The present invention relates to a method for reducing the production of methane emanating from the digestive activities of ruminants by using as active compound deoxycholic acid and/or chenodeoxycholic acid and derivatives thereof.

The present invention also relates to the use of these active compounds and derivatives thereof as components of animal feed or feed additives, as well as to compositions, feed additives and feed containing them.

The term feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal.

In the present context, a ruminant is a mammal of the order Artiodactyla that digests plant-based food by initially softening it within the animal's first stomach, known as the rumen, then regurgitating the semi-digested mass, now known as cud, and chewing it again. The process of again chewing the cud to further break down plant matter and stimulate digestion is called "ruminating". Ruminating mammals include cattle, goats, sheep, giraffes, American Bison, European bison, yaks, water buffalo, deer, camels, alpacas, llamas, wildebeest, antelope, pronghorn, and nilgai.

AdS/09.12.09 For the present purposes, Domestic cattle are the most preferred species. For the present purposes the term includes all races of domestic cattle, and all production kinds of cattle, in particular dairy cows and beef cattle.

Rumen fermentation brings some disadvantages. Methane is produced as a natural consequence of the anaerobic fermentation, which represents an energy loss to the host animal. Carbohydrate makes up 70 - 80 % of the dry matter in a typical dairy cattle ration and in spite of this the absorption of carbohydrates from the gastro-intestinal tract is normally very limited. The reason for this is the extensive fermentation of carbohydrates in the rumen resulting in production of acetate, propionate and butyrate as the main products. These products are part of the so called volatile fatty acids, VFA.

Besides the energy loss, methane is also a greenhouse gas, which is many times more potent than CO ₂ . Its concentration in the atmosphere has doubled over the last century and continues to increase alarmingly. Ruminants are the major contributors to the biogenic methane formation, and it has been estimated that the prevention of methane formation from ruminants would almost stabilize atmospheric methane concentrations.

Furthermore, the assessment of the Kyoto protocol places increased priority in decreasing methane emissions as part of a multi-gas strategy. The most effective feed additives for reducing the formation of methane contain antibiotics and ionophores which diminish the formation of microorganisms provided H ₂ to the methanogenes. However, the effect of antibiotic and ionophores on the formation of methane has some disadvantages because of rapid adaptation of the microflora and/or resistance development.

In recent years there has been an intense debate about the use of chemicals and antibiotics in feed additives and in many countries a ban on this type of additions to feed additives is being considered or already in place. Thus, there is an urgent need for agriculture to develop substances which are in line with reliable and generally accepted practice and not of a medicinal nature. The purpose of the present invention is to provide a feeding concept which is not of medicinal nature and which strongly reduces the formation of methane but without affecting microbial fermentation in a way that would be detrimental to the host animal.

The present inventors surprisingly found that the compounds specified herein after have a great potential for use in animal feed in order to essentially reduce the formation of methane and still maintain the overall microbial activity on a high level.

In particular, it has been observed that the addition of deoxycholic acid and chenodeoxycholic acid is highly effective in decreasing methane formation and methanogenic bacteria in short term in vitro experiments. Therefore and according to the inventive concept, the feeding of the animal with an active as defined hereinabove results in the inhibition of methane release with a decrease of up to 60 %.

Therefore, the present invention provides the use of deoxycholic acid and/or chenodeoxycholic acid and/or derivatives thereof for suppressing methane formation in ruminants.

Deoxycholic acid and chenodeoxycholic acid are bile acids naturally occurring in mammals. Chenodeoxycholic acid is also known as chenodesoxycholic acid. Deoxycholic acid, also known as deoxycholate, cholanoic acid, and 3α,12α- dihydroxy-5β-cholanate is one of the secondary bile acids, which are metabolic byproducts of intestinal bacteria.

The term "a derivative thereof" as used herein comprises compounds encompassed by formula 1 and 2, wherein independently from each other R1 is a hydro- gen, or an alkyl group, or an acid, or a sugar moiety; R2 is a hydrogen, or an alkyl group, or an acid, or a sugar moiety; R3 is OH, OR4, NH ₂ , NHR5, wherein R4 and R5 independently represent an ester or an amide group of the compound and are described in more detail below.

The preferred embodiments according to the invention are deoxycholic-acid 1 and chenodeoxycholic-acid 2 for which R1 and R2 is H and R3 is OH.

The term "a derivative thereof" as used herein also comprises stereoisomers of deoxycholic- and chenodeoxycholic acid and salts thereof.

Preferred cations for salt preparation may be selected from the group consisting of sodium (Na ⁺ ), potassium (K ⁺ ), lithium (Li ⁺ ), magnesium (Mg ²⁺ ), calcium (Ca ²⁺ ), barium (Ba ²⁺ ), strontium (Sr ²⁺ ), and ammonium (NH ⁴⁺ ). Salts may also be prepared from an alkali metal or an alkaline earth metal. Preferably for use, the bile salt is sodium deoxycholate or chenodeoxycholate.

Derivatives which may be seen as prodrugs of the compounds of the embodiments are also considered. The term prodrug is used for an active or inactive compound that is modified chemically through in vivo physiological action, such as hydrolysis, metabolism and the like, into a compound of the embodiments follow- ing the administration of the prodrug. For example, one may prepare an ester or an amide of the present deoxycholic- or chenodeoxycholic acid or derivatives thereof, so that the release of the actual active deoxycholic- or chenodeoxycholic acid or derivatives thereof is triggered by a chemical or enzyme catalyzed reaction.

For example, such esters can optionally include 1 -4 heteroatoms selected from oxygen, sulfur or nitrogen; alkyl groups such as methyl, ethyl, isopropyl, butyl, hexyl etc. optionally having 1 -4 heteroatoms selected from oxygen, sulfur or nitrogen; alkylphenyl groups having a total of up to 10 carbon atoms, such as, a benzyl or an 5 ethyl phenyl group optionally having 1 -4 heteroatoms at any acceptable point of substitution; and an aryl group such as a phenyl group. Example of amide includes, but is not limited to, hydroxamate. For a general review of the prodrug-approach involving esters see Svensson and Tunek "Drug Metabolism Reviews" 165 (1988) and Bundgaard "Design of Prodrugs", Elsevier (1985). Synthesis of an ester or an amide of the deoxycholic- or chenodeoxycholic acid is well known in the art. For example, an ester can be synthesized by a reaction of the corresponding acid with an alcohol in the presence of a mineral acid, in an estehfication reaction.

Prodrugs of deoxycholic acid, chenodeoxycholic acid and their derivatives also in- elude epimers that may possess opposite stereochemistry from the native molecule.

The invention further provides a method for reducing the production of methane emanating from the digestive activities of ruminants by using as active ingredients, deoxycholic acid and/or chenodeoxycholic acid and/or derivatives thereof, which are administrated to the animal.

Finally, the present invention provides animal feed additives on the basis of a compound defined above and animal feed containing as an additive such a compound or a derivative or a metabolite thereof.

The compounds of the invention are either commercially available or can easily be prepared by a skilled person using processes and methods well-known in the prior art. In particular, deoxycholic acid and chenodeoxycholic acid can be isolated and purified by methods known per se, e.g. from porcine bile acids.

Compounds according to the present invention and compositions containing them improve the performance of animals. Therefore, they can be used as feed addi- tives or for the preparation thereof and of feed by mixing or processing them with conventional animal feed or components thereof in amounts to provide the required or desired daily uptake.

The normal daily dosage of a compound according to the invention provided to an animal by feed intake depends upon the kind of animal and its condition. Normally this dosage should be in the range of from about 50 to about 1000 mg, preferably from about 100 to about 500 mg compound per kg of feed.

For the use in animal feed, however, deoxycholic acid and chenodeoxycholic acid need not be that pure; it may e.g. include other compounds and derivatives.

Deoxycholic acid and/or chenodeoxycholic acid or a derivative thereof may be used in combination with conventional ingredients present in an animal feed composition (diet) such as calcium carbonates, electrolytes such as ammonium chloride, proteins such as soya bean meal, wheat, starch, sunflower meal, corn, meat and bone meal, amino acids, animal fat, vitamins and trace minerals.

Particular examples of compositions of the invention are the following:

- An animal feed additive comprising (a) deoxycholic acid and/or chenodeoxycholic acid (b) at least one fat-soluble vitamin, (c) at least one water-soluble vitamin, (d) at least one trace mineral, and/or (e) at least one macro mineral; - An animal feed composition comprising deoxycholic acid or chenodeoxycholic acid and a crude protein content of 50 to 800 g/kg feed.

The so-called premixes are examples of animal feed additives of the invention. A premix designates a preferably uniform mixture of one or more micro-ingredients with diluent and/or carrier. Premixes are used to facilitate uniform dispersion of micro-ingredients in a larger mix.

Apart from the active ingredients of the invention, the premix of the invention contains at least one fat-soluble vitamin, and/or at least one water soluble vitamin, and/or at least one trace mineral, and/or at least one macro mineral. In other words, the premix of the invention comprises the deoxycholic acid and/or chenodeoxycholic acid together with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, trace minerals, and macro minerals.

Macro minerals may be separately added to the feed. Therefore, in a particular embodiment, the premix comprises the active ingredients of the invention together with at least one additional component selected from the group consisting of fat-soluble vitamins, water-soluble vitamins, and trace-minerals.

The following are non-exclusive lists of examples of these components:

- Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3.

- Examples of water-soluble vitamins are vitamin B12, biotin and choline, vitamin B1 , vitamin B2, vitamin B6, niacin, folic acid and pantothenate, e.g. Ca-D- panthothenate.

- Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt.

- Examples of macro minerals are calcium, phosphorus and sodium.

As regards feed compositions for ruminants such as cows, as well as ingredients thereof, the ruminant diet is usually composed of an easily degradable fraction (named concentrate) and a fiber-rich less readily degradable fraction (named hay, forage, or roughage).

Hay is made of dried grass, legume or whole cereals. Grasses include among others timothy, ryegrasses, fescues. Legumes include among others clover, lucerne or alfalfa, peas, beans and vetches. Whole cereals include among others barley, maize (corn), oat, sorghum. Other forage crops include sugarcane, kales, rapes, and cabbages. Also root crops such as turnips, swedes, mangels, fodder beet, and sugar beet (including sugar beet pulp and beet molasses) are used to feed ruminants. Still further crops are tubers such as potatoes, cassava and sweet potato. Silage is an ensiled version of the fiber-rich fraction (e.g. from grasses, legumes or whole cereals) whereby material with a high water content is treated with a controlled anaerobic fermentation process (naturally-fermented or additive treated).

Concentrate is largely made up of cereals (such as barley including brewers grain and distillers grain, maize, wheat, sorghum), but also often contain protein-rich feed ingredients such as soybean, rapeseed, palm kernel, cotton seed and sunflower. Cows may also be fed total mixed rations (TMR), where all the dietary components, e.g. forage, silage and concentrate, are mixed before serving.

As mentioned above a premix is an example of a feed additive which may comprise the active compounds according to the invention. It is understood that the compounds may be administered to the animal in different other forms. For example the compounds can also be in included in a bolus that would be placed in the rumen and that would release a defined amount of the active compounds continuously in well defined dosages over a specific period of time.

The present invention also relates to the use of deoxycholic acid and/or chenode- oxycholic acid and derivatives thereof in combination with at least one additional active substance which shows similar effects with regard to methane production in the rumen and which is selected from the group consisting of lipases, diallyl disulfide, garlic oil and allyl isothiocyanate.

Further components that could be given together with the bile acids are for exam- pie yeasts and ionophores like Monensin, Rumensin.

It is at present contemplated that diallyl disulfide, garlic oil and allyl isothiocyanate are independently administered in dosage ranges of for example 0.01 -500 mg active substance per kg feed (ppm). These compounds are either commercially available or can easily be prepared by a skilled person using processes and methods well-known in the prior art.

In the present context, a lipase is an enzyme that catalyzes the hydrolysis of ester bonds in triglyceride substrates found in oils and fats from feed leading to mono and diglycerides and free fatty acids.

In the use according to the invention, the lipase is active and effective inside the digestive system of the animal (in vivo). The lipase can be fed to the animal before, after, or - preferably - simultaneously with the diet.

The lipase belongs to the EC 3.1.1 -group of lipases, such as EC 3.1.1.3 (triacyl- glycerol lipase). The EC numbers refer to Enzyme Nomenclature 1992 from NC- IUBMB, Academic Press, San Diego, California, including supplements 1 -5 pub- lished in Eur. J. Biochem. 1994, 223, 1 - 5; Eur. J. Biochem. 1995, 232, 1 -6; Eur. J. Biochem. 1996, 237, 1 -5; Eur. J. Biochem. 1997, 250, 1 -6; and Eur. J. Biochem. 1999, 264, 610-650; respectively. The nomenclature is regularly supplemented and updated; see e.g. World Wide Web at http://www.chem.qmw.ac.uk/iubmb/enzyme.

In a preferred embodiment, the lipase is a bacterial or fungal lipase and according to the invention preferably derived from a strain of Bacillus, such as strains of Bacillus amyloliquefaciens.

For purposes of the present invention, preferred lipases are the lipases contained in the following commercial products: LIPOLASE®, Lipex®, CALA® and No- vozym® 735.

The present invention is further described by the following example which should not be construed as limiting the scope of the invention.

Examples

Example 1 : Comparative analysis of Porcine bile acids, chenodeoxycholic acid and deoxycholic acid on in vitro methane and VFA production

A modified version of the "Hohenheim Forage value Test (HFT)" is used for testing the effect of specific compounds on the rumen functions mimicked by this in-vitro system.

Principle:

Feed is given into a syringe with a composition of rumen liquor and an appropriate mixture of buffers. The solution is incubated at 39°C. After 24h the quantity (and composition) of gas produced is measured and put into a formula for conversion. Short chain fatty acids (VFAs) are also quantified.

Reagents:

Mass element solution:

- 6.2 g potassium dihydrogen phosphate (KH2PO4) - 0.6 g magnesium sulfate heptahydrate (MgSO4 ^* 7H2O)

- 9 ml concentrated phosphoric acid (1 mol/l)

- dissolved in distilled water to 1 I (pH about 1.6) Buffer solution: - 35.0 g sodium hydrogen carbonate (NaHCO3)

- 4.0 g ammonium hydrogen carbonate ((NH4)HCO3)

- dissolved in distilled water to 1 I Trace element solution:

- 13.2 g calciumchloride dihydrate (CaCI2 ^* 2H2O) - 10.0 g manganese(ll) chloride tetrahydrate (MnCI2 ^* 4H2O)

- 1.0 g cobalt(ll) chloride hexahydrate (CoCI2 ^* 6H2O)

- 8.0 g iron(lll) chloride (FeCI3 ^* 6H2O)

- dissolved in distilled water to 100 ml Sodium salt solution: - 100 mg sodium salt

- dissolved in distilled water to 100 ml Reduction solution:

- first 3 ml sodium hydroxide (c = 1 mol/l), then 427.5 mg sodium sulfide hydrate (Na2S ^* H2O) are added to 71.25 ml H2O - solution must be prepared shortly before it is added to the medium solution.

Procedure:

Sample weighing:

The feed stuff is sieved to 1 mm - usually TMR (44 % concentrate, 6 % hay, 37 % maize silage and 13 % grass silage) - and weighed exactly into 64 syringes. 4 of these syringes are the substrate controls, which display the gas production without the effect of the tested compounds. 4 other syringes are positive control, in which bromoethane sulfonate has been added to 0.1 mM. The remaining syringes contain the test substances, by groups of 4 syringes. The weighted sample is based on the dry matter of the substrate.

Preparation of the medium solution: The components are mixed in a Woulff bottle in following order:

- 711 ml water

- 0.18 ml trace element solution

- 355.5 ml buffer solution - 355.5 ml mass element solution

The completed solution is warmed up to 39 ⁰ C followed by the addition of 1.83 ml sodium salt solution and the addition of reduction solution at 36°C. The rumen liquor is added, when the indicator turns colorless.

Extraction of the rumen liquor:

750 ml of rumen liquor are added to approximately 1 ,400 ml of medium solution under continued agitation and CO2-gassing.

Filling the syringes, incubation and determining gas volumes and VFA values: The diluted rumen fluid (24 ml) is added to the glass syringe. The syringes are then incubated for 24h at 39 ⁰ C under gentle agitation. After 24h, the volume of gas produced is measured, and the percentage of methane in the gas phase is determined by gas chromatography. In parallel, an aliquot of the liquid phase is taken and transferred into sulfuric acid in order to stop fermentation. From this ali- quot, the amount of short chain fatty acids is determined by HPLC.

Results:

Porcine bile acid extract, deoxycholic acid and chenodeoxycholic acid were tested using the methodology described above.

The food fermented was artificial TMR (44 % concentrate, 6 % hay, 37 % maize silage and 13 % grass silage). All bile acids were purchased from SIGMA.

The bile acids were typically added to the fermentation syringes to a concentration of 2 and 0.5 % of dry matter (DM). This means that, as 300 mg of TMR were used, 1.5 to 6 mg of bile acids were used. Results are presented in the following tables.

Table 1 : Effect of porcine bile acids on volatile fatty acids (VFA) and methane time concentration VFA change (%) CH ₄ change (%)

Porcine bile acids 24 h 0,5 % DM -7 -11

Porcine bile acids 24 h 0,5 % DM 2 -5

Porcine bile acids 24 h 2 % DM 1 -13

Porcine bile acids 24 h 2 % DM -4 3

As visible on this table, no effect of the bile acids was detected. All effects were found to be non-statistically different from 0.

Table 2: Effect of chenodeoxycholic acid.

time concentration VFA change CH ₄ change w°) w°)

Chenodeoxycholic acid 24 h 0,5 % DM -1 1 Chenodeoxycholic acid 24 h 2 % DM -24 -43 Chenodeoxycholic acid 24 h 2 %DM -18 -58

With chenodeoxycholic acid, significant and reproducible effects could be observed. Decrease of methane (CH ₄ ) by 40-60 % and VFA by 20-25 %.

Table 3: Effect of deoxycholic acid time concentration VFA change (%) CH4 change (%)

Deoxycholic acid 24 h 0,1 % DM -7 -27

Deoxycholic acid 24 h 0,5 % DM -8 -23

Deoxycholic acid 24 h 0,5 % DM -3 -4

Deoxycholic acid 24 h 2 % DM -16 -57

Deoxycholic acid 24 h 2 % DM -8 -52

Deoxycholic acid 24 h 2 % DM -17 -26

This bile acid decreased methane by 25-55 % and VFA by less 20 %.

Example 2: Comparative analysis of chenodeoxycholic acid with bromoethanesulfonate on in vitro methane reduction.

Feed: Feed used in rumen simulation was 1 gram of dry matter and composed of the following:

- maize silage 0.5 g dry matter

- commercial compound feed Lypsy-Krossi (Suomen Rehu Ltd) 0.5g dry matter

Treatments and doses:

The rumen simulation study was accomplished with a duplicated negative control and test products at three different doses. Every treatment was simulated in six replicate vessels. Results are summarized in Table 4 according to the following nomenclature:

1- Negative control (Contr.)

2- Chenodeoxycholic acid at: 0.8mg/40ml (CDCA L); 4.0mg/40ml (CDCA M); 20mg/40ml (CDCA H) 3- 2-bromoethanesulfonate at: 0.34mg/40ml (BES L); 1.69mg/40ml (BES M); 8.44mg/40ml (BES H)

Protocol for the rumen simulation:

Individual feed components and test products were weighted in serum bottles, the bottles flushed with CO ₂ passed through a hot copper catalyst for O ₂ scavenging, and sealed with thick butyl rubber stoppers. Thirty-six ml of anaerobic, reduced, temperature adjusted (+37 ⁰ C) buffer solution was introduced into each simulation vessel under the oxygen-free CO ₂ flow. Finally, 4 ml of fresh, strained rumen fluid was added in the serum bottles, the final volume being 40 ml. This inoculation started the actual rumen simulation. Inoculation time for each vessel was registered and taken into account when sampling and stopping the fermentation.

Analyzed parameters:

Total gas production. Rumen fermentation simulation was continued for 12 hours at 37 ⁰ C. During the fermentation the total gas production was measured after 2, 5, 8 and 12 hours of simulation to reveal the general metabolic activity of the rumen microbes.

Methane and hydrogen production. All the gas produced during the 12 hours in each simulation vessel was individually collected from each of the 156 vessels into evacuated 2 liter infusion bottles, which had ethane pre-introduced as an internal standard. The analyses were performed by gas chromatography using thermal conductivity detector for hydrogen and flame ionization detector for methane and ethane.

Acid production. At 12 hours all the simulation vessels were analyzed for pH and short chain fatty acids (SCFAs). SCFAs were analyzed by gas chromatography using a packed column for the analysis of free acids. The SCFAs quantified were acetic, propionic, butyric, iso-butyhc, 2-methyl-butyric, valeric, iso-valeric and lactic acids.

Statistical analyses:

Dunnett's post hoc tests were performed for all measured parameters to determine which individual treatments differ from the control treatment. All significant test results with the risk level α = 0.05

Symbols used: ^* p < 0.05

0.01 < p < 0.05 * ^** 0.001 < p < 0.01

0.0001 < p < 0.001

Results: All results are summarized in Table 4.

Table 4: Fermentation parameters, and gas production after 8 hours fermentation

CDCA CDCA CDCA BES BES BES

(M)

Contr. (L) (H) (L) (M) (H)

Total gas production (ml) ₁₄₀ 133 130 *** 83 **** 130 **** ₁₂ 8 **** 125 at 12 h

Total VFAs (mmol/l) at 12 h 88 90 86 71 78 77 79

Acetate (mmol/l) at 12 h 45 46 44 38 37 37 38

Propionate (mmol/l) at 12 h 30 31 31 32 29 29 30

Butyrate (mmol/l) at 12 h 13 13 11 1 12 11 11

Total methane production 17 17 p _**** 0.5 ^**** (ml) after 12 h

Total Hydrogen 0 0 0.8 0 11.5 ^**** 11.5 ^**** accumulation (ml) after 12 h Example 3: Effect of chenodeoxycholic acid in an in vitro continuous rumen simulation system: "Rusitec"

In vitro system and experimental diets:

The in vitro experiment was conducted using a Rusitec continuous rumen simulation system as described in detail by Soliva & Hess (2007) "In Measuring Methane Production from Ruminants", pp. 15-135. With this in vitro system digestion of basal diets was tested at 15 g dry matter / day (DM/d) both with and without chenodeoxycholic acid supplemented at 1 % (on a DM basis) in a completely randomised design in four replicates per treatment. The basal diets composition is described in Table 5.

Table 5: Composition of the dietary substrates in basal diet

Basal diet Control

Supply to the fermenter (g DM/d)

Hay 7.47

Barley 5.23

Soybean meal 2.24

Vitamin-Mineral mixture ^* 0.08

Total dry matter supply 15.00

Analyses nutrient composition (g/kg DM)

Organic matter 826

Crude protein 182

Neutral detergent fibre (NDF) 343

^• contained (per kg) 140 g Ca; 70 g P; 80 g Na; 30 g Mg; 15 mg Se; 500'0OO IU vitamin A; 120O00 IU vitamin D ₃ ; 2'500 IU vitamin E.

Experimental procedures and sampling: In four experimental runs, each lasting for 10 days, the daily portions of experimental feeds were put into nylon bags (70 x 140 mm) with a pore size of 100 μm. Before that, hay and straw were ground to pass a 5mm sieve whereas the grains were ground to a size of 3 mm. Ruminal fluid was obtained from a lactating rumen-fistulated Brown Swiss cow which was fed hay ad libitum and concentrate (1 kg/d administered in two portions). The cow was kept according to the Swiss guidelines for animal welfare. Before inoculation, ruminal fluid was strained through four layers of medicinal gauze with a pore size of about 1 mm. At the beginning of each experimental run the fermenters were filled with 100 ml pre- warmed buffer and 900 ml strained ruminal fluid.

Thereafter, two nylon bags were administered whereby the first one was filled with solid ruminal content (about 40 g fresh matter) and the second one with the respective experimental diet. On the second experimental day the bag containing the solid ruminal content was exchanged with another bag containing the experimental diet. Each feed bag was incubated for 48 h. To maintain anaerobic conditions the system was flushed with gaseous nitrogen for 3 min after exchanging the feed bags. The incubation temperature was kept constant at 39.5°C. Buffer flow to the fermenters was continuous and averaged 397 ml/d, resulting in a dilution rate of about 40 % per day. The resulting incubation fluid outflow was collected in bottles chilled at -20 ⁰ C. Incubation fluid samples, collected directly from the fermenters via a three-way valve using a syringe equipped with a plastic tube 3 hours before exchanging the feed bags, were analysed daily for redox potential and pH using the respective electrodes connected to a pH meter (model 634; Methrom AG, Herisau, Switzerland). Part of the incubation fluid samples taken were centrifuged for 5 min at 4000 rpm (Varifugew K; Heraeus, Osterode, Germany) and the supernatant fraction was stored at -20 ⁰ C before being analysed for SCFA concentrations.

After 48 h of incubation, dietary residues were washed with cold water in a washing machine and frozen at -20 ⁰ C until nutrient analyses were performed. Later the lyophilised and ground residues were analysed for DM and organic matter, via total ash (automatically by TGA-500; Leco Corporation, St Joseph, Ml, USA), N (C/N analyser, Leco-Analysator Typ FP-2000; Leco lnstrumente GmBH, Kircheim, Germany; crude protein % 6-25 £ N) and neutral-detergent fibre. Analyses of neutral-detergent fibre were carried out with the Fibretec System M (Tecator, 1020 Hot Extraction, Hόganas, Sweden) with the addition of α-amylase but without sodium sulfite as suggested by Van Soest et al. (1991 ) J Dairy Sci. 74, 3583-3597.

Results: Results demonstrating the effect of chenodeoxycholic acid on ruminal microbes are summarized in Table 6.

Table 6: Effects of chenodeoxycholic on fermenter fluid traits, SCFA production, and rumen microbial counts. SEM, standard error of means.

Control Chenodeoxycholic acid SEM

Fermenter fluid traits

Redox potential (mV) -192 -194 5.8

PH 6.52 6.40 0.025

Ammonia (mmol/l) 11.5 8.8 0.34

Short chain fatty acids mmol/d 116 117 2.9

Ruminai microbes

Entodiniomorphs ( ^χ 10 ³ /ml) 2.37 0 0.603

Holothchs ( ^χ 10 ³ /ml) 1.18 0 0.275

Bacteria ( ^χ 10 ⁸ /ml) 5.59 6.87 0.274

As shown by this table, protozoa (Entodiniomorphs, and Holothchs) which live in symbiosis with methanogens are drastically affected by chenodeoxycholic acid.