Maataloustieteellinen Aikakauskirja Vol. 62: 51—62, 1990 The effect of cell wall degrading enzymes on the preservation of grass and on the silage intake and digestibility in sheep SEIJA JAAKKOLA Department of Animal Husbandry, University of Helsinki, SF-00710 Helsinki, Finland Abstract. Two experiments were carried out to study the effects of cell wall degrading enzymes as silage additive. A primary growth of timothy was cut with a mower and picked up with a precision-chop forage harvester in Exp. 1 (early cut), and harvested as direct-cut with a precision-chop harvester in Exp. II (late cut). The additive treatments were in Exp. I: 1) unwilted formic acid (FA) applied as AIV II (4.5 1/t), 2) wilted untreated, 3) wilted FA (4 l/t), 4) wilted enzyme A (glucose oxidase (GO) + hemicellulase (HC) 150 ml/t + cellulase (C) 200 ml/t), 5) wilted enzyme B (HC 150ml/t + C 200 ml/t); and in Exp. II: 1) untreated, 2) FA (4 l/t), 3) E2OO (C 200 ml/t + GO), 4) E4OO (C 400 ml/t + GO), 5) EBOO (C 800 ml/t + GO). The rate of application of GO was 50 000 IU/t. The silages were ensiled in pilot scale silos (3 m J) and the voluntary intake and digestibility in sheep were determined in two experiments designed as a 5 x 5 Latin square. The use of enzymes decreased the fibre content of silages, mainly the cellulose fraction, as compared with FA and untreated silages. Enzyme silages were well preserved with a low pH (3.93 —4.15), moderate ammonia N (72—119 g/kg total N) and no butyric acid. As com- pared with untreated silages (mean pH 4.6, ammonia N 131) the preservation was improved. The FA silages were also well preserved (pH 4.0, ammonia N 57) with more restricted fermen- tation than enzyme silages. FA and especially higher levels of enzymes increased the amount of effluent. In Exp. I, the digestibilities of dry matter (DM), organic matter (OM) and crude fibre were not significantly (P >0.05) affected by the silage treatment. In Exp. 11, the digesti- bility of DM and OM decreased linearly (P <0.05) with the increasing level of enzyme applica- tion. The digestibility of NDF and ADF was higher with untreated than with other silages, higher with FA than enzyme silages and decreased linearly with the increasing level of enzymes (P<0.01). Index words: grass silage, additives, enzymes, preservation, digestibility 51 JOURNAL OF AGRICULTURAL SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=LDbKnWK_qfa_XziF.hug_8sixs1qVZobdPBnT_w.Zam6_SG6VuQDOCx8Sk6yvUfqhpZamU6Rqgt71XvoPywSXXfW7we2fqa9p0_NxgFTwtska_BYyJKiJhb94oWuACdwtJg56POp-38gEuzbna8PZRHoYUDqtf7gGASywe9h_jFBRFF1Y7JPKz8pTBynp6kaNdbnsnICvGRfig Introduction The preservation of high moisture grass by ensiling is based on a reduced pH under anaerobic conditions. A high hydrogen ion concentration prevents the adverse effects of microbes and plant enzymes. Together with anaerobiosis, an adequate supply of fer- mentable substrates, mainly water soluble carbohydrates (WSC) of grass, is a major prerequisite for the preservation without ad- ditive. With acid additives the hydrogen ion concentration is increased by an extraneous addition and the natural fermentation is partly inhibited. In addition to acidification, a selec- tive bactericidal action of the undissociated acid may improve the preservation (Wool- ford 1975). Formic acid has long been known to be an efficient additive (e.g. Saue and Breirem 1969, Wilson and Wilkins 1973). However, it is unpleasant ifno hazard- ous to handle. To avoid these problems while achieving good preservation, bacterial in- oculants have been studied as additive to stimulate the fermentation, and enzymes to in- crease the amount of substrate for fermenta- tion by degrading plant cell walls (e.g. Olson and Voelker 1961, Hendersson et al. 1982, Jacobs and McAllan 1987). It has also been argued that the use of enzymes increases si- lage intake and animal performance by en- hancing the rate and/or extent of breakdown of plant structural carbohydrates in the ru- men. Results from the use of bacterial inoculants have been inconsistent (Done 1986). Seale et al. (1986) concluded that inoculants can quickly utilize available and supplied WSC in the forage, but they cannot work successful- ly to achieve satisfactory fermentation ifWSC is a limiting factor. The use of cellulolytic en- zymes has been found to reduce the cellulose content (McGullough 1964, Autrey et al. 1975, Hendersson and McDonald 1977) and the pH (Autrey et al. 1975, Hendersson and McDonald 1977) of different kinds of silages as compared with untreated silages. When comparing untreated silages with enzyme silages, no differences were observed in the digestibility of sorghum (Owen 1962) or al- falfa haylage (Jaster and More 1988). Some indication of improved digestibility of maize silage (Autrey et al. (1975) and sorghum (McCullough 1964) after cellulase treatment has been reported. However, the data con- cerning the ability of enzymes to preserve low moisture grass or improve digestibility are still inadequate. The purpose of the present experiments was to study the effects of enzymes (cellulase, hemicellulase, glucose oxidase) and formic acid as grass silage additives on the fermen- tation quality, digestibility and voluntary in- take in sheep. In addition, it was studied whether the adverse effects of postponed har- vesting on the digestibility of silage could be eliminatedby the use of enzymes. Preliminary results have been reported by Huhtanen et al. (1985). Materials and methods Silages Experiment I A primary growth of leaf stage timothy (Phleum pratense) sward was cut with a mow- er and collected with a precision-chop forage harvester. One of the silages was ensiled on 4 June as direct-cut (dry matter (DM) con- tent 153 g/kg) and the other four silages on 6 June simultaneously after a wilting period of 4 h (DM 204 g/kg). The additive treatment of the direct-cut silage was formic acid (FA) applied as AIV-II solution (800 g FA/kg + 20 g orthophosphoric acid/kg) at the rate of 4.5 1/t. The treatments of the wilted silages were as follows: untreated, FA (41/t), enzyme A (glucose oxidase (GO) + hemicellulase (HC) 150 ml/t -l- cellulase (C) 200 ml/t), en- zyme B (HC 150 ml/t + C 200 ml/t). C was produced by Trichoderma reesei, HC by Penicillium emersonii and GO by Aspergillus 52 niger. The addition of GO was 50 000 HJ/t and the activity of C 25 000 nkat (nanokatal) HEC (hydroxyethyl cellulose)/ml. The enzyme solutions were delivered dur- ing the mowing of grass. FA was delivered by watering can during the filling of the 3 m 3 pilot scale glass-fibre silos (0 1.4 m). After filling the silos were sealed with a plastic sheet, weighted (400 kg/m2 ) and stored at ambient temperature. The temperature probes were placed in the middle of the silo for monitor- ing the temperature changes. The silos were opened after 5 months of storage. Losses from the silo were measured by weighing and analyzing all the grass ensiled and the effluent and silage removed from the silo (in-out method), and by the buried bag method with 2 bags/silo (10 kg grass/bag) dis- tributed throughout the silo. Experiment II A primary growth of fully emerged timothy (840 g/kg) red clover (Trifolium pratense) sward was harvested on 26 June. Silages were harvested as direct-cut (DM 199 g/kg) with a precision-chop forage harvester and ensiled simultaneously in the same pilot scale silos as in Exp. I. The treatments were as follows: untreated, FA applied as AIV-II (4 1/t) and GO (50000 HJ/t) applied at three levels of C as E2OO (200 ml/t), E4OO (400 ml/t)and EBOO (800 ml/t). Water was delivered to the untreat- ed silage in an amount equal to that used to dilute enzyme solutions to achieve even dis- tribution. The additives were delivered during the filling of the silos. Enzyme activities were the same as in Exp. I. The silos were sealed and weighted as in Exp. I and stored at am- bient temperature for 2.5 months before the digestibility trial. Losses from the silo were measured with the in-out method as in Exp. I. Animals and experimental design Five mature male Finn sheep weighing 60—70 kg in Exp. I and 50—60 kg in Exp. II were placed in metabolism cages. The experi- ments were conducted as a 5 x 5Latin square with 21 d periods. Each period consisted of a 10day introduction with ad libitum feeding and a 4 day adaptation followed by a 7 day total collection of faeces and urine. The silages were fed in two equal portions with 20 g of commercial mineral mixture daily. In addition, the animals had free access to water. The voluntary intake was measured during the days s—lo5—10 of the ad libitum feed- ing period by offering the silages 100—150 g/ kg in excess of the previous day’s intake. Dur- ing the collection period feeding was restricted to 50 and 45 g DM/kg W 0 75 in Exp. I and 11, respectively. Sampling and chemical analyses Representative samples of grass were tak- en during the filling of the silos. As soon as the buried bags appeared from the silo, they were weighed and the content was sampled. Samples of silages, feed refusals, urine and faeces were taken in the digestibility experi- ments. Silage DM content was determined by oven drying at 105°C for 24 h and corrected for the loss of the volatile substances (lactic acid, VFA, ammonia) according to Porter et al. (1984). The fresh silage samples were analyzed for pH, water soluble carbohydrates (WSC) according to Somogyi (1945) with modifica- tions of Salo (1965), lactic acid (Barker and Summerson 1941), volatile fatty acids (Huida 1973), ammonia nitrogen (N) (McCullough 1967), and soluble and total N by the Kjeldahl method. Effluent was analyzed for pH, DM, ash and total N. Fresh urine and faecal sam- ples were analyzed for N. Feed analyses of grass, silages, refusals and faeces were made according to standard procedures. Neutral de- tergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were determined in Exp. II according to Goering and Van Soest (1970). Hemicellulose was cal- culated as a difference between NDF and ADF, and cellulose as a difference between ADF and ADL. The digestibility of the grass 53 and silages in vitro was measured according to Tilley and Terry (1963). Statistical analyses The data from the digestibility trials were tested with analyses of variance for Latin squares (Snedecor and Cochran 1967) by using a model: Yijki +Tj +Aj +Pk + e ijk. where T, A and P are the effects of treatment, animal and period, respectively. In Exp. 11, the treatment effect was further partitioned to single degree of freedom by making compar- isons of untreatedvs additive treated silages, FA vs enzyme silages, E2OO vs EBOO (linear ef- fect of enzyme level), and E4OO vs E2OO and EBOO (quadratic effect of enzyme level). Results The chemical compositions of the grasses and the silages are given in Table 1. The ac- tivity of the enzymes was evidenced in both experiments by the lower crude fibre content of enzyme silages compared with untreated and FA silages. In Exp. 11, the content of NDF and ADF decreased with the increasing level of enzyme application, the effect of en- zymes being directed mainly towards cellulose. Also FA decreased the fibre content, mainly hemicellulose, but the effect was less evident than that of the enzymes. After 5 days of storage, effluent pH was 3.2 (wilted) and 4.1 (unwilted) in FA and 4.2—4.3 in enzyme silages in Exp. I. No effluent formed from untreated silage. In Exp. 11, the pH values of effluent were 4.3 in untreated, 4.1 in FA, and 4.2—4.3 in enzyme silages. The pH of the silages treated with additives declined near 4 with the exception of E2OO (pH 4.15). The untreated silages had a mean pH value of 4.4 and 4.8 in Exp. I and 11, and a high content of acetic and propionic acid indicating both inadequate and secondary fer- mentation (Table 2). As compared with the enzymes the use of FA in both experiments resulted in more restricted fermentation, in- dicated by the lower lactic and acetic acid and higher residual WSC content in silage. In Exp. I, GO had no effect on the chemical composition or fermentation quality. In both experiments, enzyme treatment increased lac- Table 1. Chemical compositions of the grasses and silages. In dry matter (g/kg DM) DM Ash Crude EE Crude NFE NDF ADF ADL Hemi- Cellu- (g/kg) prot. fibre cell. lose Experiment I Unwilted grass 153 91 195 44 270 401 ND ND ND Formic acid 180 82 186 57 291 387 ND ND ND Wilted grass 204 85 185 33 271 426 ND ND ND Untreated 222 89 180 53 290 389 ND ND ND Formic acid 219 83 178 49 286 403 ND ND ND Enzyme A 213 87 180 63 274 396 ND ND ND Enzyme B 214 84 182 67 271 396 ND ND ND Experiment II Unwilted grass 199 67 127 30 344 433 635 336 17 299 319 Untreated 190 74 131 40 401 355 666 377 20 289 358 Formic acid 201 66 124 39 372 399 619 347 18 272 329 E2OO 207 67 128 48 361 396 609 341 20 268 320 E4OO 215 64 121 51 341 423 596 326 20 271 306 EBOO 216 64 122 51 356 408 579 315 21 265 294 EE = ether extract, NFE = nitrogen free extract, ND = not determined 54 tic acid concentration as compared with un- treated silages. In Exp. 11, the concentration of lactic acid and WSC increased with the level of enzyme application, whilst the concentra- tion of acetic and propionic acid decreased. However, the total amount of acids was not increased by doubling the amount of enzyme from 400 to 800 ml/t. As compared with untreated silage the en- zymes reduced proteolysis, the effect being most pronounced with the highest level of ap- plication. Least protein breakdown occurred, however, in the FA silages as indicated by the lowest ammonia-N content. The use of FA and enzymes increased the formation of effluent (Table 3). The effect of Table 2. Water soluble carbohydrate (WSC) and soluble N contents in the grass and the effect of additive treat- ment on the fermentation quality of silage. pH In dry matter (g/kg DM) Lactic In tot. N (g/kg) WSC Lactic Acetic Prop. But. Total NHrN Solub. acid acid acid acid acids N Experiment 1 Unwilted grass 57 336 Formic acid 4.06 15 63 18 0.8 0 82 3.5 63 605 Wilted grass 85 325 Untreated 4.40 2 93 30 0.7 0 124 3.1 113 704 Formic acid 3.98 36 64 14 0 0 78 4.6 52 589 Enzyme A 3.95 8 133 23 0 0 156 5.8 72 679 Enzyme B 3.96 9 131 23 0 0 154 5.7 72 669 Experiment 11 Unwilted grass 65 419 Untreated 4.81 1 8 61 12.8 0.1 81 0.1 148 736 Formic acid 3.98 41 52 12 0.4 0 64 4.3 57 670 E2OO 4.15 5 92 34 4.1 0 130 2.7 119 720 E4OO 3.96 9 111 28 2.2 0 141 4.0 116 717 EBOO 3.93 17 111 20 0.9 0 132 5.6 87 676 Table 3, In-silo dry matter (DM) losses (g/kg) and the amount and DM content of effluent (g/kg) from the silages treated with different additives. Effluent Other Total losses Effluent losses losses In-out Bags kg/t grass 1 DM content Experiment 1 Unwilt. formic acid 46 92 138 110 140.0 46 Wilted Untreated 0 37 37 47 0 Formic acid 8 50 58 44 5.6 62 Enzyme A ,17 88 105 67 27.2 69 Enzyme B 23 37 60 45 46.0 72 Experiment 11 Untreated 3 114 117 0 48 Formic acid 7 89 96 0 56 E2OO 21 99 120 40.3 61 E4OO 40 67 107 94.2 65 EBOO 49 72 121 107.8 71 1 The amount of additive and water delivered subtracted 55 enzymes was more profound than that of FA, and it increased with the level of enzyme ap- plication. The methods of measuring total losses were highly correlated (r =0.93) ranking the silages in a similar manner with highest losses in the unwilted FA silage. The temper- ature of silages at ensiling ranged from 17 to 20° C and the maximum temperature (20— 25°C) was reached 7 days after ensiling, be- ing highest with the untreated silages. The in- crease was 2°C smaller in theFA silages when compared with the enzyme silages. Table 4. Digestibility coefficients, voluntarysilage DM intake and nitrogen balance in sheep and in vitro digestibili- ty of silage and grass organic matter (OM) in Exp. I. Unwilted Wilted SEM Formic Untreated Formic- Enzyme A Enzyme B acid acid Dry matter 0.744 0.746 0.750 0.757 0.759 0.0039 Organic matter 0.762 0.758 0.765 0.771 0.772 0.0043 Crude protein 0.769 0.767 0.778 0.781 0.783 0.0042 Ether extract 0.740" 0.746adc 0.715" 0.794bcd 0.803»' 0.0088 Crude fibre 0.777 0.784 0.779 0.771 0.775 0.0085 N-free extract 0.743ab 0.7296 0.751ab 0.753 a 0.751 a 0.0048 Digest, in vitro Silage OM 0.734 0.729 0.736 0.747 0.746 Grass OM 0.737 0.726 0.726 0.714 0.727 Silage DM intake (g/d/kg W°") 52.4 57.2 55.7 53.9 57.9 3.97 N retained (g/d) 2.5 1.4 2.9 2.0 2.9 0.59 SEM = standard error of means Means with different letters were significantly different: a,b (P<0.05), c,d,e (P<0.01). Table 5. Digestibility coefficients, voluntary silage DM intake and nitrogen balance in sheep and in vitro digestibili- ty of silage and grass organic matter (OM) in Exp. 11. Un- Formic E2OO E4OO EBOO SEM Statistical significance treated acid of effect CI C 2 C 3 C 4 Dry matter 0.676 0.678 0.674 0.669 0.640 0.0084 NS NS * NS Organic matter 0.678 0.683 0.677 0.675 0.644 0.0083 NS NS * NS Crude protein 0.727 0.687 0.729 0.703 0.676 0.0087 NS NS ** NS Ether extract 0.723 0.717 0.765 0.791 0.758 0.0083 ** *** NS * Crude fibre 0.728 0.714 0.685 0.661 0.656 0.0119 ** •• NS NS N-free extract 0.599 0.646 0.641 0.662 0.609 0.0104 ** NS NS * NDF 0.707 0.695 0.672 0.666 0.619 0.0106 ** ** ** NS ADF 0.734 0.716 0.697 0.684 0.639 0.0106 *• ** ** NS Hemicellulose 0.672 0.667 0.641 0.645 0.595 0.0113 * ** * NS Cellulose 0.777 0.762 0.740 0.733 0.692 0.0115 »• • * NS Digest, in vitro Silage OM 0.635 0.671 0.657 0.642 0.638 Grass OM 0.689 0.689 0.689 0.689 0.689 Silage DM intake (g/d/kg W°") 41.6 52.6 55.1 51.3 56.4 2.53 **• NS NS NS N retained (g/d) 2.0 1.8 2.2 2.4 2.7 0.42 NS NS NS NS Comparisons: Cl = untreated vs additives, C 2 = FA vs enzymes, C 3 = linear effect of enzyme level, C 4 = quad- ratic effect of enzyme level Statistical significance: NS not significant, * (P<0.05), ** (P<0.01), *** (P<0.001) 56 Results concerning the digestion of differ- ent feed constituents in Exp. I are given in Ta- ble 4. No significant (P>0.05) differences were observed in the digestibility of DM, or- ganic matter (OM), crude protein and crude fibre. The digestibility of the nitrogen free extract (NFE) of the enzyme silages was sig- nificantly (Pc0.05) higher than that of the untreated silage. The significant (Pc0.05, PcO.Ol) differences in the digestibility of ether extract were mainly caused by the high concentrations of fermentationacids in the en- zyme silages and are of minor importance. In Exp. 11, no significant differences were noticed in DM, OM and crude protein digest- ibility between untreated and other orFA and enzyme silages (Table 5). However, the cor- responding digestibilities decreased (PC0.05, PcO.Ol) with the increasing level of enzyme application. The use of additives (FA and en- zymes) increased significantly (PcO.Ol) the digestibility of ether extract and NFE, and de- creased that of crude fibre, NDF, ADF, cel- lulose (PcO.Ol) and hemicellulose (Pc0.05). As compared with FA, the use of enzymes decreased (P<0.05, P< 0.01) the digestibility of all fibre fractions. Moreover, an increase in the enzyme level decreased linearly (P<0.05, PcO.Ol) the digestibility of cell wall carbo- hydrates. In Exp. I, there were no differences in the voluntary silage DM intake between the silages (Table 4) whereas in Exp. II the un- treated silage was consumed significantly (P< 0.001) less than the other silages (Ta- ble 5). No differences were observed in N retention between the silages. Discussion Plant cell wait degradation As compared with the untreated silage the content of crude fibre in enzyme silages de- creased 16—19 and 40—60 g/kg DM in Exp. I and 11, respectively, indicating mainly the breakdown of cellulose because a major part of hemicellulose is lost in the crude fibre anal- ysis. Exp. II confirmed this, since the cellu- lose content decreased 38—64 g/kg DM with the increasing enzyme level. The results are in good agreement with those of Rauramaa et al. (1987 a). The cell wall degrading ef- fect appears to be evident in most experi- ments with fibrinolytic enzymes (Autrey et al. 1975, McCullough 1964, McHan 1986, Heikkilä et al. 1987), while the material en- siled, enzyme activities and amount applied cause variation in the intensity of enzyme ef- fects. The decreasing of the NDF and ADF con- tent with increasing enzyme level is consistent with other experiments, where the amount of fermentable carbohydrates released has been proportional to the amount of enzymes applied (Henderson and McDonald 1977, Vaisto et ai. 1978, Nehring et ai. 1983, Brol- ly 1986). Hissa (1986) studied the effects of the increasing level of enzyme application up to 4 times greater level than EBOO. It was con- cluded that the degradation of cellulose fol- lowed the formula y= a —b(l e cx ), in which a, b and c are constants and x is the level of cellulase. According to that the maximal degradation of NDF could have been 75 g/ kg DM. On the other hand, Hendersson and McDonald (1977) suggest that the end prod- uct inhibition may allow hydrolysis of no more than 300—400 g cellulose/kg cellulose ensiled. Microbes, plant enzymes, acid additives or acids formed during fermentation may also decrease the cell wall content of silage. In Exp. 11, the content of hemicellulose decreased more in FA (90 g/kg) than in untreated silage (33 g/kg). Consistently, FA degraded mainly hemicellulose, which could loose up to 200 g/ kg of its content in ryegrass (Morrison 1979). On the other hand, DM losses in the form of C02 and effluent causes an increase in the fibre content of silage. The reverse effects of the losses and the above mentioned factors tend to offset each other. The fermentation quality of the silages The importance of the WSC content, buf- fering capacity and DM content of the grass 57 for preservation without additives is well de- fined (McDonald 1981). In the present study the WSC content of grass was 17 and 13 g/kg fresh weight. The quality of untreated silages obviously reflected the WSC contents being in agreement with the suggestions that WSC content of fresh grass should be at least 25— 30 g/kg to achieve satisfactory preservation (Wilkinson et al. 1983, Pettersson 1988). The amount of WSC + total acids exceeded 30 g/kg in the enzyme silages, except for E2OO (28 g/kg), but not in untreated silages (28 and 16 g/kg). These values are in agreement with the differences in fermentation quality be- tween untreated and enzyme silages, indicat- ing that the fermentable substratesreleased by enzymes accomplished the preservation. On the other hand, with grass of higher WSC con- tent, no differences were observed in fermen- tation quality between untreated and enzyme silages (Kauramaa et al. 1987 a, Toivonen 1989). Low WSC content (< 20 g/kg DM) in all enzyme silages indicated that sugars released from cell wall were fermented to acids. As compared with untreated silage the amount of lactic acid was much higher in enzyme treated silage, facilitating the achievement of low pH. The final pH was the same as in FA silages with the exception of E2OO in Exp. 11. The same positive effect of enzymes has been found also in other experiments (Autrey et al. 1975, Henderson and McDonald 1977, Nehring et al. 1983, Hendersson et al. 1987, Merry and Braithwaite 1987). Applied in conjunction with FA, enzymes may have no effect on the conversion of WSC to fermen- tation acids and thus on pH (Henderson and McDonald 1977, Vaisto et al. 1978, Brolly 1986). Measured from effluent the decrease in the pH in the first days was not more rapid in en- zyme than in untreated silages. After the first weeks, however, the enzymes proved to be ef- ficient in decreasing pH further. In untreated silages, the lack of fermentable substrates later led to increased pH due to secondary fermen- tation. With grass rich in WSC, ensiled with- out additive or with cellulase, the pH 4 was obtained after vigorous fermentation within one week (Kauramaa et al. 1987 a). As com- pared with direct acidification with FA, the natural fermentation does not reduce silage pH equally rapidly in the first hours of ensil- ing. This is obviously the main reason for the lower ammonia N contents in the FA silages as compared with enzyme treated and espe- cially with untreated silages. The better inhibi- tory effect ofFA on proteolysis as compared with enzymes agrees with the results from oth- er experiments (Kennedy 1987, Kauramaa et al. 1987 a). While containing the same total amount of acids, there was a clear difference in the pH values between E2OO and EBOO (4.15 vs 3.93), indicating different strength of lactic (pKa 3.8) and acetic acids (pKa 4.8). Gener- ally, the increase in the lactic acid/acetic acid (L/A) ratio is mainly an indicationof a more homolactic fermentation or different ferment- able substrates. Due to the breakdown of cell walls, xylose and arabinose are released from the hemicellulose fraction, while glucose is released from cellulose. Some lactic acid bac- teria are able to ferment pentose sugars to a mixture of lactic and acetic acids, homo- and heterofermentative bacteria having the same pathways (McDonald 1981). The lower L/A ratio in the FA silages than in both enzyme silages in Exp. I and in EBOO in Exp. II might suggest that proportionally more glucose was released by enzyme than FA treatment rather than indicate differences in microbial popu- lation. This is supported by the results of Kauramaa et ai. (1987 b), who found that despite of the lower L/A ratio in FA silage (2.4) than in cellulase treated silage (4.1), the number of homofermentativelactic acid bac- teria was greater in the FA silage. The origi- nal hexose sugars in acid treated silage may partially be lost in effluent due to the high level of acid application (Henderson and McDonald 1971). Low L/A ratio in E2OO and untreated silage reflects rather fermenta- tion of lactic acid to acetic acid (Woolford 1984, Lindgren et ai. 1987), degradation of 58 amino acids to acetic acid, or more heterolac- tic fermentation (McDonald 1981) than var- iation in fermentable substrate. The presence of acetic acid may also indicate the action of saccharolytic Clostridia or enterobacteria (Woolford 1984). Even with grass rich in WSC, the silage produced with cellulolytic en- zymes had a higher L/A ratio than well- preserved untreated silage (Kauramaa et al. 1987 b). The positive effect of increased enzyme ap- plication on the quality of silage has also been reported by Leatherwood et al. (1963) and Autrey et al. (1975). When the level of cel- lulase increased from 0.3 to 0.9 g/kg lucerne, the pH decreased more quickly with higher lactic acid and lower ammonia N content (Hendersson et al. 1987). Hissa (1986) found increased soluble N content with increasing level of cellulase in laboratory silos probably due to the breakdown of cell walls and subse- quent release of cell contents. However, am- monia N and true protein content were not af- fected by the level of cellulase. Increasing the level of enzyme application increased the con- tent of lactic acid; however, with EBOO the ef- fect was obviously offset by higher losses of acids in the effluent. At the same time the L/A ratio increased giving no support to the sug- gestion of Kauramaa et al. (1987 b), that when lactic acid content exceeds 80—100 g/kg DM there may occur inhibition of the end product, resulting in a change in the bacterial population or its metabolism so that relative- ly more acetic acid is produced. Glucose oxidase (GO) converts free glucose into gluconic acid (pK a 3.76), reduces pH and consumes oxygen (Heikonen et al. 1987). No differences were observed in the fermen- tation parameters of enzyme silages in Exp. I, perhaps due to the low content of WSC in grass and the low rate of application of GO. The same was noticed also by Hissa (1986). The effect of GO on oxygen consumption was not measured. The maximum temperature was slightly lower in FA silages, indicating inhibit- ing effect of FA on respiratory enzymes. In laboratory conditions, in a well-sealed, well- compacted silage, anaerobiosis is achieved as rapidly as in 30 min (Sprague 1974, Wool- ford 1984). In-silo losses In agreement with the results of Brolly (1986) and Jacobs and McAllan (1987) en- zymes increased effluent losses as compared with FA and especially untreated silage. Leatherwood et al. (1963) and Vaisto et al. (1978) observed higher amounts of free wa- ter in enzyme than in untreated silages. In contrast, no increase in the volumeof effluent with enzymes was found by Brolly (1986) with lucerne and Kennedy (1987) with grass silage. Likewise enzyme treatment, FA re- leases cell sap by penetrating beyond the epicuticular layer into the tissue and by dis- rupting mesophyll cell membranes (Winters et al. 1987). The reaction is beneficial for rapid fermentation but leads to increased effluent formation with high moisture grasses (Peder- sen et al. 1973; Bastiman 1976). Digestibility of the silages The use of additives had no significant ef- fect on the digestibility of OM, which is in agreement with the results from other studies with FA and enzyme silage in steers (Jacobs and McAllan 1987) or in sheep (Brolly 1986). EBOO silage showed no evi- dence of secondary fermentation or aerobic deterioration, and the DM losses due to homo- and heterolactic fermentation are negligible (McDonald 1981). The decreased OM digest- ibility of EBOO might have been caused by greater effluent losses. Consequently, under the conditions of experiment 11, increasing the level of enzyme application improved fermen- tation quality while increasing effluent losses and decreasing OM digestibility. This suggests that no cut-off mechanism was present to stop enzyme activity, which should be a criteria for a good enzyme system (Seale 1987). The use of enzymes clearly reduced the di- gestibility of fibre in Exp. 11, indicating a 59 change in the composition of the fibre frac- tion during ensiling. The linear decrease in the NDF and ADF digestibilities with increasing level of enzyme application suggests that the enzymes degraded the most easily digestible cell wall fraction and the remaining NDF and ADF were proportionally less digestible. The same tendency towards lower fibre digestibil- ity has been noticed also by Brolly (1986) and Heikkilä et ai. (1987). Consistent with that, the disappearance of silage from nylon bags over long incubation times in the rumen showed no improvement in the potential di- gestibility (Hissa 1986, van Vuuren et al. 1989). Obviously the enzymes were not able to degrade the ligninpolysaccharide complexes of plant cell walls, which are indigestible by rumen microbes. In conclusion, the use of enzymes as addi- tive improved the fermentation quality of grass silage as compared withuntreated silage. The present results suggest that because the digestibility of OM cannot be improved by in- creasing the level of enzyme application, the optimal level of cellulase with low DM silage is the lowest possible ensuring satisfactory preservation. References Autrey, K.M., McCaskey, T.A. & Little, J.A, 1975. Cellulose digestibility of fibrous materials treated with Trichoderma viride cellulase. J. Dairy Sci. 58; 67—71. Barker, S.B. & Summerson, W.H. 1941. The colorimet- ric determination of lactic acid in biological materi- al. J. Biol. Chera. 138: 535—554. Bastiman, B. 1976. Factors affecting silage effluent pro- duction. Exp. Husb. 31: 40—46. Brolly, R.G, 1986. The effect of in-silo addition of cel- lulases and hemicellulases on the chemical composi- tion and nutritional value of silage. A thesis for the degreeof Master of Science in the Faculty of Science, University of Glasgow. 146 p. Done, D.L. 1986. Silage inoculants A review of ex- perimental work. Research and Development in Agriculture 2: 83—87. Goering, H.R. & van Soest, P. J. 1970. Forage fibre ana- lyses. Agriculture Handbook No. 379. United States Department of Agriculture. Washington. Heikkilä, T., Väätäinen, H. & Lampila, M. 1987. Erilaiset nurmirehut lypsylehmien ruokinnassa. Suo- men maataloustieteellisen seuran tiedote 9: 43—54. Heikonen, M., Moisio, T. & Harju, M. 1987. Compo- sition and method for ensilaging fodder and grain. European patent specification, number 0108568. Henderson, A.R. & McDonald, P. 1971. Effect of for- mic acid on the fermentation of grass of low dry mat- ter content. J. Sci. Food Agric. 22; 157—163. & McDonald, P. 1977. The effect of cellulase prepa- rations on the chemical changes during the ensilage of grass in laboratory silos. J. Sci. 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Finl. 45: 438—488. Jacobs, J.L. & McAllan, A.B. 1987. Enzyme additives for silage. Digestibilities and nitrogen retention in growing steers. Eight Silage Conference. AFRC, IGAP, Hurley, pp. 107—108. Jaster,E.H. & Moore, K.J. 1988. Fermentation charac- teristics and feeding value of enzyme-treated alfalfa haylage. J. Dairy Sci. 71: 705—711. Kennedy, S.J. 1987. The effect of an enzyme additive on the preservation and nutritive value of grass fed to beef cattle. Eight Silage Conference. AFRC, IGAP, Hurley, pp. 25—26. Leatherwood, J.M., Mochrie, R.D., Stone, E.J. & Thomas, W.E. 1963. Cellulose degradation by en- zymes added to ensiled forages. J. Dairy Sci. 46: 124—127. Lindgren, S., Linovall, P. & Pettersson, K. 1987. Re- lation between chemical quality and microbial com- position in silages. Eight Silage Conference. AFRC, 60 IGAP, Hurley, pp. 11—12. Merry, R.J. & Braithwaite, G.D. 1987. The effect of enzymes and inoculants on the chemical and microbi- ological composition of grass and legume silages. Eight Silage Conference. AFRC, IGAP, Hurley, pp. 27—28. Morrison, I.M. 1979. Changes in the cell wall compo- nents of laboratory silages and the effect of various additives on these changes. J. Agric. Sci., Camb. 93: 581—586. McCullough, M.E. 1964. Influence of cellulase on silage fermentation. J. Dairy Sci. 47; 342 (Abstr.). 1967. The determination of ammonia in whole blood by direct colorimetric method. Clin. Chim. Acta 17: 297—304. McDonald, P. 1981. The biochemistry of silage. John Wiley and Sons, 226 p. McHan, F. 1986. Cellulase-treated coastal bermudagrass silage and production of soluble carbohydrates, silage acids, and digestibility. J. Dairy Sci. 69: 431—438. Nehring, K., Heinz, D. & Friedel, K. 1983. Der Einfluss von Zellulase auf die Silierung von eiweissreichen Fut- terstoffen. Arch. Tierernhr. 33: 251—258. Olson, M. & Voelker, H.H. 1961. Effectiveness of en- zyme and culture additions on the preservation and feeding value of alfalfa silage. J. Dairy Sci. 44: 1204 (Abstr.) Owen, F.G. 1962. Effect of enzymes and bacitracin on silage quality. J. Dairy Sci. 45: 934—936. Pedersen, T.A., Olsen, R.A. & Guttormsen, D.M. 1973. Numbers and types of microorganisms in silage and effluent from grass ensiled with different addi- tives. Acta Agric. Scand. 23: 109—120. Pettersson, K. 1988. Ensiling of forages. Factors affect- ing silage fermentation and quality. Swedish Univ. Agric. Sci., Dept. Anim. Nutr. Managem., Rapport 179, Uppsala (Diss.) Porter, M.G., Patterson, D.C., Steen, R.W.J. & Gor- don, F.J. 1984. Determination of dry matter and gross energy of grass silage. Seventh silage conference. Summary of Papers, p. 8. The Queens University of Belfast. Kauramaa, A., Setälä, J., Moisio, T., Heikkilä, T. & Lampila, M. 1987 a. The effect of inoculants and cel- lulase on the fermentation and microbiological com- position of grass silage. 1 Biochemical changes in the silages. J, Agric. Sci. Finl. 59: 361—370. —, Setälä, J., Moisio, T., Sivelä, S., Heikkilä, T. & ■Lampila, M. 1987b. The effect of inoculants and cel- lulase on the fermentation and microbiological com- position of grass silage. II Microbiological changes in the silages. J. Agric. Sci. Finl. 59: 371 —377. Salo, M-L. 1965. Determination of carbohydrate frac- tions in animal foods and faeces. Acta Agr. Fenn. 105: 1 102. Saue, O. & Breirem, K. 1969. Formic acid as a silage additive. Proc. 3rd Gen. Meet. Eur. Grassl. Fed., Braunschweig, pp. 161—172. Seale, D.R. 1987. Bacteria and enzymes as products to improve silage preservation. In: Developments in si- lage (ed. Wilkinson, J.M. & Stark, 8.A.), Chalcombe Pubi. pp. 47—61. —, Henderson, A.R., Petterson, K.O, & Lowe, J.F. 1986. 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Food Agric. 26: 219—228. 1984. The silage fermentation. New York, Marcel Dekker Inc., 350 p. Ms received November 10, 1990 61 SELOSTUS Kuitua hajottavien entsyymien vaikutus nurmirehun säilyvyyteen ja syöntiin sekä sulavuuteen lampaalla Seija Jaakkola Helsingin yliopisto, kolieläinlieleen laitos, 00710 Helsinki Kahdessa kokeessa tutkittiin kasvin soluseinämiä ha- jottavien entsyymien käyttöä nurmirehun säilöntäainee- na. Kokeessa 1 timoteinurmen ensimmäinen sato niitet- tiin aikaisella kasvuasteella ja korjattiin tarkkuussilp- purilla. Kokeessa 11 korjuu tehtiin myöhäisellä kasvu- asteella suoraan tarkkuussilppurilla. Säilöntäainekäsit- telyt olivat kokeessa 1:1) muurahaishappo (MH) AIV-2 liuoksena 4.51/t, 2) painorehu (ei käsittelyä), 3) MH 41/t, 4) entsyymi A (glukoosioksidaasi (GO) + hemisellulaasi (HS) 150ral/t + sellulaasi (S) 200 ml/t), 5) entsyymi B (HS 150ml/t + S 200 ml/t). Rehu 1 korjattiin tuoreena jarehut 2—5 esikuivattuna. Kokeen II käsittelyt olivat: 1) painorehu, 2) MH 4 1/t, 3) E2OO (S 200 ml/t + GO), 4) E4OO (S 400ml/t + GO), 5) EBOO (S 800 ml/t + GO). GO:n annostus oli 50 000 lU/ml. Rehut tehtiin 3 m’:n pilotsiiloihin. Vapaaehtoinen syönti ja sulavuus määri- tettiin lampailla kahdessa 5x5 latinalaisen neliön mu- kaisessa kokeessa. Entsyymien käyttö alensi säilörehun kuitupitoisuutta MH- japainorehuun verrattuna. Entsyymirehujen säilön- nällinen laatu oli hyvä pH:n vaihdellessa 3.93 ja 4.15 vä- lillä sekä ammoniakkitypen 72 —119 g/kg kok. N. Re- huissa ei ollut voihappoa. Painorehujen laatu oli selvästi entsyymirehuja heikompi (pH 4.6, ammoniakkityppi 133 g/kg kok. N). MH-rehut olivat hyvin säilyneitä fer- mentaation ollessa selvästi vähäisempää kuin entsyymi- rehuissa. Sekä MH että korkeimmat entsyymimäärät li- säsivät puristenesteen muodostumista. Kokeessa I säi- löntäaineet eivät vaikuttaneet (P>0.05) kuiva-aineen (KA), orgaanisen aineen (OA) tai raakakuidun sulavuu- teen. Kokeessa II entsyymien annostustason nosto huo- nonsi lineaarisesti (P<0,01) KA:n ja OA:n sulavuuksia. NDF:n ja ADF:n sulavuudet olivat paremmat painore- hussa kuin käsitellyissä rehuissa, paremmat MH rehussa kuin entsyymirehuissa ja huononivat lineaarisesti entsyy- mitason noustessa (P<0.01). Entsyymirehujen sulavuus oli kuitenkin selvästi huonompi vain käytettäessä korkeim- pia entsyymitasoja. 62