Enzymes as silage additive . Effect on fermentation quality , digestibility in sheep , degradability in sacco and performance in growing cattle

Etude technologique, zootechnique et physiologique de glucose oxydase et cellulase utilisees comme additifs d'ensilage en comparaison avec l'acide formique


Introduction
Silage additives used to influence fermentation can act either as stimulants by encour- aging lactic acid fermentation or as inhibitors by inhibiting completely or partially microbial growth.Fermentation stimulants can be clas- Present address: Agricultural Research Centre, SF-31600 Jokioinen, Finland sified as bacterial cultures or as carbohydrate sources.
The effects of bacterial inoculants on silage fermentation have been variable and in many experiments no benefits at all have been seen.Woolford and Sawczyc (1984) did not find any notable influence on the rate of acidification or promotion of homolactic fermen- tation by selected cultures of lactic acid bac- teriä and these also tended to increase the amount of soluble protein and deamination.
Carbohydrate rich materials such as sugars, molasses and cereals have been added to silage crop in order to supply energy for lactic acid bacteria.In his review, McDonald   (1981) notes that molasses has been shown to decrease pH and the amount of ammonia N in silage.Castle and Watson (1985) did not record any differences in the performance of dairy cows fed formic acid or molasses treated silages.A disadvantage of using carbohydrate rich materials is the relatively high concentrations required.
Forage plants have a large reserve of car- bohydrates, but these occur mainly in the form of polysaccharides which cannot be used as an energy source by lactic acid bacteria in silage.In theory, the addition of enzymes able to hydrolyse forage polysaccharides would increase the fermentation capacity of silage by releasing fermentable substrate.The cellulolytic enzymes would also promote cellular breakdown and render cell contents more ac- cessible to silage microflora.
The objectives of the present studies were to investigate the effect of enzymes on silage fermentation, degradation rate in sacco, di- gestibility in sheep and on the performance of growing bulls.The silages were made in labo- ratory, pilot and farm scale silos.
Nylon bag (porosity 40 /rm) incubations were made in the rumen of a dairy cow fed at the level of 2.8 times maintenance, with for- age 60 % of DM.The silages incubated in triplicate were withdrawn after 2,5, 9, 24 and 48 h in Exp. 1 and after 1,3, 6, 12, 24 and 48 h in Exp. 2. Residues were analysed for DM, OM and N and some treatments also for NDF.The values for degradation constants were calculated using the formula of orskov and McDonald (1979) p = a + b(I e~c l ).
The material for the feeding trial (Exp.4) was second cut timothy red clover sward (red clover 35-40 % of DM).After wilting for 4 h the grass was ensiled into a 250 t bunker silo either with formic acid (4 1/t) or 2-3 d later with enzyme mixture (GO + cellulase 150 ml/t).A feeding trial of 210 d was carried out with 24 bulls using 2x2 factorial design.Both silages were supplemented with barley or barley + fishmeal (FM).Barley was fed at the level of 40 g/kg W 0 on an air dry basis.In theFM group 0.25 kg of barley was replaced by FM.In vivo digestibility was determined at the liveweight of 250-300 kg using acid- insoluble ash as digestibility marker (Van Keulen and Young 1977).

Results and discussion
The breakdown of various fibre fractions increased during the ensilage period with increasing level of cellulase (Fig. 1).The effect was proportional to the square root of cellulase activity.In Exp. 2 with longer ensilage period and more mature material, the effect of cellulase on ADF, cellulose and crude fibre was like that in Exp. 1, but the effect on NDF was greater indicating the hydrolysis of hemi- cellulose and the hemicellulose activity of the enzyme mixture.In the normal ensilage process the breakdown of hemicellulose is caused also by plant enzymes and later, when pH is low, by acid hydrolysis (McDonald   1981).
All the silages were well preserved.Formic acid almost completely inhibited fermentation in the laboratory silos (4 kg).Presumably at the high levels of addition the bacteriostatic action of formic acid and consequent suppression of lactic acid production allowed the pH to increase in response to the release of buf- fering constituents from plant tissue.With increasing levels of cellulase the concentration of residual sugars increased and there was also some increase in the concentration of lactic acid.No effect on true protein contents or the ratio ammonia N: total N was found with increasing levels of cellulase, but the ratio solu- ble N: total N increased, presumably because of the breakdown of cell walls and subsequent release of cell contents.
The disappearance of OM from nylon bags increased with the level of cellulase addition, especially after shorter incubations (Figs. 2  and 3).After an incubation of 48 h there were no differences in the extent of OM degrada- tion between the treatments.The very rapidly degradable fraction a increased with the level of cellulase and in time degradable fraction  b decreased.The effect of treatments on c (the rate of degradation of fraction b) was incon- sistent in Exp. 1, but in Exp. 2 all enzyme treated silages had a higher rate of OM de- gradation than untreated or formic acid treated silages.Cellulase treatment had the same pattern of response on N as on OM dis- appearance.The ratio N:OM that disappea-red during the first 2 or 3 hours was 55-63 g N/kg OM in Exp. 1 and 43 -51 g in Exp.
2 being slightly lower in formic acid treated silage than in other silages.This ratio was not affected by the level of cellulase.The effect of increased N loss after short incubations on N or microbial N flow might be negative.However, Chamberlain et al. (1982) did not record any response of N flow into the duo- denum when the level of formic acid was increased from 0 to 5.9 1/t, and the undegradable fraction of silage N was increased only from 22 to 28 %.It seems that partial limita- tion of proteolysis can be achieved in the silo by the addition of high levels of formic acid, but protein spared from hydrolysis in the silo seems to be degraded by the rumen microbes.
Part of the response in the OM loss can be explained by increased N loss with higher levels of cellulase.However, the loss of N-free OM after incubations for 3, 6 and 12 h increased from 33.0 to 41.7 % (P< 0.001), from 39.9 to 46.8 % (PcO.001), and from 48.4 to 54.0 % (P<0.01) in Exp. 2.
With the highest level of cellulase applica- tion the potential degradability of NDF was decreased (Table 1), but this could be ex- plained by the breakdown of cell walls in the silo.Lag time was reduced by cellulase treat- ment, but in Exp. 1 with young grass a very high level of cellulase was needed to achieve this response.By contrast, with more mature material in Exp. 2 the same response was found with the level of 400 ml/t.
In Exp. 3 enzyme treatment decreased pH  1 On oven DM basis, FM = fishmeal, FA = formic acid Means with different letters significantly different a, b (P<0.05) and the concentrations of acetic acid and ammonia N and increased the concentrations of lactic acid and residual sugars compared with untreated control (Table 2).Enzyme treatment encouraged homofermentative lactic acid bac- teria and theratio lactic acid: acetic acid was the most favourable in enzyme silages.Apparently in the absence of fermentation inhib- itors the sugars from cell wall hydrolysis are used mainly for lactic acid production.The digestibility of OM was 76.2, 76.5, 75.8, 77.1 and 77.2 % for direct cut formic acid, pre-wilted formic acid, untreated, en- zyme A and enzyme B treated silages and N retentions were 2.5, 2.9, 1.4, 2.0 and 2.9 g/d, respectively.
Both silages in Exp. 4 were well preserved in the bunker silo.The enzyme treated silage had a higher concentration of lactic acid (11.2 vs. 8.6 %), acetic acid (3.8 vs. 2.1 ®7o), ammonia N (8.9 vs. 5.9 % of total N) and solu- ble N (49.4 vs. 41.8 % of total N) and lower concentration of sugars (1.1 vs. 0.5 %).However, DM intakes of the two silages were similar on oven DM basis (Table 3).Differ- ences between the silages in growth rate, feed conversion and carcass characteristics were small and insignificant.FM, on the other hand, improved significantly (P<0.05) the liveweight gain (Table 3).One animal in the enzyme silage + barley group had leg injuries, which partly explains the insignificant (P>0.05)interaction between the type of silage and protein supplementation.
Enzyme treatment reduced significantly (P <0.001) the digestibility of crude fibre and improved (P< 0.001) that of the ether extract compared with formic acid treated silage.The lower crude fibre digestibility is most likely explained by enzymatic cell wall digestion in the silo, but probably the slightly later har- vesting time also had some effect.It seems that the cellulase system first attacks the most easily digestible fraction of fibre in the silo and the fraction which is left is less digestible in the animal than the original fibre.There was a significant (P<0.01)interaction between the silage and protein supplementation: FM clear- ly improved the digestibility of enzyme treated silage, but had a slightly negative effect with formic acid silage.Possibly enzyme silage re- quires a different supplement than acid treated silage to optimize cell wall digestion in the rumen.
In conclusion, enzyme treatment encour- aged lactic acid fermentation and also reduced ammonia N compared with untreated control silage.No adverse effects relative to formic acid silage were found on N retention in sheep or feed intake and liveweight gain in growing bulls.The advantages of biological additives over acids are that they are safer to handle, less corrosive to machinery and easier to trans- port.

Fig
Fig. I.The effect of enzyme level on NDF A , ADF ■. cellulose • and crude fibre □ contents of si- lage (% of DM) in Exp. 2.

Table 1 .
The degradation constants of NDF disappearance from the nylon bags inExp. 1 and Exp. 2 GO = glucose oxidase, C = cellulase Fig.3.The effect of enzyme level on silage OM disap- pearance (% of total OM) from the nylon bags in Exp. 2.

Table 3 .
Feed intake and the performance data in growing bulls inExp.4.