Comparison of in vitro Fermentation with in situ Degradation to Estimate Dry Matter Degradability and Energy Protein Synchronization of Roughage Based Diets

Iranian Journal of Applied Animal Science

مقاله 3، دوره 9، شماره 1، خرداد 2019، صفحه 17-24   

نوع مقاله: Research Articles

نویسندگان

M. Aung^{* 1}؛ Y.Y. Kyawt¹؛ M.T. Htun²؛ K.S. Mu¹؛ A. Aung²

¹Department of Animal Nutrition, University of Veterinary Science, Yezin, Nay Pyi Taw, 15013, Myanmar

²Department of Physiology and Biochemistry, University of Veterinary Science, Yezin, Nay Pyi Taw, 15013, Myanmara

چکیده

This study was aimed to evaluate the correlation between in vitro fermentation and in situ degradation parameters and to predict dry matter degradability and energy protein synchronization of roughage based diets. Different inclusion of roughage in diets [roughage 50% diet (R₅₀D), roughage 60% diet (R₆₀D), roughage 65% diet (R₆₅D) and roughage 70% diet (R₇₀D)] were used to determine in vitro and in situ parameters. The relationships between in vitro and in situ parameters were analyzed by simple linear regression. The gas volumes and fermentation kinetics of R₅₀D and R₆₅D were greater (P<0.05) than those of other diets, however the lesser values of partitioning factor for microbial protein synthesis efficiency was found in R₅₀D. Although the nutrient disappearances, degradation kinetics and effective degradability were greater (P<0.05) in R₅₀D in compare with R₆₀D and R₆₅D, the lowest (P<0.05) energy protein synchronization was observed in R₅₀D. The significant correlation (P<0.05) were observed in all regression equations of in vitro gas volumes and in situ nutrient disappearances. The in situ effective dry matter degradability and energy protein synchronization were correlated (P<0.05) with in vitro fermentation kinetics and some estimated parameters such as short chain fatty acid and partitioning factor for microbial protein synthesis efficiency. Among the correlations, the greater accuracy could be achieved by inclusion of two or more parameters in regression equation. The results showed that in vitro gas production technique has the potential to predict effective dry matter degradability and energy protein synchronization of roughage based diets.

کلیدواژه‌ها

Correlation؛ degradability؛ in situ degradation؛ in vitro fermentation؛ synchronization

اصل مقاله

 INTRODUCTION

The ruminal degradation of feed is very crucial to assess its nutritional status for ruminants. Degradability of various feeds can be determined by in vivo and in situ (nylon bag) methods. The rate and extent of nutrient degradation in the rumen can influence the synchronization of ruminal available energy and protein which is one of the conceptual methods to increase the efficiency of utilization of nutrients by ruminants and it can also be determined by in situ techniques. Feeding energy and protein synchronized feeds to ruminants has the potential to increase the efficiency of ruminal microbial protein synthesis, nitrogen usage, rumen fermentation, animal performance and to decrease urinary nitrogen excretion (Sinclair et al. 1993; Cole and Todd, 2008). The in situ technique has been used for many years to estimate the rate and the extent of nutrient degradation and synchronization (Mehrez and Ørskov, 1977), however it is laborious, time consuming and expensive (Cone et al. 2002). Otherwise, the in vitro gas production technique was developed to estimate the rate and extent of nutrient fermentation and to evaluate the nutritive values of forages (Menke et al. 1979; Menke and Steingass, 1988), agro industrial by-products (Krishna and Gunther, 1987), various types of tropical feeds (Krishnamoorthy et al. 1995), compound feeds (Aiple et al. 1996), and tree and shrub legume forages (Makkar et al. 1999). Moreover, this method is less animal dependent, more appropriate for characterizing soluble or small particle feeds in comparison with in situ technique (Adesogan, 2002). These two techniques, in vitro and in situ, are well correlated with animal performance (Ørskov, 1989), feed intake (Blümmel and Ørskov, 1993), microbial protein synthesis (Krishnamoorthy et al. 1991) and in vivo digestibility (Khazaal et al. 1993). Considering the advantages of gas production technique with its simplicity of use and the possibility of processing a large number of samples in a short time, it is important to find and valid correlations between in situ dry matter degradability and in vitro fermentation parameters (Valentin et al. 1999). Moreover, the relationships between in vitro fermentation parameters and in situ degradation parameters have been investigated by many researchers (Cone et al. 2002; Kamalak et al. 2005; Ozkan and Sahin, 2006); however most of researches are emphasized on feedstuffs and results somewhat inconsistent. Moreover the information concerning the relationships between in vitro fermentation and in situ degradation parameters of roughage based diets are also limited. Therefore, this study was aimed to investigate the correlation between in vitro fermentation and in situ degradation parameters and to predict dry matter degradability and energy-protein synchronization of roughage based diets.

 

MATERIALS AND METHODS

Feed and chemical analysis

The feedstuffs used in this study were rice straw, sorghum stover, grass, cotton seed cake, chickpea meal and broken rice which are commonly used as feed for dairy cows in Myanmar. The several inclusion rates of those feedstuffs were used in diet formulation to obtain different levels of roughage and concentrate in diets. The experimental roughage based diets are roughage 50% diet (R₅₀D), roughage 60% diet (R₆₀D), roughage 65% diet (R₆₅D) and roughage 70% diet (R₇₀D). As the chemical analysis, dry matter (DM), organic matter (OM), ether extract (EE) were analyzed by the method described by AOAC (1990). Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were analyzed according to the method of Goering and van Soest (1970). All feeds were analyzed for nitrogen (N) by using Kjeldahl method (Fross 2020 digester and Foss 2100 Kjeltec distillation unit) and crude protein (CP) was calculated as 6.25 × N. The chemical compositions of experimental diets were ranged from 41.69% to 58.94% DM, 88.12% to 90.98% OM, 11.46% to 17.96% CP, 59.07% to 64.24% NDF, 34.74% to 47.98% ADF and 1.78% to 2.06% EE.

 

In vitro fermentation and measurements

Rumen fluid was obtained from a fistulated bull (320 kg BW) fed twice daily with a diet containing rice straw (60%) and concentrate (40%). The in vitro gas production method was done according to the procedures of Menke and Steingass (1988). Incubation was carried out at 39 ˚C and gas production was read at 1, 12, 24, 48 and 72 h. The rate and extent of gas production was determined by exponential model of Ørskov and McDonald (1979):

Y= a + b (1-e^-ct)

Where:

a= gas production (mL) from rapidly fermentable fraction.

b= gas production (mL) from slowly fermentable fraction.

c= constant rate of fermentation for the slowly fermentable fraction.

a + b= potential gas production (mL).

t= incubation time (h).

Y= gas production at time t.

After 24 h incubation, metabolizable energy (ME), organic matter digestibility (OMD) and short chain fatty acid (SCFA) of experimental diets were calculated as below:

ME (MJ/kg DM)= 2.20 + 0.136 Gp + 0.057 CP (Menke and Steingass, 1988).

OMD (%)= 14.88 + 0.889 Gp + 0.458 CP + 0.0651 XA (Menke and Steingass, 1988).

SCFA (mmol)= 0.0222 Gp – 0.00425 (Makkar, 2005).

Where:

CP: % crude protein in DM.

Gp: gas production from 200mg sample at 24 h.

XA: % ash in DM.

For the analysis of partitioning factor (PF) for microbial protein synthesis, another incubation was conducted with sample weights increased from 200 mg to 500 mg so as to increase the mass of residue to minimize the analytical error. The double strength medium (40 mL) was incubated with 500 mg air-dry substrate. After 24 h of incubation, the contents were centrifuged (20000 g, 30 min, 4 ˚C) and the supernatant discarded. The pellet was washed with distilled water followed by centrifugation at least 3 times. The undigested substrate and microbial mass were dried in the oven at 135 ˚C for 2 h. The residue was termed as apparent undigested residue. The residue was refluxed with neutral detergent solution (70 mL) for 1 h to determine NDF and NDF ash. From them the partitioning factor for microbial protein synthesis efficiency was calculated according to Makkar and Becker (1996) and it was described as mg of true digestible organic matter per ml of gas (true digestible OM (mg)/mL of gas).

 

In situ degradation and measurements

The in situ nutrient degradation was carried out according to the procedure described by Mehrez and Ørskov (1977). About 5 g of ground samples milled with 2 mm sieve were weighed into the nylon bags with 50 μ pore size and incubated in the rumen of fistulated bull for 1, 3, 6, 12, 24, 48 and 72 h. The animal and diet condition used in this experiment were same with the in vitro experiment. After incubation period, the nylon bags were thoroughly washed with cold, running water until it ran clear and dried at 60 ˚C for 48 h. The nutrient losses for each incubation time were determined. The degradation kinetics of nutrients was fitted to the exponential equation of Ørskov and McDonald (1979):

Y= a + b (1-e^–ct)

Where:

Y: disappearance of nutrient in rumen at time t.

a: rapidly degradable fraction.

b: slowly degradable fraction.

c: constant rate of degradation for slowly degradable fraction.

The effective degradability (Edg) of nutrients was calculated applying the equation of Ørskov and McDonald (1979):

Edg= a + (bc/(c+k))

Where:

k: rumen outflow rate of 2% per h which is at the maintenance level.

The synchronization of energy and protein was calculated from the organic matter and crude protein degradability and it was described as rumen degradable nitrogen (g) per kg of organic matter digested in the rumen (RDN (g)/OMDR (kg)).

 

Statistical analysis

Data on in vitro fermentation and in situ degradation were subjected to the standard analysis of variance using the general linear model (GLM) of SPSS (2007) for windows (version 16.0, Chicago, SPSS Inc.). Significance between individual means was identified using Duncan’s multiple range tests (Steel and Torrie, 1980). Mean differences were considered at Pin vitro fermentation and in situ degradation parameters were obtained by simple linear regression.

 

RESULTS AND DISCUSSION

The gas volumes, except at 72 h incubation time, were different (P<0.05) among the experimental diets and the highest (P<0.05) gas volume was observed in R₆₅D and R₅₀D in most of incubation times (Table 1). The DM and OM disappearances of all diets were also different (P<0.05) in every incubation times (Table 1), in which the highest value (P<0.05) was found in R₅₀D and the lowest was observed in R₆₀D and R₆₅D. The CP disappearances in early incubation time were higher (P<0.05) in R₆₅D, however R₅₀D had the greater values (P<0.05) in later incubation times. The gas production from different carbohydrate fractions and some estimated parameters were different (P<0.05) among the experimental diets (Table 2). The lowest gas production (P<0.05) from quickly soluble fraction (a_gas), the highest gas production (P<0.05) from insoluble fraction (b_gas) and potential gas production ((a+b)_gas) were observed in R₅₀D. The constant rate of fermentation (c_gas) was lowest in R₆₀D. Some estimated parameters such as ME, OMD and SCFA of R₇₀D were lesser (P<0.05) than those of other diets. Although there were no differences (P>0.05) for PF among all diets, the lowest value was found in R₅₀D. The in situ dry matter degradation kinetics and energy protein synchronization are shown in Table 3, in which the highest values (P<0.05) of rapidly degradable fraction (a_is) and constant rate of degradation (c_is) was observed in R₅₀D and the lowest was found in R₆₀D. However, the slowly degradable fraction (b_is) were not different (P>0.05) among the experimental diets. Moreover, R₅₀D also possessed the highest (P<0.05) effective dry matter degradability and the lowest (P<0.05) energy protein synchronization value. The relationships between in vitro gas volume and in situ nutrient disappearances (DM, OM and CP) of experimental diets are shown in Table 4. Correlations (P<0.05) were observed in all regression equations which compared DM, OM, CP disappearance with in vitro gas volume. The in situ effective dry matter degradability was correlated (P<0.05) with in vitro fermentation kinetics and some estimated parameters except a_gas, c_gas, ME and OMD (Table 5).

 

Table 1 In vitro gas volumes and in situ nutrient disappearances of experimental diets

 

R₅₀D: roughage 50% diet; R₆₀D: roughage 60% diet; R₆₅D: roughage 65% diet; R₇₀D: roughage 70% diet; h: hour; DM: dry matter; OM: organic matter and CP: crude protein.

SEM: standard error of the means.

* (P<0.05); ** (P<0.01) and *** (P<0.001).

NS: non significance.

 

Table 2 In vitro fermentation kinetics and some estimated parameters of experimental diets

 

R₅₀D: roughage 50% diet; R₆₀D: roughage 60% diet; R₆₅D: roughage 65% diet; R₇₀D: roughage 70% diet; a_gas: gas production (mL) from quickly soluble fraction; b_gas: gas production (mL) from insoluble fraction; c_gas: gas production rate; (a+b)_gas: potential gas production; ME: metabolizable energy; OMD: organic matter digestibility; SCFA: short chain fatty acid and PF: partitioning factor for microbial protein synthesis efficiency (true digestible OM (mg)/mL of gas).

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

* (P<0.05); ** (P<0.01) and *** (P<0.001).

NS: non significance.

 

Among the correlations, the greater percentages of coefficient of determination (R²) were observed in regression equations that included two or more in vitro fermentation parameters.

 

Table 3 In situ dry matter degradation kinetics of experimental diets

 

R₅₀D: roughage 50% diet; R₆₀D: roughage 60% diet; R₆₅D: roughage 65% diet; R₇₀D: roughage 70% diet; a_is: rapidly degradable fraction of dry matter; b_is: slowly degradable fraction of dry matter; c_is: rate of dry matter degradation; EDMdg: effective dry matter degradability and EPSync: energy protein synchronization.

The means within the same row with at least one common letter, do not have significant difference (P>0.05).

SEM: standard error of the means.

* (P<0.05); ** (P<0.01) and *** (P<0.001).

NS: non significance.

 

Table 4 Relationships between in vitro gas production and in situ nutrient disappearances

 

¹ R²: coefficient of determination and RSD: residual standard deviation.

DM_disT: dry matter disappearance at time “t”; OM_disT: organic matter disappearance at time “t”; CP_disT: crude protein disappearance at time “t” and GvT: gas volume at time “t”.

*** (P<0.001).

 

The synchronization of energy and protein was also correlated (P<0.05) with most of in vitro fermentation parameters (Table 6), however some parameter such as a_gas, c_gas, ME and OMD were not correlated (P>0.05) with synchronization. Correlations (P<0.001) with greater value of coefficient of determination (R²) were found in some relationships assembled by two or more in vitro fermentation parameters. The fermentation kinetics of feeds could be affected by carbohydrates fraction (Deaville and Givens, 2001) and the gas production of feed in buffered rumen fluid was also associated with feed fermentation and carbohydrate fraction (Sallam et al. 2008). The R₅₀D possessed the lesser gas volume form quickly soluble fraction (a_gas), greater gas volume from insoluble fraction (b_gas) and fermentation rate (c_gas), thereby increasing gas production in later incubation times and potential gas production. Conversely, the lesser values were observed in R₆₀D. The gas production from different class of feeds incubated in in vitro buffered rumen fluid was closely related to the production of SCFA which was based on carbohydrate fermentation (Blümmel et al. 1990). Therefore, R₅₀D had the greater value of SCFA, an indication of energy availability to the animal. The in vitro gas production method has been widely used to evaluate the energy value of several classes of feeds (Getachew et al. 1999; Getachew et al. 2002). The ME and OMD values of R₅₀D and R₆₀D were greater than those of R₆₅D and R₇₀D. It might be due to the different level of starch, the major carbohydrate of feeds, which were fermented by amylolytic bacteria and protozoa (Kotarski et al. 1992). The PF values were not different; however they were agreed with the theoretical range for PF (from 2.74 to 4.41). The difference in in vitro PF values reflected to in vivo microbial protein synthesis, dry matter intake and methane gas production in ruminants (Blümmel et al. 1999). Many researchers reported that the degradability of feedstuffs was influenced by the type of carbohydrate (soluble and insoluble carbohydrate). The greater dry matter effective degradability could be achieved by the feedstuffs or feed contained lesser NDF content and greater degradable fraction (Maghsoud et al. 2008). The greater values of rapidly degradable fraction (a_is) and constant rate of degradation (c_is) were observed in R₅₀D, thereby increasing dry matter disappearance and effective degradability. The synchronization is well associated with the types of carbohydrate (soluble and insoluble) which are degraded in the rumen and amount of proteins (ruminal degradable and undegradable protein) entering the rumen. The optimum synchronization achieved by energy and ruminal nitrogen supplementation has a positive effect on microbial protein synthesis (Lardy et al. 2004; Elseed, 2005). In R₅₀D, greater in energy supply (rapidly degradable fraction, ME and SCFA) and lesser ruminal nitrogen level (PF, ruminal degradable protein-data not shown) were observed, which resulted the lesser synchronization. The regression equations showing the relationship between in vitro gas volume and in situ disappearance of DM, OM and CP are given in Table 5, in which significant correlations were observed among those parameters.

 

Table 5 Estimation of dry matter degradability from in vitro fermentation parameters

 

¹ R²: coefficient of determination and RSD: residual standard deviation.

EDMdg: effective dry matter degradability; a_gas: gas production (mL) from quickly soluble fraction; b_gas: gas production (mL) from insoluble fraction; c_gas: gas production rate; (a+b)_gas: potential gas production; ME: metabolizable energy; OMD: organic matter digestibility; SCFA: short chain fatty acid and PF: partitioning factor for microbial protein synthesis efficiency (true digestible OM (mg)/Ml of gas).

* (P<0.05); ** (P<0.01) and *** (P<0.001).

NS: non significance.

 

Table 6 Estimation of energy protein synchronization from in vitro fermentation parameters

 

¹ R²: coefficient of determination and RSD: residual standard deviation.

EPSync: energy protein synchronization; a_gas: gas production (mL) from quickly soluble fraction; b_gas: gas production (mL) from insoluble fraction; c_gas: gas production rate; (a+b)_gas: potential gas production; ME: metabolizable energy; OMD: organic matter digestibility; SCFA: short chain fatty acid; PF: partitioning factor for microbial protein synthesis efficiency (true digestible OM (mg)/mL of gas).

* (P<0.05); ** (P<0.01) and *** (P<0.001).

NS: non significance.

 

This finding is consistent with the reports of researchers (Blümmel and Ørskov, 1993; Khazaal et al. 1993; Kamalak et al. 2005; Ozkan and Sahin, 2006; Sileshi et al. 1996) who observed the significant correlations between DM disappearance and in vitro gas production. In situ effective dry matter degradability (EDMdg) of roughage based diets was highly related to the gas production (b_gas) from insoluble fraction and potential gas production, (a+b)_gas. These finding were in agreement with the reports of scientists (Blümmel and Ørskov, 1993; Khazaal et al. 1993; Sileshi et al. 1996) who found a significant correlation between effective dry matter degradability and potential gas production. The inclusion of other parameters such as a_gas and c_gas to regression equation increased the percentage of variation of EDMdg expressed by gas production parameters. This result was also consistent in the reports (Kamalak et al. 2005; Ozkan and Sahin, 2006) which described that the greater correlation could be achieved with regression equations included a_gas, b_gas, c_gas and (a+b)_gas. However, there were no explanation about the relationship between EDMdg and other estimated parameters such as SCFA, ME, OMD and PF. In this study, SCFA and PF were significantly correlated with EDMdg, however no significance was found with ME and OMD. The SCFA and PF explained 84.4%, 86.2% and 92.7% of variation of EDMdg when it was combined with OMD, ME and (ME+OMD), respectively. Therefore, it was noted that the more inclusion of parameters in regression equations, the greater accuracy and variation of EDMdg could be achieved. For the synchronization of energy and protein, it was significantly correlated with the gas production (b_gas) from insoluble fraction and potential gas production, (a+b)_gas, but there were no significant relation to gas production (a_gas) from quickly soluble fraction and fermentation rate (c_gas). When two or more parameters were used in regression equations, the variation of synchronization was significantly increased from 58.5% to 75.5%. Moreover, the significant correlations were found in relationship with SCFA and PF, but not with ME and OMD alone. The SCFA and PF explained from 95.6% to 97.8% of variation of synchronization when ME, OMD and (ME+OMD) were added to the regression equation. It might be due to proper combination of parameters which are the main sources for energy and protein.

 

CONCLUSION

The correlations between the results of both in situ and in vitro techniques seem to be sufficiently strong to predict in situ disappearances of DM, OM and CP from cumulative gas volumes. Although effective dry matter degradability and energy protein synchronization were significantly correlated with in vitro fermentation kinetics (b_gas and (a+b)_gas) and some estimated parameters (SCFA and PF), the greater accuracy and variation of those parameters could be achieved by inclusion of more parameters in regression equation. Moreover, estimated parameters such as SCFA, ME, OMD and PF can give the greater percentage of correlation in compare with fermentation kinetics. It was concluded that in vitro gas production technique has the potential to predict the effective dry matter degradability and energy protein synchronization of roughage based diets.

 

ACKNOWLEDGEMENT

A special thank is extended to all laboratory staff member of Department of Physiology and Biochemistry for their assistance during this research work.

مراجع

Adesogan A.T. (2002). What are feeds worth? A critical evaluation of selected nutritive value methods. Pp. 33-47 in Proc. 13^th Ann. Florida Rumin. Nutr. Symp., Gainesville, Florida.

Aiple K.P., Steingass H. and Drochner W. (1996). Prediction of the net energy content of raw materials and compound feeds for ruminants by different laboratory methods. Arch. Anim. Nutri. 49, 213-220.

AOAC. (1990). Official Methods of Analysis. Vol. I. 15^th Ed. Association of Official Analytical Chemists, Arlington, VA, USA.

Blümmel M. and Ørskov E.R. (1993). Comparison of in vitro gas production and nylon bag degradability of roughages in predicting of food intake in cattle. Anim. Feed Sci. Technol. 40, 109-119.

Blümmel M., Mgomezulu R., Chen X.B., Makkar H.P.S., Becker K. and Ørskov E.R. (1999). The modification of in vitro gas production test to detect roughage related differences in in vivo microbial protein synthesis as estimated by the excretion of purine derivate. J. Agric. Sci. 133, 335-340.

Blümmel M., Ørskov E.R., Becker K. and Soller H. (1990). Application of the Hohenheim gas test for evaluating the kinetics of rumen fermentation. J. Anim. Physiol. Anim. Nutr. 64, 56-57.

Cole N.A. and Todd R.W. (2008). Opportunities to enhance performance and efficiency through nutrient synchrony in concentrate-fed ruminants. J. Anim. Sci. 86, 318-333.

Cone J.W., Van Gelder A.H., Bachmann H. and Hindle V. (2002). Comparison of organic matter degradation in several feedstuffs in the rumen as determined with the nylon bag and gas production techniques. Anim. Feed Sci. Technol. 96, 55-67.

Deaville E.R. and Givens D.I. (2001). Use of the automated gas production technique to determine the fermentation kinetics of carbohydrate fractions in maize silage. Anim. Feed Sci. Technol. 93, 205-215.

Elseed F.A.M.A. (2005). Effect of supplemental protein feeding frequency on ruminal characteristics and microbial N production in sheep fed treated rice straw. Small Rumin. Res. 57, 11-17.

Getachew G., Grovetto G.M., Fondivilla M., Krishnamoorthy B., Singh Sphaghero H., Steingass P.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. Anim. Feed Sci. Technol. 102, 169-180.

Getachew G., Makkar H.P.S. and Becker K. (1999). Stoichiometric relationship between short chain fatty acids and in vitro gas production in presence and polythyleneglycol for tannin containing browses. Pp. 55-61 in Proc. EAAP Satellite Symp. Gas Prod., Wageningen, The Netherlands.

Goering K.H. and van Soest P.J. (1970). Forage Fibre Analysis. Agricultural Hand Book, Washington, D.C., USA.

Kamalak A., Canbolat O., Gurbuz Y. and Ozay O. (2005). Comparison of in vitro gas production technique with in situ nylon bag technique to estimate dry matter degradation. Czech J. Anim. Sci. 50(2), 60-67.

Khazaal K., Markantonatos X., Nastis A. and Ørskov E.R. (1993). Changes with maturity in fibre composition and levels of extractable polyphenols in Greek browse: effect in vitro gas production and in sacco dry matter degradation. J. Sci. Food Agric. 63, 237-244.

Kotarski S.F., Waniska R.D. and Thurn K.K. (1992). Starch hydrolysis by the ruminant micro flora. J. Nutr. 122, 178-190.

Krishna G. and Gunther K.D. (1987). The usability Hohenheim gas test for evaluation in vitro organic matter digestibility and protein degradability at rumen level of some agro industrial by-products and wastes used as livestock feeds. Landwirtsch. Forx. 40, 281-286.

Krishnamoorthy H., Steingass H. and Menke K.H. (1991). Preliminary observations on the relationships between gas production and microbial protein synthesis in vitro. Arch. Tierernahr. 41, 521-526.

Krishnamoorthy U., Soller H., Steingass H. and Menke K. (1995). Energy and protein evaluation of tropical feedstuffs for whole tract and ruminal digestion by chemical analysis and rumen inoculums studies in vitro. Anim. Feed Sci. Technol. 52, 177-188.

Lardy G.P., Ulmer D.N., Anderson V.L. and Caton J.S. (2004). Effect of increasing level of supplemental barley on forage intake, digestibility, and ruminal fermentation in steers fed medium quality grass hay. J. Anim. Sci. 82, 3662-3668.

Maghsoud B., Akbar T., Hossein J. and Ali M.G. (2008). Evaluation of some by-products using in situ and in vitro gas production techniques. American J. Anim. Vet. Sci. 3(1), 7-12.

Makkar H.P.S. (2005). In vitro gas methods for evaluation of feeds containing phytochemicals. Anim. Feed Sci. Technol. 123, 291-302.

Makkar H.P.S. and Becker K. (1996). Nutritional value and antinutritional components of whole and ethanol extracted Moringa oleifera leaves. Anim. Feed Sci. Technol. 63, 211-228.

Makkar H.P.S., Aregheore E.M. and Becker K. (1999). Effect of saponins and plant extracts containing saponins on the recovery of ammonia during urea-ammoniation of wheat straw and fermentation kinetics of the treated straw. J. Agric. Sci. 132, 313-321.

Mehrez A.Z. and Ørskov E.R. (1977). A study of artificial fibre bag technique for determining the digestibility of feeds in the rumen. J. Agric. Sci. 88, 645-650.

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. Anim. Res. Dev. 28, 7-55.

Menke K.H., Raab L., Salewski A., Steingass H., Fritz D. and Schneider W. (1979). The estimation of the digestibility and metabolisable energy content of ruminant feeding stuffs from the gas production when they are incubated with rumen liquor. J. Agric. Sci. 93, 217-222.

Ørskov E.R. (1989). Recent advances in evaluation of roughages as feeds for ruminants. Pp. 102-108 in Advances in Animal Nutrition. D.J. Farell, Ed. University of New England Printery, Armidale, Australia.

Ørskov E.R. and McDonald P. (1979). The estimation of protein degradability in the rumen from incubation measurements weighed according to rate of passage. J. Agric. Sci. 92, 499-503.

Ozkan C.O. and Sahin M. (2006). Comparison of in situ dry matter degradation with in vitro gas production of Oak leaves supplemented with or without polyethylene glycol (PEG). Asian-Australasian J. Anim. Sci. 19(8), 1120-1126.

Sallam S.M.A., Bueno I.C.S., Godoy P.B., Nozella E.F., Vitti D.M. and Al-Abdalla S.S. (2008). Nutritive value in the value assessment of the artichoke (Cynara scolyms) by products as an alternative feed resource for ruminant. Trop. Subtrop. Agroeco. 8, 181-189.

Sileshi Z., Owen E., Dhanona M.S. and Theodorou M.K. (1996). Prediction of in situ rumen dry matter disappearance of Ethiopian forages from an in vitro gas production technique using a pressure transducer, chemical analyses or in vitro digestibility. Anim. Feed Sci. Technol. 61, 73-77.

Sinclair L.A., Garnsworth P.C., Newbold J.R. and Buttery P.J. (1993). Effect of synchronizing the rate of dietary energy and nitrogen release on rumen fermentation and microbial protein synthesis in sheep. J. Agric. Sci. 120, 251-263.

SPSS Inc. (2007). Statistical Package for Social Sciences Study. SPSS for Windows, Version 16. Chicago SPSS Inc., USA.

Steel R.G. and Torrie J.H. (1980). Principles and Procedures of Statistics. McGraw-Hill Book Company, New York.

Valentin S.F., Williams P.E.V., Forbes J.M. and Sauvant D. (1999). Comparison of the in vitro gas production technique and the nylon bag degradability technique to measure short and long term processes of degradation of maize silage in dairy cows. Anim. Feed Sci. Technol. 78, 81-99.