HK1202780B - Feed compostion for reducing ruminant methanogenesis - Google Patents

Description

Feed composition for reducing methanogenesis in ruminants

Technical Field

The present invention relates to a novel composition for reducing methanogenesis in ruminants.

Background

Methane, carbon dioxide and nitrous oxide are the main gases with greenhouse effect.

Methane (CH4) is a greenhouse gas whose atmospheric concentration has increased dramatically in the last century and is the largest potential contributor to global warming after carbon dioxide. The increase in tropospheric effect methane levels is closely associated with global expansion of the population. Thus, about 70% of methane emissions are considered to be related to human activity. Land filling of waste and agricultural practices generate methane and release it into the atmosphere, the amount of which increases due to the growing number of people in the world.

Ruminants, including cows (cattle), buffalos (buffalo), sheep and goats, have a large forestomach in which fermentation to produce methane occurs. The rumen digestive tract consists of 4 compartments of the stomach: rumen, reticulum, abomasum, and omasum. The largest and most important of them is the rumen. The rumen functions as a fermentation compartment. It contains a large population of microorganisms, including methane-producing archaea, which break down plant material. The microorganisms are commonly referred to as methanogens. The archaeal population utilizes hydrogen and carbon dioxide, i.e., anaerobic microbial fermentation products, to generate energy for growth, thereby producing methane as an end product. Eventually, methane is released from the rumen by burping.

The production of methane by cattle and sheep represents a carbon depletion pathway that reduces productivity. If the energy lost through methane synthesis can be rerouted through other biochemical pathways, typically to propionic acid synthesis, rumen fermentation can become more efficient and can be reflected in animal weight gain or improved milk production. It is cost effective for the producer and provides an effective tool for reducing methane emissions to the atmosphere. Indeed, since the lifetime of methane in the atmosphere is 12 years (whereas the lifetimes of carbon dioxide and nitrous oxide are 100 years and 120 years, respectively), reducing methane emissions can have an increasingly rapid impact on the environment.

Past studies using ruminants have demonstrated that methane production is affected by diet. By increasing the ratio of structural/non-structural (cellulosic/starch) carbohydrates, methane evolution is increased. Furthermore, the addition of a lipid source to the diet reduces intestinal methane emission. Although parallel to the reduction in methane, the high fat supplementation rate reduced rumen microbial fermentation, feed intake and fiber digestibility. A number of chemical feed additives such as antibiotics (i.e., ionophores) or pesticides are introduced into ruminant nutrition to promote growth, improve feed utilization and reduce methane production. However, concerns over the presence of chemical residues in animal products and the development of bacterial resistance to antibiotics have stimulated the search for safer natural alternatives that can be used in organic animal farms.

Plants or plant extracts comprising essential oils, tannins, saponins, flavonoids and many other plant secondary metabolites have been shown to improve ruminal microbial populations targeting specific types of ruminal metabolism. Patra A.K. and Saxenab J (2010), Phytochemistry,71(11-12):1198-222 describe the use of plant secondary metabolites to inhibit methane production in the rumen. The reference WO2005000035 relates to a method for enhancing rumen fermentation, in particular for reducing methane production, consisting of administering a soluble alfalfa extract obtained from fresh alfalfa.

Thus, there is a need for alternative ruminant feed compositions comprising compounds of natural origin and which effectively reduce methane production and are safe for use in animal farms.

Summary of The Invention

The authors of the present invention have now found that by administering a feed composition comprising a natural compound to a ruminant, methane evolution is significantly reduced.

Thus, in one aspect, the present invention relates to a method of reducing methane production in a ruminant animal, the method comprising orally administering to the ruminant animal a feed composition comprising a flavanone glycoside selected from the group consisting of neohesperidin, isochinacoside, poncirin and hesperidin, or a mixture thereof.

In a particular embodiment of the invention, the composition is a mixture comprising neohesperidin and poncirin. In a more specific embodiment, the mixture further comprises naringin. In a preferred embodiment, the mixture is a natural plant extract. In a more preferred embodiment, the plant is a citrus extract.

In a particular embodiment of the invention, the composition further comprises a carrier. In a preferred embodiment, the carrier is sepiolite.

In a particular embodiment, the composition is a mixture comprising 25-55% wt. naringin, 10-20% wt. neohesperidin, 1-5% wt. poncirin and a sufficient amount to 100% wt. of a carrier. In a preferred embodiment, the composition comprises 40-50% wt. naringin, 11-15% wt. neohesperidin, 3-5% poncirin and sufficient amounts to 100% wt. of a carrier.

In a particular embodiment of the invention, the ruminant is a calf (calf), cow (cow), buffalo, sheep, deer or goat. In a preferred embodiment, the ruminant is a calf.

In a particular embodiment, the composition of the invention is added to the feed in solid form at a concentration of 50-1000mg/Kg DM. In a preferred embodiment, the composition is added at a concentration of 200-500mg/Kg DM.

Brief Description of Drawings

Figure 1 shows a profile of biogas and methane production. The mean values used the dose in an "in vitro" simulated system using either unsupplemented quantitation (control) or supplementation with different types of flavonoids.

Detailed Description

As mentioned above, the authors of the present invention have found that by administering to ruminants a feed composition comprising flavonoids, in particular flavanulose glycosides, methane emission is significantly reduced.

Thus, in one aspect, the present invention relates to a method for reducing methane production in a ruminant animal, the method comprising orally administering to the ruminant animal a feed composition comprising a flavanone glycoside selected from the group consisting of neohesperidin, isocoryzanol, poncirin and hesperidin, or a mixture thereof.

The term "ruminant" as used herein refers to a hoofed mammal of any ruminant sub-order (Ruminantia). The mammal chews ruminants and has a stomach with 4 compartments, one of which is the rumen. This category includes deer, antelope, buffalo, cow, sheep, camel and goat.

The term "flavonoid" as used herein refers to a class of water-soluble plant pigments that produce yellow or red/blue pigmentation on petals. The term "flavanones" refers to a type of flavonoid. Flavanones are generally glycosylated with a disaccharide at position 7 to give "flavanuloglycosides".

As shown in the following examples, the inventors have surprisingly found that methane emission is significantly reduced by administering the feed composition of the present invention to ruminants.

Methane production by ruminants can be determined using methods well known in the art. For example, sulfur hexafluoride (SF6) tracer method is a technique that enables methane from individual cows to be measured using evacuated metal gas sampling canisters surrounding the cow's neck that continuously sample the expired air. Other methods include open breathing chambers, which are sealed, and climate controlled chambers, in which individual cows reside, allowing analysis of all the gases produced by the animal.

The released methane can also be determined by infrared spectroscopy, gas chromatography, mass spectrometry and tunable laser diode technology, by an adjunct technology to the fermentation equilibrium based on feed characteristics (e.g. respirometry) predictive equations, isotope tracer technology, etc.

Furthermore, methane production can be measured "in vitro". In this case, rumen fluid is collected from the animal and incubated under anaerobic conditions using an incubation medium.

In a particular embodiment of the invention, the composition is a mixture comprising neohesperidin and poncirin. In a more specific embodiment, the mixture comprises neohesperidin, poncirin and naringin. In another specific embodiment of the invention, the mixture is in the form of a natural plant extract. In a preferred embodiment, the plant extract is a citrus plant extract and more preferably a bitter orange (bitter orange) plant extract, said extract comprising different flavonoids, in particular flavanone glycosides. In a preferred embodiment, the plant extract comprises a mixture of neohesperidin, poncirin and naringin. As shown in the following examples, the plant extract is a natural plant extract comprising about 20% wt. naringin and 40% wt. bitter orange extract (25-27% naringin; 11-13% neohesperidin and 3-5% poncirin). In particular cases, the natural plant extract is commercially available ()。

Thus, according to the present invention, the flavanones of the present compositions may be obtained from plants, more specifically, from citrus plants.

All ingredients in the composition of the invention are products of natural origin and readily available. Furthermore, if the composition is in the form of a mixture, said mixture is easy to handle and can be prepared according to industrial formulation methods well known to the person skilled in the art.

The term "citrus (citrus)" as used herein refers to plants of the genus citrus. Examples of the Citrus plant include pomelo (Citrus maxima) (grapefruit), Citrus (Citrus medica) (lime), mandarin (Citrus reticulate) (tangerine), lime (Citrus aurantium) (bitter orange), bos green (Citrus latifolia) (bos green), lemon (Citrus limon) (lemon), grapefruit (Citrus paradisi) (grapefruit), sweet orange (Citrus sinensis) (sweet orange), trifoliate orange (Citrus trifoliata) (trifoliata), and the like.

Methods for isolating flavonoids from plants are well known in the art. In particular cases, bitter orange extracts can be obtained from ground citrus fruits (in particular lime) by such conventional methods as extraction, filtration, concentration, precipitation, clarification and final drying by the skilled person. The extraction process may be carried out in a glycol/water system, wherein the alcohol is selected from methanol, ethanol, propanol, and the like. Methanol is preferably used. As an alternative non-limiting example, 50g of dried bitter orange is extracted with 300ml of methanol. The suspension was centrifuged to separate the residue and the mother liquor was concentrated in vacuo to a final volume of 50 ml. The resulting liquid was allowed to stand at room temperature during 5 days, filtered to separate insoluble material, filtered again through a bed of celite, and spray dried.

In a particular embodiment, the flavanones may be obtained from the fruit of citrus plants. For example, naringin is a glycosylated flavanone derived from the pericarp of some lemon fruits such as grapefruit and bitter orange (lime). It is also found in the flesh of fruits and in the leaves, flowers and seeds of plants. Exemplary, non-limiting processes for the isolation of flavonoids according to the invention are, for example, those described in the references US2421063A and US2421062A, in which a process for recovering naringin from plant material is described, and furthermore hesperidin can be obtained according to the processes described in the references US2442110A, US2348215A and US 2400693A. Neohesperidin can likewise be obtained as described in the citrus reference US 3375242A. US3375242A describes a process for the production of neohesperidin, in which naringin is reacted with isovanillin to produce neohesperidin chalcone. The chalcone is then cyclized to give neohesperidin.

In addition, the flavanones of the present compositions are readily available because they are commercially available. For example, as shown in the accompanying examples of the present invention, isocoryzanol, neoeritrocin, and poncirin were purchased from inovidie chemical company, inc (usa). Further, as described above, the natural plant extract according to the present invention is commercially available ()。

In a particular embodiment of the invention, the composition is a mixture comprising 25-55% wt. naringin, 10-20% wt. neohesperidin, 1-5% wt. poncirin and a sufficient amount to 100% wt. of a carrier. In a more specific embodiment, the composition comprises 40-50% wt. naringin, 11-15% wt. neohesperidin, 3-5% poncirin and a sufficient amount to 100% wt. of a carrier.

According to another preferred embodiment of the invention, the composition comprises a carrier. In a particular embodiment, the carrier is sepiolite. Sepiolite is a naturally occurring clay mineral of precipitated origin. It is a non-swellable, lightweight, porous clay with a large specific surface area. Chemically, sepiolite is hydrous magnesium silicate, the particles of which each have a needle-like morphology. The high surface area and porosity of this clay accounts for its remarkable absorption capacity for liquids. These properties make it a valuable material for a wide range of applications, such as pet litter, animal feed additives, carriers, absorbents, suspending and thixotropic additives, and thickeners.

According to the method of the present invention, methane evolution/production in ruminants is reduced when animals are fed the present composition comprising flavonoids of natural origin. Feeding efficiency has an economic relevance in the breeding industry. It is known that compounds that inhibit methanogenesis in ruminants result in a shift in rumen fermentation to produce a more desirable fatty acid profile, thereby increasing the proportion of propionic acid rather than acetic acid, whereby rumen-rich viable fermentations become more efficient (see US patent nos. US3,745,221; US3,615,649; and US3,862,333). It is therefore another object of the present invention to provide a method for inhibiting methanogenesis in ruminants which has a beneficial effect on rumen microbial fermentation which increases feed utilization efficiency. As shown in the examples below, the compositions of the present invention reduce the level of methane produced and convert to volatile fatty acid production in favor of propionic acid.

Methods for determining volatile fatty acids are well known in the art. Typically, chromatographic methods are used, such as HPLC or gas chromatography with flame ionization detection.

The feeding mode is not limited to any particular mode and the feed composition of the present invention may be obtained by adding a top dressing to a compound feed or fed after mixing the feed composition of the present invention with a compound feed. Furthermore, there is no limitation on the amount fed, as long as methanogenesis is effectively reduced, while the nutritional balance is not adversely affected.

Thus, in a preferred embodiment of the invention, the composition is added to the feed in solid form. In a particular embodiment, the composition is added at a concentration of 50-1000mg/Kg DM (dry matter). In a more specific embodiment, the composition is added as a solid at a concentration of 200-500mg/Kg DM.

The composition of the invention may comprise other feed ingredients, such as vitamins, enzymes, inorganic salts, cereal flour, protein-containing ingredients, carbohydrate-containing ingredients, wheat meal and/or wheat bran.

The shape of the feed composition of the present invention is not limited to any particular form and may be any conventional feed composition form, such as powder and granules. Furthermore, the feed composition may be produced according to commonly used methods for producing compound feeds and feed supplements.

In a particular embodiment of the invention, the ruminant is a calf, cow, buffalo, sheep, deer, camel or goat. In a preferred embodiment, the ruminant is a calf.

The present invention will now be described in more detail with reference to the following examples, which are not intended to limit the scope of the present invention in any way.

Examples

Materials and methods

The method is carried out using a method based on Theodorou M K et al (1994) Animal Feed Science and Technology,48(3), p.185-197; two assays following the same protocol were designed to investigate the effect of different pure flavonoids on the rumen, according to the protocol described by Mauricio, R.M. et al (1999) Animal Feed Science and Technology 79, 321-330.

The gas production was measured by a semi-automatic manometer, and the correlation between the pressure level and the volume of gas produced was pre-calculated.

Calves receiving mixed quantitative rumen cannulas consisting essentially of concentrate (90:10) were used as a fluid donor for the rumen; the feed composition is shown in table 1.The inoculum was collected and filtered through double-layered surgical gauze and stored in a thermostatted tank. Flavonoids (table 2) +600mg concentrate (table 1) and 60mg barley straw as substrate were administered in triplicate in preheated bottles (39 ℃) and maintained under anaerobic conditions. Flavonoids Isonaringin, neoeritrocin, poncirin were purchased from Indovidi Chemical Company, Inc (USA). 10ml of rumen fluid and 40ml of incubation medium were added to a flask (McDougall, EI (1948) students on ruminal salava.1. the composition and output of sheet's salava.biochem J.43(1) 99-109). Once the bottle was full and anaerobic conditions were applied, the bottle was sealed and the incubation process was initiated in a hot water bath. Pressure readings were taken at 2, 4, 6, 8, 12, 24, 36 and 48 hours. Each sample was incubated in triplicate in two sets or batches.

TABLE 1 chemical composition of concentrate (%)

And (3) CP: crude protein; NDF: a neutral detergent fiber; DM: dry matter, ME: metabolizable energy

20% wt. naringin; 40% wt. bitter orange extract; sepiolite to 100% wt.

Table 2: flavonoid doses (mg/Kg DM) for the first and second experiments

(. about) bioflavonoid complex of citrus plants

After 12 hours incubation, 1 vial from each treatment was opened (repeat), the pH was read and the vials were sampled for volatile fatty acid (Jouany, j.p.,1982 Science des experiments 2, 131. c.c.,1996. appl.biochem.biotechnol.enzyme.eng.biotechnol.56, 49-58) and ammonia (Chaney, a.l., Marbach, e.p.,1962.clin.chem.8,130-132) analysis.

Environmental DNA was extracted using the technique proposed by Yu and Morrison (2004). Quantitative determination of bovine Streptococcus (Streptococcus bovis), Megasphaera elsdenii (Megasphaera elsdenii) and ruminant pseudomonas lunata (selenia ruminants) DNA by qPCR using specific primers (Tajima, k. et al 2001.appl. environ. micro.67, 2766-2774; ouwerkerk, D. et al 2002.J.appl.Microbiol.92, 753-758). The results were statistically analyzed using the PROC MIXED program of the SAS statistical software package (SAS,2000, User's guide: Statistics, 8 th edition inst., Inc., Cary, NC). The least significant difference was used to compare the mean. Differences between mean values where P <0.05 were accepted as significant.

As a result:

1. gas generation:

figure 1 shows the kinetics of gas and methane production when "in vitro" cultures were supplemented with different types and doses of flavonoid rumen fluid. The profile represents the average of two doses. The mean, dose and sampling time for each treatment and statistical analysis of the results are shown in table 3.

The level of gas production increases exponentially with incubation time. Addition of flavonoids significantly altered biogas production (P)<0.05) although this change does not occur uniformly among the different flavonoid species. The new eriocitrin increases gas production levels (266.7 and 253.72P) compared to the control>0.05), naringin was not changed (P)>0.05), while the remaining polyphenols reduced the average production level (P)<0.05). The lowest value corresponds to neohesperidin andmixtures (230.7 and 233.3, respectively). Isonaringin, poncirin and hesperidin (236.6, 238 and 239.6 respectively) also reduced gas production. Inclusion body levels (200 and 500mg/kg) had a significant effect on gas production (P)<0.001) but the effect varies depending on the type of flavonoid. More significant dose effects were obtained with neohesperidin.

The effect of the flavonoid on some of the fermentation activities of the archaeal (archea) population theoretically responsible for methane production was analyzed. Figure 1(b) shows the progress of methane production and table 4 provides the mean values and statistical analysis.

The average methane yield is lower than the total biogas produced. For the control, the methane yield was about 15% of the total gas production. Experimental treatments varied the mean and accumulated methane production, and these changes were different in the treatments: new eriocitrin increased (P <0.05) methane production levels compared to the control. Methanogenic activity was not altered by including hesperidin or naringin in the medium (P < 0.01). However, the neohesperidin, isocoryzanol, poncirin and Bioflavex mixture reduced methane production (P < 0.05). Neohesperidin showed the most significant reduction, a result which is also different from hesperidin (P < 0.05). In general, the dose "by itself" showed no significant difference, except in the case of neohesperidin, where methane production was more reduced by increasing the dose.

The present experimental design allows to determine whether the effect of flavonoids on methane production results from a general reduction in microbial activity and whether biogas production or conversely flavonoids have a specific influence on the methanogenic (archaeal) population. For this purpose, a statistical analysis of the methane contribution in the total gas production is shown in table 5. The presence of flavonoids in the culture medium reduced the methane contribution in the total biogas production (P <0.05), although the effects mentioned above were not uniform.

The inclusion of new eriocitrin (table 5) significantly increased the methane ratio, while the presence of Bioflavex and neohesperidin significantly decreased this ratio (13.70 vs. 13.66 and 14.58 for neohesperidin, Bioflavex and controls, respectively). The remaining polyphenols significantly reduced methanogenic activity, however, the reported differences were not statistically significant. The new eriocitrin and Bioflavex mixture dose (500 vs 200mg/kg DM) tended to inhibit methane production, but the remaining FL-species did not show any effect, which is reflected in a significant interaction of dose x FL-species type (P < 0.05).

In the second test, sepiolite (as filler) and CBC (bioflavonoid complex of citrus plants) were tested with negative (no flavonoids, control) and two positive references (containing flavonoid sources of neohesperidin and Bioflavex). The effect of the excipient (sepiolite) was zero in the gas and methane production project (tables 6 and 7), while CBC reduced gas production moderately, but no change was detected in methane production.

TABLE 3 cumulative production (72h) and mean, dose and incubation time of biogas in "in vitro" rumen fluid medium supplemented with different types of flavonoids.

¹SEM: standard error of mean

²Dosage: 0.2g/kg DM y 0.5g/kg DM substrate

P <0.05, P <0.01, P <0.001 and ns are not significant

The mean values (a, b, c, d) with different indices represent significant differences between these mean values (P < 0.05).

Table 4 average and accumulated methane production (72 hours), dose and incubation time in "in vitro" rumen fluid medium supplemented with different types of flavonoids (treatment).

¹SEM: standard error of mean

²Dosage: 0.2g/kg DM y 0.5g/kg DM substrate

P <0.05, P <0.01, P <0.001 and ns are not significant

The mean values (a, b, c, d) with different indices represent significant differences between these mean values (P < 0.05).

Table 5 methane ratio, dose and incubation time in biogas produced in "in vitro" rumen fluid medium supplemented with different types of flavonoids (treatments).

¹SEM: standard error of mean

²Dosage: 0.2g/kg DM y 0.5g/kg DM substrate

P <0.05, P <0.01, P <0.001 and ns are not significant

The mean values (a, b, c, d) with different indices represent significant differences between these mean values (P < 0.05).

TABLE 6 cumulative production of biogas (72h) and mean, dose and incubation time in "in vitro" rumen fluid medium supplemented with different types of flavonoids [ treated ].

¹SEM: standard error of mean

²Dosage: 0.2g/kg DM y 0.5g/kg DM substrate

P <0.05, P <0.01, P <0.001 and ns are not significant

The mean values (a, b, c, d) with different indices represent significant differences between these mean values (P < 0.05).

TABLE 7 cumulative production of biogas (72h) and mean, dose and incubation time in "in vitro" rumen fluid medium supplemented with different types of flavonoids [ treated ].

¹SEM: standard error of mean

²Dosage: 0.2g/kg DM y 0.5g/kg DM substrate

P <0.05, P <0.01, P <0.001 and ns are not significant

The mean values (a, b, c, d) with different indices represent significant differences between these mean values (P < 0.05).

2. Characterization of rumen fermentation

2.1 concentration of VFA and Ammonia

Volatile Fatty Acids (VFA) and ammonia (N-NH) in ` in vitro ` medium with or without flavonoids (type and dosage concerned)₃) The average concentration of (a) is shown in table 8. (VFA and N-NH)₃) As shown in the first corresponding column after its progression through the incubation time for each flavonoid type and dose. Apparently, Bioflavex showed higher average and cumulative concentration of VFA; however, the difference did not reach statistical significance (P)>0.05). The ammonia level exceeded a threshold level to ensure proper microbial fermentation (50 mg/L). Apparently, the new North American eriocitrin (227.84mg/L) and Bioflavex cocktail (209.92mg/L) showed the highest and lowest mean values, respectively.

The initial VFA concentration [ constant value recorded at t ═ 0 ] increased. The increase between 0 and 12h was higher than the values recorded between 12 and 72h, which reflects a gradual substrate fermentation during the incubation time [ i.e.the average VFA concentration (mmol/L) increased to 2.1 mmol/h over the first period (0-12 h) and after this period these increases dropped to an average of 0.2 mmol/h ]. The increase in VFA concentration is not reflected in an increase in medium acidity, depending on the buffering activity of the mineral mixture. The average pH values were 6.81, 6.77. + -. 0.0034y 6.73. + -. 0.0033 at 0, 12 and 72 hours, respectively. The stability of the medium was confirmed by the standard error of the strict mean values.

2.2 molar ratio of VFA

Media supplementation with a carbohydrate source (consisting primarily of starch; i.e., concentrate) resulted in a significant change in the VFA profile, which resulted in an increase in the ratio of propionic (20.03, 28.20, and 26.45) and butyric (9.07, 9.88, and 10.45 at 0, 12, and 72 hours, respectively), while a decrease in the acetic acid ratio was observed (mol/100 mol; 62.5, 55.86, and 55.86). However, the increase is not uniform among the different flavonoid types. The propionic acid ratio in the culture medium was improved over the control due to naringin, isonaringin, poncirin, Bioflavex mixture and neohesperidin, but the rest was unchanged. It should be noted that in neohesperidin, naringin and Bioflavex, the response to incubation time was also significantly dose-regulated (D x H: P < 0.009). In general, a negative correlation was observed between methane production (table 5) and propionic acid ratio (table 9), containing neoeriocitrin increased the methane ratio, while the opposite results were exact in the case of neohesperidin and Bioflavex, which apparently inhibited methane evolution (13.70 and 13.66 and 14.58 for neohesperidin, Bioflavex and control, respectively), thereby improving the propionic acid ratio (25.7 and 25.8 and 24.4(P <0.1 and 23.7(P <0.05), respectively, for neohesperidin, Bioflavex and control and neoeritrocin propionic acid).

TABLE 8 volatile fatty acid concentration (VFA; mmol/l) and ammonia (N-NH 3; mg/l) in rumen fluid cultures not supplemented (control) or supplemented with different types and doses of flavonoids.

¹SEM: standard error of mean

²Dosage: 0.2g/kg DM y 0.5g/kg DM substrate

P <0.05, P <0.01, P <0.001 and ns are not significant

The mean values (a, b, c, d) with different indices represent significant differences between these mean values (P < 0.05).

2.1. Lactic acid concentration and microbial profile.

The correlation between ruminal lactic acid concentration and acidotic dysfunction has been experimentally confirmed. The lactic acid concentration values and the bacterial titers for lactic acid production (s.bovis) or consumption (s.ruminants, s.lunantium and megasphaera elsdenii (m.elsdenii)) from the 12 hour incubated bottles are shown in table 10.

The effect on lactic acid concentration using different flavonoids was modest and the mere presence of neohesperidin, hesperidin and Bioflavex tended to mitigate the increase recorded over the incubation period ([ c ] t ═ 0: 22.16 mg/l). The previously described changes in fermentation conditions (tables 8 and 9) resulted in an increase in microbial DNA concentration, however, when compared to control, neoeriocitrin, poncirin and hesperidin titers, the increase was only statistically significant in the case of neohesperidin. The treatment of this experiment did not alter the streptococcus bovis and ruminant selenomonas lunatus titers, however, according to the results obtained from the previous experiment, both the neohesperidin and Bioflavex mixture improved the recorded megasphaera elsdenii titers compared to the recorded control values.

Table 10: effect of flavonoids in lactic acid concentration and bacterial DNA concentration (mg/ml) as determined by qPCR and relative quantification of ruminal populations of ruminant selenomonas ruminalis, streptococcus bovis and megasphaera elsdenii at 12 hour incubation in "in vitro" rumen fluid cultures either unsupplemented (control) or supplemented with different types and doses of flavonoids.

¹SEM: standard error of mean

P <0.05, P <0.01, P <0.001 and ns are not significant

The mean values (a, b, c, d) with different indices represent significant differences between these mean values (P < 0.05).

Claims (13)

1.A method for reducing methane production in a ruminant animal comprising orally administering to the ruminant animal a feed composition comprising a flavanone glycoside selected from the group consisting of neohesperidin, isocoryzanin, poncirin, hesperidin and mixtures thereof.

2. The method of claim 1, wherein the feed composition is a mixture comprising neohesperidin and poncirin.

3. The method of claim 2, wherein the mixture further comprises naringin.

4. The method of claim 1, wherein the mixture is a natural plant extract.

5. The method of claim 4, wherein the plant extract is a citrus extract.

6. The method of any one of claims 1-5, wherein the composition further comprises a carrier.

7. The method of claim 6, wherein the composition is a mixture comprising 25-55% wt. naringin, 10-20% wt. neohesperidin, 1-5% wt. poncirin and a sufficient amount to 100% wt. of a carrier.

8. The method of claim 7, wherein the composition comprises 40-50% wt. naringin, 11-15% wt. neohesperidin, 3-5% wt. poncirin and a sufficient amount to 100% wt. of a carrier.

9. The method of claim 6, wherein the carrier is sepiolite.

10. The method of claim 1, wherein the ruminant is a calf, cow, buffalo, sheep, deer, camel, or goat.

11. The method of claim 10, wherein the ruminant is a calf.

12. The method of claim 1, wherein the composition is added to the feed in solid form at a concentration of 50-1000mg/Kg dry matter.

13. The method according to claim 12, wherein the composition is added to the feed in solid form at a concentration of 200-500mg/Kg dry matter.