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Comparative analysis of silage fermentation and in vitro digestibility of tropical grass prepared with Acremonium and Tricoderma species producing cellulases.


Purple Guinea grass (Panicum maximum cv. TD 58) and Napier grass (Pennisetum purpureum xPennisetum americanum cv. Pak Chong 1) are widespread throughout tropical and subtropical areas. They adapt and grow well in a variety of soil types, are tolerant to different conditions and require quite low inputs for growth [1-3]. Both grasses are important roughage sources for ruminant feed because of high dry matter biomass production; ranged from 15 to 18 t/ha per year for Guinea grass [1] and 63 to 87 t/ha per year for Napier grass [3,4]. However, both grasses are seasonal with high dry matter yield over the short rainy season; thus, silage making is an increasingly important method to preserve fresh forages for feeding ruminants year-round [5]. It is well known to be difficult to make good quality silage with these species because they are usually low in water soluble carbohydrate (WSC) content, insufficient for rapid decrease of silage pH. They are also low in dry matter and high in fiber content which promotes the growth of clostridia and decreases nutrient utilization of ruminants [6-8].

Cellulase enzymes are popular biological products for improving the fermentation quality of silages. It is widely accepted that they can improve the fermentation process of both grasses and legumes and thus enhance cattle performance [6-8]. The two main reasons for cellulase enzyme addition in silage making are firstly, to enhance plant fiber degradation to increase the content of WSC as a lactic acid bacteria (LAB) substrate to produce lactic acid and enhance the fermentation process [9-11] and secondly, to break down the structural carbohydrate component thus improving silage digestibility [10,12,13]. Many microorganisms that produce various cellulolytic enzymes have been studied for several decades. The genus of Acremonium and Trichoderma has been especially famous for producing cellulolytic enzymes with relatively high enzymatic activity [14]. There is increasing interest in applying cellulase enzymes at ensiling due to reports that it can improve the fermentation quality of silage [15,16].

However, there is limited information available for comparative analysis of silage prepared with these cellulases produced from Acremonium and Trichoderma. The objectives of this study were to determine the fermentation quality and in vitro digestibility of tropical grass silage prepared with two cellulases at different concentration.


Forage sample and silage preparation

Purple Guinea grass (Panicum maximum cv. TD 58) and Napier (Pennisetum purpureumxPennisetum americanum cv. Pak Chong 1) grass were grown in a Korat soil series (Oxic Paleustults) at the experimental farm, Faculty of Agriculture, Khon Kaen University, Thailand in May 2014. Before planting, the plot for Guinea grass was ploughed twice and harrowed once. Root stock of purple Guinea grass was planted by hand with a spacing of 75x75 cm in row and between rows in a 10x 30 m of the rectangular plot. The plot was fertilized with cattle manure at a rate of 24,000 kg/ha which 4 separate equal portions of 6,000 kg/ha split applied for 4 cuts [16,17]. For Napier grass, one week before planting, the plot was ploughed and fertilized with basal fertilizers of NPK (15-15-15) and cattle manure at 300 and 12,500 kg/ha, respectively. The 11,111 stem cuttings of Napier grass were planted by hand with a spacing of 120x75 cm in row and between rows in a total area of 1,600 [m.sup.2]. Two weeks after planting, the plot was weeded out and nitrogen fertilizer (urea) applied at a rate of 60 kg/ha [4]. On 20 April 2015, guinea grass was cut to adjust the height to 10 cm above ground level and Napier grass was cut close to the soil surface. After 60 d of regrowth, both grasses were harvested in early morning and immediately chopped using a forage chopper (Supachai, Kanchanaburi, Thailand) to 1 cm particle size. Half of each chopped grass sample was wilted for 6 h in the shade to study the effect of moisture adjustment. Both fresh or wilted forages were then treated as follows: control (untreated), Acremonium cellulase (AC, Meiji Seika Pharma Co., Ltd, Tokyo, Japan): AC 0.0025%, AC 0.005%, AC 0.01%, Trichoderma cellulase (TC, Meiji Seika Pharma Co., Ltd, Japan): TC 0.0025%, TC 0.005%, and TC 0.01%. The production strain, main composition and carboxymethyl-cellulase (CMCase) of AC were Acremonium cellulolyticus, glucanase and pectinase, and 7,350 U/g; for TC they were Tricoderma viride, xylanase and glucanase, and 2,720 U/g, respectively. Treatments were ensiled using a small scale fermentation system [18]. Both AC and TC cellulases were added as % of fresh matter (FM). The design of this experiment was a 2 (two grasses)x2 (fresh vs wilted)x2 (two enzymes)x3 (three application levels) factorial arrangement in a completely randomized design with triplicates for each treatment. The silage was prepared with a laboratory-scale fermentation method [16]. The mixed grass with additive (1,000 g) was packed into a synthetic silo laminated from nylon and polyethylene (Hiryu KN type, Asahikasei, Tokyo, Japan), and sealed by using a vacuum sealer (SQ-303, Asahi Kasei Pax Corp., Japan). All silos were stored at room temperature (25[degrees]C to 37[degrees]C). On 30 d of ensiling, silos were opened for evaluation of fermentation products, chemical composition, and in vitro digestibility.

Microorganism analysis

Microorganism numbers were analyzed using the plate count method as described by [19], and reported as colony-forming unit (cfu)/g of FM. Fresh or wilted forage samples (1,000 g) or silage samples (200 g) were sub-sampled for 10 g samples. Then, each sample (10 g) was homogenized with 90 mL sterilized distilled water, and serial dilutions in 0.85% sodium chloride solution at [10.sup.-1] to [10.sup.-5]. Each dilution (20 [micro]L) was dropped and spread on prepared agar plates. MRS agar (Difco Lab-oratories, Detroit, MI, USA) was used for the LAB count, which the agar plates were put into an anaerobic box (TE-HER Hard Anaerobox, ANX-1; Hirosawa Ltd., Tokyo, Japan), and incubation at 30[degrees]C for 48 h in an incubator. Blue light broth agar (Nissui-seiyaku Ltd., Tokyo, Japan) was used for coliform bacteria counts after incubating at 30[degrees]C for 48 h. Nutrient agar (Difco, USA) and potato dextrose agar (Nissui-seiyaku, Japan) were used for aerobic bacteria, yeast, and mold. Mold was counted after 2 d of incubation. Yeasts were distinguished from molds or bacteria by colony appearance and cell morphology observation.

Chemical composition

Cold water extraction of silage samples was used for fermentation end-product analysis following the method of Cai [20]. Ten grams of wet silage was soaked with 90 mL sterilized distilled water, and then, incubated at 4[degrees]C in a refrigerator overnight. Silage pH was measured immediately after incubating using a glass electrode pH meter (FiveGo; Mettler Toledo, Greifensee, Switzerland), and 10 mL of cold water extract was sampled and stored at -20[degrees]C for further analysis of organic acids and ammonia nitrogen concentrations. Organic acid content of silages was measured by HPLC methods [20]. Ammonia nitrogen concentration was analyzed using a spectrophotometer (UV/VIS Spectrometer, PG Instruments Ltd., London, UK) [21]. The lactate buffering capacity (LBC) of forages was analyzed by the titration method viz. 10 g of sample homogenized with distilled water, titrated with 0.1 M hydrochloric acid (HCl) to decrease pH from initial pH to 3, then titrated to pH 6 with 0.1 M sodium hydroxide (NaOH) [13]. The WSC content of forage was extracted and measured following the methods of [16].

Forage and silage samples on 30 d of ensiling were dried at 60[degrees]C for 48 h in a hot air oven, and ground through a 1 mm mesh screen. Dry matter (DM), organic matter (OM), crude protein (CP), and ether extract (EE) were analyzed following methods 934.01, 942.05, 976.05, and 920.39, respectively [22]. Neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were analyzed using a fiber analyzer (ANKOM 200, ANKOM Technology, New York, USA), based on the method of [23].

Animal care

The animal experiment was approved by the Animal Care and Use Committee of Khon Kaen University (KKU), Khon Kaen, Thailand and performed at KKU in August 2015. The experiment was performed according to recommendations proposed by the European Commission [24] and to minimize the suffering of animals.

In vitro digestibility analysis

In vitro digestibility of DM (IVDMD) and OM (IVOMD) were measured after incubating samples in buffered rumen fluid at 6 and 48 h. The buffered rumen inoculum was prepared following the method of [25]. Rumen fluid was collected from two dairy steers by a stomach tube sucker before morning feeding. The pH of rumen fluid was 7.2. Ground silage samples (0.5 g) was put into serum bottles of 50 mL capacity (3 replications per sample). The bottles were closed by a rubber stopper with an aluminum seal cap. The buffered rumen inoculum (40 mL) was injected into each sample bottle using a 60 mL syringe (Nipro Thailand corporation, Ltd., Phra Nakhon Si Ayutthaya, Thailand) with 18 gaugex3.5 cm needle (Nipro Corporation, Osaka, Japan), and flushed with carbon dioxide gas to produce an anaerobic condition. All samples were incubated in a water bath at 39[degrees]C, swirled by hand and the head space gas production released using a 21 gaugex3.5 cm needle (Nipro, Japan) at 2 h intervals. Two blanks of 40 mL of rumen inoculum per bottle were also incubated.

The animals were housed in individual pen equipped with mineral blocks (FNZ Red Lick, Thai Serve Co. Ltd., Buriram, Thailand; mineral composition: NaCl 930.00 g, Mg 2.00 g, Zn 0.77 g, Mn 0.50 g, Co 18.00 g, I 0.05 g, Se 0.01 g, Cu 0.22 g, another 2.50 g) and fresh water, and fed concentrate (16% CP, 11.0 MJ/kg of metabolizable energy, feed ingredient including: 10% palm kernel meal, 7% coconut meal, 6% rice bran, 70% cassava chip, 4% urea, 1% sulfur, 1% mineral premix, and 1% salt on a DM basis) at 0.5% of body weight and ad libitum rice straw daily. The diet was fed in two equal meals at 08:00 and 16:00 h.

Statistical analysis

Data on fermentation products, chemical composition, and in vitro digestibility of silages at 30 d after fermentation were analyzed using a completely randomized design with a 2x2x 2x3 (forages [A]xmoisture adjustment [B]xenzymes [C]x application levels [D]) factorial treatment combinations. The analysis of variance procedure of SAS version 6.12 (SAS Institute Inc., Cary, NC, USA) was used for the analysis and the statistical model is as follows:

[mathematical expression not reproducible]

Where [Y.sub.ijklm] = observation; [mu] = overall mean, [[alpha].sub.i] = forage effect (i = Guinea grass and Napier grass), [[beta].sub.j] = moisture adjustment effect (j = fresh and wilted), [[gamma].sub.k] = enzyme effect (AC and TC), [[delta].sub.1] = application levels (0.0025%, 0.005%, and 0.01%), [alpha][beta][gamma][[delta].sub.ijkl] = forages x moisture adjustment x enzymes x application levels, and [[epsilon].sub.ijklm] = error. The significant difference among treatment means was tested by Duncan's new multiple range test at p = 0.05 [26].


Microorganism population and chemical composition of forages

The microorganism counts of forages are shown in Table 1. Fresh Guinea grass and fresh Napier grass showed similar numbers of LAB ([10.sup.3] cfu/g of FM), aerobic bacteria ([10.sup.7] cfu/g of FM), and coliform bacteria ([10.sup.7] cfu/g of FM). The counts of yeasts and molds of both fresh grasses ranged from [10.sup.4] to [10.sup.5] and [10.sup.2] to [10.sup.3] cfu/g of FM, respectively. After wilting process, the counts of LAB, yeasts and molds of both grasses increased from [10.sup.4] to [10.sup.5], [10.sup.5] to [10.sup.6], and [10.sup.4] cfu/g of FM, respectively, the counts of coliform bacteria and aerobic bacteria did not change by the wilting process.

The chemical composition and LBC of Guinea grass and Napier grass are shown in Table 2. The OM content was higher (p<0.01) and WSC content was lower (p<0.01) in Napier grasses than those in Guinea grasses. During the wilting process, the DM of Guinea grass and Napier grass significantly (p<0.01) increased by 7% and 8%, respectively, but the contents of ADL and LBC contents did not differ. Compared with fresh Guinea grass, the contents of CP and ADF were higher (p<0.01), but the NDF content was lower in wilting Guinea grass. Also, wilted Napier grass showed higher (p<0.01) CP content and lower EE content than fresh Napier grass.

Fermentation quality of silages

The DM, pH, and fermentation products of Guinea grass and Napier grass silages at 30 d of ensiling are shown in Table 3. The interaction between A, B, C, and D influenced (p<0.01) the silage DM, pH, all organic acid contents, and ammonia nitrogen content. Guinea grass silage treated with AC 0.01% showed good quality with lower (p<0.01) pH and ammonia nitrogen, and higher (p<0.01) lactic acid content than other silages. Regarding the differences in production strain (A. cellulolyticus vs T. viride), main composition (glucanase and pectinase, vs xylanase and glucanase), and CMCase (7,350 vs 2,720 U/g) between AC and TC, the highest (p<0.01) pH and ammonia nitrogen content with lowest (p<0.01) lactic acid content were found in wilted Guinea grass silages treated with TC 0.05%. The AC treatments improved grass silage quality more than TC treatments. The addition level of AC at 0.01% improved fermentation quality more than 0.005% and 0.0025%.

Microorganism counts of silages

The microbiological analysis of Guinea grass and Napier grass silages at 30 d of ensiling are shown in Table 4. The A, B, C, D and their interaction (AxBxCxD) were not significantly different (p<0.05) in LAB count, aerobic bacteria and yeasts. Coliform bacteria and molds were below the detectable level (102 cfu/g of FM) in all silages. The LAB population of all silages was dominant ranging from [10.sup.7] to [10.sup.8] cfu/g of FM. Aerobic bacteria were [10.sup.3] to [10.sup.4] cfu/g of FM and yeast were [10.sup.2] to [10.sup.4] cfu/g of FM.

Chemical composition of silages

The chemical compositions of Guinea grass and Napier grass silages at 30 d of ensiling are shown in Table 5. The significances of AxBxCxD (p<0.001) influenced all chemical compositions. The OM content of AC or TC treated Guinea grass silage were higher (p<0.01) than other silages. The wilted Napier grass silages treated with AC 0.01% showed higher (p<0.01) CP content and lower (p<0.01) NDF and ADF content than other silages. The highest (p<0.01) fiber contents were observed in TC treated fresh Guinea grass silages. When silages treated with AC, the CP, EE, and ADL contents were significantly (p< 0.01) higher and the NDF and ADF contents were significantly (p<0.01) lower than TC treatments; and the application levels of enzymes at 0.01% decreased fiber content more than 0.005% and 0.0025%.

In vitro digestibility of silages

The in vitro digestibility of Guinea grass and Napier grass silages at 30 d of ensiling are shown in Table 6. Fresh Napier grass silages treated with AC 0.01% had higher (p<0.05) IVDMD than other treatments. Napier grass silages had higher (p<0.01) IVDMD and IVOMD than Guinea grass silages. The wilted grasses silages had higher (p<0.05) IVOMD than fresh grasses silages. When silages treated with AC, the IVDMD and IVOMD at 6 h after incubation were significantly higher (p<0.01) than those of TC treatments. The IVDMD and IVOMD of 0.01% cellulase treated silage at 6 and 48 h after incubation were greater (p<0.05) than those of 0.0025%. The significances of AxBxCxD (p<0.05) influenced IVDMD at 48 h after incubation in buffered rumen fluid, while IVDMD at 6 h after incubation and IVOMD at both 6 and 48 h after incubation did not.


Generally, the DM content of forages influences fermentation quality of the silage; and optimal DM content ranges from 30% to 40% for good quality silage making [13,27]. If dry matter content is less than 20%, the fermentation process would be dominated by clostridium, resulting in low quality silage production [28]. In this study, the DM content of fresh Guinea grass and Napier grass were lower than 18%, which was not ideal to preserve the forage. When both grasses were wilted for 6 h in the field, DM contents of Guinea grass and Napier grass were higher than 21%, much more suitable for ensiling. The decreasing of moisture during the wilting process under this experiment is negligible and less than those of the optimal DM content ranges. In addition, the wilted both grasses as shown Table 2 had a higher CP and lower ADF compared with that of fresh forage. However, we cannot fully explain the mechanism of these effects, this may be influenced by the climatic conditions such as low temperature and high humidity during the wilting process. Future study is necessary to study the effect of climatic condition on the chemical composition of forage during wilting process.

The CP, NDF, and ADF of Guinea grass and Napier grass used in this study were similar to our previous work which reported that Guinea grass and Napier grass harvested at 60 d of regrowth were lower in CP contents, but high in fiber [16]. The WSC in the ensiling material also plays an important role in silage fermentation and it is the main energy source for LAB growth to produce lactic acid [29,30]. Wilkinson [27] suggested that WSC needs to be greater than 10% of DM for good fermentation. However, the WSC contents of fresh forages in this present study were less than 2.7% of DM (Table 2). Although a lower WSC content was found in Guinea grass compared to Napier grass, it would be difficult to make good quality silage from both grasses [5].

At 30 d of fermentation, Guinea grass silages were poor quality. As shown in Tables 1, 2, both fresh and wilted Guinea grasses had low LAB counts and WSC content which were not sufficient to produce more lactic acid for good fermentation. Thus, the pH of Guinea grass silages did not decrease below 4.2, which cannot inhibit the growth of harmful bacteria, especially clostridia [13]. As a result, a harmful fermentation could occur with clostridia using WSC, amino acid, and other organic acids to produce butyric acid and ammonia nitrogen.

Some protein of forages breaks down to simpler or nonprotein nitrogen during wilting process, result in decreased true protein solubility [31]. Nevertheless, this is consistent with previous studies [32] that total protein content of intact asparagus spear and excised tip sections increased approximately 10% after 6 to 12 h of harvested. Also, our results indicated that wilted forages silages were lower (p<0.01) lactic acid content than fresh forage silages. Consistently, our previous study [16] evidenced a lower lactic acid concentration in silages prepared from wilted forages compared to fresh forages. The significantly higher DM in wilted forages silages may attribute to decrease LAB growth and activity during silage fermentation.

When grass silages treated with AC 0.01%, their fermentation quality were more improved compared to other treatments. Our findings are in agreement with [9,33-35]. that the addition of cellulase enzyme resulted in a decrease of pH and increased lactic acid content in sorghum straw, oil palm frond and mixed hulless-barley straw with corn silages. This could be attributed to the cellulase enzyme degrading plant cellulose so increasing the WSC, an essential substrate for LAB growth and more lactic acid production. As a result, the pH of silages decrease rapidly inhibiting the growth of clostridia and preventing proteolysis in the silo [33,36]. Some studies [15,29,34,37] reported that addition of cellulase resulted in a decrease in fiber content of mixed silages of hulless-barley straw and corn, wheat straw, Leymus chinensis, and Napier grass silage. On the other hand, cellulase addition had no effect on NDF and ADF content of barley and vetch silage [38]. In the present study, the AC improved silage fermentation more than TC, and the AC 0.01% cellulase effectively increased CP content and reduced NDF and ADF contents of silages. Perhaps the type, composition and enzyme activity of cellulase may affect cellulose degradation and silage fermentation. Generally, cellulases catalyze the hydrolysis of cellulose, which are mainly three types: endoglucanases, cellobiohydrolases and [beta]-glucosidases, and it is used for any occurring mixture or complex of various such enzymes, that act serially or synergistically to decompose cellulosic material. Acremonium cellulolyticus-producing cellulases contain a strong glucanase and pectinase, and cellulase produced by Trichoderma viride contains mainly xylanase and glucanase. Therefore, Acremonium cellulolyticus-producing cellulase is said to be more potent than Trichoderma viride-producing cellulase. This indicated that AC is more effective for improvement of silage fermentation than TC, and its addition concentration is necessary to be 0.01% on a FM basis.

The digestibility of roughage also plays an important role in animal production [10,29]. The IVDMD and IVOMD depend on physical characteristics of forage, especially the fiber content, low NDF and ADF contents resulted in a rapid increase in digestibility of DM and OM [29,39]. In the present study, Napier grass silages had higher IVDMD and IVOMD when compared to Guinea grass. This could be attributed to the differences in chemical composition of Guinea grass and Napier grass. Guinea grass had higher fiber content than Napier grass as shown in Table 2. Furthermore, the cellulase enzyme addition, especially AC 0.01% treated silages had greater IVDMD and IVOMD values than other enzyme application levels in both 6 and 48 h of incubation (Table 6). This study is consistent with previous studies reported by [10,40] that the addition of an exogenous cellulase enzyme could increase IVDMD of forage crops, but inconsistent with [29] that cellulase addition did not increase the IVDMD of Leymus chinensis after 48 h of incubation. The greater effect of AC 0.01% treatment in the present study due to the composition of enzymes. The AC used in this study has both glucanase and pectinase; the synergistic effects of these enzymes probably increase silage digestibility. The CMCase activity is also higher in AC (7,350 U/g) than TC (2,720U/g).

These results confirmed that Napier grass has more suitable ensiling characteristics than Guinea grass, and the wilted silage can decrease the feed nutrient loss and improve the in vitro digestibility. The addition of AC 0.01% cellulase (73.5 U/g of FM) was effective in improving the silage fermentation and promoting the in vitro degradation of tropical grass.


Silage fermentation and in vitro digestibility of Guinea grass and Napier grass prepared with cellulase enzyme at different addition concentration were studied. The AC has the potential to improve the fermentation quality, chemical composition and in vitro degradation of Guinea grass and Napier grass, but the TC did not affect the tropical silage fermentation. The AC addition at 0.01% (73.5 U/g) of FM was the most promising for improving silage fermentation containing low WSC.


We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.


This work was supported by the Project "The Establishment of the Sustainable and Independent Farm Household Economy in the Rural Areas of Indo-China", Japan International Research Center for Agricultural Sciences (JIRCAS), Japan. Acknowledgments are extended to Meiji Seika Pharma Company, Ltd., Tokyo, Japan for providing the commercial cellulase enzymes.


[1.] Phitsuwan P, Charupongrat S, Klednark R, Ratanakhanokchai K. Structural features and enzymatic digestibility of Napier grass fibre treated with aqueous ammonia. J Ind Eng Chem 2015;32:360-364.

[2.] Reddy KO, Maheswari CU, Shukla M, Rajulu AV. Chemical composition and structural characterization of Napier grass fibers. Mater Lett 2012;67:35-38.

[3.] Hare M, Tatsapong P, Phengphet S. Herbage yield and quality of Brachiaria cultivars, Paspalum atratum and Panicum maximum in north-east Thailand. Trop Grassl 2009;43:65-72.

[4.] Kiyothong K. Manual for planting Napier pakchong 1. Nakhonrajasrima, Thailand: Department of Livestock Development, Thailand; 2014.

[5.] Pholsen S, Khota W, Pang H, Higgs D, Cai Y. Characterization and application of lactic acid bacteria for tropical silage preparation. Anim Sci J 2016;87:1202-1211.

[6.] Zhu Y, Nishino N, Xusheng G. Chemical changes during ensilage and in sacco degradation of two tropical grasses: Rhodesgrass and guineagrass treated with cell wall-degrading enzymes. Asian-Australas J Anim Sci 2011;24:214-221.

[7.] Yitbarek M, Tamir B. Silage additives: review. Open J Appl Sci 2014;4:258-274.

[8.] Takekhalaf M, Torbatinejad NM, Zerehdaran S, Toghdory AH. Effect of treated alfalfa silage with lactobacillus plant arum and sugar beet pulp molasses on performance of Holstein dairy cows. J Appl Environ Biol Sci 2015;5:254-259.

[9.] Xing L, Chen LJ, Han LJ. The effect of an inoculant and enzymes on fermentation and nutritive value of sorghum straw silages. Bioresour Technol 2009;100:488-491.

[10.] Li M, Zi X, Zhou H, Hou G, Cai Y. Effects of sucrose, glucose, molasses and cellulase on fermentation quality and in vitro gas production of king grass silage. Anim Feed Sci Technol 2014;197:206-212.

[11.] Eun J-S, Beauchemin KA. Relationship between enzymic activities and in vitro degradation of alfalfa hay and corn silage. Anim Feed Sci Technol 2008;145:53-67.

[12.] Ridla M, Uchida S. Effect of combined treatment of lactic acid bacteria and cell wall degrading enzymes on fermentation and composition of rhodesgrass (Chloris gayana Kunth.) silage. Asian-Australas J Anim Sci 1998;11:522-529.

[13.] McDonald P, Henderson A, Heron S. The biochemistry of silage. Marlow, UK: Chalcombe Publications; 1991.

[14.] Li X, Yang H, Roy B, et al. Enhanced cellulase production of the Trichoderma viride mutated by microwave and ultraviolet. Microbiol Res 2010;165:190-198.

[15.] Desta ST, Yuan XJ, Li J, Shao T. Ensiling characteristics, structural and nonstructural carbohydrate composition and enzymatic digestibility of Napier grass ensiled with additives. Bioresour Technol 2016;221:447-454.

[16.] Khota W, Pholsen S, Higgs D, Cai Y. Natural lactic acid bacteria population of tropical grasses and their fermentation factor analysis of silage prepared with cellulase and inoculant. J Dairy Sci 2016;99:9768-9781.

[17.] Yoottasanong C, Pholsen S, Higgs DEB. Dry matter yields and forage quality of grass alone and grass plus legume mixture in relation to cattle manure rates and production methods. Pakistan J Biol Sci 2015;18:324-332.

[18.] Cai Y, Kumai S, Ogawa M, Benno Y, Nakase T. Characterization and identification of Pediococcus species isolated from forage crops and their application for silage preparation. Appl Environ Microbiol 1999;65:2901-2906.

[19.] Kozaki M, Uchimura T, Okada S. Experimental manual for lactic acid bacteria. Tokyo, Japan: Asakurasyoten; 1992. p. 29-72.

[20.] Cai Y. Analysis method for silage. In: Japanese Society of Grassland Science, editor. Field and Laboratory methods for grassland science. Tokyo, Japan: Tosho Printing Co. Ltd.; 2004.

[21.] Fawcett JK, Scott JE. A rapid and precise method for the determination of urea. J Clin Pathol 1960;13:156-159.

[22.] AOAC. Official methods of analysis. 15th edn. Arlington, VA, USA: Association of Official Analytical Chemists; 1990.

[23.] Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 1991;74:3583-3597.

[24.] European Commission. "European Union Strategy for the Protection and Welfare of Animals 2012-2015", Communication from the Commission to the European Parliament. The Council and the European Economic and Social Committee; 2002. COM 6 final/2, 15 Feb, 2012.

[25.] Makkar HP, Blummel M, Becker K. Formation of complexes between polyvinyl pyrrolidones or polyethylene glycols and tannins, and their implication in gas production and true digestibility in in vitro techniques. Br J Nutr 1995;73:897-913.

[26.] Steel RGD, Torrie JH. Principles and procedures of statistics: a biometrical approach. 2nd ed. New York, NY, USA: McGrawHill Book Co. Inc.; 1980.

[27.] Wilkinson M. Silage UK. Great Britain: Cambrian printers; 1990.

[28.] Nkosi BD, Meeske R, Langa T, Motiang MD, Mutavhatsindi TF. The influence of ensiling potato hash waste with enzyme/bacterial inoculant mixtures on the fermentation characteristics, aerobic stability and nutrient digestion of the resultant silages by rams. Small Rumin Res 2015;127:28-35.

[29.] Zhang Q, Yu Z, Yang H, Na RS. The effects of stage of growth and additives with or without cellulase on fermentation and in vitro degradation characteristics of Leymus chinensis silage. Grass Forage Sci 2016;71:595-606.

[30.] Cai Y, Benno Y, Ogawa M, Kumai S. Effect of applying lactic acid bacteria isolated from forage crops on fermentation characteristics and aerobic deterioration of silage. J Dairy Sci 1999; 82:520-526.

[31.] Rotz CA, Muck RE. Changes in forage quality during harvest and storage. In: Fahey GC, Collins M, Mertens DR, Moser LE, editors. Forage quality, evaluation, and utilization. Madison, USA: American Society of Agronomy, Crop Science Society of America, Soil Science Society of America; 1994. pp. 828-68.

[32.] King GA, Woollard DC, Irving DE, Borst WM. Physiological changes in asparagus spear tips after harvest. Physiol Plant 1990;80:393-400.

[33.] Oladosu Y, Rafii MY, Abdullah N, et al. Fermentation quality and additives: a case of rice straw silage. Biomed Res Int 2016; 2016:1-14.

[34.] Guo G, Yuan X, Li L, Wen A, Shao T. Effects of fibrolytic enzymes, molasses and lactic acid bacteria on fermentation quality of mixed silage of corn and hulless-barely straw in the Tibetan Plateau. Grassl Sci 2014;60:240-6.

[35.] Ebrahimi M, Rajion MA, YongMeng G, et al. The effects of adding lactic acid bacteria and cellulase in oil palm (Elais guineensis Jacq.) frond silages on fermentation quality, chemical composition and in vitro digestibility. Ital J Anim Sci 2014;13: 557-62.

[36.] Nkosi BD, Vadlani PV, Brijwani K, Nanjunda A, Meeske R. Effects of bacterial inoculants and an enzyme on the fermen tation quality and aerobic stability of ensiled whole-crop sweet sorghum. S Afr J Anim Sci 2012;42:232-40.

[37.] Ni K, Wang Y, Pang H, Cai Y. Effect of cellulase and lactic acid bacteria on fermentation quality and chemical composition of wheat straw silage. Am J Plant Sci 2014;5:1877-84.

[38.] Kung L, Carmean B, Tung R. Microbial inoculation or cellulase enzyme treatment of barley and vetch silage harvested at three maturities. J Dairy Sci 1990;73:1304-11.

[39.] Huhtanen P, Rinne M, Nousiainen J. Evaluation of the factors affecting silage intake of dairy cows: a revision of the relative silage dry-matter intake index. Animal 2007;1:758-70.

[40.] Moharrery A, Hvelplund T, Weisbjerg MR. Effect of forage type, harvesting time and exogenous enzyme application on degradation characteristics measured using in vitro technique. Anim Feed Sci Technol 2009;153:178-92.

Waroon Khota (1), Suradej Pholsen (1), David Higgs (2), and Yimin Cai (3),*

* Corresponding Author: Yimin Cai Tel: +81-298386365, Fax: +81-298386653, E-mail:

(1) Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand

(2) Department of Biological and Environmental Sciences, University of Hertfordshire, AL10 9AB, UK

(3) Japan International Research Center for Agricultural Science (JIRCAS), Tsukuba, Ibaraki 305-8686, Japan


Waroon Khota

Suradej Pholsen

David Higgs

Yimin Cai

Submitted Jan 22, 2018; Revised Mar 22, 2018; Accepted May 11, 2018
Table 1. Microorganism counts (cfu/g of FM) of Guinea grass and
Napier grass before ensiling

Items           Lactic acid        Coliform         Aerobic
                  bacteria         bacteria         bacteria

Guinea grass
  Fresh        1.8x[10.sup.3]   1.5x[10.sup.7]   2.6x[10.sup.7]
  Wilted       1.2x[10.sup.4]   4.2x[10.sup.7]   2.4x[10.sup.7]
Napier grass
  Fresh        2.5x[10.sup.3]   8.1x[10.sup.7]   1.1x[10.sup.7]
  Wilted       3.5x[10.sup.5]   6.2x[10.sup.7]   2.0x[10.sup.7]

Items              Yeast             Mold

Guinea grass
  Fresh        2.9x[10.sup.4]   1.3x[10.sup.3]
  Wilted       1.5x[10.sup.5]   1.6x[10.sup.4]
Napier grass
  Fresh        1.5x[10.sup.5]   2.5x[10.sup.2]
  Wilted       2.0x[10.sup.6]   2.9x[10.sup.4]

cfu, colony-forming unit; FM, fresh matter.

Table 2. Chemical composition (% of DM) and lactate buffer capacity
(LBC, mE/kg of DM) of Guinea grass and Napier grass before ensiling

Items              DM           OM          CP          EE

Guinea grass
  Fresh        17.81 (c)    90.16 (a)    7.49 (c)    1.65 (b)
  Wilted       24.78 (a)    89.76 (a)    8.21 (b)    1.63 (b)
Napier grass
  Fresh        13.08 (e)    87.23 (b)    7.19 (c)    2.07 (a)
  Wilted       21.86 (b)    87.62 (b)    8.70 (a)    1.66 (b)
SEM               1.02        0.154        0.119       0.091
p-value          0.001        <0.001      <0.001       0.022

Items             NDF          ADF        ADL       WSC       LBC

Guinea grass
  Fresh        77.47 (a)    48.26 (b)    3.32    0.48 (b)    760.81
  Wilted       76.24 (b)    51.45 (a)    2.76    0.39 (b)    526.64
Napier grass
  Fresh        71.14 (c)    42.81 (c)    3.18    2.59 (a)    722.48
  Wilted       70.82 (c)    39.11 (d)    2.92    2.46 (a)    622.15
SEM              0.237        0.452      3.32      0.058     75.167
p-value          <0.001       <0.001     0.411    <0.001     0.110

DM, dry matter; OM, organic matter; CP, crude protein; EE, ether
extract; NDF, neutral detergent fiber; ADF, acid detergent fiber;
ADL, acid detergent lignin; WSC, water soluble
carbohydrate; SEM, standard error of the mean.

(a-d) Means within columns with different superscripts differ at
p <0.05.

Table 3. Dry matter (%), pH and fermentation products (g/kg of DM)
of Guinea grass and Napier grass silages at 30 d after fermentation

Treatments                      DM            pH            acid

Fresh Guinea
  AC 0.0025%                18.43 (c)     4.56 (bc)      0.10 (h)
  AC 0.005%                 17.09 (cd)     4.49 (c)      0.52 (gh)
  AC 0.01%                  18.34 (c)      3.55 (i)      6.71 (ab)
  TC 0.0025%                17.09 (cd)    4.59 (bc)      0.00 (h)
  TC 0.005%                 18.18 (c)     4.63 (bc)      0.01 (h)
  TC 0.01%                  17.60 (c)     4.60 (bc)      0.00 (h)
Wilted Guinea
  AC 0.0025%                23.84 (a)     4.66 (bc)     1.33 (efgh)
  AC 0.005%                 23.46 (a)      4.49 (c)     2.78 (def)
  AC 0.01%                  23.48 (a)     4.25 (de)     2.07 (efgh)
  TC 0.0025%                24.07 (a)      4.90 (a)     1.75 (efgh)
  TC 0.005%                 23.29 (a)      5.05 (a)      0.47 (gh)
  TC 0.01%                  23.65 (a)      4.91 (a)     0.71 (fgh)
Fresh Napier
  AC 0.0025%                13.51 (ef)    3.89 (gh)     2.69 (defg)
  AC 0.005%                 12.64 (f)      3.78 (h)      7.56 (a)
  AC 0.01%                  15.06 (de)    3.87 (gh)      5.52 (ab)
  TC 0.0025%                18.08 (c)     4.23 (de)     4.73 (bcd)
  TC 0.005%                 14.18 (ef)    4.08 (ef)      5.51 (ab)
  TC 0.01%                  13.79 (ef)    4.03 (fg)      5.36 (bc)
Wilted Napier
  AC 0.0025%                23.46 (a)      4.33 (d)     0.76 (fgh)
  AC 0.005%                 22.52 (ab)    4.17 (def)    4.85 (bcd)
  AC 0.01%                  23.58 (a)     4.09 (ef)      5.07 (bc)
  TC 0.0025%                22.08 (ab)     4.68 (b)     3.27 (cde)
  TC 0.005%                   23.61a      4.64 (bc)     3.26 (cde)
  TC 0.01%                  21.06 (b)      4.68 (b)     3.19 (cde)
SEM                            0.826         0.062         0.781
Grasses means
  Guinea grass              20.71 (a)      4.56 (a)      1.37 (b)
  Napier grass              18.63 (b)      4.21 (b)      4.31 (a)
Moisture adjust
  Fresh                     16.17 (b)      4.19 (b)      3.23 (a)
  Wilted                    23.18 (a)      4.57 (a)      2.46 (b)
  AC                           19.62       4.18 (b)      3.33 (a)
  TC                           19.73       4.58 (a)      2.35 (b)
Application levels
  0.0025%                      20.07       4.48 (a)      1.83 (c)
  0.005%                       19.37       4.42 (b)      3.12 (b)
  0.01%                        19.57       4.25 (c)      3.58 (a)
Significance of main effect and interaction
  Grasses means (A)           <0.001        <0.001         <0.001
  Moisture adjust (B)         <0.001        <0.002         0.008
  Enzymes (C)                   712         <0.003         0.001
  Application levels (D)       0.141        <0.004         <0.001
  AxBxCxD                      0.001        <0.005         <0.001

                               Acetic       Propionic      Butyric
Treatments                      acid          acid           acid

Fresh Guinea
  AC 0.0025%                 4.42 (b)       0.21 (b)      3.09 (a)
  AC 0.005%                  3.92 (c)       0.13 (c)      3.28 (a)
  AC 0.01%                   1.79 (ef)     0.02 (efg)    0.18 (defg)
  TC 0.0025%                 5.27 (a)       0.32 (a)      2.66 (b)
  TC 0.005%                  4.71 (b)       0.35 (a)      2.23 (c)
  TC 0.01%                   5.18 (a)       0.21 (b)      2.62 (b)
Wilted Guinea
  AC 0.0025%                 1.15 (hi)     0.03 (efg)    0.18 (defg)
  AC 0.005%                  1.74 (fg)     0.03 (efg)     0.42 (d)
  AC 0.01%                   0.88 (i)      0.02 (efg)    0.06 (efg)
  TC 0.0025%                 2.31 (d)       0.06 (d)     0.24 (defg)
  TC 0.005%                 1.29 (fghi)    0.05 (de)      0.42 (d)
  TC 0.01%                   1.18 (hi)     0.04 (def)    0.21 (defg)
Fresh Napier
  AC 0.0025%                1.55 (fgh)     0.02 (efg)     0.01 (g)
  AC 0.005%                 1 35 (fghi)     0.00 (g)      0.03 (fg)
  AC 0.01%                  1.21 (ghi)     0.01 (fg)      0.03 (fg)
  TC 0.0025%                1.68 (fgh)     0.01 (fg)      0.04 (fg)
  TC 0.005%                 1.47 (fgh)     0.01 (fg)      0.04 (fg)
  TC 0.01%                  1.22 (ghi)      0.00 (g)      0.02 (g)
Wilted Napier
  AC 0.0025%                1.57 (fgh)     0.01 (fg)      0.01 (g)
  AC 0.005%                 1.48 (fgh)     0.01 (fg)      0.01 (g)
  AC 0.01%                  1.42 (fgh)      0.00 (g)      0.01 (g)
  TC 0.0025%                 2.48 (d)      0.02 (efg)     0.35 (d)
  TC 0.005%                  2.23 (de)     0.01 (fg)     0.08 (efg)
  TC 0.01%                   2.51 (d)       0.00 (g)     0.33 (def)
SEM                            0.188          0.012         0.105
Grasses means
  Guinea grass               2.82 (a)       0.12 (a)      1.30 (a)
  Napier grass               1.68 (b)       0.01 (b)      0.08 (b)
Moisture adjust
  Fresh                      2.82 (a)       0.11 (a)      1.19 (a)
  Wilted                     1.69 (b)       0.02 (b)      0.19 (b)
  AC                         1.87 (b)       0.04 (b)      0.61 (b)
  TC                         2.63 (a)       0.09 (a)      0.77 (a)
Application levels
  0.0025%                    2.56 (a)       0.09 (a)      0.82 (a)
  0.005%                     2.27 (b)       0.07 (b)      0.81 (a)
  0.01%                      1.92 (c)       0.04 (c)      0.43 (b)
Significance of main effect and interaction
  Grasses means (A)            <0.001        <0.001         <0.001
  Moisture adjust (B)          0.008         <0.001         <0.001
  Enzymes (C)                  0.001         <0.001         <0.001
  Application levels (D)       <0.001        <0.001         <0.001
  AxBxCxD                      <0.001        <0.001         <0.001

Treatments                    nitrogen

Fresh Guinea
  AC 0.0025%                0.99 (efgh)
  AC 0.005%                 0.88 (fghi)
  AC 0.01%                    0.18 (j)
  TC 0.0025%                1.49 (bcde)
  TC 0.005%                 1.20 (cdef)
  TC 0.01%                   1.63 (bcd)
Wilted Guinea
  AC 0.0025%                 1.78 (bc)
  AC 0.005%                 1.56 (bcde)
  AC 0.01%                   1.69 (bc)
  TC 0.0025%                  2.08 (b)
  TC 0.005%                   2.74 (a)
  TC 0.01%                   1.74 (bc)
Fresh Napier
  AC 0.0025%                0.42 (ghij)
  AC 0.005%                  0.32 (ij)
  AC 0.01%                    0.27 (j)
  TC 0.0025%                0.76 (fghij)
  TC 0.005%                 0.65 (fghij)
  TC 0.01%                   0.40 (hij)
Wilted Napier
  AC 0.0025%                0.74 (fghij)
  AC 0.005%                 0.67 (fghij)
  AC 0.01%                  0.77 (fghij)
  TC 0.0025%                 1.03 (efg)
  TC 0.005%                  1.04 (ef)
  TC 0.01%                   1.07 (def)
SEM                             0.21
Grasses means
  Guinea grass                1.50 (a)
  Napier grass                0.68 (b)
Moisture adjust
  Fresh                       0.77 (b)
  Wilted                      1.41 (a)
  AC                          0.86 (b)
  TC                          1.32 (a)
Application levels
  0.0025%                       1.16
  0.005%                        1.13
  0.01%                         0.97
Significance of main effect and interaction
  Grasses means (A)            <0.001
  Moisture adjust (B)          <0.001
  Enzymes (C)                  <0.001
  Application levels (D)        0.087
  AxBxCxD                       0.007

DM, dry matter; AC, Acremonium cellulase (Meiji Seika Pharma Co.
Ltd., Tokyo, Japan); TC, Trichoderma cellulase (Meiji Seika
Pharma Co. Ltd., Japan); SEM, standard error of the mean.

(a-j) Means within columns with different superscripts differ at
p <0.05.
Values are means of three silage samples.

Table 4. Microbiological analysis of Guinea grass and Napier grass
silages at 30 d after fermentation

                                  Microorganism (cfu/g of FM)

Treatments                  Lactic acid     Coliform      Aerobic
                              bacteria      bacteria      bacteria
Fresh Guinea
  AC 0.0025%               9.0x[10.sup.7]      ND      1.4x[10.sup.4]
  AC 0.005%                3.9x[10.sup.8]      ND      1.2x[10.sup.4]
  AC 0.01%                 2.8x[10.sup.8]      ND      1.5x[10.sup.4]
  TC 0.0025%               1.2x[10.sup.8]      ND      3.5x[10.sup.3]
  TC 0.005%                7.9x[10.sup.7]      ND      0.9x[10.sup.4]
  TC 0.01%                 2.6x[10.sup.8]      ND      1.9x[10.sup.4]
Wilted Guinea
  AC 0.0025%               2.4x[10.sup.8]      ND      8.1x[10.sup.3]
  AC 0.005%                 7x[10.sup.7]       ND      1.6x[10.sup.4]
  AC 0.01%                 1.5x[10.sup.8]      ND      8.0x[10.sup.3]
  TC 0.0025%               1.2x[10.sup.8]      ND      6.8x[10.sup.3]
  TC 0.005%                3.7x[10.sup.8]      ND      1.8x[10.sup.4]
  TC 0.01%                 1.7x[10.sup.8]      ND      1.6x[10.sup.4]
Fresh Napier
  AC 0.0025%               3.0x[10.sup.7]      ND      1.8x[10.sup.4]
  AC 0.005%                1.6x[10.sup.8]      ND      3.4x[10.sup.4]
  AC 0.01%                 1.5x[10.sup.8]      ND      1.6x[10.sup.4]
  TC 0.0025%               4.7x[10.sup.7]      ND      8.0x[10.sup.4]
  TC 0.005%                1.5x[10.sup.8]      ND      1.0x[10.sup.4]
  TC 0.01%                 1.6x[10.sup.8]      ND      7.8x[10.sup.3]
Wilted Napier
  AC 0.0025%               8.1x[10.sup.7]      ND      7.8x[10.sup.3]
  AC 0.005%                1.8x[10.sup.8]      ND      2.1x[10.sup.4]
  AC 0.01%                 1.4x[10.sup.8]      ND      1.5x[10.sup.4]
  TC 0.0025%               7.6x[10.sup.8]      ND      6.6x[10.sup.3]
  TC 0.005%                1.0x[10.sup.8]      ND      4.7x[10.sup.3]
  TC 0.01%                 7.3x[10.sup.8]      ND      2.6x[10.sup.4]
SEM                            1.331           ND          2.037
Grasses means
  Guinea grass             1.9x[10.sup.8]      ND      1.2x[10.sup.4]
  Napier grass             1.1x[10.sup.8]      ND      2.1x[10.sup.4]
Moisture adjust
  Fresh                    1.6x[10.sup.8]      ND      1.9x[10.sup.4]
  Wilted                   1.4x[10.sup.8]      ND      1.3x[10.sup.4]
  AC                       1.6x[10.sup.8]      ND      1.5x[10.sup.4]
  TC                       1.4x[10.sup.8]      ND      1.7x[10.sup.4]
Application levels
  0.0025%                  1.0x[10.sup.8]      ND      1.8x[10.sup.4]
  0.005%                   1.9x[10.sup.8]      ND      1.6x[10.sup.4]
  0.01%                    1.7x[10.sup.8]      ND      1.5x[10.sup.4]
Significance of main effect and interaction
  Grasses means (A)            0.088           ND          0.239
  Moisture adjust (B)          0.779           ND          0.341
  Enzymes (C)                  0.675           ND          0.790
  Application levels (D)       0.271           ND          0.943
  AxBxCxD                      0.939           ND          0.660

                                (cfu/g of FM)

Treatments                     Yeast        Mold

Fresh Guinea
  AC 0.0025%                     ND          ND
  AC 0.005%                      ND          ND
  AC 0.01%                       ND          ND
  TC 0.0025%               1.9x[10.sup.2]    ND
  TC 0.005%                1.7x[10.sup.2]    ND
  TC 0.01%                 2.3x[10.sup.2]    ND
Wilted Guinea
  AC 0.0025%                     ND          ND
  AC 0.005%                      ND          ND
  AC 0.01%                       ND          ND
  TC 0.0025%               4.6x[10.sup.2]    ND
  TC 0.005%                5.6x[10.sup.2]    ND
  TC 0.01%                 1.9x[10.sup.2]    ND
Fresh Napier
  AC 0.0025%                     ND          ND
  AC 0.005%                7.2x[10.sup.2]    ND
  AC 0.01%                 1.0x[10.sup.2]    ND
  TC 0.0025%                     ND          ND
  TC 0.005%                1.4x[10.sup.2]    ND
  TC 0.01%                 1.6x[10.sup.2]    ND
Wilted Napier
  AC 0.0025%               1.0x[10.sup.4]    ND
  AC 0.005%                1.0x[10.sup.3]    ND
  AC 0.01%                 4.9x[10.sup.3]    ND
  TC 0.0025%               7.8x[10.sup.3]    ND
  TC 0.005%                5.1x[10.sup.3]    ND
  TC 0.01%                 1.4x[10.sup.4]    ND
SEM                            57.895        ND
Grasses means
  Guinea grass             1.6x[10.sup.2]    ND
  Napier grass             4.8x[10.sup.3]    ND
Moisture adjust
  Fresh                    1.3x[10.sup.2]    ND
  Wilted                   3.7x[10.sup.3]    ND
  AC                       1.4x[10.sup.3]    ND
  TC                       3.6x[10.sup.3]    ND
Application levels
  0.0025%                  2.3x[10.sup.3]    ND
  0.005%                   2.7x[10.sup.3]    ND
  0.01%                    2.5x[10.sup.3]    ND
Significance of main effect and interaction
  Grasses means (A)            0.250         ND
  Moisture adjust (B)          0.243         ND
  Enzymes (C)                  0.294         ND
  Application levels (D)       0.991         ND
  AxBxCxD                      0.878         ND

FM, fresh matter; ND, not detected; AC, Acremonium cellulase
(Meiji Seika Pharma Co. Ltd., Tokyo, Japan); TC, Trichoderma
cellulase (Meiji Seika Pharma Co. Ltd., Japan); SEM, standard
error of the mean.
Values are means of 3 silage samples.

Table 5. Chemical compositions (% of DM) of Guinea grass and Napier
grass silages at 30 d after fermentation

Treatments                     OM           CP              EE

Fresh Guinea
  AC 0.0025%               90.91 (a)    6.64 (ef)      2.85 (abcde)
  AC 0.005%                90.54 (a)    6.26 (fg)      2.78 (abcde)
  AC 0.01%                 90.83 (a)    6.76 (de)       2.36 (fg)
  TC 0.0025%               90.51 (a)    5.84 (hi)     2 72 (abcdefg)
  TC 0.005%                90.31 (a)    6.26 (fg)      2.64 (cdefg)
  TC 0.01%                 90.60 (a)    5.69 (ijk)    2.68 (bcdefg)
Wilted Guinea
  AC 0.0025%               89.18 (b)    7.33 (bc)      2 92 (abcd)
  AC 0.005%                89.16 (b)    7.34 (bc)       2.97 (abc)
  AC 0.01%                 89.26 (b)    6.91 (de)       3.08 (ab)
  TC 0.0025%               89.05 (b)    7.05 (cd)     2.68 (bcdefg)
  TC 0.005%                89.01 (b)     7.64 (b)      2.84 (abcde)
  TC 0.01%                 89.28 (b)     7.71 (b)       3.09 (ab)
Fresh Napier
  AC 0.0025%               86.05 (d)    5.85 (hij)    2 74 (abcdef)
  AC 0.005%                87.22 (c)    5.56 (jk)       2.98 (abc)
  AC 0.01%                 86.77 (c)    5.97 (ghi)      3.02 (abc)
  TC 0.0025%               85.72 (d)    6.14 (gh)     2 75 (abcdef)
  TC 0.005%                84.59 (e)    5.55 (jk)        2.32 (g)
  TC 0.01%                 85.84 (d)     5.34 (k)       2.46 (efg)
Wilted Napier
  AC 0.0025%               85.46 (d)     7.67 (b)      2.90 (abcd)
  AC 0.005%                85.39 (d)     7.71 (b)     2.69 (bcdefg)
  AC 0.01%                 85.89 (d)     8.25 (a)      2.89 (abcd)
  TC 0.0025%               85.98 (d)     7.59 (b)     2 71 (abcdefg)
  TC 0.005%                85.50 (d)     7.51 (b)        3.12 (a)
  TC 0.01%                 85.50 (d)     7.64 (b)      2 53 (defg)
SEM                          0.265         0.148           0.141
Grasses means
  Guinea grass             89.89 (a)       6.79             2.8
  Napier grass             85.83 (b)       6.73            2.76
Moisture adjust
  Fresh                    88.32 (a)     5.99 (b)        2.69 (b)
  Wilted                   87.39 (b)     7.53 (a)        2.87 (a)
  AC                       88.06 (a)     6.85 (a)        2.85 (a)
  TC                       87.66 (b)     6.66 (b)        2.71 (b)
Application levels
  0.0025%                  87.86 (a)       6.77            2.78
  0.005%                   87.72 (b)       6.73            2.79
  0.01%                    87.99 (a)       6.78            2.76
Significance of main effect and interaction
  Grasses means (A)          <0.001        0.306           0.396
  Moisture adjust (B)        <0.001        <.001           0.001
  Enzymes (C)                <0.001        0.001           0.009
  Application levels (D)     0.059         0.666           0.881
  AxBxCxD                    <0.001       <0.001           0.002

Treatments                     NDF            ADF

Fresh Guinea
  AC 0.0025%               75.58 (ab)     50.29 (cde)
  AC 0.005%                 74.82 (b)     49.54 (de)
  AC 0.01%                 71.04 (de)      46.17 (f)
  TC 0.0025%               75.36 (ab)     51.84 (ab)
  TC 0.005%                75.12 (ab)     50.97 (bc)
  TC 0.01%                  76.48 (a)      52.67 (a)
Wilted Guinea
  AC 0.0025%                70.69 (e)     49.63 (de)
  AC 0.005%                 70.30 (e)      49.12 (e)
  AC 0.01%                  68.46 (f)      47.31 (f)
  TC 0.0025%               72.22 (cd)     50.56 (bcd)
  TC 0.005%                 72.52 (c)     50.67 (bcd)
  TC 0.01%                 72.42 (cd)     51.42 (abc)
Fresh Napier
  AC 0.0025%               65.27 (ij)      41.62 (h)
  AC 0.005%                67.64 (fg)      43.01 (g)
  AC 0.01%                 66.57 (ghi)    42.10 (gh)
  TC 0.0025%               67.10 (fgh)     43.03 (g)
  TC 0.005%                65.24 (ij)     42.01 (gh)
  TC 0.01%                 66.29 (ghi)     43.17 (g)
Wilted Napier
  AC 0.0025%               64.49 (jk)      38.82 (j)
  AC 0.005%                63.34 (kl)     37.97 (jk)
  AC 0.01%                  62.79 (l)      36.89 (k)
  TC 0.0025%               64.32 (jk)      40.05 (i)
  TC 0.005%                66.42 (ghi)     40.26 (i)
  TC 0.01%                 66.09 (hi)      40.28 (i)
SEM                           0.551          0.489
Grasses means
  Guinea grass              72.92 (a)      50.02 (a)
  Napier grass              65.46 (b)      40.77 (b)
Moisture adjust
  Fresh                     70.54 (a)      46.37 (a)
  Wilted                    67.84 (b)      44.41 (b)
  AC                        68.42 (b)      44.37 (b)
  TC                        69.97 (a)      46.41 (a)
Application levels
  0.0025%                   69.38 (a)      45.73 (a)
  0.005%                    69.43 (a)      45.44 (a)
  0.01%                     68.77 (b)      45.00 (b)
Significance of main effect and interaction
  Grasses means (A)           <0.001         <0.001
  Moisture adjust (B)         <0.001         <0.001
  Enzymes (C)                 <0.001         <0.001
  Application levels (D)      0.013          0.005
  AxBxCxD                     <0.001         <0.001

Treatments                      ADL

Fresh Guinea
  AC 0.0025%               4.18 (bcdef)
  AC 0.005%                 4.57 (abcd)
  AC 0.01%                 3 91 (cdefgh)
  TC 0.0025%                 5.22 (a)
  TC 0.005%                4.34 (abcde)
  TC 0.01%                   4.87 (ab)
Wilted Guinea
  AC 0.0025%                 2.64 (j)
  AC 0.005%                4.14 (bcdef)
  AC 0.01%                  4.67 (abc)
  TC 0.0025%               3.99 (bcdefg)
  TC 0.005%                3.54 (efghij)
  TC 0.01%                   2.92 (ij)
Fresh Napier
  AC 0.0025%               3.69 (defghi)
  AC 0.005%                 4.31 (bcde)
  AC 0.01%                  4.68 (abc)
  TC 0.0025%                 2.83 (ij)
  TC 0.005%                  2.65 (j)
  TC 0.01%                   2.81 (ij)
Wilted Napier
  AC 0.0025%                3.02 (hij)
  AC 0.005%                3 37 (fghij)
  AC 0.01%                 3.28 (fghij)
  TC 0.0025%                 2.85 (ij)
  TC 0.005%                 3.04 (hij)
  TC 0.01%                  3 17 (ghij)
SEM                            0.322
Grasses means
  Guinea grass               4.08 (a)
  Napier grass               3.31 (b)
Moisture adjust
  Fresh                      4.00 (a)
  Wilted                     3.39 (b)
  AC                         3.87 (a)
  TC                         3.52 (b)
Application levels
  0.0025%                       3.55
  0.005%                        3.74
  0.01%                         3.79
Significance of main effect and interaction
  Grasses means (A)            <0.001
  Moisture adjust (B)          <0.001
  Enzymes (C)                  0.003
  Application levels (D)       0.207
  AxBxCxD                      <0.001

DM, dry matter; OM, organic matter; CP, crude protein; EE, ether
extract; NDF, neutral detergent fiber; ADF, acid detergent fiber;
ADL, acid detergent lignin; AC, Acremonium cellulase (Meiji Seika
Pharma Co. Ltd., Tokyo, Japan); TC, Trichoderma cellulase (Meiji
Seika Pharma Co. Ltd., Japan); SEM, standard error of the mean.

(a-l) Means within columns with different superscripts differ at p
<0.05. Values are means of three silage samples.

Table 6. In vitro dry matter digestibility (IVDMD, % of DM) and in
vitro organic matter digestibility (IVOMD, % of DM) at 6 and 48h
of incubation of Guinea grass and Napier grass silages at 30
d after fermentation


Treatments                       6 h              48 h

Fresh Guinea
  AC 0.0025%               30.02 (abcdef)    55.62 (fghij)
  AC 0.005%                30.46 (abcdef)     54.85 (ghij)
  AC 0.01%                  32 21 (abcd)     6076 (cdefgb)
  TC 0.0025%                26.69 (cdef)       54.05 (ij)
  TC 0.005%                  25.16 (ef)       54.43 (hij)
  TC 0.01%                  26.36 (def)        52.19 (j)
Wilted Guinea
  AC 0.0025%               27.99 (bcdef)     59.48 (defghi)
  AC 0.005%                31 14 (abcdef)    57.38 (efghij)
  AC 0.01%                   34.59 (ab)      59.44 (defghi)
  TC 0.0025%                 24.83 (f)       57.12 (efghij)
  TC 0.005%                 32.68 (abcd)     58.51 (defghi)
  TC 0.01%                 29 52 (abcdef)    59.23 (defghi)
Fresh Napier
  AC 0.0025%               29.12 (abcdef)    57.35 (efghij)
  AC 0.005%                 33.22 (abcd)      66.26 (abc)
  AC 0.01%                   35.60 (a)         69.68 (a)
  TC 0.0025%                 33.87 (ab)        68.12 (ab)
  TC 0.005%                30.19 (abcdef)     61.45 (cdef)
  TC 0.01%                 31.96 (abcde)      62.42 (bcde)
Wilted Napier
  AC 0.0025%                32.47 (abcd)      62.34 (bcde)
  AC 0.005%                 33.53 (abc)       64.55 (abcd)
  AC 0.01%                   35.02 (ab)       66.69 (abc)
  TC 0.0025%               31.77 (abcdef)     63.98 (abcd)
  TC 0.005%                  34.15 (ab)      61.13 (cdefg)
  TC 0.01%                  32.94 (abcd)      64.84 (abcd)
SEM                             2.392             2.184
Grasses means
  Guinea grass               29.31 (b)         38.35 (b)
  Napier grass               32.82 (a)         41.67 (a)
Moisture adjust
  Fresh                         30.41             59.76
  Wilted                        31.72             61.22
  AC                         32.11 (a)            61.2
  TC                         30.01 (b)            59.79
Application levels
  0.0025%                    29.60 (b)         59.76 (b)
  0.005%                     31.32 (ab)        59.82 (b)
  0.01%                      32.27 (a)         61.90 (a)
Significance of main effect and interaction
  Grasses means (A)            <0.001            <0.001
  Moisture adjust (B)           0.12'             0.065
  Enzymes (C)                   0.016             0.074
  Application levels (D)        0.04              0.044
  AxBxCxD                       0.445            <0.001


Treatments                      6 h            48 h

Fresh Guinea
  AC 0.0025%               36.98 (bcd)      64.85 (ef)
  AC 0.005%                 36.01 (cd)      64.38 (ef)
  AC 0.01%                 42.65 (abc)     67.17 (cdef)
  TC 0.0025%                36.29 (cd)      64.09 (ef)
  TC 0.005%                 35.81 (cd)      64.54 (ef)
  TC 0.01%                  36.06 (cd)      61.68 (f)
Wilted Guinea
  AC 0.0025%               38.17 (bcd)     68.73 (bcde)
  AC 0.005%                40.96 (abcd)    66.62 (def)
  AC 0.01%                  44.23 (ab)     71.89 (abcd)
  TC 0.0025%                34.91 (d)      66.21 (def)
  TC 0.005%                39.29 (bcd)     68.12 (bcde)
  TC 0.01%                 38.83 (bcd)     68.23 (bcde)
Fresh Napier
  AC 0.0025%               38.57 (bcd)     67.31 (cdef)
  AC 0.005%                42.98 (abc)     73.32 (abc)
  AC 0.01%                 41.33 (abcd)     75.77 (a)
  TC 0.0025%               41.14 (abcd)     73.74 (ab)
  TC 0.005%                38.92 (bcd)     72.08 (abcd)
  TC 0.01%                 40.61 (abcd)    73.35 (abc)
Wilted Napier
  AC 0.0025%               42.58 (abc)     71.56 (abcd)
  AC 0.005%                41.62 (abcd)     73.84 (ab)
  AC 0.01%                  47.15 (a)       76.07 (a)
  TC 0.0025%               4071 (abcd)     73.38 (abc)
  TC 0.005%                 4276 (abc)     71.10 (abcd)
  TC 0.01%                 41.65 (abcd)     74.19 (ab)
SEM                            2.499           2.165
Grasses means
  Guinea grass              56.92 (b)       66.38 (b)
  Napier grass              64.07 (a)       72.97 (a)
Moisture adjust
  Fresh                     38.95 (b)       68.52 (b)
  Wilted                    41.07 (a)       70.83 (a)
  AC                        41.10 (a)          70.12
  TC                        38.92 (b)          69.23
Application levels
  0.0025%                   38.67 (b)       68.73 (b)
  0.005%                    39.79 (ab)      69.25 (ab)
  0.01%                     41.56 (a)       71.04 (a)
Significance of main effect and interaction
  Grasses means (A)           <0.001          <0.001
  Moisture adjust (B)          0.02            0.004
  Enzymes (C)                  0.017           0.246
  Application levels (D)       0.034           0.044
  AxBxCxD                      0.787           0.264

AC, Acremonium cellulase (Meiji Seika Pharma Co. Ltd., Tokyo, Japan);
TC, Trichoderma cellulase (Meiji Seika Pharma Co. Ltd., Japan);
SEM, standard error of the mean.

(a-h) Means within columns with different superscripts differ at
p <0.05. Values are means of three silage samples.
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Article Details
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Author:Khota, Waroon; Pholsen, Suradej; Higgs, David; Cai, Yimin
Publication:Asian - Australasian Journal of Animal Sciences
Article Type:Report
Geographic Code:9THAI
Date:Dec 1, 2018
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