USE OF GOSSYPOL AND A METHANE INHIBITOR FOR REDUCING THE FORMATION OF METHANE EMANATING FROM THE DIGESTIVE ACTIVITIES OF RUMINANTS

Abstract

The present invention relates to the field of reduction of methane emission in ruminants. Particularly, it relates to the administration of gossypol and a methane inhibitor to a ruminant for reducing the production of methane emanating from the digestive activities of said ruminant.

Claims

1. Use of gossypol and a methane inhibitor for reducing the formation of methane emanating from the digestive activities of ruminants, wherein the methane inhibitor is administered to the ruminant in an amount selected in the range from 0.01 to 100 g methane inhibitor/animal/day and gossypol is administered to the ruminant in an amount selected in the range from 0.05 to 100 g gossypol/animal/day.

2. The use according to claim 1, wherein the methane inhibitor is administered to the ruminant in an amount selected in the range from 0.01 to 50 g methane inhibitor/animal/day, most preferably in the range from 0.01 to 25 g methane inhibitor/animal/day.

3. The use according to claim 1, wherein gossypol is administered to the ruminant in an amount selected in the range from 0.1 to 50 g gossypol/animal/day, most preferably from 0.2 to 25 g gossypol/animal/day.

4. The use according to claim 1, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 100 and 0.1, preferably between 75 to 0.25, more preferably between 60 to 0.35, most preferably between 50 to 0.5.

5. The use according to claim 1, wherein the ruminant animal is selected from the group consisting of domestic cattle, most preferably from beef cattle or dairy cows.

6. The use according to claim 1, wherein the methane inhibitor is selected from the group consisting of eugenol, tannic acid, allicin, bromoform, chloroform and propanediol mononitrate.

7. A method for reducing the production of methane emanating from the digestive activities of ruminants, said method comprising orally administering to the ruminant an effective amount of gossypol and a methane inhibitor, wherein the effective amount of the methane inhibitor is selected in the range 0.01 to 100 g methane inhibitor/animal/day and the effective amount of gossypol is selected in the range from 0.05 to 100 g gossypol/animal/day.

8. The method according to claim 7, wherein the effective amount of the methane inhibitor is selected in the range from 0.01 to 50 g methane inhibitor/animal/day, most preferably in the range from 0.01 to 25 g methane inhibitor/animal/day.

9. The method according to claim 7, wherein the effective amount of gossypol is selected in the range from 0.1 to 50 g gossypol/animal/day, most preferably from 0.02 to 25 g gossypol/animal/day.

10. A feed composition or feed additive comprising gossypol and a methane inhibitor, wherein the feed composition is a ruminant feed comprising gossypol in an amount selected in the range from 25 mg to about 10 g gossypol per kg dry matter feed and the methane inhibitor in an amount selected in the range from 1 mg to 5 g methane inhibitor per kg dry matter feed.

11. The ruminant feed according to claim 10, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 100 and 0.1, preferably between 75 to 0.25, more preferably between 60 to 0.35, most preferably between 50 to 0.5.

12. The ruminant feed according to claim 10, wherein the methane inhibitor is selected from the group consisting of eugenol, tannic acid, allicin, bromoform, chloroform and propanediol mononitrate.

13. The feed composition according to claim 10, wherein the feed composition is a mineral premix, a vitamin premix or a bolus.

14. Use of gossypol to enhance the methane reducing properties of a methane inhibitor in ruminants.

15. Use according to claim 14, wherein the molar ratio of gossypol to the methane inhibitor is comprised between 100 and 0.1, preferably between 75 to 0.25, more preferably between 60 to 0.35, most preferably between 50 to 0.5.

Description

EXAMPLES

[0076] In vitro test for methane production: 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.

[0077] Principle: Feed is gadded 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 gas phase produced is measured and put into a formula for conversion.

Reagents:

Mass Element Solution:

[0078] 6.2 g potassium dihydrogen phosphate (KH.sub.2PO.sub.4) [0079] 0.6 g magnesium sulfate heptahydrate (MgSO.sub.4*7H.sub.2O) [0080] 9 ml concentrated phosphoric acid (1 mol/1) [0081] dissolved in distilled water to 1 l (pH about 1.6)

Buffer Solution:

[0082] 35.0 g sodium hydrogen carbonate (NaHCO.sub.3) [0083] 4.0 g ammonium hydrogen carbonate ((NH.sub.4)HCO.sub.3) [0084] dissolved in distilled water to 1 l

Trace Element Solution:

[0085] 13.2 g calcium chloride dihydrate (CaCl.sub.2*2H.sub.2O) [0086] 10.0 g manganese(II) chloride tetrahydrate (MnCl.sub.2*4H.sub.2O) [0087] 1.0 g cobalt(II) chloride hexahydrate (CoCl.sub.2*6H.sub.2O) [0088] 8.0 g iron(III) chloride (FeCl.sub.3*6H.sub.2O) [0089] dissolved in distilled water to 100 ml

Sodium Salt Solution:

[0090] 100 mg sodium salt [0091] dissolved in distilled water to 100 ml

Reduction Solution:

[0092] first 3 ml sodium hydroxide (c=1 mol/1), then 427.5 mg sodium sulfide hydrate (Na.sub.2S*H.sub.2O) are added to 71.25 ml H.sub.2O [0093] solution must be prepared shortly before it is added to the medium solution

Procedure:

[0094] Sample weighing: The feed stuff (i.e. TMR (44% concentrate, 6% hay, 37% maize silage and 13% grass silage) is sieved to 1 mm 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 (3-NOP, 10 ?M). 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, in the amounts as indicated in table 1.

Preparation of the Medium Solution:

[0095] The components are mixed in a Woulff bottle in following order: [0096] 711 ml water [0097] 0.18 ml trace element solution [0098] 355.5 ml buffer solution [0099] 355.5 ml mass element solution [0100] 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.

[0101] Extraction of the rumen liquor: 750 ml of rumen liquor are added to approximately 1,400 ml of medium solution under continued agitation and CO.sub.2-gassing.

[0102] 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 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.

Results

[0103] The food fermented was artificial TMR (44% concentrate, 6% hay, 37% maize silage and 13% grass silage). Gossypol was obtained from Sigma-Aldrich (G8761), and used at a concentration as outlined in table 1. 3-Nitrooxy-propanol (3-NOP) was used at the concentration as outlined in table 1.

[0104] The results are presented in the following Table 1. Clear synergistic effects were obtained for the methane reduction when propanediol mononitrate was combined with gossypol, which translates into additional performance benefit for the animal.

TABLE-US-00001 TABLE 1 Effect on Methane production resulting from the average of four to eight experiments with either gossypol (G), propanediol mononitrate (3-NOP), or combination of both. 3- Total methane change Ratio NOP G [% vs. control] Synergy [G/3- # [?M] [?M] expected* found [%] NOP] 1 (Control) 2 (Positive 10 ?98 control) 3 (Ref) 5 ?54 4 (Ref) 2.5 ?12 5 (Ref) 100 ?69 6 (Ref) 20 ?15 7 (Ref) 10 ?11 8 (Ref) 5 ?1 9 (Inv) 2.5 100 ?81 ?98 +21 40:1 10 (Inv) 5 20 ?69 ?95 +38 4:1 11 (Inv) 2.5 20 ?27 ?46 +70 8:1 12 (Inv) 5 5 ?55 ?90 +63 1:1 13 (Inv) 5 2.5 ?54 ?89 +65 1:2 14 (Inv) 2.5 5 ?13 ?19 +46 2:1 *expected = sum of individual contribution of gossypol and PDMN #synergy = found/expected *100%

TABLE-US-00002 TABLE 2 Correlation of in vitro data to respective dosage/feeding regime 3-NOP G Dosage.sup.? Dosage? # [?M] [?M] [g/animal/d] [g/kg feed] 2 (Positive control) 10 2 0.1 3 (Ref) 5 1 0.05 4 (Ref) 2.5 0.5 0.03 5 (Ref) 100 86 4.3 6 (Ref) 20 17 0.9 7 (Ref) 10 9 0.4 8 (Ref) 5 4 0.2 .sup.?based on correlation of in vitro/in vivo data ?based on the respective average feed intake per animal/day