Methods of reducing methane emissions from livestock and improving animal productivity

Abstract

The present invention relates to feed additives, specifically a combination of feed additives that can be used to both reduce cattle methane emissions, and also increase animal productivity. This increase in productivity may be achieved through a reduction in both enteric methane emissions and also a reduction in the accumulation of hydrogen in the rumen.

Description

[0001] Methods of reducing methane emissions from livestock and improving animal productivity

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to feed additives, specifically a combination of feed additives that can be used to both reduce cattle methane emissions, and also increase animal productivity, which may be achieved through a reduction in accumulation of hydrogen in the rumen.

[0004] BACKGROUND OF THE INVENTION

[0005] Reducing methane emissions is a critical step in addressing climate change. Methane is a potent greenhouse gas which efficiently traps radiation with a global warming potential 28-fold greater than carbon dioxide in a 100-year horizon (IPPCC AR6, 2022). While the atmospheric lifespan of methane is shorter than that of carbon dioxide, the warming effects of methane are significant. Thus, reducing methane emissions provides a means by which the rate of global warming can be slowed.

[0006] Methane originating from livestock production accounts for around a third of anthropogenic methane emissions, with enteric methane from ruminants, particularly cattle, accounting for the majority of these emissions. Methane is a by-product of fermentation of feeds during digestion, thus representing a loss of energy. Consequently, decreasing methane emissions holds benefits for improving livestock productivity as well as environmental advantages. Feed additives like red seaweed (e.g. Asparagopsis taxiformis), plant extracts i.e. phytogenic feeds, and 3-Nitrooxypropanol (3-NOP) have proven effective in reducing enteric methane emissions when fed regularly as part of an animal's diet. Such feed additives are commercially available. For example, Bovaer™10 (DSM Nutritional Products Ltd., Basel, Switzerland) comprises 10% 3-NOP on a silicate carrier and on average, reduces enteric methane emissions by 30% from dairy cows and 45% from beef cattle. Other feed ingredients based on nitrate containing compounds (Silvair™’ Cargill USA) have also been shown to have a methane mitigating effect when included in ruminant diets. Despite the positive impact of existing feed additives such as Bovaer™10 on enteric methane emissions, the additional energy available through the reduction in losses via methane production (which is between 2 and 12% of gross energy intake lost due to methane production), is not redirected to other metabolic pathways leading to more productivity, i.e. , milk production, in dairy cows. That is, the supplementation of animal feed with Bovaer™10 had no effect on animal productivity.

[0007] Furthermore, hydrogen is the primary substrate for ruminal methanogenesis. Therefore, if methanogenesis is inhibited, without alternative hydrogen sinks, there tends to be an accumulation of hydrogen in the rumen. While there is known to be a negative relationship between methane emissions and milk production due to the effective loss of energy, hydrogen accumulation in the rumen can also disrupt the rumen microbiota and inhibit re-oxidation of NADH, adversely affecting fermentation and conversely leading to reduced feed efficiency. Ultimately, this has the effect of reducing cattle productivity, which can be exhibited through a decrease in milk production in dairy cattle.

[0008] Therefore, it is desirable to find a means of reducing cattle methane emissions and also hydrogen accumulation, to both mitigate the environmental impact of keeping livestock, and to improve livestock productivity. The present invention addresses this need.

[0009] SUMMARY OF THE INVENTION

[0010] In one aspect of the invention, there is provided a feed additive comprising a combination of a methane inhibitor and at least one yeast.

[0011] In an embodiment, the yeast stimulates the production of acetate in the rumen of an animal.

[0012] In one embodiment, the yeast is a live yeast. In one embodiment, the yeast is selected from Saccharomyces cerevisiae, Kluyvermyces species, Pichia species, such as Pichia jadinii (formerly Candida util us) and Trichoderma. In one embodiment, the yeast is Saccharomyces cerevisiae. In one embodiment, the yeast is LEVUCELL SC ® (Saccharomyces cerevisiae CNCM 1-1077) commercially available in animal nutrition as a probiotic to help improve rumen fermentation and stabilise the gut microflora (see “Safety and efficacy of Levucell SC® (Saccharomyces cerevisiae CNCM 1-1077) as a feed additive for calves and minor ruminant species and camelids at the same developmental stage", EFSA J. 2019 Jun 12; 17(6)). The recommended dose rate is 1x10 e10 cfu / cow / day equating to 0.5 g of live yeast cells / cow / day. During in vitro and in vivo rumen incubations this strain has been shown to stimulate acetate production from the rumen microbiota leading to a change in the VFA profile.

[0013] The methane inhibitor can either be a natural or synthetic compound and contain the active ingredients 3-nitrooxypropanol, bromoform, iodoform or other halogenated compounds. In one embodiment, the methane inhibitor is or comprises 3- nitrooxypropanol.

[0014] In a further embodiment, the feed additive may comprise one or more further methane inhibitors such as, but not limited to, nitrate, tannins, essential oils derived from plants, and synthetic essential oils.

[0015] In another aspect of the invention, there is provided a method of reducing enteric methane emissions and maintaining animal productivity, wherein the method comprises administering the feed additive of the invention to the animal / livestock.

[0016] In a further aspect of the invention, there is provided the use of a feed additive of the invention to reduce methane emissions and maintain animal productivity.

[0017] Where the term “administered” is used herein it is also meant fed. Although other routes of administration are included in the scope of the present invention.

[0018] In one embodiment, the animal / livestock is cattle. Alternatively, the livestock is a sheep or a goat.

[0019] In one embodiment, maintaining animal productivity comprises maintaining milk production.

[0020] In a further embodiment, the method further decreases hydrogen accumulation. Alternatively, in another aspect of the invention there is provided a method of decreasing hydrogen accumulation in livestock (particularly in the rumen of livestock). BRIEF DESCRIPTION OF THE FIGURES

[0021] The invention is further described in the following non-limiting figures:

[0022] Figure 1A shows the effect of different yeast alone or in combination with a methane inhibitor on methane inhibition in a high starch diet. Figure 1 B shows the effect of different yeast alone or in combination with a methane inhibitor on methane inhibition in a high fibre diet.

[0023] Figure 2A shows the effect of different yeast alone or in combination with a methane inhibitor on hydrogen accumulation in a high starch diet. Figure 2B shows the effect of different yeast alone or in combination with a methane inhibitor on hydrogen accumulation in a high fibre diet.

[0024] Figure 3A shows the effect of yeast alone or in combination with a methane inhibitor on dry matter disappearance in a high starch diet. Figure 3B shows the effect of yeast alone or in combination with a methane inhibitor on dry matter disappearance (DMD) in a high fibre diet.

[0025] DETAILED DESCRIPTION OF THE FIGURES

[0026] The following embodiments apply to all aspects of the invention.

[0027] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0028] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry and biochemistry, which are within the skill of the art. Such techniques are explained fully in the literature. The object of the invention is to negate the negative effects on rumen fermentation that are observed when rumen methanogenesis is inhibited. As discussed above, these effects may, in part, arise through reducing hydrogen accumulation. In the present invention, it has been found that a combination of a methane inhibitor and at least one probiotic yeast, as described below, can be used as a feed additive to reduce methane emissions, reduce enteric hydrogen accumulation and consequently maintain milk production.

[0029] Accordingly, in one aspect of the invention, there is provided a feed additive comprising a combination of a methane inhibitor and at least one probiotic yeast. Preferably, the probiotic yeast is a live yeast, and more preferably, the yeast is Saccharomyces cerevisiae. Preferably, the feed additive is a livestock feed additive.

[0030] As used herein “a feed composition” may be considered to be a combination of ingredients and their proportions that comprise an animal feed. A feed composition may also be referred to as a nutritional supplement. A nutritional supplement is a composition that can be added to the diet to provide macro and / or micro-nutrients.

[0031] As used herein a “methane inhibitor” is any agent that is capable of inhibiting the ability of rumen-dwelling methanogens to produce methane. In one embodiment the agent is a chemical agent. Suitable examples include, but are not limited to, 3-nitrooxypropanol (3- NOP), nitrate, tannins, bromoform or other halogenated containing compounds, essential oils. In one embodiment, the methane inhibitor is or comprises 3- nitrooxypropanol (3-NOP), as shown below:

[0032] In one embodiment, the feed composition comprises Bovaer™ (3-NOP), and in particular, Bovaer™ 10.

[0033] In one embodiment, the methane inhibitor, such as 3-NOP, is mixed into the animal feed (mineral feed, mash feed or concentrate feed, basal mixed ration) at dosage equivalent to between 60 mg 3-NOP / kg total Dry Matter Intake to 150 mg 3-NOP / kg total Dry Matter Intake. Preferably, the methane inhibitor, such as 3-NOP is added to the animal feed at a dose equivalent to 60 to 90 mg 3-NOP / kg total Dry Matter Intake.

[0034] In one embodiment, the yeast is Saccharomyces cerevisiae. More preferably, the yeast is live.

[0035] In one embodiment, the yeast stimulates acetate production in the rumen. That, is the yeast stimulates the production of acetate by the rumen microbiota leading to a change in the VFA profile. By “stimulates” production, may be meant that the level of acetate produced in the rumen following administration of the feed additive is higher than the level of acetate produced in the rumen without administration of the feed additive of the invention - or following administration of a feed additive comprising a different yeast. Alternatively, the level of acetate in the rumen can be compared to a standard or predetermined level. In one embodiment, acetate production is increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or more. The level of acetate in the rumen of an animal may be determined by collecting a sample of rumen fluid, and analysing the rumen fluid for volatile fatty acids (VFAs) using, for example, gas chromatography. In a further embodiment, the yeast does not simulate or does not significantly stimulate the production of propionate in the rumen.

[0036] In one embodiment of the invention, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Kluyvermyces species, Pichia species, such as Pichia jadinii (formerly Candida util us) and Trichoderma.

[0037] In one embodiment, the yeast is LEVUCELL SC ® (Saccharomyces cerevisiae CNCM I- 1077) commercially available in animal nutrition as a probiotic to help improve rumen fermentation and stabilise the gut microflora. The recommended dose rate is 1x10 e10 cfu / cow / day equating to 0.5 g of live yeast cells.

[0038] In a specific embodiment, the feed additive comprises a combination of 3-NOP at a dose of 60 to 150 mg 3-NOP / kg total Dry Matter Intake, preferably 60 to 90 mg 3-NOP / kg total Dry Matter Intake, and is LEVUCELL SC ® (Saccharomyces cerevisiae CNCM I- 1077), at a dose of 1x10 e10 cfu / cow / day equating to 0.5 g of live yeast cells. In one embodiment, the yeast is added either in the basal ration or delivered within the concentrate feed pellet delivered via the VMS (Voluntary Milking System) or via Out of Parlour Concentrate pellet feeders.

[0039] The feed additive may further comprise additional nutrients, trace elements, minerals, vitamins, feed ingredients, feed by-products, stabilisers, inert carriers, fillers. Typical components of animal feed are cereal grains such as maize, sorghum, wheat, rice, oats, barley, corn and millet; brans, such as wheat bran, maize bran and de-oiled rice bran; protein meals / cakes such as rapeseed meal / cake, soybean meal, cottonseed meal / cake, groundnut meal / cake, coconut meal / cake, palm kernel meal / cake, sesame cake, linseed cake, maize germ oil cake, maize gluten meal, sunflower meal, kardi meal and guar meal; and vitamins and minerals, such as calcite powder, salt, di-calcium phosphate and vitamins A, D3 and E. Preferably, carriers used in the present invention are either corn meal or spring barley and mineral mix.

[0040] In another aspect of the invention, there is provided a method of reducing livestock methane emissions and increasing animal productivity, the method comprising administering the feed additive of the invention to livestock.

[0041] In another aspect of the invention there is provided the use of a feed additive of the invention to reduce methane emissions and increase animal / livestock productivity.

[0042] By a “reduction in methane emissions” is meant a reduction in methane emissions / production by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or more compared to methane emissions from livestock fed with an un-supplemented diet (i.e. where the feed additive of the invention is not added to the animal’s standard feed) or compared to methane emissions from livestock fed a diet supplemented with yeast only or supplemented with a methane inhibitor and a yeast that is not LEVUCELL SC ® (Saccharomyces cerevisiae CNCM 1-1077). Preferably, methane emissions are reduced by more than 30%. The level of methane emitted can be measured using standard techniques in the art. In one embodiment, the level of methane may be reduced in strict anaerobic or aerobic conditions or atmospheric conditions. There are several ways to measure methane emissions from animals known in the art, these include: respiration chambers, the SF6- technique, breath sampling during milking and feeding, the GreenFeed method and use of a laser methane detector.

[0043] The respiration chamber method is the gold standard technique for measuring methane in livestock. A single animal (or more) is confined in a chamber for between 2 and 7 days. Concentration of methane is measured at the air inlet and outlet vents of the chamber. The difference between outlet and inlet concentrations is multiplied by airflow to indicate methane emissions rate. Respiration chambers are available in a variety of materials with different chamber sizes and air flow rates.

[0044] In the SF6 tracer gas technique, air is sampled near the animal’s nostrils through a tube attached to a halter and connected to an evacuated canister worn around the animal’s neck or on its back. A capillary tube or orifice plate is used to restrict airflow through the tube so that the canister is between 50% and 70% full after approximately 24 hours. A permeation tube containing SF6 is placed into the rumen of each animal. The predetermined release rate of SF6 is multiplied by the ratio of methane to SF6 concentrations in the canister to calculate methane emission rate.

[0045] In methods to measure methane concentration in the breath of cows during milking and / or feeding (also known as “sniffer methods”) air is sampled near the animal’s nostrils through a tube fixed in a feed bin and connected directly to a gas analyser. The feed bin may be in an automatic milking station or in a concentrate feeding station. A variety of different gas analysers may be used such as: Nondispersive Infrared (NDIR), Fourier- transform infrared (FTIR) or photoacoustic infrared (PAIR). Methane concentration measured during a sampling visit of typically between 3 and 10 min may be specified as the overall mean, or the mean of eructation peaks. CO2 can be used as a tracer gas and daily methane output can be calculated according to the ratio of methane to CO2 and daily CO2 output predicted from performance of the animal.

[0046] GreenFeed is a method of measuring methane through breath samples, whereby breath samples are provided when animals visit a bait station. Samples of breath from individual animals are taken several times per day for short periods (3 to 7 min). GreenFeed is a portable standalone system used in barn and pasture applications, and incorporates an extractor fan to ensure active airflow and head position sensing for representative breath sampling. The GreenFeed unit is fitted with several different calibrated sensors (CH4, CO2, H2 and O2) to be able to measure enteric emissions of each gas. Measurements are pre-processed by the manufacturer, and data are available in real time through a web-based data management system. As GreenFeed captures a high proportion of emitted air and measures airflow, which can be calibrated using a tracer gas, methane emission is estimated as a flux at each visit. Providing visits occur throughout the 24 h, methane emission can be estimated directly as g / day (Garnsworthy PC, Difford GF, Bell MJ, Bayat AR, Huhtanen P, Kuhla B, Lassen J, Peiren N, Pszczola M, Sorg D, Visker MHPW, Yan T. Comparison of Methods to Measure Methane for Use in Genetic Evaluation of Dairy Cattle. Animals (Basel). 2019 Oct 21 ;9(10):837).

[0047] The laser methane detector (LMD) is a highly responsive, hand-held device that is pointed at an animal’s nostrils and measures methane column density along the length of the laser beam. Typically, animals are restrained either manually or in head yokes at a feed fence for the required length of time. The operator has to stand at the same distance (1 to 3 m) from each animal every time throughout. The LMD can be used in the animal’s normal environment, although for consistency restraint is required during measurement.

[0048] In terms of “animal productivity” this may refer to the level of livestock productivity. By this it is meant the level of productivity of milk, meat or other products. In one embodiment, livestock productivity refers to the level of milk production - also referred to as milk yield. Milk yield can be measured as a volume over a given time. Typically, milk yield is measured as liters produced per day. As used herein “maintaining animal productivity” or “maintaining milk production” is meant that there is no or no statistically significant reduction in milk yield or average volume compared to the milk yield or average volume produced by the livestock that is not fed a feed composition of the invention. This may be at a given time-point - for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or so on of administration of the feed additive of the invention. This can be measured by measuring the fat and protein-corrected milk yield (FPCM) / kg / day each week that the animal is fed (nor not) the feed additive of the invention.

[0049] By a “reduction in hydrogen production or accumulation” is meant a reduction in the levels of enteric hydrogen by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or more compared to the level of enteric hydrogen produced in livestock fed an un- supplemented diet (i.e. where the feed additive of the invention is not added to the animal’s standard feed) or compared to the level of enteric hydrogen produced in livestock where the animal is fed a diet supplemented with a methane inhibitor only or supplemented with a methane inhibitor and a yeast that is not LEVUCELL SC ® (Saccharomyces cerevisiae CNCM 1-1077). Preferably hydrogen production / accumulation is reduced by more than 30% or more than 35% The level of hydrogen production can be measured using standard techniques in the art. Examples include measuring the amount of hydrogen produced when a sample of rumen fluid / digesta (as described below) is supplemented with a control or the feed additive of the invention. Alternative methods of measuring enteric hydrogen accumulation in an animal include use of the GreenFeed system described above.

[0050] In one embodiment, the feed additive is added before, concurrently or following the animal’s normal feed.

[0051] In one embodiment, the method further increases the production of volatile fatty acids (VFAs) in the livestock.

[0052] The invention is now described in the following non-limiting example.

[0053] EXAMPLE 1 Ex vivo model to measure effect of test items on rumen fermentation.

[0054] The aim of the current example was to examine the effect of combining both feed additives (yeast and 3-NOP) together to determine whether it would still be possible to achieve a good rumen fermentation profile with high nutrient digestion yet still to be able to reduce methane emissions and reduce hydrogen accumulation in the rumen.

[0055] As used herein in all examples, “yeast 3” is LEVUCELL SC ® (Saccharomyces cerevisiae CNCM 1-1077) commercially available in animal nutrition, as described above (“Safety and efficacy of Levucell SC® (Saccharomyces cerevisiae CNCM 1-1077) as a feed additive for calves and minor ruminant species and camelids at the same developmental stage", EFSA J. 2019 Jun 12; 17(6)).

[0056] Yeast 1 is an active dry yeast from Saccharomyces cerevisiae, and specifically the strain Y1242, available at the National Collection of Yeast Cultures (NCYC) under reference NCYC 1242. Yeast 2 is an active dry yeast from Saccharomyces cerevisiae, and specifically the strain MLICL 39885 commercially available under Biosprint ®. Yeast 4 is an active dry yeast from Saccharomyces cerevisiae, and specifically the strain CNCM I- 4407, commercially available under Actisaf®. Yeast 5 is an inactive dry yeast of unknown strain. Yeast 1 , 2, 4 and 5 can be characterised as yeasts that stimulate the production of propionate (rather than acetate) in the rumen of an animal (e.g. a cow).

[0057] An ex vivo rumen model was developed. The model was used to test the effect of adding the 5 different commercially available yeast products (4 active dry yeasts and 1 inactive dry yeast) either individually or in combination with a known methane mitigator (3-NOP) versus a control (no supplementation) on gas production (total, methane, H2 and CO2), dry matter disappearance and fibre breakdown, volatile fatty acid production (VFA) and rumen fermentation profile. The dose rates of the test items investigated were based on those recommended by the manufacturer.

[0058] Briefly fresh rumen digesta was mixed with artificial saliva (ratio 1 :2) and 50 ml placed into an incubation bottle under anaerobic conditions to mimic rumen digestion. To each incubation bottle a small feed bag of known weight that had been prepared prior to starting the incubation was added that also contained a known weight of feed. The feed added was approximately 0.7g of either a high starch diet (50% forage: 50% concentrate, starch content 29%, NDF (natural detergent fibre) content 28%) or a high fibre diet (70% forage: 30% concentrate, starch content of 19%, NDF content of 36%). To each bottle the respective treatment was added and then the bottles were capped, mixed, and incubated at 39°C with shaking. At 3, 6 and 24 h the total gas pressure was measured and a small sample of fermentation headspace gas was collected and measured for its percentage composition of CH4, H2, CO2. After 24 h incubation, the fermentation was stopped. Gas production was measured and the headspace gas composition was analysed using a GC calibrated with known gas standards. The feed bags were removed, washed, dried at 60°C for 48 h and then weighed. Dry matter disappearance of the feed was calculated using the following equation:- 100* ((W1 - W2) / W1)) where W1 = Initial weight of feed+feed bag prior to incubation and W2 = Final weight of feed + feed bag) following incubation and drying Ten ml of incubation liquid were removed, acidified and then frozen until later analysis of VFA composition using a GC. Data showed that as expected, including the methane inhibitor in the incubation led to a reduction in methane production and an increase in hydrogen accumulation. Including the yeast products in the incubations led to variable effects on the fermentation profile, gas production and gas composition. Depending on the yeast product used, either an increase in methane emissions, no effect on methane emissions or a decrease in methane was observed, and very little hydrogen was accumulated. When the different yeast products were added in combination with the methane inhibitor, surprisingly, only one combination of the methane inhibitor + yeast 3 led to a reduction in the accumulation of hydrogen. As shown in Figure 2, combining Yeast 3 with the methane inhibitor led to a significant reduction in methane and most notably a reduction in hydrogen accumulation, indicating that hydrogen had been re-channelled.

[0059] Moreover, as shown in Figure 3, the combination of methane inhibitor + yeast 3 led to an increase in dry matter disappearance (DMD) of the High Fibre diet of more than 10% or an increase in DMD of the High starch diet of more than 3% when compared to the respective control incubation. Dry matter disappearance (DMD) refers to a reduction in the dry matter content of feed ingredients after digestion. Essentially, DMD is a measure of how much of the dry matter of a feed is broken down and used by the animal in the digestive system. The DMD is a common measurement to evaluate the effectiveness of a feed additive. A higher DMD indicates better digestibility and nutrient availability.

Claims

CLAIMS:

1. A feed additive comprising a combination of a methane inhibitor and at least one yeast, wherein preferably the yeast stimulates the production of acetate in the rumen of an animal.

2. The feed additive of claim 1 , wherein the yeast is live.

3. The feed additive of claim 1 or 2, wherein the yeast is selected from Saccharomyces cerevisiae, Kluyvermyces species, Pichia species, and Trichoderma.

4. The feed additive of claim 3, wherein the yeast is Saccharomyces cerevisiae, preferably wherein the yeast is LEVUCELL SC ® (Saccharomyces cerevisiae CNCM 1-1077).

5. The feed additive of any preceding claim, wherein the methane inhibitor is a natural or synthetic compound.

6. The feed additive of claim 5, wherein the methane inhibitor is selected from 3- nitrooxypropanol, bromoform, iodoform or other halogenated compounds.

7. The feed additive of claim 6, wherein the methane inhibitor is 3-nitrooxypropanol (3-NOP).

8. The feed additive of any of claims 1 to 7, wherein the feed additive further comprises a carrier, wherein the carrier comprises a cereal grain and / or at least one vitamin.

9. A method of reducing livestock methane emissions and maintaining animal productivity, the method comprising administering the feed additive of any of claims 1 to 8 to the livestock.

10. The method of claim 9, wherein methane emissions are reduced by at least 20%, preferably at least 30% compared to methane emissions in livestock not administered the feed additive of claims 1 to 8.11 . The method of claim 9 or 10, wherein the livestock is a cattle.

12. The method of any of claims 9 or 11 , wherein maintaining animal productivity comprises maintaining milk yield.

13. The method of any of claims 9 to 12, wherein the method reduces enteric hydrogen accumulation.

14. Use of the feed additive of any of claims 1 to 8 to reduce methane emissions and maintain animal productivity.

15. Use of claim 14, wherein the livestock is a cattle.

16. Use of claim 14 or 15 wherein maintaining animal productivity comprises maintaining milk yield.

17. Use of any of claims 14 to 16, wherein the method further decreases enteric hydrogen accumulation.

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