In vitro Assessment of the Effect of Plant Extracts on Digestibility, Estimated Energy Value, Microbial Mass and Rumen Fermentation Kinetics

Document Type: Research Articles

Authors

1 Department of Animal Science, Faculty of Agriculture, Razi University, Kermanshah, Iran

2 Department of Animal Science, College of Agriculture, University of Kurdistan, Sanandaj, Iran

Abstract

Three ethanol extracts, chamomile (CHA), clove (CLO) and tarragon (TAR), were tested at five doses (0, 250, 500, 750 and 1000 µL/L) to determine their effects on in vitro organic matter digestibility (IVOMD), metabolizable energy (ME), net energy of lactation (NEL), short-chain fatty acids (SCFA), microbial mass (MM) and rumen fermentation kinetics of a 40:60 forage: concentrate diet using in vitro gas production. These three extracts had significant effects on gas production kinetics. CHA (at 500 µL/L dose) and CLO (at 1000 µL/L dose) decreased (P<0.05) potential gas production. The initial gas production rate constants (c) was increased (P<0.05). However, CHA, CLO and TAR ethanol decreased (P<0.05) later gas production rate constants (d). Lag time (h) was decreased (P<0.05) due to addition of CHA and TAR ethanol (at 750 µL/L dose), and CLO (at 500 and 1000 µL/L doses). TAR and CLO ethanol did not affect fermentation rate (h-1), but CHA at 1000 µL/L increased it. The TAR and CLO ethanol did not affect IVOMD, ME, NEL, SCFA and microbial mass. However, organic matter digestibility, ME, NEL, SCFA and microbial mass were increased by addition of CHA ethanol at 750 and 1000 µL/L doses. Results suggest that CHA, CLO and TAR ethanol extracts at appropriate doses may have potential to improve the rumen fermentation kinetics and nutritive value of ruminant diets due to secondary metabolites contents.

Keywords


INTRODUCTION

In the recent years, the use of plant extracts in dairy cattle rations have been considered worldwide by ruminant nutritionists especially after the prohibition of growth promoting antibiotics by the (EC number 1831/2003; European Union, 2003), because plant extracts were believed to be natural, safe and efficient without negative side effects. Secondary metabolites present in the natural plant extracts can modify rumen fermentation kinetics and improve milk production in dairy cattle (Alexander et al. 2007; Benchaar et al. 2008; Hart et al. 2008; Naseri et al. 2012; Naseri et al. 2015). It has also been observed that secondary metabolites suppressed protozoal populations, increased bacterial and fungal populations, propionate production, microbial yield and efficiency of microbial protein synthesis (EMPS), increased dietary dry matter (DM), organic matter (OM) and neutral detergent fibre (NDF) degradation and reduced dietary crude protein (CP) degradation and methanogenesis. A number of fast and cost-effective in vitro gas measurement methods have been used by several groups to evaluate the nutritional value of feedstuffs and kinetics of rumen fermentation (Getachew et al. 1998; Getachew et al. 2004; Makkar, 2005; Mirzaei-Aghsaghali et al. 2011a; Naseri et al. 2015). These methods can provide useful data on fermentation kinetics of feedstuffs, prediction of feed intake (Khazaal et al. 1995; Mirzaei-Aghsaghali et al. 2011b), digestibility, and microbial nitrogen supply, amount of short-chain fatty acids, carbon dioxides and metabolizable energy of feeds for ruminants (Menke and Steingass, 1988; Babayemi, 2007; Mirzaei-Aghsaghali et al. 2008b; Mirzaei-Aghsaghali et al. 2008a; Maheri-Sis et al. 2008; Maheri-Sis et al. 2007). The ease of measuring fermentation end-products makes these methods more preferable (Makkar, 2005). This work aimed to evaluate the in vitro gas production kinetics and estimate the in vitro organic matter digestibility (IVOMD), metabolizable energy (ME), short-chain fatty acids (SCFA), net energy of lactation (NEl) and microbial protein production of high-concentrate diet for dairy cattle after supplementing the feed material with ethanol extract of chamomilla (Matricaria chamomilla), clove (Syzygium aromaticum) and tarragon (Artemisia dracunculus).

 

MATERIALS AND METHODS

Selection of plants

Three medicinal plants: chamomile, clove and tarragon were selected on the basis of their traditional usage for the various digestive ailments, and in the light of recent literature (Patra, 2011).

 

Preparation of plant extract

Chamomile and tarragon leaves used in this study were collected at vegetative stage from Abidar Mountains and clove buds were purchased from local markets in Sanandaj (longitude 46.99 ˚E, latitude 35.32˚N and Köppen-Geiger climate), Iran. Approximately 100 g of fresh chamomile and tarragon leaves were cut into small pieces, placed into a blender (Saya Quick, QMC-20) and added 80 mL 70% ethanol then they were well blended three times for 5 minutes per time. The blended material was squeezed through four layers of muslin cloth into the labeled beaker and fibrous materials discarded. The combined filtrate was filtered using Whatman No.1 filter paper, and then transferred to a round-bottom Buchi flask. Also, the clove buds crushed into small pieces, oven-dried at 39 ˚C and ground to pass a 1mm screen. Fifty of ground sample was weighed into a 250 mL conical flask and added 200 mL 70% ethanol. The extraction was completed by placing the flasks in a shaker at 22 ˚C and 200 rpm for 24 h. Contents of the flask were squeezed through four layers of muslin cloth into the labeled beaker and fibrous materials discarded. The combined liquid phase was filtered using Whatman No.1 filter paper and then transferred to a round-bottom Buchi flask. Finally, ethanol was evaporated by using a vacuum evaporator (Heidolph Laborota 4011 digital) at 40-50 ˚C until the ethanol-streak stopped on the side of the bottle. The remaining concentrate was resuspended in 10 mL water, transferred into 10 mL sterile anaerobic crimped serum vials, and stored at -20 ˚C.

 

Inoculum and substrate

The inoculum was prepared according to the method of Tilley and Terry (1963). Briefly, rumen fluid was obtained from three rumen cannulated rams before the morning feeding. The rumen fluid was mixed on volume basis then it was bubbled with CO2 for approximately 2 min and strained through four layers of cheese cloth. The incubation inoculum was prepared by diluting the fluid inoculum with the buffer (Tilley and Terry, 1963) in a 1:4 (V/V) ratio and stirring in a water bath at 39 ˚C with purging CO2 until its use. The ration of the rams consisted of 40% alfalfa, 35% barley grain, 15% corn grain, 9% soybean meal, 0.5% salt and 0.5% vitamin-mineral premix. The substrate used in the in vitro ruminal fermentation was at 40:60 forage:concentrate ratio, formulated for dairy cattle (Table 1), oven dried (at 39 ˚C for 72 h) and finely ground to pass through a 1 mm screen.

 

In vitro gas production

The method used for gas production measurements was as described by Theodorou et al. (1994). Approximately 250 mg dry matter (DM) of substrate was weighed into 100 mL sterile tubes, kept at 39 ˚C. Plant extracts were added at different volumes (0, 250, 500, 750 and 1000 µL/L). Each sample was incubated in three replicates. Thirty milliliters of incubation inoculum (in the proportion of 20% rumen fluid+80% buffer) prepared (as described in the inoculum and substrate) and by flushing CO2 before was anaerobically dispensed in each tube at 39 ˚C. The samples were swirled to mix the contents and placed in ashaker incubator (Thermoshaker Gerhardt) at 39 ˚C (Blümmel and Ørskov, 1993). The pressure of gas produced in each tube was recorded using a pressure transducer (Testo 512; Testo Inc., Germany) at 0, 2, 4, 8, 16, 24, 48 and 72nd h of incubation. To estimate the kinetics of gas production, data on cumulative gas volume produced were fitted using the generalized Mitscherlich model, proposed by France et al. (1993):

Where:

G (mL): denotes cumulative gas production at time t.

A (mL): asymptotic gas production.

c (h-1): initial gas production rate constant.

d (h-1/2): later gas production rate constant rate constants.

L (h): lag time.

 

Calculation

The half-life (t1/2, h) of the degradable fraction of substrate was calculated as the time taken for gas accumulation to reach 50% of its asymptotic value. The fractional degradation rate at t1/2 (µ1/2, h-1) was calculated as:

The metabolizable energy (MJ/kg DM) content of the substrate and in vitro organic matter digestibility were calculated using the equations below (Menke et al. 1979) as:

ME (MJ/Kg DM)= 2.20 + 0.136 GP + 0.0057 CP + 0.00029 EE2

IVOMD (%)= 14.88 + 0.889 GP + 0.45 CP + 0.0651 XA

Where:

GP: 24 h net gas production (mL/250 mg-1).

CP: crude protein (%).

EE: ether extract (%).

XA: ash content (%).

 

Short-chain fatty acid (SCFA) content was calculated using the equation of Makkar (2005); Maheri-Sis et al. (2007) and Maheri-Sis et al. (2008):

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

Where:

GP: 24 h net gas production (mL/250 mg-1).

Net energy for lactation (NEL) was calculated using the equation of Abas et al. (2005) as follows:

NEL (MJ/kg DM)= 0.115 GP + 0.0054 CP + 0.014 EE -0.0054 CA - 0.36

Microbial mass (mg) was estimated using equation of Blummel et al. (1997):

Microbial mass (mg)= mg substrate truly degraded (OMD) - (GP×stoichiometrical factor)

The stoichiometrical factor was 2.20.

 

Chemical analysis

The substrate was analysed for DM (24 h at 103 ˚C), ash and organic matter (OM) (4 h at 550 ˚C), CP content was adapted for an automatic distiller Kjeldahl apparatus (Kjeltec Auto 1030 Analyser; Tecator, Höganäs, Sweden) and using CuSO4/Se as catalyst instead of CuSO4/TiO2, ether extract using petroleum ether for distillation instead of diethyl ether (AOAC, 1990). The neutral detergent fibre (NDF) contents were determined as described (Van Soest et al. 1991).

 

Statistical analysis

Data were subjected to analysis of variance (ANOVA) using the general linear model (GLM). Significant differences between individual means were identified using Duncan’s test (all pairwise multiple comparison procedures). All statements of significance were based on a probability of (P<0.05) (SAS, 1996).

 

RESULTS AND DISCUSSION

Chemical composition

The chemical composition of diet which used as fermentation substrate is shown in Table 1.

 

Table 1 Chemical composition (g/kg DM) of substrate used for in vitro gas production

 

 

Effect of plant ethanol extracts on in vitro rumen fermentation kinetics

Effect of ethanol extracts of chamomille, clove and tarragon on in vitro fermentation kinetics is presented in Tables 2, 3 and 4, respectively. Potential gas production (A) decreased (by 7%) significantly (P<0.05) by the addition of chamomile and clove extracts at 500 and 1000 (µl/L) doses, respectively. In addition, 500 and 750 µL/L doses of tarragon extract were also found to be effective in decreasing potential gas production (A) by 8% (P=0.07). The main active compounds of chamomile, clove and tarragon extract were terpenoids α-bisabolol and chamazulene, eugenol (phenylpropanoid) and methyleugenol, respectively (Janmejai et al. 2010; Jamalian et al. 2012; Renata and Grażyna, 2014). These active compounds are known as of plant secondary metabolites, which include terpenoids, alkaloids and phenolics present in the essential oil fraction of many plants (Sallam et al. 2011). Essential oils have antimicrobial activities against both gram-negative and gram-positive bacteria, a property that has been attributed to the presence of terpenoid and phenolic compounds (Conner, 1993; Dorman and Deans, 2000; Calsamiglia et al. 2007).

 

Table 2 Parameters estimated by fitting generalized mitscherlich model to gas production values, recorded for a high-concentrate diet for dairy cattle treated with different levels (0, 250, 500, 750 and 1000 µL/L) of ethanol Chamomile (Matricaria chamomilla) extract

 

A: asymptotic gas production; c (h-1): initial gas production rate constant; d (h-1/2): later gas production rate constant rate constants and L (h): lag time.

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

SEM: standard error of the means.

 

Table 3 Parameters estimated by fitting generalized mitscherlich model to gas production values, recorded for a high-concentrate diet for dairy cattle treated with different levels (0, 250, 500, 750 and 1000 µL/L) of ethanol Clove (Syzygium aromaticum) extract

 

A: asymptotic gas production; c (h-1): initial gas production rate constant; d (h-1/2): later gas production rate constant rate constants and L (h): lag time.

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

SEM: standard error of the means.

 

Table 4 Parameters estimated by fitting generalized mitscherlich model to gas production values, recorded for a high-concentrate diet for dairy cattle treated with different levels (0, 250, 500, 750 and 1000 µL/L) of ethanol Tarragon (Artemisia dracunculus) extract

 

A: asymptotic gas production; c (h-1): initial gas production rate constant; d (h-1/2): later gas production rate constant rate constants and L (h): lag time.

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

SEM: standard error of the means.

 

Debashis-Roy et al. (2015) have reported that eugenol hadamore effective antimicrobial potential in comparison with other non phenolic plant secondary metabolites because of the presence of a hydroxyl group in its phenolic structure and resulted in the loss of integrity of bacterial cell membrane and ultimately in reduction in glucose-uptake of bacteria. It has also been demonstrated that a-bisabolol and Chamazulene had the strongest activity against both gram-positive and gram-negative bacteria (Janmejai et al. 2010). However, decrease in potential gas production may be due to their secondary metabolites. In the present study, it was evidenced that other kinetic parameters of fermentation also affected. Overall, initial gas production rate constant (c) increased (P<0.05) due to addition of plant ethanol extracts to medium. But, ethanol extracts decreased (P<0.05) later gas production rate constant (d). Chamomile extract at 750 µL/L, clove extract at 1000 µL/L and tarragon extract at 750 µL/L had the lowest lag time, resulting in a faster rate of fermentation.

 

Effect of plant ethanol extracts on in vitro OM digestibility, estimated energy value and microbial mass

In vitro OM digestibility, estimated energy value and microbial mass results were presented (Tables 5, 6 and 7).

 

Table 5 Predictions of in vitro organic matter digestibility (IVOMD), metabolizable energy (ME), short-chain fatty acids (SCFA), net energy lactation (NEL) and microbial mass estimation (MM) for a high-concentrate diet for dairy cattle treated with different levels (0, 250, 500, 750 and 1000 µl/L) of ethanol Chamomile (Matricaria chamomilla) extract

 

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

SEM: standard error of the means.

 

Table 6 Predictions of in vitro organic matter digestibility (IVOMD), metabolizable energy (ME), short-chain fatty acids (SCFA), net energy lactation (NEL) and microbial mass estimation (MM) for a high-concentrate diet for dairy cattle treated with different levels (0, 250, 500, 750 and 1000 µl/L) of ethanol Clove (Syzygium aromaticum) extract

 

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

SEM: standard error of the means.

 

Table 7 Predictions of in vitro organic matter digestibility (IVOMD), metabolizable energy (ME), short-chain fatty acids (SCFA), net energy lactation (NEL), and microbial massestimation (MM) for a high-concentrate diet for dairy cattle treated with different levels (0, 250, 500, 750 and 1000 µl/L) of ethanol Tarragon (Artemisia dracunculus) extract

 

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

SEM: standard error of the means.

 

Chamomile extract at 1000 µL/L increased (approximately 5 to 8%) significantly (P<0.05) IVOMD, metabolizable energy, SCFA, net energy lactation and microbial mass. Ethanol extracts of clove and tarragon did not affect in vitro OM digestibility of substrate, estimated energy value and microbial mass. The results of GP measurement revealed that chamomile ethanol extract at 1000 µL/L resulted in an increase in GP compared with the control, which was consistent with an increase in IVOMD, metabolizable energy, SCFA and NEL. However, an increase in OM digestibility because of the addition of chamomile ethanol extract at high dose could also be attributed to stimulated bacterial activity (Naseri et al. 2012), which results in an increase in potential gas production. Generally, medicinal plants or their extracts usually yield complex mixtures of biochemical so that identification of the phytochemical fractions that might be involved in the effects observed was not possible (Scehovic, 1999). However, three explanations can be made as follows: (1) the inhibitory or stimulatory action of plant secondary metabolites (PSM) on some rumen microorganisms; (2) the effect of the degradation products of PSM and (3) direct action of other secondary metabolites. Therefore, in the current study, our observations possibly might have resulted from the inhibitory or stimulatory action of PSM, especially from the presence of essential oils (EOs) on some rumen microorganisms.

 

CONCLUSION

In vitro effect of ethanol extracts of chamomilla (Matricaria chamomilla), clove (Syzygium aromaticum) and tarragon (Artemisia dracunculus) at differing concentrations on organic matter digestibility, estimated energy value, microbial mass, and rumen fermentation kinetics of a high-concentrate diet for dairy cattle, suggested that chamomile, clove and tarragon extracts have potential to alter rumen fermentation kinetics. However, these findings should be considered preliminary and further investigation should be undertaken which also use in vivo methods in order to better assess the value of these plant extracts as feed additives to improve the yield of dairy products.

 

ACKNOWLEDGEMENT

The authors thank the University of Razi (Kermanshah, Iran) for the financial support.

Abas I., Ozpinar H., Can-Kutay H. and Kahraman R. (2005). Determination of the metabolizable energy (ME) and net energy lactation (NEL) contents of some feeds in the Marmara region by in vitro gas technique. Turkish J. Vet. Anim. Sci. 29, 751-757.

Alexander G., Singh B., Sahoo A. and Bhat T.K. (2007). In vitro screening of plant extracts to enhance the efficiency of utilization of energy and nitrogen in ruminant diets. Anim. Feed Sci. Technol. 145, 229-242.

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

Babayemi O.J. (2007). In vitro fermentation characteristics and acceptability by West African dwarf goats of some dry season forages. African J. Biotechnol. 6(10), 1260-1265.

Benchaar C., Calsamiglia S.,Chaves A.V., Fraser G.R., Colombatto D., McAllister T.A. and Beauchemin K.A. (2008). A review of plant-derived essential oils in ruminant nutrition and production. Anim. Feed Sci. Technol. 145, 209-228.

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

Blümmel M., Makkar H.P.S. and Becker K. (1997). The relationship between in vitro gas production, in vitro microbial mass yield and 15N incorporation and its implications for the prediction of voluntary feed intake of roughages. British J. Nutr. 77, 911-921.

Calsamiglia S., Busquet M., Cardozo P.W., Castillejos L. and Ferret A. (2007). Invited review: essential oils as modifiers of rumen microbial fermentation. J. Dairy Sci. 90, 2580-2595.

Conner D.E. (1993). Naturally occurring compounds. Pp. 441-468 in Antimicrobials in Foods. P.M. Davidson and A.L. Branen, Eds. Marcel Dekker, New York, USA.

Debashis R.,Tomar S.K. and Vinod K. (2015). Rumen modulatory effect of thyme, clove and peppermint oils in vitro using buffalo rumen liquor. Vet. World. 8(2), 203-207.

Dorman H.J.D. and Deans S.G. (2000). Antimicrobial agents from plants: antibacterial activity of plant volatile oils. J. Appl. Microbiol. 88, 308-316.

European Union. (2003). Regulation (EC) No 1831/2003 of the European Parliament and the Council of 22 September 2003 on additives for use in animal nutrition.

France J., Dijkstra J., Dhanoa M.S., Theodorou M.K., Lister S.J., Davies D.R. and Isac D.A. (1993). A model to interpret gas accumulation profiles associated with in vitro degradation of ruminant feeds. J. Theor. Biol. 163, 99-111.

Getachew G., Blummel M., Makkar H.P.S. and Becker K. (1998). In vitro gas measuring techniques for assessment of nutritional quality of feeds: a review. Anim. Feed Sci. Technol. 72, 261-281.

Getachew G., Depeters E.J. and Robinson P.H. (2004). In vitro gas production provides effective method for assessing ruminant feeds. California Agric. 58(1), 54-58.

Hart K.J., Yanez-Ruiz D.R., Duval S.M., McEwan N.R. and Newbold C.J. (2008). Plant extracts to manipulate rumen fermentation. Anim. Feed Sci. Technol. 147, 8-35.

Jamalian A., Shams-Ghahfarokhi M., Jaimand K., Pashootan N., Amani A. and Razzaghi-Abyaneh M. (2012). Chemical composition and antifungal activity of Matricaria recutita flower essential oil against medically important dermatophytes and soil-borne pathogens. J. Mycol. Med. 22, 308-315.

Janmejai K., Eswar S. and Sanjay G. (2010). Chamomile: a herbal medicine of the past with bright future. Mol. Med. Rep. 3(6), 895-901.

Khazaal K., Dentinho M.T., Ribeiro R. and Ørskov E.R. (1995). Prediction of apparent digestibility and voluntary intake of hays fed to sheep: comparison between using fibre components, in vitro digestibility or characteristics of gas production or nylon bag degradation. J. Anim. Sci. 61, 527-538.

Maheri-Sis N., Chamani M., Sadeghi A.A., Mirza-Aghazadeh A. and Aghajanzadeh-Golshani A. (2008). Nutritional evaluation of kabuli and desi type chickpeas (Cicer arietinum) for ruminants using in vitro gas production technique. African J. Biotechnol. 7(16), 2946-2951.

Maheri-Sis N., Chamani M., Sadeghi A.A., Mirza-Aghazadeh A. and Safaei A.A. (2007). Nutritional evaluation of chickpea wastes for ruminants using in vitro gas production technique. J. Anim. Vet. Adv. 6(12), 1453-1457.

Makkar H.P.S. (2005). In vitro gas methods for evaluation of feeds containing phytochemicals. Anim. Feed Sci. Technol. 123, 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. Anim. Res. Dev. 28, 47-55.

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 feedstuffs from the gas production when they are incubated with rumen liquor. J. Agric. Sci. 93, 217-222.

Mirzaei-Aghsaghali A. and Maheri-Sis N. (2008a). Nutritive value of some agro-industrial by-products for ruminants: a review. World J. Zool. 3(2), 40-46.

Mirzaei-Aghsaghali A., Maheri-Sis N., Mansouri H., Ebrahim R.M., Aghajanzadeh-Golshani A. and Cheraghi H. (2011b). Evaluating nutrItional value of sugar beet pulp for ruminant animals using in vitro gas production technique. Int. J. Res. 3(2), 147-152.

Mirzaei-Aghsaghali A., Maheri-Sis N., Mirza-Aghazadeh A., Safaei A.R. and Aghajanzadeh-Golshani A. (2008b). Nutritive value of alfalfa varieties for ruminants with emphasis of different measuring methods: a review. Res. J. Biol. Sci. 3(10), 1227-1241.

Mirzaei-Aghsaghali A., Maheri-Sis N., Mansouri H., Razeghi M.E., Shayegh J. and Aghajanzadeh-Golshani A. (2011a). Evaluating nutritional value of apple pomace for ruminants using in vitro gas production technique. Ann. Biol. Res. 2, 100-106.

Naseri V., Hozhabri F. and Kafilzadeh F. (2012). Assessment of in vitro digestibility and fermentation parameters of alfalfa hay based diet following direct incorporation of fenugreek seed (Trigonella foenum) and asparagus root (Asparagus officinalis). J. Anim. Physiol. Anim. Nutr. 97(4), 773-784.

Naseri V., Kafilzadeh F. and Hozhabri F. (2015). Fenugreek seed (Trigonella foenum-graecum) and Asparagus Root (Asparagus officinalis) effects on digestion and kinetics of gas production of alfalfa hay using in vitro technique. Iranian J. Appl. Anim. Sci. 5(3), 185-188.

Patra A.K. (2011). Effects of essential oils on rumen fermentation, microbialecology and ruminant production. Asian J. Anim. Vet. Adv. 6, 416-428.

Renata N. and Grażyna Z. (2014). Herb yield and bioactive compounds of Tarragon (Artemisia dracunculus) as influenced by plant density. Acta. Sci. Pol. Hortorum Cultus. 13(2), 207-221.

Sallam S.M.A., Abdelgaleil S.A.M., Bueno I.C.S., Nassera M.E.A., Araujo R.C. and Abdalla A.L. (2011). Effect of essential oils on ruminal fermentation, microbial population and methane emission in vitro. Options Méditerran. 57, 149-156.

SAS Institute. (1996). SAS®/STAT Software, Release 6.11. SAS Institute, Inc., Cary, NC. USA.

 Scehovic J. (1999). Evaluation in vitro de l’activité de la popula-tion microbienne du rumen en présence d’extraits végétaux. Rev. Suisse. Agric. 31, 89-93.

Theodorou M.K., Williams B.A., Dhanoa M.S., McAllan A.B. and France J. (1994). A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Anim. Feed Sci. Technol. 48, 185-197.

Tilley J.M.A. and Terry R.A. (1963). A two-stage technique for the in vitro digestion of forage crops. J. British Grassland. Soc. 18, 104-111.

Van Soest P.J., Robertson J.B. and Lewis B.A. (1991). Carbohydrate methodology, metabolism and nutritional implications in dairy cattle. J. Dairy Sci. 74, 3583-3597.