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Effects on In Vitro Methane Production, Digestibility and Fermentation Characteristics of Various Untreated and Urea Treated Maize Stover Based Complete Diets

Sudheer babu Arumbaka Madhu Mohini Datta Goutam Mondal Sujata Pandita
Vol 7(8), 245-253

A study was undertaken to evaluate untreated and 4% urea treated maize stover based various complete diets for efficient methane mitigation, optimum degradability and fermentation characteristics using in vitro gas production technique.

Keywords : Maize Stover Complete Diets Urea Treatment Methane In Vitro Gas Production Technique


Cattle and buffalo rearing is a major activity of rural India, primarily for milk and is as a source of secondary income and provides the livestock holders, a sustainable livelihood, on day to day basis. Production of 17.00 million tonnes of milk and per capita availability of 130 g of milk /day in the year 1950-51 has improved to the tune of 132.4 million tonnes of milk and 299 g milk /day respectively, in the year 2012-2013, on all India basis (GOI, 2014). By 2020 India would require a total 526 million tonnes (Mt) of dry matter, 855 Mt of green fodder, and 56 Mt of concentrate feed (comprising 27.4 Mt of cereals, 4.0 Mt of pulses, 20.6 Mt of oilseeds, oilcakes and meals, and 3.6 Mt of manufactured feed). In terms of nutrients, this translates into 738 Mt of dry matter, 379 Mt of total digestible nutrients and 32 Mt of digestible crude protein (Dikshit and Birthal, 2010). In the present scenario with a healthy and steady growth in the aspect of supply and demand for milk the ruminant production systems based on grazing are unlikely to sustain for long, unless supplemented by stall feeding apart from utilizing feed agricultural crop residues as total mixed rations or complete diets. Besides, feeding a balanced diet it considerably minimizes the labor requirement reduces wastage and it facilitates the efficient utilization of the crop residues under predetermined optimum ratio of concentrate to roughage. It is an established fact, that the nutritional value of cereal crop residues to ruminants is constrained by low nitrogen and high fibre contents. To increase the nutrient availability from these crop residues and poor quality roughages, processing methods like physical (grinding, pelleting, steam treatment, extrusion), and chemical (alkali treatment and urea ammoniation) are available. Methane production is an inescapable consequence of the fermentation of carbohydrates in the rumen. Methane emissions from ruminant livestock in 2014 was 97.1 million tonnes (MT) CH4 or 2.72 Gigatonnes (Gt) CO2-eq (1 MT = 1012 g, 1 Gt = 1015 g) which accounted for 47%-54% of all non-CO2 GHG emissions from the agricultural sector (Dangal et al., 2017).

Therefore, the present study was undertaken to know the optimum level of maize stover and 4% urea treated maize stover inclusion in complete diets formulated (through digestibility, methane production and fermentation characteristics variables) along with oat grass and concentrate with various levels of roughage to concentrate ratio using in vitro gas production technique (IVGPT).

Materials and Methods

Ethical Approval

The experimental protocol was approved by Institute Ethics committee of Animal Experiments, National Dairy Research Institute, Karnal, India.

Sample Preparation

MS, UMS, oat grass and concentrate mixture (Table 1) were ground using Wiley mill having 1 mm screen. Four types of experimental diets (Table 2) i.e. two maize stover (MS) based high fibre (HFD) and low fiber (LFD) complete diets and two similar 4% urea treated maize stover (UMS) based high fibre (U-HFD) and low fiber (U-LFD) complete diets were formulated along with oat grass and concentrate in the following roughage, green to concentrate ratio (HFD= R80:G10:C10; U-HFD=UR80:G10:C10; LFD= R50:G10:C40; U-LFD= UR50:G10:C40, where R: Maize stover, UR: 4% Urea treated maize stover, G: Oat grass, C: Concentrate). A 0.2 g of oven dried each complete diet were weighed in triplicate for in vitro gas evaluation. The chemical compositions of the four complete diets are presented in Table 2.

Table 1: Chemical composition of feed ingredients (% DM basis) used for preparing various diets

Parameters Maize Stover (MS) 4% Urea treated maize stover (UMS) Oat Grass Concentrate
Organic matter 91.83 90.81 89.52 94.45
Crude protein 5.35 8.8 7.52 20.61
Crude fibre 36.97 34.96 36.21 16.85
Ether extract 1.65 1.55 2.58 2.92
Neutral detergent fibre 72.26 73.8 64.75 39.89
Acid detergent fiber 53.88 52.66 43.5 19.03

*Each value is average of three observations

Table 2: Chemical composition of the diets (% DM basis)

Parameters HFD U-HFD LFD U-LFD
R80:G10:C10 UR80:G10:C10 R50:G10:C40 UR50:G10:C40
OM 90.81 91.47 89.29 91.23
CP 6.23 10.44 10.58 12.9
CF 34.17 33.5 25.08 24.89
EE 1.51 1.21 1.56 1.25
NDF 69.66 71.22 65.06 66.93
ADF 52.12 51.6 38.75 36.13

HFD= MS based high fibre diet, U-HFD= UMS based high fibre diet, LFD= MS based low fibre diet, U-LFD= UMS based low fibre diet; R: Maize stover, UR: 4% Urea treated maize stover, G: Oat grass, C: Concentrate

Collection of Rumen Liquor (RL) and Media Preparation

Rumen contents were collected 2hr after morning feeding from two rumen fistulated steers (approximately 400 kg body weight) fed maize stover ad libitum and concentrate (2.0 kg in equal proportions at 10:00 h and 16:00 h), mixed and strained through two layers of cheesecloth into pre-warmed thermo-flask with an O2-free headspace. Ruminal feed particles were allowed to settle to the bottom (5 min), and finally ruminal fluid was strained through two layers of nylon cloth (50 mm pore size). Particle-free ruminal fluid was mixed with the buffer solution (Menke and Steingass, 1988) in a proportion 1: 2 (v/v) at 39°C, anaerobically.

Two hundred milligrams of the various diets were accurately weighed into 100 ml calibrated glass syringes (Fortuna Optima, Germany). The syringes were pre-warmed (39°C) prior to the dispensing of 30 ml of buffered rumen liquor into each one. The syringes were incubated in water bath at 39±0.5°C for 24 h. Three blanks containing only 30 ml of buffered rumen fluid were included to correct gas production values for the gas released from endogenous substrates. The corrected gas values (net gas) were used for calculation of partitioning factor and microbial biomass production. After 24 hr incubation, fermentation was arrested by chilling the syringes to 4°C, and the quantities of fermentation products were determined in each syringe. The total and net gas values were recorded and a suitable aliquot of gas was withdrawn from the tip of the incubation syringes using gas tight syringe. Thereafter, the syringe contents were transferred to centrifuge tubes and were centrifuged at 1600 g for 10 min and an aliquot of supernatant was acidified with equal volume of 0.5 M HCl for measuring ammonia nitrogen (N) and kept at -20°C. Another aliquot of supernatant (4 ml) was added to 25% m-phosphoric acid (2 ml), kept overnight at 4°C and centrifuged at 1600 g for 15 min and supernatant was stored at minus 20°C for volatile fatty acids (VFA) analysis. The pellets were transferred to spoutless beakers (500 ml) by dissolving with 70 ml of neutral detergent solution. Beakers were kept on heater and refluxed at 100°C for 1 hr from when the boiling started. The contents in the beakers were filtered under vacuum through pre-weighed sintered (G1) crucibles and washed with hot water. The crucibles containing residue was oven dried (65°C for 48 hr), weighed and the dried residue was ashed at 550°C.

Measurement of In vitro Parameters

Feed samples were analyzed for proximate principles (AOAC, 2005) and fiber fractions (Van Soest et al. 1991). The gas production was recorded at 24 hr of incubation. Methane analyzed with Gas Chromatograph (GC) (Nucon 5700, India) fitted with stainless steel column packed with Porapak-N and Flame Ionization Detector (FID). The temperature of injector, column and detector was 40º, 50º and 100oC, respectively. The flow rate of carrier gas (nitrogen), air and fuel gas (IOLAR grade hydrogen) through the column was 30, 300 and 30 ml/minGas sample (2 ml) from the syringe (5 ml) was injected into the GC through injection port. The standard gas used for methane estimation (Spantech House, Surrey, England) composed of 50% methane and 50% CO2. For the estimation of ammonia nitrogen concentration (Raab et al., 1983), acidified supernatant (5 ml) was mixed with 10 ml of NaOH (1 N) and immediately steam distilled using KEL PLUS – N analyser (Pelican, India). The ammonia evolved was collected in boric acid solution (20% w/v) having mixed indicator and titrated against N/100 sulfuric acid.Individual VFA (acetate, propionate and butyrate) in the samples were determined using GC with FID and stainless steel column, packed with chromosorb 101 mesh 80–100 (length 1.5 m, o.d 3.175 mm and i.d. 2 mm). Analytical conditions for fractionation of VFA were as follows: injection port temperature, 210°C; column temperature, 180°C and detector temperature, 230°C. The flow rate of carrier gas (nitrogen) was 40 ml/min. In vitro digestible DM (IVDMD) and truly digestible OM (TDOM) were calculated from the disappearances of DM and OM. Partitioning factor (PF) and microbial biomass production (MBP) were calculated based on truly degraded organic matter (TDOM) as described by Blummel et al. (1999) and Blummel et al. (2005).

Statistical Analysis

The data obtained in this study were subjected to one way analysis of variance using statistical analysis systems statistical software package version 8.2 (SAS Institute, Cary, NC, USA).The differences between the means were tested for significance at 5 % level (P<0.05) according to DMRT (Duncan, 1955).


In vitro total gas production in (ml/g DM) at 24 h incubation was (P=0.024) highest in LFD and lowest in U-HFD with HFD and U-LFD having statistically similar values (Table 3). Methane expressed as (ml/100 mg DDM) was (P=0.016) lowest in U-LFD and higher in HFD, LFD while U-HFD was comparable to U-LFD, LFD and HFD. The IVDMD (%) and TOMD (%) were (P=0.001) higher in LFD and U-LFD while lower in HFD and U-HFD. The MBP (mg) recorded highest (P=0.043) in U-LFD and lowest in HFD. The ammonia nitrogen levels showed a linear (P=0.001) trend (HFD<U-HFD<LFD<U-LFD) in the diets. Propionate (mM/L) levels were higher (P= 0.014) in U-LFD, U-HFD and LFD with lowest in HFD while butyrate was lower (P=0.001) in U-LFD and LFD while higher in HFD and U-HFD diets. The acetate to propionate ratio (A/P ratio) was lowest in UMS based U-LFD and U-HFD diets while higher value was observed in HFD the LFD had statistically similar value comparable to the rest of the three diets (Table 4).

Table 3: In vitro gas and methane production, digestibility, PF and MBP in MS and UMS based diets incubated for 24h

R80:G10:C10 UR80:G10:C10 R50:G10:C40 UR50:G10:C40 SEM P value
Total gas (ml/g DM) 185.52ab 173.51a 211.67b 200.63ab 5.33 0.024
CH4 (%) 24.94 25.35 26.26 22.44 0.58 0.086
CH4 (ml/24 h) 9.26 8.8 11.12 8.98 0.34 0.029
CH4 (ml/100mg DDM) 9.16b 8.12ab 8.59b 6.70a 0.33 0.016
CH4 (g/kg DM incubated) 25.1 25.74 31.44 26.34 1.04 0.09
CH4 (g/kg IVDMD) 49.67 47.49 48.58 39.28 1.73 0.111
CH4 (g/kg TDOM) 48.26 45.2 50.39 38.28 1.83 0.068
IVDMD % 50.54a 54.29a 64.73b 67.05b 2.14 <0.001
TOMD % 52.75a 56.26a 64.14b 68.09b 1.9 <0.001
PF 3.54 3.83 3.56 3.85 0.09 0.532
MBP (mg) 35.63a 43.99ab 43.78ab 53.55b 2.43 0.043

a,b Values bearing different superscripts in a row differ significantly (P<0.01), SEM=Standard error of means;

HFD= MS based high fibre diet, U-HFD= UMS based high fibre diet, LFD= MS based low fibre diet, U-LFD= UMS based low fibre diet. R: Maize stover, UR: 4% Urea treated maize stover, G: Oat grass, C: Concentrate. DDM = Digestible dry matter, IVDMD = In vitro dry matter digested, TOMD = True organic matter digestibility, TDOM=truly digestible organic matter, PF= Partition factor, MBP= Microbial biomass production

Table 4: In vitro ammonia nitrogen and VFA’s production in various untreated and 4% urea treated maize stover based rations incubated for 24hr

Parameter HFD U-HFD LFD U-LFD SEM P value
R80:G10:C10 UR80:G10:C10 R50:G10:C40 UR50:G10:C40
Ammonia nitrogen (mg/dl) 8.70a 11.35b 13.11c 18.50d 1.09 <0.001
Volatile Fatty Acids
Acetate (mM/L) 37.18 33.2 35.52 31.96 0.85 0.11
Propionate (mM/L) 10.79a 15.06b 13.83ab 15.73b 0.67 0.014
Butyrate (mM/L) 8.96b 8.71b 7.03a 6.82a 0.31 <0.001
A/P ratio 3.45b 2.22a 2.61ab 2.03a 0.18 0.003

a,b,c,d Values bearing different superscripts in a row differ significantly (P<0.01), SEM=Standard error of means


There was decrease in total gas production (GP) by 6.47 per cent for U-HFD compared to HFD and 5.22 per cent for U-LFD compared to LFD. GP reflects the apparent substrate degradability (Pashaei et al., 2010). GP showed reduction as the difference in the protein content of the feed evaluated increased and the fibre content decreased as inferred by Lopez et al. (1998). It was of the opinion that the gas production technique lacks the complexity of the animal, and cannot be indicative of the complex interactions between the OM and microbial population compartmentalization (Dijkstra et al., 2005). Methane expressed as (ml/100 mg DDM) when calculated per unit of IVDMD % the trend observed for the methane mitigation potential was (U-LFD<LFD<U-HFD<HFD) indicating that low fermentation and organic matter digestibility results in higher methane emissions (Gemeda and Hassan, 2015) and on supplementation with concentrate improves digestibility and enhance propionate production. Increasing protein in the diet is expected to decrease methane emission because of direct negative association of protein with methane or the replacement in the diet of methanogenic carbohydrate with protein (Pelchen and Peters 1998).

It can be observed that the IVDMD and TOMD %, though were statistically similar within high fiber and low fiber diets, numerically there was an increase of digestibility of U-HFD and U-LFD over the MS based diets as the alkali pre treatment of maize stover increased the extent (P =0.001) of in vitro DM disappearance (Wang et al., 2004). Ammonia nitrogen showed a linear increasing trend (HFD<U-HFD<LFD<U-LFD) with the increase in protein (Chen et al., 2010; Ghorbani et al., 2011) and changing levels of concentrate (Pina et al., 2009; Agle et al., 2010) in the diets and might have resulted in active degradation of protein and hydrolysis of non-protein nitrogen substances (urea treated stover is enriched with nitrogen, Manyuchi et al., 1992). The U-HFD showed an increase of 23.46 per cent compared to HFD and the U-LFD showed an increase of 22.32 per cent compared to LFD for the MBP synthesis values. This concurrent numerical increase of the MBP synthesis values might be due to synchronization between liberated ammonia and availability of fermentable carbohydrates consequent to higher digestibility. It has been reported, by earlier studies that in low quality roughage diets, the utilization of urea for microbial protein synthesis is primarily limited by the low availability of fermentable energy (Farid et al., 1986). In this context, it can be inferred that the efficiency of utilization of the ammonia nitrogen was improved with stover being treated with urea and with higher degradability of dry matter. The high ammonia levels, in urea treated stover based diets imply that nitrogen incorporated during treatment is readily available for use by rumen microbes (Manyuchi et al., 1992).

The IVFA (propionate and butyrate) and A/P ratio were significantly affected by increased protein level and varying roughage to concentrate ratio. In contrast, Norrapoke et al. (2012) reported that increasing CP had no significant effect on the VFA, while the difference of roughage to concentrate ratios, significantly affected total VFA production. When rumen fermentation conditions are optimal, the acetate to propionate ratio should be greater than 2.1:1 (McDonald et al., 1995). In the present study, acetate to propionate ratios was within the conditions of optimal fermentation and a lower A/P generally reflects improved nutritional value of a feed while higher A/P ratios may have resulted from altered growth rates of bacteria (Russell and Wallace, 1997) or perhaps a shift in bacteria populations.


Based on the results, it can be concluded that among the diets studied urea treated maize stover based low fiber diet showed optimum digestibility and methane mitigation followed by LFD which can be further evaluated In vivo.


The authors are thankful to the authorities of ICAR-NDRI, Karnal, for providing assistance and infrastructure to carry out this investigation. Appreciation is expressed to staff members and research scholars of Animal Nutrition Department, ICAR-NDRI, Karnal for their cooperation and support for smooth conduct of this study.

Competing Interests

The authors declare that they have no competing interests.


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