably enzyme activity within the rumen was not easily increased by a simple addition of exogenous enzyme products.In vivo study with Korean Native goats showed that methods of enzyme application did not affect ruminal fermentation and enzymes activity, and digestibility of nutrients in the total tract.While, in situ study showed that levels and pre-treatment times of enzyme affect DM, NDF and ADF degradation, indicating that this is still possibility for improved nutrient digestibility by exogenous hydrolytic enzymes.More detailed studies on method of application and, dietary and animal factors are warranted, as improved nutrient degradation resulted by exogenous enzyme under certain condition.  Interaction between level and pre-treatment time of enzyme. 9NS=Not significant (p>0.05),* p<0.05, ** p<0.01 and *** p<0.001.

Table 1 .
Ruminal fermentation characteristics in Korean Native goats supplemented with exogenous enzyme Diets

Table 2 .
Effect of enzyme supplementation on DM, crude protein, NDF and ADF digestibility.