Livestock Research for Rural Development 22 (11) 2010 Notes to Authors LRRD Newsletter

Citation of this paper

Evaluation of feed mixture interactions by using in vitro gas production method

R Arhab*,**, K Laadjimi*, D Driss*, B Djabri* and H Bousseboua**

* Faculté des Sciences Exactes et des Sciences de la Nature et de la Vie, Département de Biologie, Université de Tébessa, route de Constantine 12002, Tébessa, Algérie
** Laboratoire de Génie Microbiologique et applications, Faculté des Sciences de la Nature et de la Vie, Université Mentouri, Constantine, Algérie


The aim of the present study was to evaluate interactions occurring between microbial degradation of three substrates: Triticosecale wittmack (triticale) as nitrogen source, Hordeum vulgare (barely) as carbon source and commercial concentrate as supplement. Associative effects of feed mixtures were evaluated in vitro by using batch systems (in vitro gas production technique). The model included the principle effects of single feeds and multiples interaction of feed mixtures. The mixtures were prepared on the basis of 3x3 Latin square design and included three percentages 0, 25 and 50%. Cumulative gas production (CGP) was recorded at 2, 4, 6, 8, 24, 48 and 72 hours of incubation. The qualitative analysis of gas produced (carbon dioxide (CO2) and methane (CH4)) was recorded after 24 h of incubation and measured according to procedure described by Jouany (1994). Cumulative gas production profiles were fitted to the exponential model Y = a + b (1 – e-ct). At the end of incubation, pH values were noted and in vitro dry matter digestibility (IVDMD) was determinate.


Total gas production recorded after 72 hours of incubation were significantly similar among mixtures (P > 0.05). However, the cumulative gas production at the earlier hours of fermentation (2-8 hours) was significantly different (P < 0.05). The higher gas production was noted for the mixture containing Hordeum (50%) and the lowest were noted for Triticosecale and commercial concentrate at the same level. The carbon dioxide and methane productions were significantly comparable between all mixtures (P > 0.05). The same pattern was observed for IVDMD. The values recorded were 76.7, 77.7 and 76.6% for M1 (25% Triticosecale wittmack  + 25% Hordeum vulgare+ 50% concentrate), M2 (25% Triticosecale wittmack  + 50% Hordeum vulgare+ 25% concentrate) and M3 (50% Triticosecale wittmack  + 25% Hordeum vulgare+ 25% concentrate) respectively. Rate of gas production, deduced from the exponential model, was significantly different among mixtures (P < 0.05). Mixture containeing Hordeum (M2) was faster degraded by ruminal microbiota than Triticosecale mixture (M1). Positive and negative associative effects were found for all mixtures. It was concluded that nitrogen and carbon sources for all mixtures fermented by ruminal microbiota were synergic. However, the higher ruminal rate degradation of Hordeum limit its incorporation at high level in ruminants ration.

Key words: concentrate, Hordeum vulgare, ruminal microbiota, interactions, in vitro fermentation, Triticosecale wittmack


Animal performance is the most direct measure in the evaluation of feed quality. However, performance data are insufficient to determine and explain possible interactions that may take place in the ruminal environment. Feeds are commonly evaluated as single entities; despite the fact an animal is always fed a mixture of ingredients (Sandoval et al 2002; Robinson et al 2009). The classic methods for evaluating feed mixtures were in vivo techniques. However, these methods are time consuming, laborious, expensive and require large quantities of feed. Now, there are substituted by in vitro gas methods (Makkar 2005) which appear to be more suitable for use in developing countries. In this context, this study was assessed to evaluate interactions between microbial degradation of three substrates: Triticosecale wittmack, Hordeum vulgare and commercial concentrate, and to investigate associative effects of feed mixtures incubated using in vitro gas production technique.


Material and methods 



Three substrates were tested: Triticosecale wittmack (triticale), Hordeum vulgare (barley) and commercial concentrate (soybean, wheat, corn, barley and minerals). These substrates were sun dried, ground to pass 1mm sieve and stored in plastic flasks at room temperature for further analyses. The chemical composition of triticale and barley were illustrated in Table 1. The mixtures were prepared on the basis 3x3 Latin square at three levels 0, 25 and 50%.    

Table 1.  Chemical composition (g/100 g DM) of the single substrates.


Dry matter

Crude protein

Crude fiber


Soluble sugars


Ether extract

Triticosecale wittmack








Hordeum vulgare








Gas production measurements


Inoculum preparation


Rumen fluid was obtained from two healthy sheep fed with vetch-oat hay as basal diet. It was strained trough 4 layers of cheesecloth and stored in thermos containers saturated with carbon dioxide (CO2) and maintained at 39°C.


The inoculum was prepared following the procedures of Menke et al (1979). It consisted of the rumen liquor mixed with anaerobic artificial saliva (1: 2 v/v). The latter was prepared as described by Menke and Steingass (1988).


Gas production measurements


The syringes, prewarmed at 39°C and contained two hundred grams of single substrates or mixtures, were inoculated with 30 ml inoculum under continuous CO2 reflux. They were incubated in an incubator at 39°C for 72 hours. The gas production of each syringe was recorded after 2, 4, 6, 8, 24, 48 and 72 hours of fermentation. To prevent the gas volume in the syringes from exceeding 60 ml, the pistons were moved back to the 30 ml piston after 12 hours of fermentation. Each substrate was incubated in triplicate in three different runs in order to generate 9 measurements per substrate sample. Each run included in triplicate, a blank (syringes incubated with the inoculum alone).


The carbon dioxide and methane productions were evaluated after 24h of incubation by injection in each syringe 4 ml of sodium hydroxide (NaOH, 10N) (Jouany 1994).


 In vitro dry matter digestibility determination


At the end of incubation, the content of each syringe were centrifuged at 12000 rpm for 20mn and the residues were dried at 80°C until constant weight. The IVDMD was assessed by difference between initial and final amounts of samples (correcting for blanks).


Gas production data calculation and statistical analysis


Mean gas production data of blanks were subtracted from the recorded gas production of single substrates and mixtures to get net gas production values. These calculated CGP were fitted with the monomolecular model (Ørskov and McDonald 1979):



  where "a" is the gas production from the readily fermented fraction, "b" the gas production from the slowly fermented fraction and "c" the rate of fermentation.


The metabolisable energy and organic matter digestibility of samples can be estimated by 24 hour gas production and chemical composition.


The data for single substrates and mixtures (pH, CGP, CO2, CH4 and IVDMD) were analyzed by ANOVA employing the SAS software (1990). The means were compared by Scott-Knott’s test at the level of 5%, using the entirely random design.


The associative effects observed from the mixtures were analysed by model based on differences noted between experimental data and predicted data. From fitted parameters a prediction of CGP was made for all mixtures. The prediction was done solving from exponential model for each observed time using the fitted parameters obtained to the inclusion level of each component of the mixtures, was taken and use as data for fitting the new profile (predicted). Parameters obtained from fitting predicted valued from a mixture were compared with their correspondent original profiles (experimental data from the mixtures) using 95% confidence interval (CI) as indicator of departure from linearity.


Results and discussion  

In vitro gas production parameters and exponential model characteristics


The mean values of pH, CGP and exponential model characteristics were illustrated in Table 2.

Table 2.  In vitro fermentation parameters and exponential characteristics of single substrates and mixtures



CGP, ml

A, ml

B, ml

C, %/h


Single substrates







Triticosecale wittmack



- 8,60b




Hordeum vulgare



- 7,50a





























- 4,47







- 4,66







- 5,28


















CGP: cumulative gas production; a: gas production from the readily fermented fraction; b: gas production from the slowly fermented fraction; c: rate of fermentation; IVDMD: in vitro dry matter digestibility; M1: 25% Triticosecale wittmack  + 25% Hordeum vulgare + 50% concentrate; M2: 25% Triticosecale wittmack  + 50% Hordeum vulgare + 25% concentrate; M3: 50% Triticosecale wittmack  + 25% Hordeum vulgare + 25% concentrate; Pr: probability; S.E.M.: standard error of means; a, b, cmeans in the same column with different superscripts are significantly different.

The pH values for single substrates and mixtures were significantly similar (P > 0.05). After 72 hours of fermentation, the CPG for the single substrates was significantly different (P < 0,001). The highest value was observed for Triticosecale wittmack  (60.67 ± 2.24ml) and the lowest for concentrate (50.06 ± 2.57ml). This difference was possibly related to their chemical composition. Effectively, Triticosecale wittmack  and Hordeum vulgare were richer in starch and soluble sugars than concentrate. However, there were no significant differences in CGP between all mixtures (P > 0.05).


The exponential model parameters for single substrates were significantly different (P < 0,001) (Table 2). The higher potential gas production (a+b) was observed for Triticosecale wittmack   and  Hordeum vulgare (59,49 ± 2,27 and 58,11 ± 2,77 ml, respectively) and the lowest was recorded for concentrate (49,42 ± 2,69ml). Hordeum vulgare and Triticosecale wittmack  were faster degraded by ruminale microbiota (0.203 and 0,185 ml/h) than concentrate (0.1187 ml/h). For mixtures, all parameters, except rate gas production, were statistically comparable (P > 0.05). Similarly, the rate degradation of mixture which contains 50% of Hordeum vulgare was higher than mixture which prepared with 50% Triticosecale wittmack  (P < 0.05).


The negative values of soluble fraction (a) have also been reported by other authors working under the same conditions (Sandoval et al 2002). They are associated to latency phase and they could be explained by the necessary time to ruminal microbiota to degrade soluble fraction and then to adhere to cellulosic fraction of the substrate.


In vitro dry matter digestibility (IVDMD)


IVDMD was not affected by the increased proportion of single substrates in the total mixtures (P > 0.05) (table 2). These values were comprised between 76.9 and 77.7%. The same pattern was also noted for single substrates among which the digestibility coefficient varied between 72.9 and 79.9%. These results agree with those mentioned in vitro by Umucalilar et al (2002).


Gas production profile


Gas production kinetics follows an ascending pattern for the different mixtures (Figures 1 and 2).  

Figure 1.  Experimental gas production kinetics and exponential model profile of single substrates

Figure 2.  Experimental gas production kinetics and exponential model profile of  mixtures

The fermentation is relatively intensive during the first 24 hours of incubation, after which it reaches a stationary phase. The kinetics of gas production appears to be determined by two distinct phases; the first one corresponds to the degradation of the soluble fraction of the tested mixtures and the second to the insoluble but potentially fermentable fraction. The same profile in the gas production kinetics between the 3 mixtures is probably due to their chemical composition. This last indicates that Triticosecale wittmack  and Hordeum vulgare were rich in starch and amino acids. Besides, their cell walls are less lignified because grains of grass have a less levels of lignin in their composition and their starch is rapidly fermentable (Sauvant 1997). This result could also be explained by the fact that Triticosecale wittmack, Hordeum vulgare belong to gramineous family which were less rich in secondary compounds (phenolic substances and tannins) (Arhab et al 2006; Haddi et al 2003).

Qualitative gas production analysis


The quantitative analysis of gas production reveals that the mixtures degradation was similar and the dominant gas released is CO2 beyond 24 hours of incubation (Figure 3).

Figure 3.  Qualitative gas production analysis of single substrates and mixtures

The gas production during fermentation is correlated with both the quantitative and qualitative production of volatile fatty acids (Orskov and Ryle 1990). Numerous authors suggest that the degradation of substrate rich in starch and soluble sugars favors the production of propionic and butyric acids. Otherwise, the fermentation of fibrous substrates produces acetic acid itself, being associated with an important production of H2 which induces an increased production of gas in the form of CH4 (Wolin 1975; Orskov and Ryle 1990). This leads us to deduce that the degradation of Triticosecale wittmack  and Hordeum vulgare, which are rich in starch and soluble sugars might favor the production of propionic and butyric acids.


Associative effects


Associative effects of mixtures of Triticosecale wittmack , Hordeum vulgare and concentrate were shown in Figure 4.

Figure 4.  Associative effects for mixtures at various times of incubation

The synchrony occurred at different times during the incubation period: 2, 6, 8, 48 and 72 hours and was characterized by changes in the fermentation kinetics of mixture. Associative effects were shown to vary with time and with single substrate level included in the mixture. These results were agreed with that observed by Robinson et al (2009). These authors noted that these associative effects varied with time and were more expressed at earlier times of incubations.


The amount of nutrients which a ruminant can extract from one feed can be modified by the type and quantity of other consumed the same day. These interactive processes can have substantial consequences for intake and digestibility of feeds and for animal performance. In general, associative effects between components of a mixed diet occur when, as a consequence of the interactive processes, the nutritional value of the mixture is not equal to the sum of its individual components. These effects can be positive (synergistic) or negative (antagonist) (Rosales  and Gill 1998). In our studies, associative effects, in terms of digestion and gas production, are related to the effect of the using of Triticosecale wittmack, Hordeum vulgare as rapidly fermentable carbohydrates. Mixtures showed positive and negative effects, these effects were possibly due to synchrony or asynchrony in the release of protein contents in mixtures.




Arhab R, Macheboeuf D, Doreau M and Bousseboua H 2006 Nutritive value of date palm leaves and Aristida pungens estimated by chemical, in vitro and in situ methods. Tropical and Subtropical Agrosystems  6: 167-175


Haddi M L, Filacorda S, Meniai K, Rollin F and Susmel P 2003 In vitro fermentation kinetics of some halophyte shrubs sampled at three stages of maturity. Animal Feed Science and Technology 104: 215-225.


Jouany J P 1994 Les fermentations dans le rumen et leur optimisation. INRA Productions Animales 7(3): 207-225.


Makkar H P S 2005 In vitro gas methods for evaluation of feeds containing phytochemicals. Animal Feed Science and Technology 123-124: 291-302.


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 K H, Raab L, Salewski A, Steingass H, Fritz D, Schneider W 1979 The estimation of the digestibility and metabolizable energy content of ruminant feedingstuffs from the gas production when they are incubated with rumen liquor. Journal of Agricultural Science  Cambridge 97: 217-222.


Orskov E R and Mc Donald I 1979 The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. Journal of Agricultural Science Cambridge 92: 499-503.


Orskov E R and Ryle M 1990 Energy nutrition of rumen microorganisms. In: Energy Nutrition in Ruminants. Elsevier Science (Editors), New York, USA, pp. 10-28.


Robinson P H, Getachew G and Cone J W 2009 Evaluation of the extent of associative effects of two groups of four feeds using an in vitro gas production procedure. Animal Feed Science and Technology 150: 9-17.


Rosales M and Gill M 1998 Tree mixtures within integrated farming systems. Livestock Research for Rural Development 9: 415-424.


Sandoval-Castro C A, Captillo-Leal C, Cetina-Gongora R and Ramirez-Aviles L 2002 A mixture simplex design to study associative effects with an in vitro gas production technique. Animal  Feed Science and Technology 101: 191-200.


Sauvant D 1997 Conséquences digestives et zootechniques des variations de la vitesse de digestion de l'amidon chez les ruminants. INRA Production Animale 10: 287-300.


Statistical Analysis System Institute Inc 1990 SAS/STAT® user’s guide Int Volume 1, version 6, Fourth Edition, Cary, NC, USA.


Umicalilar H. D, Coskum B Gülşen N 2002 In situ rumen degradation and in vitro gas production of some selected grains from Turkey. Journal of Animal Physiology and Animal Nutrition 86: 288-297.


Wolin  M.J 1975 Interaction between the bacterial species in the rumen. In: Digestion and Metabolisme in the Ruminant. Mc Donald J W and Warner A C I (Editors), pp. 134-148.

Received 5 January 2010; Accepted 21 October 2010; Published 1 November 2010

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