Livestock Research for Rural Development 26 (1) 2014 Guide for preparation of papers LRRD Newsletter

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Methods of adding exogenous fibrolytic enzymes to rice (Oryza sativa L.) straw and in vitro ruminal fermentation parameters

D A Lorenzo, Á Ojeda, D Vargas and J L Gil*

Facultad de Agronomía. Universidad Central de Venezuela. Maracay, Venezuela,
* Instituto Nacional de Investigaciones Agrícolas. CENIAP Maracay, Venezuela.


To evaluate the effect of a commercial exogenous fibrolytic enzymes (EFE) on some in vitro ruminal fermentation parameters of rice straw (Oryza sativa L.), gas production (3, 6, 9, 12, 16, 24, 36, 48, 60, 72 and 96 h) and apparent digestibility (24 and 48 h) were measured on treatments that included EFE at 0.04 and 0.08% on dry basis, with (Inc) or without (NoInc) incubation with EFE at room temperature for 24 h before the in vitro evaluation started. A control treatment without EFE or incubation was also included.

Independently of EFE level, NoInc increased the gas production compared to control at 12 (52.4%) and 48 (18.1 %) h. After 24 h of fermentation, the gas production was higher in EFE 0.08% than EFE 0.04% in a range of 5.0 to 8.9%. Inc treatment reduced the gas production compared to NoInc at 24 (33.5%) and 96 (12.7%) h, while the lag time was reduced in EFE 0.08%. In relation to the Control, the methods of adding the fibrolytic enzymes did not affect the apparent dry matter digestibility, which increased only 0.2 MJ EM/kg DM in NoInc. In conclusion, the EFE adding increased the gas production without affecting the apparent dry matter digestibility during the first 48 h of fermentation, while the incubation before the in vitro fermentation reduced both parameters, independently of exogenous fibrolytic enzymes addition.

Keywords: fiber, gas production, incubation, ruminants


In most tropical countries, the ruminant feed is based on fibrous resources with a cell wall content between 40 and 70% of dry matter, of which less than 50% is quickly digested, which generates high excretion of nutrients to the environment and low productivity in their production systems (Devendra 2000). The use of exogenous fibrolytic enzymes (EFE) is an alternative to increase the ruminal hydrolysis of cell wall contents due to increase in the microbial binding capacity to digest, stimulation of microbial populations, and the synergy between the enzymes synthesized in the rumen and polysaccharides contained in such products (Beauchemin et al 2004).

However, the responses obtained with the inclusion of EFE in cattle rations are inconsistent. Several studies have shown increases in vitro gas production during the first 16 h of incubation (Lewis et al 1996, Colombatto et al 2007), and improved ruminal digestibility of dry matter and cell wall (Gómez-Vazquez et al 2011, Yang et al 2011), although these results differ from those which did not change gas production (Yescas-Yescas et al 2004) and digestibility of low quality forages (Avellaneda et al 2009). This variability is associated with factors such as the species and physiological status, characteristics of the substrate used, composition of the enzyme product, amount of enzyme applied and method of application, among others (Hussain et al 2008, Gallardo et al 2010).

The aim of this study was to determine the effect of inclusion level and incubation time with the substrate of a commercial source of EFE on gas production and apparent in vitro ruminal digestibility of rice straw (Oryza sativa L.).

Materials and methods

Study location

The test was carried out at the Laboratory of Animal Nutrition, Agronomy School of the Universidad Central de Venezuela (10 º 16 ' 20" N and 67° 36' 35" W), located at an altitude of 455 meters, with an average temperature of 25°C and a relative humidity of 75% (INIA 2012).


As fibrous resource was used rice straw [89.2% dry matter (DM), 3.5% crude protein (CP), 72.2% neutral detergent fiber (NDF), and 8.2% ash] manually chopped up to 2.5 cm long. The EFE source was generated by solid state fermentation with Aspergillus niger according to the manufacturer's specifications (Allzyme SSF®, Alltech Inc., Nicholasville, KY, EUA). This EFE has fibrolytic activity primarily (cellulase, β-glucanase, and pentosanase), although it also contains other enzymes (phytase, protease, and amylase) in less proportion.

The experiment was conducted as a complete randomize design in a 2 x 2 factorial arrangement. Treatments consisted of EFE addition at 0.04% (EFE-0.04) or 0.08% (EFE-0.08) DM at the beginning of the in vitro evaluation (NoInc) or incubated (Inc) with the fibrous resource previously for 24 h at room temperature. Additionally, a control with untreated rice straw was included. For the incubation, the EFE was dissolved in sufficient water to reach 70% moisture in the substrate, and the resulting material was stored in plastic containers of 500 cm3 capacity. After the incubation period, the material was dried at 65°C in a forced ventilation oven for subsequent milling (Tecator, Cyclotec 1093) through a 1mm mesh.

Gas production and in vitro apparent digestibility

Gas production was measured using amber glass bottles with a capacity of 130 cm3. To each, and according to the protocol described by Theodorou et al (1994), were added 0.75 g of substrate, 67 ml of solutions (minerals and sodium bicarbonate buffer), and EFE according to treatments. Inoculum was obtained from two cows (Bos taurus x Bos indicus) cannulated in the rumen and 485.5 ± 3.5 kg body weight, grazing pastures of cultivated grasses from genera Brachiaria, Cynodon, Digitaria and Panicum, and a supplement supply at 2 kg/animal/day (90.1% DM, 16.9% CP, 37.2% NDF and 7.6% ash). Gas production was recorded with a pressure transducer (Red Lion®, model DP5-1/8 DIN) at 3, 6, 9, 12, 16, 24, 36, 48, 60, 72 and 96 h.

Considered four repetitions per treatment, apparent digestibility of dry matter at 24 and 48 h was evaluated by filtering the contents of each glass bottles in porcelain crucibles (pore # 1) previously weighed, which were subsequently dried at 105°C in an oven with forced ventilation. The gas produced was determined by the transformation of pressure readings (psi) to volume (ml) according to Giraldo et al (2006), while the parameters of the gas production kinetics were estimated with an exponential model developed by France et al (1993) using the NLIN procedure of SAS (2008). The metabolizable energy content (ME) was estimated according to Menke et al (1979).

Statistical analysis

All statistical analyzes were performed using the GLM procedure of SAS (2008). The evaluated effects were EFE (0.04 and 0.08%), incubation (Inc and NoInc) and their interaction (EFE x Incubation). Additionally it was performed a linear orthogonal contrast to compare the Control and other treatments. If case of statistical differences (P <0.05) among means, comparison was made by Tukey test (P≤0.05).

Results and discussion

There was an effect of the inclusion level EFE on the in vitro gas production (Table 1 and Figure 1). EFE-0.04 was higher by 60.4 and 21.1% at 6 and 12 h; however, from this last record to the end of the evaluation, EFE-0.08 led to an increase in gas production of 5.94 ± 1.6%. Incubation of fibrous resource with EFE decreased gas production by 33.5 and 12.7% for 24 and 96 h, respectively. Regardless of the level of inclusion of EFE, the substrate incubation did not generate variation of in vitro gas production compared to Control; however, NoInc treatment caused an increase regards Control of 52.4% control at 12 h and 20.9 ± 4.1% during the remainder of the evaluation. As proposed by Moharrery et al (2009), to prolong the enzyme-substrate interaction prior to in vitro fermentation of a fibrous material can generate a complex between EFE and structural carbohydrates, reducing the enzyme hydrolytic activity.

Table 1. Accumulated in vitro gas production (ml/g DM) and estimated parameters in rice straw treated with exogenous fibrolytic enzymes (EFE)
Time Parameter³
Treatment¹ 6 12 24 48 72 96 a b T
EFE-0.04 0.83 7.21* 19.4* 37.3* 49.0* 55.6* 61.9* 0.031* 3.71
EFE-0.08 0.35* 5.63* 19.9* 39.7* 52.5* 59.3* 64.2* 0.038* 1.92*
EFE-0.04 0.87 3.92 13.0 31.2 43.3 49.2 54.8* 0.040* 3.84
EFE-0.08 0.71 4.03 14.2 33.0 44.4 59.1 54.3* 0.041 1.64*
Control² 0.64 4.26 15.5 32.6 43.0 47.6 50.8 0.044 3.15
SEM 0.12 0.34 0.57 0,99 0.86 0.74 1.15 0.0001 0.422
EFE <0.001 <0.05 <0.001 <0.001 <0.001 <0.001 NS NS <0.001
Incubation NS <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 NS
EFE*Incubation NS NS NS NS NS NS NS NS <0.01
Control² vs rest <0.05 <0.001 <0.001 <0.01 <0.001 <0.001 <0.001 <0.001 <0.01
¹EFE added to 0.04 (EFE-0.04) or 0.08% (EFE-0.08) on dry matter basis to the beginning of the evaluation in vitro (NoInc) or incubated (Inc) previously during 24 h with the fibrous resource.
²Untreated rice straw.
³a: gas production potential (ml), b: fractional rate of gas production (ml/h) and T: lag time. SEM: Standard error of the mean
*Means with asterisk indicate significant differences compared to control

There was no effect of EFE on the potential gas production and fractional rate, but EFE-0.08 reduced the time of colonization (lag time) by 52.6%. Substrate incubation decreased the gas production by 13.5%, even with an increase in the fractional rate of 33.3%. Regarding the Control, regardless of the level of inclusion of EFE, there was an increase in gas production of 7.9 and 21.9% for Inc and NoInc, respectively. The lag time decreased with the highest level of EFE, which showed a higher efficiency of microbial colonization of the substrate mediated by the increase in the concentration of exogenous enzymes in the rumen (Avellaneda et al 2009, Gallardo et al 2010). Because of its high solubility, EFE frequently generate increases in gas production in short periods of fermentation (Gado et al 2009, Moharrery et al 2009), since the rumen tends to inactivate after the first 6 h of fermentation (Colombatto et al 2007). In this study the effect lasted for 96 h, which could be associated with a positive relationship between EFE enzyme profile and structural carbohydrate content in the substrate (Jalilvand et al 2008).

Figure 1. Impact of the incorporation of exogenous fibrolytic enzymes (EFE) and the
incubation (Inc) on in vitro gas production (ml/g DM) of rice straw.

In general, the mode of action by which exogenous enzymes can improve the ruminal degradation of fiber resources is still subject of speculation, indicating that the mechanism might be related to higher microbial growth due to an increase in the availability of soluble carbohydrates product of enzyme activity, which may further promote a higher rate of synthesis of glicocalyx, and thus a greater capacity for microbial adhesion to the substrate (Van de Vyver and Useni 2012). There was no effect of inclusion level of EFE on the apparent in vitro digestibility of dry matter and metabolizable energy content, while Inc effect was consistent with previous results, which showed a decrease (P<0.001) in the digestibility when compared to NoInc (Table 2). Regarding the Control, EFE application methods did not affect the digestibility of dry matter, showing just an increase of 0.2 MJ ME/kg DM in NoInc.

Table 2. In vitro dry matter digestibility and metabolizable energy content of rice straw treated
Digestibility, % ME³
Efects¹ 24 h 48 h (MJ/kg DM)
EFE 0.04 14.5 29.7 5.93*
EFE 0.08 15.1 30.8 5.92*
EFE 0.04 11.8 27.0 5.64
EFE 0.08 11.2 26.9 5.61
Control² 12.5 27.7 5.73
SEM 1.18 1.41 0.31
Incubation <0.001 <0.001 NS
EFE*Incubation <0.001 NS NS
Control vs rest NS NS <0.05
¹EFE added to 0.04 (EFE-0.04) or 0.08% (EFE-0.08) dry matter basis to the beginning of the evaluation in vitro (NoInc) or incubated (Inc) previously during 24 h with the fibrous resource.
²Untreated rice straw.
³Metabolizable energy content as estimated by Menke et al 1979

This is consistent with several studies which indicate that dry matter digestibility is not affected by the addition of enzymes to diets with low quality forages (Yescas-Yescas et al 2004, Avellaneda et al 2009, Martins et al 2010). In this sense, Gallardo et al (2010) suggest the need to deepen the relationship between the molecular structure of cellulose and exocellulase content of EFE, determinant factors of fibrolytic activity of exogenous enzymes.

It is good to note that there was no causal relationship between gas production and in vitro apparent digestibility of dry matter, confirming that increased gas production does not necessarily indicate greater efficiency by microorganisms in substrate utilization (Posada and Noguera 2005). In this regard, the relationship between substrate truly degraded (mg) to volume of gas produced (ml) may reflect variations in the microbial biomass production, a so-called partition factor. This factor decreases with the incubation time, so that gas production and microbial biomass per unit of degraded substrate is not constant, and may even be an inverse relationship between them (Blümmel et al 1999).



The authors wish to thank Alltech Venezuela, SCS for the financial support to conduct this research.


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Received 13 October 2013; Accepted 1 December 2013; Published 1 January 2014

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