Livestock Research for Rural Development 27 (3) 2015 Guide for preparation of papers LRRD Newsletter

Citation of this paper

Enhanced in vitro fermentation parameters of guinea grass ecotype ‘A’ (Panicum maximum) and rice straw (Oryza sativa) with supplementation of exogenous fibrolytic enzymes

S Sujani, I N Pathirana and R T Seresinhe

Department of Animal Science, Faculty of Agriculture, University of Ruhuna, Kamburupitiya, Sri Lanka


A study was conducted to evaluate the use of exogenous enzymes as a potential means of improving the rumen fermentation of guinea grass ecotype ‘A’ (Panicum maximum) rice straw (Oryza sativa). The enzymes used for the in vitro incubations were characterized for Cellulase (CE), Xylanase (XY). Enzymes were supplemented separately (CE and XY) as levels of enzymes in 5, 10, 20, 40 and 0 l for 500 mg ground (1mm) substrates dry matter. Anaerobic buffer medium and strained ruminal fluid (42 ml) were added to the in vitro incubations (followed by 24 h pre incubation of substrate and enzyme) in triplicates and in vitro gas production (IVGP) was measured at 2, 4, 8, 12, 18, 24 and 48 h of incubation. At the end of incubation in vitro rumen dry matter disappearance (IVRDMD), ammonia nitrogen (NH3-N), short chain fatty acid (SCFA) and metabolozable energy (ME) were estimated.

All enzyme treatments except for some instances significantly increased (p<0.05) IVGP while there was no significant effect on IVRDMD irrespective of enzyme or substrate. The total NH3-N in fermentation liquid was significantly increased by both enzymes irrespective of substrate. Calculated values for SCFA and ME also have significantly enhanced with enzyme supplementation. Therefore it can be concludedthat the use of fibrolytic enzymes as an effective way to improve the ruminal fermentation characteristics of guinea grass ecotype ‘A’ and rice straw.

Key words: gas production, metabolizable energy, rumen ammonia-nitrogen, rumen dry matter disappearance, short chain fatty acid


Livestock industry is an integral part of tropical agriculture. From whole picture of tropical livestock, ruminant animal plays a vital role by providing food, manure and draught power. These tropical ruminant animal production systems are characterized with low productivity, mainly due to the poor status of feeding regimes (Seresinhe et al 2012). Most of these production systems depend on year around grazing on natural forage or agro industrial by product (Seresinhe and Pathirana 2008) for animal’s feed requirement. This situation is clearly noticeable in Sri Lanka where the main feed resources for ruminants are wild Guinea grass (ecotype A) (Panicum maximum) and rice straw (Oryza sativa), most abundant agricultural by product. Low digestibility and crude protein level that will lead to the lower animal performance and productivity are major problems associated with above mentioned feed sources (Aganga and Tshwenyane 2004). Several physical, chemical and biological methods have been developed to improve the nutritive value of feed stuffs (Wanapat et al 2009). Supplementation of ruminant feeds with exogenous fibrolytic enzymes, a biological approach as to improve rumen performance has attracted a distinct attention in recent years. Promising results have obtained both under in vitro (Elghandour et al 2013) and in vivo (Salem et al 2013) experiments. Despite large number of studies conducted, most have focused on temperate feed stuffs and with a poor attention to the tropical feed resources. Therefore this study was undertaken to investigate the effect of supplementing fibrolytic enzymes with wild guinea grass and rice straw on rumen performance under in vitro conditions.

Materials and methods

Substrate, Enzymes and Treatments

Guinea grass ecotype ‘A’ (Panicum maximum) and rice straw (Oryza sativa) were collected and used as the substrate after drying at 55C for 48 hours and grinding to pass a 1 mm screen. Proximate analysis of dry matter, ash, neutral detergent fiber (NDF), and nitrogen, N (Kjeldahl method) were done according to the AOAC (1990). CP was calculated as N 6.25.

Two fibrolytic enzymes namely cellulase (CE; E.C., Dyadic International, Inc., Jupiter, FL, USA) with activity of 115,000 to 140,000 cellulase units/g and xylanase (XY; E.C., Dyadic International, Inc., Jupiter, FL, USA) with activity of 34,000 to 41,000 xylanase units/g [In a preliminary study activity of enzymes was assayed following the procedures of Wood and Bhatt (1988)]. Four different doses of enzymes were evaluated [5 (CE1 and XY1), 10 (CE2 and XY2), 20 (CE3 and XY3) and 40 (CE4 and XY4 L] along with control (C, Control, no enzyme) and blank (no substrate, only buffered rumen fluid).

In vitro gas production technique

In vitro fermentation procedure and preparation of buffer and mineral solutions were done according to the procedures demonstrated by Menke and Steingas (1988). Samples (500 mg) of substrate were accurately weighted into glass bottles (120 ml) and supplemented with previously mentioned four doses of diluted enzyme and kept them for the pre-incubation for 24 h. For the in vitro incubation procedure, the medium of 1 liter volume was prepared with 2.5 g of tryptone (Sigma-Aldrich, Co., 3050 Spruce Street, St. Louis, MO, USA) dissolved in 500 ml distilled water, 0.125 ml of micro mineral solution, 250 ml of buffer solution and 1.25 ml of 0.1% (w/v) resazurin (Fluka AG, CH-9470 Buchs, Switzerland) solution. The medium was mixed in a container which kept in a water bath (39C) while bubbling CO2 through the solution for 45 minutes. L-cysteine hydrochloride (0.313 g) (Sigma-Aldrich, Co., 3050 Spruce Street, St. Louis, MO, USA) and sodium sulphide (0.313 g) (Park Scientific Limited, Northampton, UK) were directly added to the medium and further bubbled with CO2 for 15 min. At this point rumen fluid was collected from a donor heifer, maintained on natural grazing at the farm of the Faculty of Agriculture, University of Ruhuna, Kaburupitiya, Sri Lanka through an esophageal suction method. Collected rumen fluid was transferred to a pre-warmed flask and strained through four layer cheese cloth. All the laboratory handlings of rumen fluid were carried out under continuous flow of CO2 and 39C of temperature. Prepared rumen fluid was added to the medium in a ratio of 1:4 (rumen fluid: medium) and flushing of CO2 was continued until the solution turned to grey or clear, after which 42 ml of medium were pipetted into each incubation bottle, containing the pre-incubated substrate, and the bottles were immediately crimp sealed with a rubber stopper and placed in the water bath with shaker at 39C.

Measurements and data collection

The gas production was recorded at 2, 4, 8, 12, 18, 24 and 48 h within the incubation period. After 48 h, bottles were removed from the shaker and placed on ice to terminate the reaction. Remaining solid portions were separately prepared to determine IVRDMD while the aliquots of the filtrates were stored at 20C until analyzed for NH3-N.

Chemical Analysis

At the end of 48 h incubation solid portions were separately analyzed to determine IVRDMD with oven dry method (550C, 48 hours). Liquid portion was analyzed for NH3-N (Kjeltec System 1002, Tecator AB, Hoganas, Sweden) (AOAC, 1990).


Total SCFA acids were calculated according to the following equation derived by Getachew et al (2002).

SCFA (mmol/500 mg DM)=0.0222 GP-0.00425

Metabolizable energy (ME, MJ kg-1 DM) was calculated according to Menke et al (1979) as:

ME=2.20+0.136 GP (mL 0.5 g-1 DM)+0.057 CP (g kg-1 DM)

Experimental design and statistical analysis

Experiment was carried out in a randomized complete block design with three replicates for each treatment per run in three consecutive runs for each substrate.

Data of IVGP, IVRDMD, NH3-N production, SCFA and ME were subjected to standard analysis of variance using the general linear model of SPSS version 20.0 (IBM Cooperation, Somers, NY, USA). Significance between individual means was identified using least significance difference (LSD) test. The significance of means were considered at p<0.05.


Chemical composition of two substrates, guinea grass and rice straw are presented in Table 1.

Table 1. Proximate composition of two substrates
Substrate DM (g/ kg) Ash (g/ kg DM) CP (g/ kg DM) NDF (g/ kg DM)
Guinea grass 2383.37 93.50.35 95.92.1 7002.71
Rice straw 9211.4 1812.9 46.31.4 6923.9
Values are means of three replicates SE

The in vitro gas production (IVGP) (ml/ 200 mg DM/ 48 h and rumen ammonia nitrogen production (NH3-N) (mg/ 100 ml) of two substrates in response to the enzyme treatments are presented by Figure 1A, 1B and Figure 2 respectively. As figures illustrate the stronger effect of XY than CE is noticeable irrespective of substrate proclaiming XY as the most effective enzyme in means of IVGP. Conferring to the data it is visible that lower doses of CE failed to show any significant influence on IVGP of guinea grass. In response to the all enzyme treatments NH3-N production in both substrates significantly enhanced where the effect of XY on guinea grass was more prominentwhile CE was more effective with rice straw (Figure 2).

Figure 1A. IVGP of Guinea grass in response to the supplementation of CE and XY. Figure 1B. IVGP of rice straw in response to the supplementation of CE and XY.
0: Control; 5: CE1 or XY1; 10: CE2 or XY2; 20: CE3 or XY3; 40: CE4 or XY4, CE: cellulase; XY: xylanase, GG: guinea grass; RS: rice straw

Figure 2. NH3-N production of guinea grass and rice straw in response to the supplementation of CE and XY.
0: Control; 5: CE1 or XY1; 10: CE2 or XY2; 20: CE3 or XY3; 40: CE4 or XY4, CE: cellulase; XY: xylanase, GG: guinea grass; RS: rice straw

None of the treatments showed any enhancement of IVRDMD for both substrate (Table 2). Production of short chain fatty acid (SCFA) (mmol/ 500 mg DM) and metabolizable energy (ME) content (MJ/ kg DM) of two substrates upon treatments of enzymes are reported in Table 2. Except for some instances most of the enzyme treatments have improved the SCFA production and ME content of substrates.The effect of XY was more prominent.

Table 2. IVRDMD andcalculated values of SCFA and ME of two substrates in response to the cellulase (CE) and xylanase (XY) supplementation after 48 h incubation
Substrate/Treatment IVRDMD
(mmol / 500 mg DM)
(MJ / kg DM)
Guinea grass
CE1 60.9a 1.10a 8.94a
CE2 59a 1.10a 9.08a
CE3 59.1a 1.14b 9.26a
CE4 62.5a 1.13b 9.62b
XY1 64.5a 1.22b 9.84b
XY2 64.6a 1.25c 10.0bc
XY3 64.5a 1.32d 10.4c
XY4 67.3b 1.36e 10.7c
Control 61.9a 1.04a 8.80a
SEM 0.8481 0.0215 0.2966
P value 0.173 0.03 0.024
Rice straw
CE1 51.3a 0.88b 7.75a
CE2 49.3a 0.91c 7.91b
CE3 51.2a 0.93d 8.08b
CE4 51.1a 1.06e 8.86c
XY1 47.7a 0.98b 8.18a
XY2 47.7a 1.03bc 8.42b
XY3 48.6a 1.06c 8.59b
XY4 49.6a 1.18d 9.33b
Control 48.3a 0.83a 7.55a
SEM 0.7745 0.0176 0.2404
P value 0.297 0.000 0.01
Values are mean of six replicates of two runs.
Means with different superscripts (a, b, c and d) are significantly different (p<0.05);
CE1-CE4, cellulase enzyme treatment 1 to 4;
XY1-XY4, xylanase enzyme treatment 1 to 4; 1, 5; 2, 10; 3, 20 and 4, 40 l of enzyme


Gases, short chain fatty acid and microbial cells are the end products of rumen fermentation. In vitro gas production is considered as an indirect measurement of rumen fermentation therefore conversing it serves as a better way to quantify nutrient utilization (Sommart et al 2001). Eun and Beauchemin (2007) obtained significantly enhanced gas production in response to the supplementation of exogenous fibrolytic enzymes with the alfalfa hay as similar to the present study results. In contrast to the present results Giraldo et al (2007) found that there was no significant effect of enzyme supplementation on IVGP of a diet containing 60% of grass hay. In some other studies conducted by Rodrigues et al (2008) and Yang et al (2011), they reported increased IVGP for rice straw supplemented with fibrolytic enzymes.According to the existing body of knowledge a strong relationship between measured IVGP and rumen dry matter disappearance is established (Chumpawadee et al 2005). Therefore significantly enhanced IVGP should be an indirect measurement of positive changes of IVRDMD, though the IVRDMD of the present study remained unchanged. With the unaffected IVRDMD it can be assume that the effect of enzymes on fiber digestibility mainly xylose (fraction of hemicellulose) and cellulose was not significant. Conversing the result in present study, the significance activity of XY suggests that it has a higher resistant to degradation within rumen conditions than in CE which is less stable under rumen (Morgavi et al 2000 b).

As theory implies the level of ammonia in rumen liquor is an indicator of nutritional conditions that many types of rumen micro-organisms utilize ammonia as a source of N. According to the well documented studies dietary protein is fermented in the rumen to simpler N compounds and re-incorporated; primarily as NH3-N which acts as an indicator of microbial nitrogen synthesis. As NH3-N is the primary N source of most rumen organisms increased NH3-N could be resulted from improved microbial activities (Seresinhe et al 2012). Several researches have proclaimed no effect of fibrolytic enzyme supplementation on ruminal NH3-N production both under in vivo (Giraldo et al 2008) and in vitro (Wang et al 2001) conditions and even some researches experienced the reduction of NH3-N production compared to the control (Gaafar et al 2010). It can be assumed that supplementation of enzymes, more specifically XY has encouraged the colonization and proliferation of the ammonia producing bacteria. Elevated NH3-N in ruminal fluid suggests the preferential use of peptides or amino acids by ruminal microbes (Satter and Slyter 1974).

Conferring to the Blummel et al (1999) the IVGP in the followed gas production technique for the present study mirror SCFA production very closely. The production of gas, both quantitatively and qualitatively is the result of SCFA production. Therefore the increased IVGP may be due to the enhanced SCFA production upon enzyme supplementation. However the effects of enzyme supplementation on rumen SCFA production seem to be inconsistent (Eun at al 2007) in the literature, even some studies yield increased SCFA with enzyme treatments. Eun and Beauchemin (2007) reported no effect of fibrolytic enzymes on rumen SCFA profile. Tricario et al (2005) experienced that addition of xylanase and endoglucanase increased total VFA production from the fescue hay-based diet.Changes in SCFA as a direct effect of adding exogenous fibrolytic enzymes have been reported, implying that these enzymes could affect microbial growth and/or shift the metabolic pathways by which specific microbes utilize substrates (Eun and Beauchemin 2008). It can be assumed that relatively lower amount of calculated SCFA for rice straw is due to the low carbohydrate percentage.

A strong correlation between ME values measured in vivo and predicted from 24 h in vitro gas production and chemical composition of feed is reported by Menke and Steingass (1988). Referring to that verdict the in vitro gas production method has been widely used to evaluate the energy value of several classes of feed (Getachew et al 2002). Therefore the equation developed by Menke and Steingass (1979) is used to estimate the ME of two substrates after enzyme supplementation. By observing the calculated ME values it is noticeable that exogenous fibrolytic enzymes more specifically XY has a good potential to enhance energy content of tropical roughage feedstuffs.

In an overall view it can be stated that there was a marked effect of fibrolytic enzymes on rice straw than on guinea grass and it can be converse with the aid of following theories. As stated by Waghorn and MacNabb (2003) the esterified bonds between cellulose, hemicellulose, and lignin restrict the digestion of recalcitrant cereal straws by ruminal microorganisms. Conferring to the finding of Bhat and Hazelwood (2001) it can be assumed that treatments of CE and XY might have acted on β 1–4 linkages of cellulose and hemicellulose (xylan), to release soluble sugars and thus facilitating the growth of microbes.



Aganga A A and Tshwenyane S 2004 Potentials of guinea grass (Panicum maximum) as forage crop in livestock production. Pakistan Journal of Nutrition 3, 1 – 4

AOAC 1990 Official Methods of Analysis (15thEd.). Association of Official Analytical Chemists, Arlington, Va, USA

Bhat M K and Hazelwood G P 2001 Enzymology and other characteristics of cellulases and xylanases. In: Enzymes in Farm Animal Nutrition. Eds M.R. Bedford and G.G. Partridge. CABI Publishing; Oxon, UK.

Blummel M, Aiple KP, Steingass H and Becker K 1999 A note on the stoichiometrical relationship of short chain fatty acid productuin and gas formation in vitro in feedstuffs of widely different quality. Journal of Animal Physiology and Nutrition 81, 157-67

Chumpawadee S, Sommart K, Vanpralub T and Pattaarajinda 2005 Nutritional evaluation of non-forage high fibrous tropical feeds for ruminant using in vitro gas production technique. Pakistan Journal of Nutrition 4, 298-303

Elghandour M M Y, Salem A Z M, Gonzalez-Ronquillo M, Brquez J L, Gado H M, Odongo N E and Penuelas C G 2013 Effects of exogenous enzymes on in vitro gas production kinetics and ruminal fermentation of four fibrous feeds. Animal Feed Science and Technology 179, 46– 53

Eun J S and Beauchemin K A 2007 Assessment of the efficacy of varying experimental exogenousfibrolyticenzymes using in vitro fermentation characteristics. Animal Feed Science and Technology 132, 298–315

Eun J S and Beauchemin K A 2008Relationship between enzymatic activities and in vitro degradation of alfalfa hay and corn silage. Animal Feed Science and Technology 14, 53-67

Eun J S, Beauchemin K A and Schulze H 2007 Use of exogenous fibrolytic enzymes to enhance in vitro fermentation of alfalfa hay and corn silage. Journal of Dairy Science 90, 1440-1451

Gaafar H M A, Abdel-Raouf E M and El-Reidy F A 2010 Effect of fibrolytic enzyme supplementation and fiber content of total mixed ration on productive performance of lactating buffaloes. Slovakian Journal of Animal Science 43, 147 – 153

Getachew G, Crovetto G M, Fondivila M, Krishnamoorthy U, Singh B, Spaghero M,Steingass H, Robinson P H and Kailas M M 2002 Laboratory variation of 24 h in vitro gas production and estimated metabolizable energy values of ruminant feeds. Animal Feed Science and Technology 102, 169-180

Giraldo L A, Tejido M L, Ranilla M J and Carro M D 2007 Effects of exogenous cellulase supplementation on microbial growth and ruminal fermentation of a high-forage diet in Rusitec fermenters. Journal of Animal Science 85, 1962-1970

Giraldo L A, Tejidoa M L, Ranillaa M J and Carroa M D 2008 Effects of exogenous fibrolytic enzymes on in vitroruminal fermentation of substrates with different forage: concentrate ratios. Animal Feed Science and Technology 141, 306–325

Menke K H and Steingass H 1988 Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development 28, 7–55

Menke KH, Raab L, Salewski A, Steingass H, Fritz D and Schneider W 1979 The estimation of digestibility and metabolizable energy content of ruminant feeding stuffs from the gas production when they are incubated with rumen liquor in vitro. Journal of Agricultural Science 93, 217- 222

Morgavi D P, Newbold C J, Beever D E and Wallace R J 2000b Stability and stabilization of potential feed additive enzymes in rumen fluid. Enzyme Microbiology Technology 26, 171-177

Rodrigues M A M, Pinto P, Bezerra R M F, Dias A A, Guedes C V M, Cardoso V M G, Cone J W, Ferreira L M M, Colaco J and Sequeira C A 2008 Effect of enzyme extracts isolated from white-rot fungi on chemical composition and in vitro digestibility of wheat straw. Animal Feed Science and Technology 141, 326-338

Salem A Z M, Gado H M, Colombatto D and Elghandour M M Y 2013 Effects of exogenous enzymes on nutrient digestibility, ruminal fermentation and growth performance in beef steers. Livestock Science 154, 69–73

Satter L D and Slyter L L 1974 Effect of ammonia concentration on rumen microbial protein production in vitro. British Journal of Nutrition 32, 199

Seresinhe T, Madushika S A C, Seresinhe Y, Lal P K and Orskov E R 2012 Effects of tropical high tannin non legume and low tannin legume browse mixtures on fermentation parameters and methanogenesis using gas production technique. Asian- Australasian Journal of Animal Science 25, 1404-1410

Seresinhe T and Pathirana K K 2008 Effect of supplementation of straw based diets on the digestibilityand microbial nitrogen production of cross bred cattle. World Journal of Agricultural Sciences 04, 745-751

Sommart K, Parker D S, Wanapat M and Rowlinson P 2000 Fermentation characteristics and microbial protein synthesis in an in vitro system using cassava, rice straw and dried ruzi grass as substrates. Asian-Australasian Journal of Animal Science 13, 1084-1093

Tricario J M and Dawson K A 2005 Influence of supplemental endoglucanase or xylanase on volatile fatty acid production from ruminant feed by ruminalin vitro cultures. In: Archives of animal nutrition. Department of Animal Sciences, University of Kentucky, Lexington, USA. 59, 325-34

Waghorn G C and McNabb W C 2003 Consequences of plant phenolic compounds for productivity and health of ruminants.Proceedings of the Nutrition Society. 62, 383–392.

Wanapat M, Polyrach S, Boonnop K, Mapato C and Cherdthong A 2009 Effect of treating rice straw with urea and calcium hydroxide upon intake, digestibility, rumen fermentation and milk yield of dairy cows. Livestock Science 125, 238-243

Wang Y, McAllister T A, Rode L M, Beauchemin K A, Morgavi D P, Nsereko V L, Iwaasa A D and Yang W 2001 Effects of an exogenous enzyme preparation on microbial protein synthesis, enzyme activity and attachment to feed in the Rumen Simulation Technique (Rusitec). British Journal of Nutrition 85, 325–332

Wood T M and Bhat K M 1988 Methods for measuring cellulase activities. In: W. A. Wood, S. T. Kellogg (Editors). Methods in enzymology. Academic Press Inc., New York, NY, pp. 87-112

Yang H E, Son Y S and Beauchemin K A 2011 Effects of exogenous enzymes on ruminal fermentation and degradability of alfalfa hay and rice straw. Asian-Australasian Journal of Animal Science 24, 56 – 64.

Received 18 December 2014; Accepted 27 December 2014; Published 3 March 2015

Go to top