Use of nitrooxy organic molecules in feed for reducing methane emission in ruminants, and/or to improve ruminant performance

Abstract

The present invention relates to a method for reducing the production of methane emanating from the digestive activities of a ruminant and/or for improving ruminant animal performance by using, as active compound at least one organic molecule substituted at any position with at least one nitrooxy group, or a salt thereof, which is administrated to the animal together with the feed. The invention also relates to the use of these compounds in feed and feed additives such as premix, concentrates and total mixed ration (TMR) or in the form of a bolus.

Claims

1. A feed composition or feed additive for a ruminant comprising an amount between about 1 mg to about 10 g/kg of feed of 3-nitrooxy propanol sufficient to reduce production of methane emanating from digestive activities of a ruminant by at least 10% calculated in liters per kilogram of dry matter intake when measured in a metabolic chamber.

2. A feed composition or feed additive for reducing the production of methane emanating from the digestive activities of ruminants, wherein the feed composition comprises an amount between about 1 mg/kg of feed to about 10 g/kg of feed sufficient to reduce production of methane emanating from the digestive activities of a ruminant of at least one active organic molecule or a salt thereof selected from the group consisting of 3-nitrooxypropanol, rac-4-phenylbutane-1,2-diyldinitrate, 2-(hydroxymethyl)-2-(nitrooxymethyl)-1,3-propanediol, N-ethyl-3-nitrooxy-propionic sulfonyl amide, 5-nitrooxy-pentanenitrile, 5-nitrooxy-pentane, 3-nitrooxy-propyl propionate, 1,3-bis-nitrooxypropane, 1,4-bis-nitrooxybutane, 1,5-bis-nitrooxypentane, 3-nitrooxy-propyl benzoate, 3-nitrooxy-propyl hexanoate, 3-nitrooxy-propyl 5-nitrooxy-hexanoate, benzylnitrate, isosorbid-dinitrate, N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide, and bis-(2-nitrooxyethyl) ether.

3. The feed composition or feed additive according to claim 2 which is a mineral premix, a vitamin premix, or a premix including vitamins and minerals or a bolus.

4. The feed composition or feed additive according to claim 2, wherein the at least one active organic molecule is 3-nitrooxy propanol.

5. The feed composition or feed additive according to claim 2, wherein the at least one active organic molecule is a mixture of 3-nitrooxy propanol and 1,3-bis-nitrooxypropane.

Description

EXAMPLES

Example 1

In Vitro Test for Methane Production

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

(2) Principle:

(3) 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 8 hours the quantity (and composition) of methane produced is measured and put into a formula for conversion.

(4) Reagents:

(5) Mass Element Solution:

(6) 6.2 g potassium dihydrogen phosphate (KH.sub.2PO.sub.4) 0.6 g magnesium sulfate heptahydrate (MgSO.sub.4*7H.sub.2O) 9 ml concentrated phosphoric acid (1 mol/l) dissolved in distilled water to 1 l (pH about 1.6)
Buffer Solution: 35.0 g sodium hydrogen carbonate (NaHCO.sub.3) 4.0 g ammonium hydrogen carbonate ((NH.sub.4)HCO.sub.3) dissolved in distilled water to 1 l
Trace Element Solution: 13.2 g calcium chloride dihydrate (CaCl.sub.2*2H.sub.2O) 10.0 g manganese(II) chloride tetrahydrate (MnCl.sub.2*4H.sub.2O) 1.0 g cobalt(II) chloride hexahydrate (CoCl.sub.2*6H.sub.2O) 8.0 g iron(III) chloride (FeCl.sub.3*6H.sub.2O) 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 (Na.sub.2S*H.sub.2O) are added to 71.25 ml H.sub.2O solution must be prepared shortly before it is added to the medium solution
Procedure:
Sample Weighing:

(7) The feed stuff is sieved to 1 mmusually 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. When needed, 4 syringes contain a carrier control (if the test compounds need a carrier). The remaining syringes contain the test substances, by groups of 4 syringes.

(8) Preparation of the Medium Solution:

(9) 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

(10) The completed solution is warmed up to 39 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 colourless.

(11) Extraction of the Rumen Liquor:

(12) 750 ml of rumen liquor are added to approximately 1,400 ml of medium solution under continued agitation and CO.sub.2-gassing.

(13) Filling the Syringes, Incubation and Determining Gas Volumes and VFA Values:

(14) The diluted rumen fluid (24 ml) is added to the glass syringe. The syringes are then incubated for 8 hours at 39 C. under gentle agitation. After 8 hours, the volume of gas produced is measured, and the percentage of methane in the gas phase is determined by gas chromatography.

(15) Results

(16) The food fermented was artificial TMR (44% concentrate, 6% hay, 37% maize silage and 13% grass silage). The compounds produced as described in examples 2 to 14 were added to the fermentation syringes to a concentration of 2 to 0.005 of dry matter (DM). The results are presented in the following table.

(17) TABLE-US-00003 TABLE 3 Methane reduction effect resulting from the average of two experiments with some compounds according to the present invention (an integer in the column effect on methanogenesis change (%) means a reduction in methane produced when compared to control; no value means that the concentration was not tested) effect on methanogenesis (%) 2% 1% 0.5% 0.25% 0.1% 0.05% 0.01% 0.005% Structure DM DM DM DM DM DM DM DM 100 100 100 100 79 20 10 4 85 6 99 99 24 10 99 95 12 7 0 100 100 33 4 100 100 21 6 99 100 98 29 100 100 92 16 100 100 45 6 99 99 11 98 99 42 100 100 37 3 100 0 100 100 99 64 3

Example 2

Comparative Example: In Vitro Test for Methane Production

(18) The same in vitro assay as described in example 1 has been performed with a series of molecules, wherein the nitrooxy group has been replaced by different organic groups. Moreover, the inorganic salt Na NO3 has also been tested. See results in Table 4. This data demonstrates that a significant methane reduction activity is only observed when the Nitrooxy group is present in the series.

(19) TABLE-US-00004 TABLE 4 Methane reduction effect resulting from the average of two experiments with 3-nitrooxypropanol according to the present inventionin comparison with similar compounds in which the nitrooxy group has been replaced. (An integer in the column effect on methanogenesis change (%) means a reduction in methane produced when compared to control; no value means that the concentration was not tested) effect on methanogenesis (%) 0.5% 0.1% 0.05% 0.01% Structure 2% DM DM DM DM DM 100 100 100 79 2 8 6 2 Na NO3 23 2

Example 3

Synthesis of 3-Nitrooxypropanol

(20) ##STR00057##

(21) 50.1 mmol 3-Bromopropanol dissolved in 100 ml acetonitrile and 125.25 mmol silver nitrite were added into a flask protected from light. This suspension was stirred for 21 hours at 70 C. After cooling to room temperature the suspension was filtrated and concentrated in vacuo. The residue was dissolved in Water and extracted two times with TMBE. The organic phases were washed with water and brine, combined, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo leaving 5.63 g.

(22) The crude product was purified by flash chromatography on silica gel using heptane/ethyl acetate 2:1; Yield: 4.82 g (38.8 mmol, 77.4%).

Example 4

Synthesis of 2-(Hydroxymethyl)-2-(nitrooxymethyl)-1,3-propanediol

(23) ##STR00058##

(24) 5 mmol 2-(Bromomethyl)-2-(hydroxymethyl)-1,3-propanediol dissolved in 20 ml acetonitrile and 15 mmol silver nitrite were added into a flask protected from light. This suspension was stirred for 24 hours at 70 C. After cooling to room temperature the suspension was filtrated and the solvent was removed in vacuo leaving 3.05 g.

(25) The crude product was purified by flash chromatography on silica gel using dichloromethane/methanol 50:1; Yield: 0.36 g (1.99 mmol, 40.2%).

Example 5

Synthesis of rac-4-Phenylbutane-1,2-diyldinitrate

(26) ##STR00059##

(27) 7.5 mmol 4-Phenyl-1-buten dissolved in 40 ml acetonitrile, 20.3 mmol silver nitrite and 7.5 mmol lode were added into a flask protected from light. This suspension was stirred for 30 minutes at 25 C. and then for 16 hours at 79 C. After cooling to room temperature the suspension was filtrated and washed with Ethyl acetate. The filtrate was extracted three times with water and washed brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo leaving 1.92 g.

(28) The crude product was purified by flash chromatography on silica gel using Hexane/Ethyl acetate 10:1; Yield: 0.52 g (2.03 mmol, 27%).

Example 6

Synthesis of N-Ethyl-3-nitrooxy-propionic sulfonyl amide

(29) ##STR00060##

(30) In a flask 17 mmol 3-chloropropionic sulfonyl chloride were dissolved in 5 ml Tetrahydrofurane. 33.3 mmol Ethylamine were added over a period of 45 minutes. After that, the solvent was removed in vacuo. The residue was dissolved in water, extracted three times with ethyl acetate. The combined organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo.

(31) The residue was dissolved in 50 ml acetonitrile and 60 mmol silver nitrite were added into a flask protected from light. This suspension was stirred for 41 hours at 70 C. After cooling to room temperature the suspension was filtrated and concentrated in vacuo. The residue was dissolved in dichloromethane and extracted with Water. The water phase was washed again with two times with dichloromethane. The combined organic phase was washed with water and brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo; Yield: 3.05 g (14.5 mmol; 84.5%).

Example 7

Synthesis of 3-Nitrooxy-propyl propionate

(32) ##STR00061##

(33) 9.1 mmol Propionyl chloride were dissolved in 10 ml TMBE and cooled to 3 C. 8.25 mmol 3-Nitrooxypropanol and 9.1 mmol triethylamine in 5 ml TMBE were dropped over a period of 5 min at 3 to 6 C. After 2 hours and 30 minutes stirring without cooling the reaction mixture were extracted with 1N HCl, twice with water, washed with brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo leaving 1.35 g.

(34) The crude product was purified by flash chromatography on silica gel using Hexane/Ethyl acetate 4:1; Yield: 1.14 g (6.4 mmol, 78.0%).

Example 8

Synthesis of 3-Nitrooxy-propyl benzoate

(35) ##STR00062##

(36) 16.5 mmol 3-Nitrooxypropanol dissolved in 10 ml TMBE and 18.2 mmol Triethylamine were cooled to 3 C. 18.2 mmol benzoylchloride in 5 ml TMBE were dropped over a period of 7 minutes at 3 to 6 C. After 24 hours and 30 minutes stirring without cooling, the reaction mixture was extracted with sated. NaHCO.sub.3, water, 1N HCl, twice with water, washed with brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo leaving 3.3 g.

(37) The crude product was purified by flash chromatography on silica gel using a gradient of Hexane/Ethyl acetate from 1:0 to 2:1; Yield: 0.66 g (2.9 mmol, 17.7%).

Example 9

Synthesis of 3-Nitrooxy-propyl hexanoate

(38) ##STR00063##

(39) 20 mmol 3-Nitrooxypropanol dissolved in 10 ml Diethylether and 20 mmol Triethylamine were cooled to 0 C. 18.2 mmol hexoylchlorid were dropped over a period of 5 minutes at 0 to 5 C. After 19 hours stirring without cooling, the reaction mixture was extracted with 1N HCl, twice with water, washed with brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo leaving 3.1 g.

(40) The crude product was purified by flash chromatography on silica gel using Heptane/Ethyl acetate 4:1; Yield: 2.4 g (10.9 mmol, 60.0%).

Example 10

Synthesis of 3-Nitrooxy-propyl 5-nitrooxy-hexanoate

(41) ##STR00064##

(42) 20 mmol 3-Nitrooxypropanol dissolved in 10 ml Diethylether and 20 mmol Triethylamine were cooled to 0 C. 18.2 mmol 5-nitrooxypentoylchlorid were dropped over a period of 5 min at 0 to 5 C. After stirring over night without cooling, the reaction mixture was extracted with 1N HCl, twice with water, washed with brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo.

(43) The crude product was purified by flash chromatography on silica gel using Heptane/Ethyl acetate 4:1; Yield: 2.4 g (9.1 mmol, 50.0%).

Example 11

Synthesis of Benzylnitrate

(44) ##STR00065##

(45) 10 mmol Benzylbromide dissolved in 80 ml acetonitrile and 25 mmol silver nitrite were added into a flask protected from light. This suspension was stirred for 5 hours at 70 C. After cooling to room temperature the suspension was filtrated and concentrated in vacuo. The residue was dissolved in dichloromethane and extracted with Water. The water phase was washed again with two times with dichloromethane. The combined organic phase was washed with water and brine, dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo; Yield: 1.55 g (10.1 mmol; 100%).

Example 12

Synthesis of 1,3-bis-Nitrooxy-propane

(46) ##STR00066##

(47) To a solution of 1,3-dibromopropane (2.00 g, 1.0 eq) in 20.0 mL of dry acetonitrile was added Silver Nitrate (3.70 g, 2.2 eq). The reaction mixture was heated at 70 C. for 2 hours in the dark. The resulting mixture was filtered off through celite and the filtrate was concentrated. The residue was dissolved into water (50.0 mL), extracted with dichloromethane (250.0 mL), dried over magnesium sulfate and solvents were evaporated under vacuum to afford 1.44 g of compound as a colorless liquid (Yield=87%).

Example 13

Synthesis of 1,4-bis-Nitrooxy-butane

(48) ##STR00067##

(49) To a solution of 1,4-dibromobutane (2.00 g, 1.0 eq) in 20.0 mL of dry acetonitrile was added Silver Nitrate (3.50 g, 2.2 eq). The reaction mixture was heated at 70 C. for 2 hours in the dark. The resulting mixture was filtered off through celite and the filtrate was concentrated. The residue was dissolved into water (50.0 mL), extracted with dichloromethane (250.0 mL) and dried over magnesium sulphate. Solvents were evaporated under vacuum to afford 1.49 g of compound as a colorless liquid (Yield=89%).

Example 14

Synthesis of 1,5-bis-Nitrooxy-pentane

(50) ##STR00068##

(51) To a solution of 1,5-dibromopentane (2.00 g, 1.0 eq) in 20.0 mL of dry acetonitrile was added Silver Nitrate (3.30 g, 2.2 eq). The reaction mixture was heated at 70 C. for 2 hours in the dark. The resulting mixture was filtered off through celite and the filtrate was concentrated. The residue was dissolved into water (50.0 mL), extracted with dichloromethane (250.0 mL) and dried over magnesium sulphate. Solvents were evaporated under vacuum to afford 1.38 g of compound as a colorless liquid (Yield=82%).

Example 15

Synthesis of 5-Nitrooxy-pentanenitrile

(52) ##STR00069##

(53) To a solution of 5-bromovaleronitrile (4.00 g, 1.0 eq) in 40.0 mL of dry acetonitrile was added Silver Nitrate (4.60 g, 1.1 eq). The reaction mixture was heated at 70 C. for 2 hours in the dark. The resulting mixture was filtered off through celite and the filtrate was concentrated. The residue was dissolved into water (50.0 mL), extracted with dichloromethane (250.0 mL) and dried over magnesium sulphate. Solvents were evaporated under vacuum to afford 3.56 g of compound as a colorless liquid (Yield=99%).

Example 16

In Vivo Effect of 3-Nitrooxypropanol Compared to ethyl-3-nitrooxypropionate

(54) Material and Methods

(55) 10 sheep were cannulated in the rumen. The trial started one month after the surgical operation. There were 3 treatments: control, additive 1 and additive 2, both at a single dose. Additive 1 is ethyl-3-nitrooxypropionate, and additive 2 is 3-nitrooxypropanol of the present invention. The experimental design consisted of a 33 Latin square with 3 sheep per treatment in each period and 3 consecutive periods. Each period included 28 days of adaptation to the treatment plus two consecutive days of methane measurements in chambers and collection of rumen samples. Over the course of the adaptation phase, a medium term one day methane measurement was done at day 14. In addition, during days 22 and 23 samples of alfalfa hay and oats, placed in nylon bags, were incubated in the rumen of sheep to determine the dry matter ruminal degradation. During the two days of methane measurements in chambers (days 29 and 30) rumen contents samples were collected two hours after the morning feeding, sub-sampled and immediately frozen prior DNA extraction and determination of volatile fatty acids and ammonia nitrogen concentration. Experimental animals were randomly allocated in three sub-groups of 3 animals each and were randomly assigned one of the three treatments (control, additive 1 and additive 2). The 3 sub-groups started the adaptation to the diet with a gap of two days so they were in the same adaptation day prior methane measurement in the chambers. Animals were individually held in cages with constant access to fresh water. A diet consisting of alfalfa hay chopped at 15-20 cm and oats in a 60:40 ratio plus mineral-vitamin supplement was provided to the animals at approximately 1.1 times the energy maintenance level in two equal meals at 900 and 1400 hours. Fresh matter intake was monitored daily for each animal throughout the trial.

(56) The additive was provided twice a day through the ruminal cannula at the same time as the feed. The corresponding amount to each additive (100 mg per animal and day for both additives) was pipetted into 10 grams of grounded oats and wrapped in cellulose paper immediately before it was placed in the rumen. Since the active molecule is volatile the previously mentioned procedure was carried out in a cold room at 4 C.

(57) Methane Measurement and Samples Collection

(58) A set of four methane chambers was used. On days 14, 29 and 30 animals were placed in the chambers for methane measurements. Each chamber measured 1.8 m wide1.8 m deep1.5 m tall. Chamber air temperature was maintained between 15 and 20 C. Within each chamber, the animals were individually restrained in the same cages as during adaptation. Interruptions occurred daily at 0900 hours, when the chamber floor was cleaned, and the animals were fed. These interruptions had little impact on the daily methane emissions because fluxes were calculated three times per day and then averaged to derive the 23-h emission value.

(59) Airflow and concentration of methane was measured for the inflow and outflow ducts of each chamber. Air velocity was continuously monitored over the day in the exhaust duct for each chamber. The air stream in each of the 4 ducts (chambers 1, 2 and 3 and background) was sub-sampled, and methane concentration was measured continuously using a gas analyzer ADM MGA3000 (Spurling works, Herts, UK). It took 11 min to sequentially sample the airflow in all inflow and exhausts ducts in the chambers (3 min in chambers 1, 2, 3, 2 min for background). In summary, the flux of methane for each chamber was calculated for each measuring day from the difference of fresh-air inflow and chamber exhaust methane concentrations and mean air velocities.

(60) Rumen Samples Analysis

(61) Samples of rumen contents were freeze-dried and thoroughly mixed by physical disruption using a bead beater (Mini-bead Beater; BioSpec Products, Bartlesville, Okla., USA) before DNA extraction, which was performed from approximately 50 mg of sample using the QIAamp DNA Stool Mini Kit (Qiagen Ltd, West Sussex, UK) following the manufacturer's instructions with the modification that a higher temperature (95 C.) was used for lysis incubation. DNA samples were used as templates for quantitative real-time PCR (qPCR) amplification. The abundance of total bacteria, total protozoa and total methanogenic archaea were quantified by Real TimePCR (qPCR). Different primer sets were used to amplify 16S rRNA gene-targeted total bacteria (Maeda et al., 2003), and 18S rRNA gene-targeted total protozoa (Sylvester et al 2005). Primers designed for the detection of methanogenic archaea were targeted against the methyl coenzyme-M reductase (mcrA) gene (Denman et al., 2007). The amplifications mixture contained 11.5 l 2RT-PCR supermix BioRad (Bio-Rad Laboratories Inc., Hercules, Calif., USA), 0.4 l of each primer and 0.5 l of sample in a final volume of 23 l. The amplification efficiency was evaluated for each pair of primers with the following program: a 5 min cycle at 95 C., 40 cycles at 95 C. for 15 s, 60 C. for 30 s, 72 C. for 55 s and, 75 C. during 6 s for fluorescent emission measures. The melting curve was built by increasing temperature from 55 C. to 95 C. and readings were taken every 5 C. Amplification of each target group was carried out with the following program: a 5 min cycle at 95 C., 40 cycles at 95 C. for 15 s, 15 s at 60 C. and 72 C. for 45 s (including the fluorescence emission measuring) and a melting curve with a set point temperature of 45 C. and end temperature of 95 C. The absolute amount of bacteria, protozoa and methanogenic archaea, expressed as the number of DNA copies, was determined by using the plasmid pCR4-TOPO (Invitrogen, Carlsbad, Calif., USA) as standard. The PCR product obtained using the respective set of primers was purified and then cloned into pCR 4-TOPO plasmid (Invitrogen, Carlsbad, Calif., USA) to produce recombinant plasmids. A single colony, verified for the expected insert using PCR, was grown in solid media with antibiotics and X-gal overnight. Afterwards, a screening of transformed E. coli colonies was done and some of the positive ones were randomly selected. After checking the presence of the inserted fragment in the colonies by PCR, massive culture of positive colonies was done in liquid media overnight. Plasmids belonging to these cultures were extracted using the Pure Link Miniprep kit (Invitrogen, Carlsbad, Calif., USA) and then sequenced to verify the presence of the fragment inserted. The number of 16S rRNA gene copies present in the plasmid extracts was calculated using the plasmid DNA concentration and the molecular mass of the vector with the insert. The concentrated plasmid was serially diluted (10-fold) to provide a range of 10.sup.8 to 10.sup.2 copies to generate a standard curve.

(62) A relative abundance quantification was used for methanogenic archaea and protozoa as described by Denman and McSweeny (2006) using the 16sRNA as reference gene. Volatile fatty acids were analysed by gas chromatography and ammonia N concentration by colorimetry following the protocols established in our laboratory (Martin-Garcia et al., 2004).

(63) Rumen Degradability

(64) Three grams of 2 mm ground feed were placed in 5 cm10 cm nylon bags with a pore size of 50 m (#R510 Ankom in situ bags, Macedon N.Y.). The two ingredients used in the animals' diets were tested: oats and alfalfa hay. Bags with oats were incubated in the rumen for 24 hours, while those with alfalfa hay for 48 hours. The incubations times were chosen based on average residing times in the rumen of different feedstuffs. On days 22 and 23 two bags per feed and animal and period were. Bags were placed in the rumen immediately before the morning feeding. At 24 or 48 hours they were taken out of the rumen, washed with cold water and frozen at 20 C. At the end of every period the frozen bags were washed in a washing machine using a short cold water program including two bags per feed that had not been incubated in the rumen to account for solubility. After washing, the bags were placed in the oven at 60 C. for 48 hours. Rumen degradability (%) was calculated as the loss of dry matter over the incubation time.

(65) Experimental Animals Care

(66) All management and experimental procedures to the sheep were carried out by trained personnel in strict accordance with the Spanish guidelines (Act No. 1201/2005 of 10 Oct. 2005) for experimental animal protection. The temperature, humidity and air turn out in chambers were carefully monitored considering the animal welfare conditions. CO.sub.2 concentration was also continuously monitored in order to keep it within the limits that ensured a good air quality and renovation rate. Animals didn't show any stressed behaviour while they were allocated in chambers.

(67) Statistical Analysis

(68) Individual methane emissions, VFA profiles, ratio of acetate to propionate, ammonia N concentration, log.sub.10 transformations of concentration of total bacteria, total protozoa and methanogenic archaea and the relative abundance were analyzed for effect of including the additive. The standard error of the mean (SEM) was computed for each analysis. Means were further compared using a least significant difference (LSD) test.

(69) Results

(70) Dry matter intake was not affected (P>0.05) by the treatment and only slight reduction in intakes were observed when the animals were introduced in the methane chambers on days 14 and 30.

(71) As described for intakes, the body weight (as an average of weights recorded prior and after chamber measurements) was not different (P>0.05) among treatments (Table 5). Methane emissions, expressed as liters per kg of fresh matter intake, were significantly (P=0.020) reduced on day 14 when both additives were incorporated in the diet. The reduction observed against the control was 14% and 23%, respectively, for additives 1 and 2. When methane emissions were recorded two weeks later, on days 29 and 30, there was still a numerically reduction, although it did not reach the statistical significance (P=0.061 and 0.183 for days 29 and 30, respectively). If the measurements recorded during the last two consecutive days are pooled together the effect of the addition shows a similar tendency (P=0.092) as the values considered separately.

(72) TABLE-US-00005 TABLE 5 Effect of the addition of additives 1 and 2 on body weights, intakes and methane emissions by sheep measured on days 14, 29 and 30 after commencing the treatment. Addi- P Time Item Control Additive 1 tive 2 SEM value day 14 intake, kg/day 0.819 0.849 0.867 CH4 l, day 24.6 21.9 20.0 CH4 l/kg intake 29.9 25.6 22.5 2.31 0.020 day 29 intake, kg/day 0.856 0.944 0.922 CH4 l, day 22.0 20.9 18.3 CH4 l/kg intake 25.8 21.7 19.6 2.12 0.061 day 30 intake, kg/day 0.760 0.925 0.747 CH4 l, day 22.7 21.8 19.7 CH4 l/kg intake 29.8 23.2 25.6 2.34 0.183 days intake, kg/day 0.780 0.933 0.823 29-30 CH4 l, day 21.8 21.5 19.1 CH4 l/kg intake 28.2 22.6 23.1 2.17 0.092 .sup.a,bValues in a row not sharing a common superscript letters significantly differ, P < 0.05. *Average of weighing prior and after chamber measurements. SEM: Standard Error of the Means

(73) TABLE-US-00006 TABLE 6 Effect of the addition of additive 1 and 2 on volatile fatty acid profile (mol/100 mol), ammonia N concentration (mg/100 ml) and dry matter degradation (DMD, %) of oats (24 hours) and alfalfa hay (48 hours) in the rumen of sheep. Control Additive 1 Additive 2 SEM P value Acetate 69.2.sup.c 67.5.sup.b 64.5.sup.a 0.742 0.007 Propionate 14.3.sup.a 16.6.sup.a 17.5.sup.b 1.030 0.004 Butirate 2.08 2.05 2.11 0.818 0.353 iso-butirate 11.2 10.1 12.3 0.201 0.995 Valerate 1.91 1.94 1.82 0.194 0.100 iso-valerate 1.47 1.79 1.82 0.281 0.908 Total 57.4 58.2 57.1 5.193 0.995 C2/C3 4.91.sup.b 4.09.sup.a 3.89.sup.a 0.262 0.002 NNH.sub.3 100.1 97.3 104.1 9.157 0.924 DMD alfalfa 78.6 78.3 78.8 1.22 0.725 hay DMD oats 74.2 74.0 70.6 2.02 0.167 .sup.a,bValues in a row not sharing a common superscript letters significantly differ, P < 005. SEM: Standard Error of the Means

(74) The study of the rumen fermentation parameters from rumen samples collected on days 29 and 30 showed a shift in the fermentation pathways (Table 5) towards a more propionate type profile in the rumen of animals receiving both additives in comparison to the control. As a consequence, in both treatments the acetate to propionate ratio was significantly (P=0.002) reduced. The concentration of ammonia N was similar among treatments and within the range expected for the diet supplied to the animals.

(75) The in sacco degradation study on days 22.sup.nd and 23.sup.rd showed no effect of the additive treatment on the rumen degradability of both alfalfa hay and oats.

(76) TABLE-US-00007 TABLE 7 Effect of the addition of additives 1 and 2 on the concentration (log copy gene numbers/g fresh matter) of total bacteria (16S rRNA), protozoa (18S rRNA) and methanogenic archaea (mcr gene) in the rumen of sheep. The relative abundance (Ct) in relation to total bacteria is also shown for protozoa and methanogens. Control Additive 1 Additive 2 Error P value Total 7.45 * 10.sup.10 9.08 * 10.sup.10 9.74 * 10.sup.10 bacteria log10 10.8 10.9 11.0 0.123 0.607 Total 2.84 * 10.sup.10 1.87 * 10.sup.10 2.51 * 10.sup.10 protozoa log 10 10.4 10.2 10.2 0.212 0.702 Ct 1.65 1.58 1.55 0.267 0.984 Archaea 3.54 * 10.sup.8.sup. 2.86 * 10.sup.8.sup. 2.86 * 10.sup.8.sup. log10 8.54 8.45 8.34 0.133 0.511 Ct 0.028 0.022 0.020 0.005 0.602

(77) Total and relative concentration of the analysed microbial groups in the rumen showed no difference (P>0.05) among treatments. When the abundance of both protozoa and methanogenic archaea were expressed relative to total bacteria the same lack of effect was observed.

(78) Conclusions

(79) The use of both additives resulted in a significant reduction of methane production and, according to the VFA profiles, a shift in the metabolic pathways involved in H.sub.2 transferring was promoted by additives as well. The objective of this trial was to confirm whether the treatment of animals for a month showed a persistence of the results observed over two weeks treatment. This is essential when assessing the suitability of the practical use of a feed additive. In this study both additives showed effect over a month treatment in methane emissions that was further confirmed by a shift in the fermentation pattern.

(80) On the other hand, a change in the fermentation pattern might be not only due to a reduction in methane production but also to a lower fibre degradation which, in turn, would produce less acetate and therefore lowered acetate to propionate ratio. In order to rule out this occurring, a rumen degradability assessment was carried out by incubating nylon bags with both oats and alfalfa hay in the rumen of the animals.

(81) The results showed no such effect on dry matter degradation which is also supported by the same bacterial and protozoa biomass recorded in animals receiving the additives compared to those with no treatment.

Example 17

In Vivo Effect of 3-Nitrooxypropanol in Dairy Cows

(82) Material and Methods

(83) Animals: Six rumen fistulated lactating Holstein X Friesian dairy cows of second or greater parity and weighing from 550 to 800 kg were used for the study. Cows were in mid lactation at the start of the study.

(84) Experimental diets: A single total mixed ration (TMR) diet was provided to all cows throughout the study. Cows were fed ad libitum (5% refusals) for the duration of the trial.

(85) Experimental design: Beginning in mid lactation (with milk yields of 30 liters or more), the six cows were randomly assigned to one of the three supplement treatments in a 33 Latin Square design (Table 8). Treatment periods were 5 weeks in duration.

(86) TABLE-US-00008 TABLE 8 Experimental design Cow Pair 1 Pair 2 Pair 3 Square 1 Square 2 Cow Cow Period 888 Cow 989 Cow 973 1000 Cow 1030 Cow 1060 1 1 2 3 1 2 3 2 2 3 1 2 3 1 3 3 1 2 3 1 2 Diets: 1Control 23-Nitrooxypropanol (500 mg/day) 33-Nitrooxypropanol (2500 mg/day)

(87) Dosing of 3-Nitrooxypropanol or placebo: The doses of 3-Nitrooxypropanol or placebo was administered to the animals via the rumen cannula at feeding time in the morning and evening.

(88) Period Design: As only two cows can be housed in the indirect calorimeters at any one time cows were run in pairs staggered by one week. At the end of week 4 animals were moved to the indirect calorimeters and held in individual tie stalls where four complete 24 hr measurements of respiratory exchange (methane and carbon dioxide production and oxygen consumption) were obtained (Cammell et al., 2000).

(89) Results

(90) Feed Intake: There was no significant effect of the product (3-Nitrooxypropanol) on daily dry matter intake (DMI) (see table 9).

(91) Methane Production: Methane production (liters/d) and methane yield (liters/kg DMI) were significantly reduced by the 3-Nitrooxypropanol. Methane production was 93 and 90% of control values when the 500 and 2500 mg/d doses were given, respectively (see table 9). As regards methane yield, the corresponding values were 96 and 93% of control methane yield, respectively, for the low and high doses.

(92) TABLE-US-00009 TABLE 9 Effects of DSM product fed at two doses. Daily dose, mg/d 0 500 2500 SEM DMI, kg/d 18.9 18.8 18.5 0.7 CH.sub.4, L/d 594 555 536 15.3 CH.sub.4, g/d 425 398 384 11.0 CH.sub.4, L/kg DMI 31.3 29.9 29.2 1.2

(93) Large variations were observed between animals some showing more response that some others. These results show the potential of the compounds of the present invention in reducing methane production in dairy cows, and shed light on further improving the feeding regimen.