### INTRODUCTION

_{4}production in rumen fermentation is important for identifying the strategies for mitigating CH

_{4}production from ruminants. Many studies indicated that the nutrient composition of feeds is closely correlated with the CH

_{4}production in rumen fermentation. Since there is a correlation between the feed composition and the CH

_{4}production, the feed composition is used to predict the CH

_{4}production. The dietary variables including dry matter intake (DMI) (Kriss, 1930; Axelsson, 1949; Mills et al., 2003; Ellis et al., 2007), energy digestibility (Blaxter and Clapperton, 1965), digestible carbohydrates (dCHO) (Bratzler and Forbes, 1940), intakes of non-structural carbohydrate (NSC), hemicellulose and cellulose (Moe and Tyrell, 1979) and digestible crude protein (CP), ether extract (EE), crude fibre and N-free extract of the diets (Jentsch et al., 2007) etc. were used in CH

_{4}predicting models. The use of different dietary variables in different models affected the accuracy of CH

_{4}predicting models. Screening the dietary parameters which are easily determined and closely correlated with the CH

_{4}production would be helpful for accurately modeling and predicting the CH

_{4}production from ruminants.

*in situ*undegraded dietary protein (UDP) for ruminants (Shannak et al., 2000) etc. Since CH

_{4}is one of the important products of microbial fermentation of carbohydrates in the rumen, it could be speculated that the CH

_{4}production in rumen fermentation could be closely correlated with the CNCPS carbohydrate fractions.

_{4}production from feeds for cattle and the CNCPS carbohydrate fractions, and the suitability of the CNCPS carbohydrate fractions as dietary variables in modeling CH

_{4}production in rumen fermentation.

### MATERIALS AND METHODS

### Animals and feeding management

*Jinniuweiye*Science and Technology Co., Ltd., Beijing, China), were used as the donors of rumen fluid. The daily ration for the cattle included 6.0 kg Chinese wildrye and 2.0 kg concentrate mixture. The concentrate mixture was composed of 58% corn, 20% soybean meal, 18% wheat bran, 2% calcium hydrogen phosphate, 1% sodium chloride, and 1% trace element mixture. The cattle were fed twice daily at 07:00 h and 17:00 h, in two equal meals, and had free access to fresh drinking water. The management of the cattle was according to The Administration Regulations on Laboratory Animals (The Administrative Department of Beijing Municipal Science and Technology, 2002).

### Feed samples

### *In vitro* incubation

_{4}production of feed samples. Glass syringes with a calibrated volume of 100 mL were used as the incubation vessels.

### Determinations and analysis

*Ruili*Analytical Instruments, China) after converting starch to glucose using an enzyme kit containing thermostable α-amylase and amyloglucosidase (Megazyme International Ireland Ltd., Wicklow, Ireland; Method 996.11, AOAC, 1990).

_{4}and CO

_{2}in the gas samples were analyzed using gas chromatography (TP-2060T, Beijing

*Beifen Tianpu*Instrument Technology Co., Ltd., Beijing, China). The conditions for the analysis were as following: TCD detector, TDX-01 column, size 1 m×2 mm×3 mm, column temperature 70°C, detector temperature 100°C. The carrying gas was argon, with the flowing rate of 30 mL/min. The standard gas used was composed of 26.796% CH

_{4}, 65.300% CO

_{2}, 0.605% O

_{2}, 7.100% N

_{2}and 0.199% H

_{2}(v/v).

### Calculation and statistical analysis

_{1}, starch and pectin; CB

_{2}, available cell wall; CC, unavailable cell wall; NSC, non-structural carbohydrate; CHO, carbohydrate; CP, crude protein; NDICP, neutral detergent insoluble crude protein. The unit for all the CNCPS fractions is % DM.

_{4}, CO

_{2}or total gas production of feed samples (mL) was calculated as following:

_{sample}refers to the CH

_{4}production of feed sample in 48 h; Y

_{total}, the CH

_{4}, CO

_{2}or total gas production of incubation in 48 h; Y

_{blank}, the CH

_{4}, CO

_{2}or total gas production of the blank in 48 h. The CH

_{4}, CO

_{2}and total gas production and the pH for modeling were listed in Table 3.

_{4}, CO

_{2}and total gas production (mL) and the CNCPS carbohydrate fractions (g) was analyzed using the following equation:

*y*refers to the CH

_{4}, CO

_{2}or total gas production;

*a*refers to a constant;

*b*,

_{1}*b*, and

_{2}*b*refer to coefficients.

_{3}_{4}, CO

_{2}and total gas production and the CNCPS carbohydrate fractions. The components, the nutrient composition and the CH

_{4}, CO

_{2}and total gas production of the rations for validation were listed in Table 4, 5 and 6, respectively.

_{4}, CO

_{2}and total gas production was evaluated in three ways. The observed and the predicted CH

_{4}, CO

_{2}and total gas production were compared using the paired

*t*-test; the relationships between the observed and the predicted CH

_{4}, CO

_{2}and total gas production were analysed using the equation:

*x*refers to the observed CH

_{4}, CO

_{2}or total gas production, mL/g DM;

*y*refers to the predicted CH

_{4}, CO

_{2}or total gas production, mL/g DM; The root mean square prediction error (RMSPE) between the observed and the predicted CH

_{4}, CO

_{2}or total gas production was also calculated for evaluating the multiple regression relationship established in the trial. The RMSPE was calculated as:

*i*= 1, 2, …,

*n*;

*O*refers to the observed value;

_{i}*P*, the predicted value;

_{i}*n*, the number of determinations. RMSPE%, the percentage of the prediction error/the average observed value.

### RESULTS

### *In vitro* incubation

*in vitro*incubation for 48 h, the pH value of the incubation residue was within the range of 6.40 to 6.80, and the microscopic check indicated that the rumen microorganisms were active.

### Relationships between the CH_{4}, CO_{2}, and total gas production and the CNCPS carbohydrate fractions

_{4}production were shown in Table 2 and 3.

_{4}, CO

_{2}and total gas production (mL) and the CNCPS carbohydrate fractions CA, CB

_{1}and CB

_{2}(g).

##### (Equation I)

### Validation of the equations between the CH_{4}, CO_{2} and total gas production and the CNCPS carbohydrate fractions

*t*-test showed that no difference was found between the observed and the predicted CH

_{4}, CO

_{2}and total gas production based on the Equation I (p = 0.443), Equation II (p = 0.150) and Equation III (p = 0.326), respectively. Significant linear regression relationship was found between the observed and the predicted CH

_{4}production based on Equation I (R

^{2}= 0.94, p<0.0001, n = 10, Figure 1), between the observed and the predicted CO

_{2}production based on Equation II (R

^{2}= 0.77, p = 0.0008, n = 10, Figure 2) and between the observed and the predicted total gas production based on Equation III (R

^{2}= 0.87, p< 0.0001, n = 10, Figure 3). The RMSPE% of Equations I, II, and III was found to be 3.82%, 8.16% and 5.93%, respectively.

### DISCUSSION

### Measurement of gas production

*in vivo*digestibility of feed was obtained after 45 to 52 h of

*in vitro*fermentation (Prasad et al., 1994; Liu et al., 2002), the gas production of the rations during the 48 h

*in vitro*incubation was believed to be close to that of the actual rumen fermentation.

*in vitro*gas production of ruminant feeds abundant in CP such as soybean meal (CP 51.3% DM) and cell mass from lysine production (CMLP) (CP 72.3% DM) was significantly lower than the theoretical value, indicating that the nitrogenous compounds of the feeds interfered with the acid-base reaction, increased the pH and reduced the indirect gas production. In this case, the

*in vitro*gas test might be not a suitable technique for measuring the gas production. In the present trial, the CP content of the mixed rations for modeling was from 6.44 to 15.04% and that for validation was from 6.50 to 14.20%, which was in a moderate range, it could be believed that the

*in vitro*gas test was suitable for the gas measurements and the results were reliable.

### Relationships between the CH_{4}, CO_{2} and total gas production and the CNCPS carbohydrate fractions

_{1}(starch and pectin), CB

_{2}(available fibre) and CC (unavailable fibre) (Sniffen et al., 1992). Fractions CA, CB

_{1}and CB

_{2}can be fermented in the rumen at fast, moderate and slow speed, respectively, whereas fraction CC is not fermentable in the rumen. The classification of the CNCPS carbohydrate fractions reflects the carbohydrate composition as well as the fermentative characteristics of the carbohydrates. Significant positive regression relationships found in the present trial between the CH

_{4}production (Equation I), the CO

_{2}production (Equation II), the total gas production (Equation III) and the CNCPS carbohydrate fractions CA, CB

_{1}and CB

_{2}indicated that the CNCPS carbohydrate fractions were suitable parameters for predicting the CH

_{4}production, the CO

_{2}production and the total gas production.

_{4}production and the lignin intake of Holstein cows.

_{2}and H

_{2}which are used for CH

_{4}production, whereas fermentation of sugars and NSC mainly produces propionate accompanied with an uptake of H

_{2}. It could be presumed that the fermentation of fraction CB

_{2}would produce more CH

_{4}than that of fractions A and CB

_{1}. In Equation I, however, the coefficients of CA and CB

_{1,}CB

_{2}are 89.16, 124.10, and 30.58 mL/g, respectively, indicating that fraction CB

_{2}produced less CH

_{4}than fractions CA and CB

_{1}. The reason for the results could be that CB

_{2}was the available fibre with slower fermentation rate than fractions CA and CB

_{1}.

_{4}production of Holstein cows and found a regression relationship between the CH

_{4}production and the intakes of the soluble residue, hemicellulose and cellulose (R

^{2}= 0.67) and the regression relationship became closer when the apparently digested soluble residue, hemicellulose and cellulose were used as the dietary variables (R

^{2}= 0.73). Jentsch et al. (2007) found a regression relationship between the CH

_{4}production and the intakes of CP, crude fat, crude fibre and N-free extract of cattle (R

^{2}= 0.859). The regression relationship also became closer when the digestible nutrients including CP, EE, crude fibre and N-free extract were used as the dietary variables (R

^{2}= 0.896). In Equation I of the present trial, the determination coefficient (R

^{2}= 0.81) between the CH

_{4}production and the CNCPS fractions CA and CB

_{1,}CB

_{2}was between the determination coefficient of Moe and Tyrell (1979) and that of Jentsch et al. (2007), indicating that Equation I was reliable for predicting the CH

_{4}production.

### Validation of the equations established in the trial

^{2}) between the observed and the predicted CH

_{4}, CO

_{2}or total gas production was high. The RMSPE% of Equations I, II and III was lower than that of Moe and Tyrrell (1979) (34%), Ellis et al. (2007) (14.4%) and Blaxter and Clapperton (1965) (36.5%). The high determination coefficient and the low RMSPE% indicate that Equations I, II, and III are reliable for predicting the CH

_{4}CO

_{2}or total gas production of rations with the roughage/concentrate ratios within the range of 10:90, 20:80, 30:70, 40:60, and 50:50 for cattle. The trial demonstrated that the CNCPS fractions CA and CB

_{1,}CB

_{2}are suitable dietary variables for modeling CH

_{4}production.

*in vitro*measurement of gas production. Since rumen is a dynamic system with the passage and digestion of digesta taking place simultaneously, and the substrate for methanogenesis shifts from hydrogen to formate after 10 h of

*in vitro*batch culture (Seo et al., 2009), this trial suffers from the limitations of the 48 h

*in vitro*incubation. The equations need to be validated using

*in vivo*trials for predicting the CH

_{4}CO

_{2}and total gas production from rumen fermentation of cattle.

### CONCLUSION

_{4}production and the CNCPS carbohydrate fractions CA, CB

_{1}and CB

_{2}of rations for cattle over a wide range of concentrate/roughage ratios. Evaluation results demonstrated that the

*in vitro*CH

_{4}production of rations for cattle could be accurately predicted based on the CNCPS carbohydrate fractions CA, CB

_{1}and CB

_{2}using Equation I. To utilize the equation for predicting the CH

_{4}production from rumen fermentation of cattle, it is necessary to validate the equation using

*in vivo*trials.