AU2022100024B4 - Improvements to devices and methods for delivery of substances to animals - Google Patents

Abstract

The present invention relates to bolus devices for the improved delivery of hydrophobic substances to ruminant animals. In particular, hydrophobic substances to be delivered include haloforms such as bromoform, for use to inhibit methane production in ruminants. The bolus comprises a core comprisingthe haloform and a carrier such as paraffin waxes, beeswax, carnauba wax or castor wax. The core is surrounded by a housing made from a plastic material such as poly lactic acid (PLA), poly glycolic acid (PGA), poly lactic glycolic acid (PLGA), polypropylene, SLA polymer, ABS, or a combination thereof.

Description

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IMPROVEMENTS TO DEVICES AND METHODS FOR DELIVERY OF SUBSTANCES TO ANIMALS

Field of Invention

[0001] The present invention relates to improvements in devices and methods for delivery of

substances to animals, and in particular to devices and methods for administering at least one advantageous substance to an animal, and methods of manufacturing the devices.

Background to the Invention

[0002] In farming it is often necessary to deliver substances to animals. This can be for any of

various purposes, including but not limited to treatment or prevention of disease and to

increase animal production.

[0003] There are various devices and methods to deliver substances such as medicament to animals. However, one class of compounds that are difficult to deliver to animals are hydrophobic compounds. The properties of these compounds present challenges to developing

technology for the controlled release of these hydrophobic substances, particularly via an

animal's stomach.

[0004] One specific purpose to administer substances to animals is to reduce the adverse

effects of agriculture. For instance, various methane and nitrification inhibitors are known to be administered to animals to reduce or mitigate the adverse effects of the methane and nitrate

containing compounds produced by the animals.

[0005] However, despite current efforts, climate change is creating a wide range of

environmental and social impacts globally. It is widely understood that these impacts will only continue to increase over time. As a result, there has been a global push to reduce harmful

greenhouse gas (GHG) emissions in an effort to avoid the worst effects of climate change.

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[0006] The agricultural sector is considered to be a major source of GHG emissions. Total emissions of methane from global livestock accounts for an estimated 7.1 gigatons of C0 2

equivalent per year, representing 14.5% of all anthropogenic GHG emissions. Therefore, this

sector will play a key role in reducing overall GHG emissions.

[0007] The main GHGs released by agriculture are methane (CH 4 ) and nitrous oxide (N 2 0), with the main source of methane emission attributed to livestock. Most methane is emitted when

cattle burp. The amount of methane produced for each farm is directly related to the total animal feed intake.

[0008] Countries which have a strong agricultural sector such as New Zealand, face challenging

goals of reducing agricultural emissions. For instance, the New Zealand government has

introduced policies aimed to reduce methane emission by 24-50% before 2050. In New Zealand livestock methane production is estimated to comprises as much as half of the country's total

GHG emissions. The reduction of methane is a critical component of meeting targets for emissions of GHGs and reducing the effects of global warming.

[0009] Release of GHGs by animals also has adverse effects on animal productivity. Any feed

that is converted to a compound which is subsequently expired or released by the animal is an energy source that has not been converted to a productive use. Accordingly, for efficiency, it is

important to optimise conversion of feeds into animal productivity in the form of weight gain or

milk production.

Object of the Invention

[00010] It is an object of the present invention to provide improved devices and methods to deliver substances to an animal, e.g. hydrophobic substances and / or methane inhibitors.

[00011] It is an object of the invention to provide devices and methods to reduce

emission of GHGs.

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[00012] It is an object of the invention to provide devices and methods to improve or

optimise animal productivity.

[00013] Alternatively, it is an object of the invention to provide devices and methods to improve animal production gains e.g. through reduction of methane production.

[00014] It is an object of the invention to provide a formulation to reduce emission of

GHGs by one or more animals e.g. a ruminant animal.

[00015] It is an object of the invention to provide devices and methods that can release substances at different rates over a period of time.

[00016] Alternatively, it is an object of the invention to provide methods of manufacturing devices to deliver substances to an animal e.g. substances to reduce emission of

GHGs.

[00017] Alternatively, it is an object of the invention to overcome some of the

disadvantages of the prior art.

[00018] Alternatively, it is an object of the present invention to provide the public with a

useful choice.

Summary of the Invention

[00019] According to one aspect of the invention, there is provided a bolus configured

for administration to an animal, wherein said bolus is configured to release a hydrophobic substance to the animal over a period of time.

[00020] According to one aspect of the invention, there is provided a bolus for

administration to a ruminant animal, wherein said bolus is configured to release an effective amount of the substance.

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[00021] According to a further aspect of the invention, there is provided a method for

reducing emission of gas from a ruminant animal, the method comprising the step of

administering to said ruminant animal a bolus comprising at least one inhibiting agent.

[00022] According to another aspect of the invention, there is provided a use of a methane inhibitor and a carrier in a bolus for reducing methane production in a ruminant

animal.

[00023] According to another aspect of the invention, there is provided a use of a methane inhibitor and a carrier in a bolus for reducing methane emission from a ruminant

animal.

[00024] According to another aspect of the invention, there is provided a use of a

haloform in the manufacture of a bolus for reducing the emission of one or more greenhouse gases ("GHGs") from a ruminant animal.

[00025] In a preferred embodiment, the bolus may be configured to be administered to a

ruminant, the ruminant may include beef or dairy cows, sheep, goats, buffalo, deer, elk, giraffes or camels.

[00026] In one embodiment, the bolus may be adapted to reduce the release of one or more greenhouse gases ("GHGs") from the ruminant.

[00027] In another embodiment, the bolus may be a slow-release bolus, configured to

release the at least one inhibiting agent in the ruminant animal over a period of time e.g. in the animal's rumen.

[00028] According to a further aspect, there is provided a bolus for administration to a ruminant animal, wherein the bolus comprises:

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a core, wherein the core includes at least one substance to be administered to the ruminant animal mixed with a carrier; and

a housing which covers at least a portion of the core;

wherein, the bolus is configured to release the substance through the housing over a period of time.

[00029] In another aspect of the invention, there is provided a bolus comprising

a core which contains a substance to be administered to an animal, and a housing which at least partially covers a portion of the core;

wherein the housing is formed from at least one polylactic acid (PLA).

[00030] In a further aspect of the invention, there is provided a bolus comprising

a core, wherein the core comprises a mixture of at least one wax and a haloform.

[00031] The inventors have surprisingly found that the technology described herein may provide a number of benefits. These benefits may be the result of the unique synergistic

interactions between different aspects of the technology. The technology of the present

invention is therefore described based on the inventor's current understanding of these interactions. It should be appreciated any aspect described herein, or the interaction of two or

more aspects, may form a distinct invention.

[00032] Throughout the present specification reference will be made to the term "substance" or "substance to be administered to an animal". This should be understood as

meaning any substance which provides benefits to the animal e.g. a drug for treatment or

prevention of disease, which improves animal productivity, or mitigates at least one adverse effect of agriculture.

[00033] In preferred embodiments, the substance may be hydrophobic substance.

[00034] In particularly preferred embodiments the hydrophobic substance may be an

inhibiting agent. Reference will be made herein to the substance as an inhibiting agent.

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However, this should not be seen as limiting on the scope of the present invention and alternatives are envisaged for the e.g. it may be a hydrophilic substance.

[00035] In an embodiment, the at least one inhibiting agent may be a methane inhibitor.

The use of a methane inhibitor may provide a number of advantages. For instance, a methane inhibitor will reduce, or eliminate, production of methane by the ruminant e.g. in the rumen. As a result, there is less methane in the rumen which could be emitted by the ruminant and

therefore emission of GHGs are effectively reduced.

[00036] In addition, reducing production of methane may provide animal production

benefits. For instance, reduction of methane ensures that relatively more of the feed ingested is available for digestion and conversion into protein (either milk or meat). As a result, farmers

may be able to improve efficiency by either securing greater productivity for a given feed volume or reduce feed accordingly.

[00037] In an embodiment, the methane inhibitor may be a haloform.

[00038] In a preferred embodiment, the methane inhibitor may be selected from the list of chloroform, bromoform, iodoform, or combinations thereof.

[00039] In a particularly preferred form, the haloform may be bromoform (CHBr 3 ). The

use of bromoform may provide a number of advantages. For instance, t has a high efficacy for a

relatively small dose, which enables one device to deliver sufficient amounts of the inhibiting agent over an extended period of time. In addition, bromoform also has a relatively high

density which adds to the overall weight of the bolus and allows for the bolus to be retained in the rumen i.e. it sinks to the ventral part of the rumen rather than floats as this can reduce

regurgitation.

[00040] However, despite these advantages the inventors have faced a number of

challenges and problems to developing a bolus for the controlled release of a haloform, particularly bromoform, to a ruminant.

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[00041] In a further embodiment, the bolus may comprise a core.

[00042] The core may be formed by the inhibiting agent mixed with a carrier.

[00043] However, in alternative embodiments, the inhibiting agent may be provided in a

substantially pure form e.g. is not mixed with a carrier.

[00044] In embodiments, the carrier may have a structure which promotes or facilitates affinity for the carrier by the inhibiting agent. For instance, the carrier may have polar

functional groups.

[00045] In embodiments, the carrier may be a relatively polar substance e.g. it has a

relatively high %w/w of polar functional groups. The inventors have surprisingly found that the carrier and the inhibiting agent can interact with each other, and the interaction can affect the

release rate of the inhibiting agent from the bolus. This aspect of the invention should become clearer from the following description.

[00046] Examples of suitable functional groups for the carrier to include are ester, fatty acids, fatty alcohols, carbonyls and fatty amines. Without being limited to a specific mechanism,

the inventors believe that the inhibiting agents may interact with polar functional groups in waxes, potentially via creation of hydrogen bonds. The amount of polar functional groups

present in the carrier will affect the affinity of the carrier and the inhibiting agent for each

other.

[00047] The inventors have found that a range of substances may be suitable for use as a

carrier in the present invention. For instance, the carrier may be selected from the list of waxes, myristic acid, stearic acid, steryl alcohol, cetyl alcohol, cetosteryl alcohol or a combination

thereof.

[00048] In a particularly preferred embodiment, the carrier may be a waxy substance.

For example, the carrier may be selected from the list of bee's wax, paraffin wax, PEG4000, Carnauba, castor wax, Candellila, Jojoba, or Lanolin or a combination thereof.

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[00049] In another embodiment, the carrier may comprise a mixture of two or more

components. For example, the carrier may comprise a mixture of at least one relatively polar

substance with a relatively non-polar substance. For instance, in some forms the carrier may include a mixture of paraffin wax (a mixture of alkanes with no polar functional groups) and

castor wax and / or carnauba wax (which have a relatively high amount of polar functional groups). As a result, the overall polarity of the carrier may be adjusted to achieve a required

affinity for the inhibiting agent. This can be used to achieved a desired release rate for the inhibiting agent.

[00050] Additionally to the above, solid carriers such as powdered activated carbon, zeolite or bentonite may also be used a carrier. Accordingly, the discussion herein should not be

seen as limiting on the scope of the present invention.

[00051] In a further embodiment, the carrier may also include one or more additional

components. For example, additional components such as elemental zinc or zinc oxide may be incorporated. The additional components may be used to achieve a desired density for the core

and / or bolus.

[00052] It should also be understood that additional components may be added to a

cavity of the bolus separate to, and not mixed with, the carrier. This may be particularly beneficial where the additional components are not required to form a core with a desired

release profile, but where the density of the bolus needs to be adjusted to a desired amount.

[00053] Other suitable additives for incorporation into the carrier may also include

colloidal silicon dioxide, charcoal, bentonite and zeolite(s).

[00054] Further aspects of the carrier and its affect on the release of the inhibiting agent

from the bolus, together with the interaction of the carrier and housing, should become clearer from the following description.

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[00055] In a preferred embodiment, the carrier may have a melting point between substantially 50-90°C.

[00056] In a particularly preferred embodiment, the carrier has a melting point which is less than the boiling point of the inhibiting agent. This may be useful as the carrier can be

melted and mixed with the inhibiting agent without substantial loss of the inhibiting agent due to evaporation.

[00057] In a preferred embodiment, the core may have a melting point greater than

370 C.

[00058] In a particularly preferred embodiment, the core may have a melting point

greater than 40°C.

[00059] The melting point of the core may be beneficial to the function of the present

technology in several ways. For instance, having a melting point above 370 C, and more preferably 40 0C, can assist the carrier in stabilising the inhibiting agent when the bolus is in the

rumen. This could be beneficial to control release of the inhibiting agent e.g. movement of the inhibiting agent through the material forming the housing.

[00060] In an embodiment, the bolus may be adapted to reach a maximum release rate

of approximately 0.05g to 2g of bromoform per day into the rumen.

[00061] In a particularly preferred embodiment, the bolus may be adapted to reach a

maximum release rate of approximately 0.1 to 0.5g of bromoform per day into the rumen.

[00062] The inventors have found that the rate of release of the inhibiting agent into the

rumen increases overtime. This may be the result of several factors. Therefore, the rate of release starts from zero on administration of the animal and increases to a maximum. However,

the foregoing should not be seen as limiting, and other release rates are envisaged as within the scope of the present invention.

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[00063] In a further embodiment, the bolus may include a housing.

[00064] Throughout the present specification, reference to the term "housing"should be

understood as meaning a structure which can receive and hold a core containing the at least one inhibiting agent.

[00065] In preferred embodiments, the housing comprises a body which has a cavity in

which a core is located.

[00066] However, it should also be understood that the housing may take other forms. For instance, the housing may include two or more cavities which can each receive and hold a

separate core.

[00067] In one embodiment, the housing may include an open end.

[00068] The bolus may be used with an open end e.g. administered to an animal with the end open. As a result, in these embodiments the open end provides an opening to in use

expose the contents of the core to fluids in the rumen.

[00069] In yet a further embodiment, the housing may completely cover and surround

the core e.g. it has a sealed cavity in which the core is located.

[00070] For instance, the bolus may include a housing with a cavity in which at least a

portion of the core can be located, and an open end to facilitate insertion of the core into the

cavity. A cap can be used to cover the open end.

[00071] The cap may be formed separately of the housing and releasably or permanently

secured thereto. Alternatively, the cap may be formed integrally to the housing.

[00072] In yet a further embodiment, the housing may be provided in at least two-parts, each of which has a cavity to receive a respective portion of the core. Together the at least two parts completely surround the core and define a closed and sealed cavity in which the core is

located.

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[00073] In yet further embodiments, the housing may be formed around the core e.g. by

moulding. Alternatively, the housing and cap may together define a substantially closed and

sealed cavity in which the core is located.

[00074] The inventors believe that the provision of a substantially or completely closed and sealed cavity can assist in achieving a desired controlled release of the inhibiting agent

from the bolus of the present invention. For instance, in such an embodiment, the inhibiting

agent must pass through the material forming the housing e.g. by mass diffusion.

[00075] In embodiments, the housing may be configured to have sufficient structural integrity to remain intact for a predetermined period of time.

[00076] In a preferred embodiment, the housing may be configured to degrade over a

predetermined period of time.

[00077] Throughout the present specification, reference to the term "predetermined period of time" should be understood as meaning the period of time over which the inhibiting

agent is to be released to the animal.

[00078] In a particularly preferred embodiment, the predetermined period of time may be at least two months, preferably six months, and more preferably 12 months.

[00079] The inventors have surprisingly found that housings of the present invention

may assist with the controlled release of the inhibiting agent. For instance, the housing is able to withstand the conditions in the rumen for the predetermined period of time. During this

time, the housing protects the core from fluid in the rumen, yet can facilitate or contribute to the controlled release of the inhibiting agent. However, the design of the housing may allow

the housing to disintegrate or degrade over the predetermined period of time. This can contribute to mitigating adverse effects of device administration to an animal, and could also

ensure that an animal can be treated with multiple bolus e.g. a second bolus is administered at or towards, or after, the end of the predetermined period of time.

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[00080] In embodiments of the invention, the thickness of the housing may be selected to contribute to the rate of release of the inhibiting agent. For instance, the inventors have

identified that thickness of the housing can affect the rate of release of the inhibiting agent

from the bolus. In these embodiments, a relatively thicker housing will have a relatively slower release rate than a relatively thinner housing.

[00081] In a preferred embodiment, the housing may have a thickness of at least 1mm.

[00082] In yet a further preferred embodiment, the housing may have a thickness of less

than 3 mm.

[00083] In yet another preferred embodiment, the housing may have a thickness of

between 1.5 to 2 mm.

[00084] The thickness of the housing may be particularly important for achieving a

desired controlled release for the inhibiting agent in embodiments such as those where the

core is entirely encapsulated by the housing. This should become clearer from the following discussion.

[00085] In an embodiment, the dimensions of the cavity may vary along the length of the

housing.

[00086] In a preferred embodiment, the cavity includes at least two regions which have a

different cross-sectional area to each other e.g. a first region having a first cross-sectional area and a second area having a second cross-sectional area.

[00087] In a particularly preferred embodiment, the first region has a relatively smaller

cross-sectional area and the second region has a relatively larger cross-sectional area.

[00088] In yet a further preferred embodiment, the first region may be located closer to the open end than the second region.

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[00089] Having a cavity with regions having different cross-sectional areas to each other

may facilitate more controlled release of the inhibiting agent(s) to better meet an animal's

requirements. For instance, a relatively smaller across-sectional area can be provided closer to

the open end to deliver a relatively smaller dose of the inhibiting agent(s), whereas the relatively larger cross-sectional area may be provided closer to the distal end; this may be

useful where the dose of the inhibiting agent needs to increase over time e.g. due to animal growth.

[00090] It should also be understood that the reverse arrangement may be provided e.g.

the relatively larger cross-sectional area is provided closer to the open end and the relatively smaller cross -sectional area may be provided closer to the distal end. This arrangement may be

useful where an initially higher dose of the inhibiting agent(s) is required, to be followed by a

subsequently smaller dose at a subsequent time. For instance, this arrangement may be used where an animal has a high demand for the inhibiting agent e.g. at periods of relatively high

feed intake and energy requirements such as during milking but to be followed by a period of relatively low feed intake e.g. during the dry-period.

[00091] Furthermore, it should be understood that the cross-sectional area of the cavity

may increase gradually and continuously from the first region to the second region e.g. there is no defined "step" between the first region and the second region.

[00092] In other embodiments, the housing may include a third region having a third cross sectional area. This may be further used to control the dose of the inhibiting agent(s) to

the animal. Accordingly, the foregoing should not be seen as limiting on the scope of the present technology.

[00093] In an embodiment, the thickness of a wall of the housing may vary along the

length of the housing. In such an embodiment, the wall thickness at or towards one end of the housing may be thicker than at the distal end. For example, the thickness of the wall at or

towards the open end may be thinner in size than that of the distal end.

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[00094] This arrangement may be particularly beneficial in assisting to control release of the inhibiting agent(s) over time. For instance, the relatively thinner wall(s) will degrade

relatively quicker than the relatively thicker wall(s). This structure can be used to control the

rate of degradation of the housing along its length. For instance, it may be used to ensure that the open end is the only site at which fluids in the rumen are able to come into contact with,

and erode, the core.

[00095] In preferred embodiments, the housing made be made from a material through

which the inhibiting agent can migrate in use e.g. by a mass diffusion process.

[00096] In a preferred embodiment, the housing may be made from at least one plastic material. For instance, the housing may be made from a degradable plastic or material that

degrades over time in the rumen.

[00097] In a particularly preferred embodiment the housing may be made from a material selected from the list of one or more of poly lactic acid (PLA), poly glycolic acid (PGA),

poly lactic glycolic acid (PLGA), polypropylene, Polycaprolactone (PCL), poly(d-lactic acid) (PDLA), Polybutylene succinate (PBS), Polybutylene adipate terephthalate (PBAT), SLA polymer,

ABS, or a combination thereof.

[00098] In addition, the housing may also be made from a non-biodegradable material, such as EVA, silicons, acrylates etc. As a result, the discussion herein should not be seen as

limiting on the scope of the present invention.

[00099] In addition, the material from which the housing is made may include one or

more other compounds e.g. plasticisers, hardeners, colourants etc.

[000100] However, in alternate embodiments, the housing may be made from one or

more non-adsorbent materials i.e. a material into which, or through which, the inhibiting agent does not migrate. Using a non-absorbent material for the housing can assist with controlling

the rate of release of the inhibiting agent(s) in certain embodiments such as an open-ended bolus. For instance, in these embodiments, the concentration of the inhibiting agent(s) in the

core is not decreased by their absorption into the housing material.

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[000101] In some embodiments, the bolus may include a barrier layer. In these

embodiments, the barrier layer may be positioned between at least a portion of the core and

the housing. For instance, the barrier layer can minimise, or completely prevent, contact between the portion of the core and the housing. This can be useful to prevent dissolution of

the inhibiting agent (or other compounds) to better control the release of the inhibiting agent(s) and improve the stability of the device. This could be particularly useful where the

inhibiting agent(s) has a high solubility in the material(s) from which the housing is made.

[000102] Alternatively, in an embodiment where the barrier layer is provided between only a portion of the core and the housing, it may reduce but not completely prevent, migration

of the inhibiting agent into the housing. In effect, the barrier layer reduces the contact area

between the core and the housing and so therefore may reduce the release rate of inhibiting agent than were the barrier layer not provided.

[000103] Alternatively, the bolus may not include a barrier layer. This configuration may be useful where the inhibiting agent(s) has a relatively low solubility in the material from which

the housing is constructed. It may also be useful where the composition of the housing and / or

carrier are selected to control the release rate e.g. The rate of diffusion of the inhibiting agent through the housing.

[000104] In another embodiment, the bolus may be adapted to have rates of dissolution of the core and the housing which provide substantially uniform dissolution of both

components in the rumen overtime.

[000105] In one embodiment, the cavity in the housing may provide a reservoir configured to receive an amount of the inhibiting agent(s). For instance, the reservoir may be a closed

cavity in the housing which can receive and hold the amount of the inhibiting agent.

[000106] In one embodiment, the bolus may include a dispensing mechanism.

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[000107] Inone embodiment, the carrier may have a relatively higher affinity for the

inhibiting agent compared to the affinity of the housing for the inhibiting agent. As discussed

elsewhere in this document, this may be achieved by the relative polarity of the substances

forming the carrier and the housing, and matching these materials appropriately to the inhibiting agent.

[000108] In another embodiment, the housing may be formed from a substance having a

Shore D hardness of at least 40. In such an embodiment, it is believed that having a housing with a lower Shore D hardness of 40 to result in a bolus that is too soft, which could hinder

administration of the bolus to an animal or lead to it being otherwise damaged or prematurely degraded before the full amount of inhibiting agent is administered.

[000109] In a further embodiment, the housing may be formed from a substance having a Shore D hardnessofless than 80.

[000110] In another embodiment, the housing may be configured to facilitate the controlled release the inhibiting agent from the core. Without being limited to a specific

mechanism, the inventors postulate that the inhibiting agent may be released through the

housing by the mechanism of mass diffusion.

[000111] At present, it is understood that controlled release of the inhibiting agent

through the housing may be influenced by a number of factors. For example, the affinity of the inhibiting agent for the carrier may play a role in the diffusion of the inhibiting agent through

the housing. It is understood that more polar carriers or carriers containing a high degree of polar functional groups will have a higher affinity with the inhibiting agent than less polar

carriers or carriers with a lower degree of functional groups.

[000112] The relative affinity of the materials forming the housing and the core for the inhibiting agent may also affect controlled release of the inhibiting agent from the core. For

example, having a housing with a relatively lower affinity for the inhibiting agent compared to the affinity of the carrier for the inhibiting agent, could be a factor in controlling the rate of

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release of the inhibiting agent from the core. These aspects of the invention should become clearer from the description herein.

[000113] Throughout the present specification, reference to the term "release mechanism"should be understood as meaning an arrangement to release a predetermined

amount of the inhibiting agent (s) over time. For instance, the release mechanism may comprise a valve arrangement which can release an amount of the inhibiting agent(s) via an

outlet. Alternatively, the release mechanism may be a syringe-type mechanism having a plunger and actuator; over time, the actuator moves the plunger in the reservoir to drive the

inhibiting agent(s) out of the reservoir.

[000114] Further aspects of the invention, which should be considered in all its novel

aspects, will become apparent to those skilled in the art upon reading of the following description which provides at least one example of a practical application of the invention.

Brief Description of the Drawings

[000115] One or more embodiments of the invention will be described below by way of

example only, and without intending to be limiting, with reference to the following drawings, in which:

[000116] Figure 1A is a front view of a bolus in accordance with one aspect of the invention.

[000117] Figure 1B is a perspective cross sectional view of the bolus of Figure 1A.

[000118] Figure 2A is a front view of an alternative embodiment of a bolus in accordance

with a further aspect of the invention.

[000119] Figure 2B is a perspective cross sectional view of the bolus of Figure 2A.

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[000120] Figure 3A is a front view of an alternative embodiment of a bolus in accordance with a further aspect of the invention.

[000121] Figure 3B is a perspective cross sectional view of the bolus of figure 3A.

[000122] Figure 4A is a front view of an alternative embodiment of a bolus in accordance with a further aspect of the invention.

[000123] Figure 4B is a perspective cross sectional view of the bolus of Figure 4A.

[000124] Figure 5A is a front view of an alternative embodiment of a bolus in accordance

with a further aspect of the invention.

[000125] Figure 6A is a front cross sectional-view of an alternative embodiment of a bolus

in accordance with a further aspect of the invention.

[000126] Figure 6B is a perspective cross-sectional view of the bolus of Figure 6A.

[000127] Figure 7 is a flow diagram showing representative steps in a method of manufacturing a bolus in according with an aspect of the invention.

[000128] Figure 8 is a graph showing the daily diffusion/release rate of bromoform from bolus in the media.

[000129] Figure 9 is a graph showing variability in the diffusion results.

[000130] Figure 10 is a graph showing the concentration of Bromoform in a diffusion

media overtime.

[000131] Figure 11 is a graph showing the mass of Bromoform released (%) over time.

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[000132] Figure 12 is a graph showing the release rates of bromoform from different carriers in open top falcon tubes.

[000133] Figure 13A is a graph showing the release rate of bromoform from paraffin wax as a carrier.

[000134] Figure 13B is a graph showing the release rate of bromoform from carnauba wax

as a carrier.

[000135] Figure 13C is a graph showing the release rate of bromoform from Beeswax as a carrier.

[000136] Figure 14 is a graph showing the average release rate of bromoform for a reinforced bolus in accordance with an embodiment of the present invention.

[000137] Figure 15A is a side view showing a reinforced bolus design in accordance with an alternative embodiment of the present invention.

[000138] Figure 15B is a side cross section view of a reinforced bolus design in accordance with an alternative embodiment of the present invention.

[000139] Figure 15C is a side cross section view of a reinforced bolus design in accordance with an alternative embodiment of the present invention.

[000140] Figure 15D is a cross section view of the internal structure of a reinforced bolus

design in accordance with an alternative embodiment of the present invention.

Brief Description of Preferred Embodiments of the Invention

[000141] The present invention relates to devices and methods to deliver substances to animals, particularly hydrophobic substances to animals. In preferred forms, the substance is an

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inhibiting agent such as a methane inhibitor. The present invention is exemplified with reference to a preferred embodiment. However, this should not be seen as limiting on the

scope of the invention. One skilled in the art would understand how to apply the teachings

herein to devices for delivery of other substances to animals.

[000142] Referring first to Figures 1A and 1B, there is provided a bolus (100). The bolus (100) is configured to reduce or eliminate release of one or more greenhouse gases ("GHGs")

from a ruminant animal. For instance, the bolus (100) may reduce or eliminate production of GHGs by the ruminant animal, and therefore reduce the gases which are released by the

animal.

[000143] In addition, or in the alternative, the bolus (100) may improve animal production

by preventing the conversion of feed into one or more GHGs from a ruminant animal.

[000144] The bolus (100) includes a core (110) and a housing (120).

[000145] In some embodiments, the bolus (100) also includes a barrier layer (130). The

barrier layer (130) is configured to separate the core (110) from the housing (120).

[000146] The housing (120) is generally cylindrical and has an open end indicated

generally as (60), and a rounded, closed end (170). The open end (160) can allow fluids in the

ruminant animal's rumen to contact the core (110).

[000147] Further aspects of the bolus (100) should become clearer from the following discussion.

Core

[000148] The core (110) includes at least one inhibiting agent and can be optionally mixed

with a suitable carrier(s). Particularly preferred carriers include PEG4000,PEG400, natural and

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synthetic waxes, fatty acids, fatty alcohols, fatty amines, phospholipids-lecithin, and adsorbents, and combinations thereof.

[000149] Suitable waxes include beeswax, paraffin, castor wax, Carnauba wax, Candellila wax, Jojoba wax, and Lanolin.

[000150] In addition, minerals such as zeolite, bentonite, kaolin, activated carbon or a

combination thereof may also be suitably mixed with the inhibiting agent. It is also possible to include other compounds such a zinc (i.e. in powdered form) or zinc oxide.

[000151] Alternatively, the core (110) may include a concentrated (substantially pure) form of the inhibiting agent.

[000152] In a preferred embodiment, the inhibiting agent is a methane inhibiting agent.

Particularly preferred forms include haloforms e.g. halomethanes such as bromoform (CHBr 3)

as is discussed in more detail below.

[000153] It should be appreciated by a person skilled in the art that other carriers may be selected or used depending on the application. It is envisioned that certain carriers can be

selected in order to provide a desired release profile for the inhibiting agent, or alternatively provide the desired physical properties of the core material -density or volume etc.

[000154] In preferred embodiments the carrier used in the present invention is a natural waxy substance, with a preferred melting point between 50-90C, or more preferably 60-80C.

[000155] It was found by the inventors that having a carrier with this melting point range

allowed for melting of the carrier and mixing with the inhibiting agent(s) to form a homogenous core (110), and to subsequently solidify at room temperature.

[000156] A particularly preferred carrier is a mixture containing castor was with one or

more of paraffin wax, beeswax, and carnauba wax.

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[000157] It should be appreciated that the ratio of carrier to inhibiting agent may be

chosen to optimise the function of the bolus (100) e.g. to suit the desired release profile for the

inhibiting agent(s).

[000158] When formed, the core (comprising both the carrier and inhibiting agent(s)) preferably has a melting point of at least 450 C. Having this minimum melting point will assist

with ensuring that the core (110) does not melt when the bolus (100) has been administered to the ruminant animal. In addition, it will assist to ensure that the bolus (100) is unlikely to melt

on inadvertent exposure to elevated temperatures e.g. those temperatures that could reasonably be experienced during transport and/or storage.

[000159] It should be appreciated that the range of melting points for the core (110) may be adapted by varying the ratio of inhibiting agent(s) to carrier forming the core (110).

[000160] A preferred ratio of inhibiting agent to carrier may include substantially 80:20 w/w% to substantially 50:50 w/w%, or preferably substantially 70:30 w/w% to substantially

:40 w/w %, or more preferably substantially 66:33 w/w%.

Inhibiting agent(s)

[000161] In a preferred embodiment, the inhibiting agent is one or more methane inhibiting compounds.

[000162] Suitable methane inhibitors include haloforms such as bromoform, chloroform, iodoform and combinations thereof. It is envisioned that any methane inhibitor that is suitable for internal administration to a ruminant animal may be used with the present invention.

[000163] The inventors have surprisingly found that bromoform is a particularly well

suited for use in a bolus (100) according to the present invention. Accordingly, reference herein will be made to the inhibiting agent(s) as bromoform. However, this should not be seen as

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limiting on the scope of the present invention as alternatives are also envisaged as being within the scope of the present invention.

[000164] Bromoform is reactive and has a short half-life in animals (0.8 hrs in rats, 1.2 hours in mice, US Dept of Health, 2003). It is a liquid at room temperature and is denser than

water. Previous trials demonstrated no residues in meat and tissue from slaughtered steers, after 48 hour with holding period (Kinley et al. Mitigating the carbon footprint and improving

productivity of ruminant livestock agriculture using a red seaweed, Journal of Cleaner Production 259 (2020) 120836), and no significant increase in the level in milk (Roque et al.

Inclusion of Asparagopsis armata in lactating dairy cows' diet reduces enteric methane emission by over 50 percent; Journal of Cleaner Production 234 (2019) 132-138).

[000165] Bromoform has a relatively high efficacy e.g. effect per administered dose. This enables sufficient quantities to be provided in a core (110) to manufacture a bolus (100) which

can deliver controlled release of the inhibiting agent over an extended term.

[000166] Additionally, bromoform also has a relatively high density. This can assist with

achieving a higher retention of the bolus (100) in the rumen, as the density of the bolus can be

optimised to promote the bolus (100) sinking to the ventral part of the rumen, rather than floating.

[000167] The above points notwithstanding, there is a prevailing concern about using

bromoform in animals. The compound is thought to have adverse effects such as being carcinogenic at certain exposure levels.

[000168] In addition, there are technical challenges which must be addressed to enable bromoform to be administered to animals. These include the volatility of the substance, and its

ability to dissolve substances which could be used for its delivery. Furthermore, achieving a precise (and relatively low) dose rate over a period of time is a challenge.

Housing

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[000169] The housing (120) includes a cavity (not numbered in the Figures) which is sized and dimensioned to receive the core (110). The housing (120) forms the external structure of

the bolus (100).

[000170] The housing (120) is configured to provide structural integrity for the bolus (100)

but yet is also adapted to degrade overtime. Degradation of the housing (120) can facilitate release of the inhibiting agent over the predetermined period of time.

[000171] The housing (120) is preferably non-toxic and resists erosion in the rumen of the

ruminant for a sufficient period of time to facilitate release of inhibiting agent from the core (110) at the desired rate. It should be appreciated by the person skilled in the art that the

dissolution rate of the housing (120) and the core (110) can be configured to allow the

controlled release of the inhibiting agent in the ruminant animal's rumen.

[000172] Preferably, the housing (120) is composed of a biodegradable, non-absorbent material, or a material which is otherwise compatible with waste disposal in slaughter facilities. It should be appreciated that any material that is suitable for internal administration to a

ruminant animal with the desired dissolution rates can be used with the present invention.

[000173] In a preferred embodiment, the housing (120) is preferably selected from a

biodegradable material, particularly preferred biodegradable materials include polymers such

as polylactic acid (PLA), polyglycolic acid (PGA), polylactic glycolic acid (PLGA), polypropylene, SLA polymer, ABS and combinations thereof.

[000174] In a preferred embodiment the housing (120) is composed of PLA. PLA is

available in three forms, D-, L- and a racemic mixture of both D and L. All three types of PLA may be used in the housing (120) of the present invention.

[000175] In a preferred form, PLA is preferred as it degrades into lactic acid and is

commonly used as medical implants. Depending on the type of PLA used, PLA breaks down inside the body within six months to two years.

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[000176] It should be appreciated by the person skilled in the art that other suitable

biodegradable materials can be used as the housing (120).

[000177] In an optional embodiment, further fillers, binders, surfactants, active agents

and/or absorbents may be included in the bolus of the present invention.

[000178] As can be seen in Figures 1A and 1B, the bolus (100) has a substantially cylindrical form. The housing (120) includes a smooth external surface to assist with ingestion

of the bolus (100) by the ruminant animal.

[000179] It should be appreciated by the person skilled in the art that the size, thickness

and/or dimensions of the bolus (100), including the core (110), barrier layer (130) if provided, and the housing (120) can be adjusted depending on the dose of inhibiting agent to be

delivered to the ruminant, without departing from the spirit and scope of the invention. For example, a smaller size bolus (100) can be adapted for use in smaller ruminant animals such as sheep or goats, while a larger sized bolus (100) can be used in larger ruminant animals such as

cattle.

[000180] Additionally, the housing (120) may be also be configured to control the release

rates of the core (110) and/or degradation of the bolus (100). For example, the internal cross

sectional area of the cavity may be adapted to control the amount of the core (110) present in the bolus (100). In such an embodiment, the internal volume of the cavity may be adapted to

increase in size from the open end (160) to the closed end (170). This may be useful for increasing the amount of inhibiting agent(s) over time. This may account for animal growth

where feed intake of the animal increases.

[000181] Additionally, or alternatively, the cross-sectional thickness of the wall(s) forming the housing (120) may increase along the length of the housing (120). For instance, the wall(s)

may be a thicker at one end of the housing (120) than the other. In such an embodiment, the thickness of the wall at the open end (160) may be thinner in size than towards closed end

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(170). This can assist with providing controlled dissolution of the core formulation from the bolus.

Barrier layer

[000182] The barrier layer (130) is an optional component of the bolus (100) of the present invention and may be included to provide additional stability to the bolus (100). The

barrier layer (130) can be configured to partially or completely prevent contact between the core (110) and the housing (120). The barrier layer (130) is preferably selected from a waxy

material, epoxy or a silicon material.

[000183] It should be appreciated by the person skilled in the art, the barrier (130) layer

may be selected dependent on the required application and/or release profile. For example, where further control of the release rate of the inhibiting agent is required, choosing a barrier

layer (130) material, shape and configuration can facilitate achieving the release required profile.

Method of Treatment

[000184] The bolus (100) is delivered orally into the rumen of the ruminant animal to be

treated, entering the rumen via the oesophagus. In the rumen, stomach fluids (and other

matter such as plant fibre mat) act to erode or dissolve the core (110) to release the inhibiting agent overtime.

[000185] The open end (160) allows stomach fluids and fibrous matter to come into

contact with the core (110). In addition, it assists to control release of the core (110) therefrom to the rumen.

[000186] The core (110) and the housing (120) are designed to facilitate release of the

inhibiting agent over a period of time for which an animal is to be treated according to a method disclosed herein.

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[000187] The bolus (100) is adapted to release the inhibiting agent over a period of at least six months, preferably 12 months, and potentially up to two years.

[000188] Preferably, the release rates of the inhibiting agent may be calculated based on the weight of the ruminant animal to be treated and the type of inhibiting agent used. As such, it will be appreciated that the desired release rates may vary from animal to animal. Typically, the desired release rates may be calculated on an amount of inhibiting agent/weight of animal. Alternatively, the desired release rates may also be calculated based on the amount of feed consumed by the animal. Particularly preferred release rates for bromoform include from approximately 0.1- approximately 0.5 g/day, and more preferably approximately 0.2 g/day.

[000189] Additionally, it should be appreciated by a person skilled in the art that a ruminant animal can be treated by multiple boluses (100) according to the present invention in order to achieve a preferred dosage of the inhibiting agent. This can allow a bolus (100) to be manufactured which has a concentration and total load of the inhibiting agent. Multiple of those bolus (100) can be administered to an animal concurrently or sequentially. This will allow the desired dosage to be provided to the animal. This can be particularly beneficial to allow the bolus (100) to be used with animals requiring different doses of inhibiting agent e.g. larger or smaller animals, or to compensate for natural growth over time.

[000190] The bolus (100) is adapted to deliver a dose of inhibiting agent directly into the rumen of the animal. For instance, bromoform may be released at a rate at which it can effectively reduce or eliminate methane production during digestion. That will reduce the emission of greenhouse gases by the animal and therefore reduce the environmental impacts of agriculture.

[000191] In addition, the bolus (100) may improve the ruminant's conversion of feed for animal production. For example, by reducing methane production during digestion, it is believed that this may lead to more efficient utilisation of ingested feed, and result in improved growth and weight gain, or other production such as milk production. In addition, the

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compositions for the core and synergistic effects arising from the combination of carrier and inhibiting agent(s) may enable the provision of a slow-release, long term delivery device to

improve animal productivity and / or reduce emission of greenhouse gases.

First Alternate Housing Embodiment

[000192] Referring now to Figure 2A-2B which shows an alternative embodiment of a

bolus (200) according to an embodiment of the invention.

[000193] Aspects of the bolus (200) are similar to those of the bolus (100), and therefore like references refer to like components.

[000194] A series of ribs (240) are provided along an external surface of the housing (120). The ribs (240) may provide additional structural strength to the bolus (200), and can assist to

prevent it rupturing if the core (110) were to swell. Additionally, or alternatively, the (240) ribs may also assist the administration of the bolus (200) to the ruminant animal.

[000195] As illustrated, the ribs (240) are provided as a series of concentric "hoops".

However, the ribs (240) could be a series of parallel or non-parallel ribs (not illustrated) which extend along the length of the bolus (200)

Second Alternate Housing Embodiment

[000196] Referring now to Figures 3A-3B which show an alternative embodiment of a bolus (300) according to an embodiment of the invention.

[000197] Aspects of the bolus (300) are similar to those of the bolus (100) described

above, and therefore like references refer to like components.

[000198] The bolus (300) includes additional features on the external surface of the housing (120), including depressions or grooves (350).

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[000199] The grooves (350) may promote portions of the housing (120) breaking away as

it degrades. This can be used to further control the release profile for the inhibiting agent.

Third Alternate Housing Embodiment

[000200] Referring now to Figures 4A-4B which show an alternative embodiment of a

bolus (400) according to an embodiment of the invention.

[000201] Aspects of the bolus (400) are similar to those of the bolus (100) described above, and therefore like references refer to like components.

[000202] The bolus (400) includes a housing (120) which has a cavity (not illustrated in the Figures) that is configured to receive and hold the core (110).

[000203] The housing (120) tapers along its length. For instance, the distance between the external surfaces of distal sides of the housing (120) increases along the length of the bolus

(400). For instance, as is indicated in Figure 4A, the width (X) is less than the width (Y).

[000204] Alternatively, the bolus (400) may have side walls of substantially constant

thickness, but which are structured and orientated to define a taper for the bolus (400).

[000205] This configuration may allow for better controlled degradation of the core (110)

and thereby provide additional control for release of the inhibiting agent.

Fourth Alternate Housing Embodiment

[000206] Referring now to Figure 5A which shows an alternative embodiment of a bolus (500) according to an embodiment of the invention.

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[000207] Aspects of the bolus (500) are similar to those described above, and therefore like references refer to like components.

[000208] The bolus (500) includes a reservoir (580) adapted to hold a relatively concentrated form of the inhibiting agent e.g. bromoform in a substantially pure, liquid form.

[000209] The bolus (500) includes a dispensing mechanism which is configured to

dispense predetermined dose(s) of the inhibiting agent from the reservoir (580).

[000210] In the illustrated embodiment, the dispensing mechanism is a pump (590) in communication with a valve. At predetermined times, the pump (590) dispenses a dose of the

inhibiting agent via the valve (590), to release the inhibiting agent to the rumen to which the

bolus (500) has been administered.

[000211] The dispensing mechanism may be configured to release a consistent e.g. the same, amount of the inhibiting agent at defined intervals.

[000212] Alternatively, the dispensing mechanism may be configured to vary the amount

of inhibiting agent released at different times. This may be useful to enable the bolus (500) to provide an effective amount of inhibiting agent which accounts for growth of the animal. In

addition, or alternatively, it may compensate for other factors changes e.g. seasonal variations

in methane production which would necessitate a higher dose of inhibiting agent.

[000213] In a further embodiment, the bolus (500) may include sensors (not shown). For example, temperature sensors may be included within the bolus (500). Additionally, or

alternatively, other sensors may also be included in the bolus, such as locomotion and pH. The addition of such sensors can provide valuable information on the feed intake of the animal and

assess whether the amount of inhibiting agent is sufficient for the animal.

Fifth Alternate Housing Embodiment

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[000214] Referring now to Figures 6A and 6B which show an alternative embodiment of a bolus (600) according to an embodiment of the invention.

[000215] The bolus (600) can be adapted to include additional features within the cavity of the housing, such as grooves or ribs (680) formed on an inner wall of the housing (120) that

defines the cavity.

[000216] Aspects of the bolus (600) are similar to those of the bolus (100), and therefore like references refer to like components.

[000217] A series of ribs (680) are provided along an internal surface of the housing (120).

The ribs (680) may provide additional structural strength to the bolus (600), and/or provide

additional means to retain the contents of the core formulation within the cavity of the housing. Additionally, or alternatively, the (680) ribs may also assist with the retention of the

core within the housing. Further, the ribs may also provide controlled dissolution of the core formation from the bolus (600) to the ruminant animal.

[000218] In one embodiment, the external surface of the housing will remain smooth or

uniform.

Sixth Alternate Housing Embodiment

[000219] Referring now to Figures 15A to 15D which show a further embodiment of a

bolus (700) according to an aspect of the present invention.

[000220] The bolus (700) can be adapted to include additional features with the internal reinforcing structure on the housing.

[000221] Aspects of the bolus (700) are similar to those of the bolus (100), and therefore

like references refer to like components.

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[000222] The bolus (700) includes at least one reinforcing rib (710) located inside a cavity (unnumbered) defined by the housing structure. A cap (720) may also be provided e.g. releasably attached to the bolus (700) to close the open end of the bolus (700). Attachment may be provided by a friction fit arrangement, or a screw thread arrangement in which corresponding screw threads on the housing and cap engage each other. Alternatively, the cap may be attached to the housing by an adhesive or other mechanical fastener.

[000223] The reinforcing rib(s) (720) may improve the structural integrity of the bolus (700) and assist it to hold its shape. .

Method of Manufacture

[000224] Referring now to Figure 7, which is a flow chart showing representative steps in a method of manufacturing (800) a bolus e.g. (100), (200), (300), (400), according to the present invention.

[000225] In general terms, the method includes the step (810) of forming the housing (120) and the step (820) forming a core (110).

Housing

[000226] Forming the housing (120) may occur using any technique as should be known to one skilled in the art. For instance, a suitable material may be extruded into a desired shape defining a cavity. Alternatively, an additive layering manufacturing process could also be used to build the housing shape defining a cavity. It is also envisaged that a moulding process could be used e.g. a sacrificial moulding or injection moulding process, 3D printing or hot melt extrusion processes may be used.

Core

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[000227] In step 820, the core (110) is manufactured.

[000228] Step 820 may include one or more of the following steps:

[000229] Step 822 which involves melting a carrier material to provide a melted carrier material;

[000230] Step 824 which involves adding the inhibiting agent(s) to the melted carrier

material;

[000231] Step 826- which involves mixing the inhibiting agent and the melted carrier

material to create a substantially homogenous mixture;

[000232] Step 828 which involves forming the substantially homogeneous mixture into a desired shape.

[000233] It should be understood that the substantially homogenous mixture contains the

inhibiting agent(s) at a concentration sufficient to achieve the desired release profile for the inhibiting agent on administration of the device to a ruminant animal. The concentration can be

varied according to the type of ruminant animal to be treated, the shape and dimensions of the

device, or the required release profile to be achieved.

[000234] It should be understood that the step of forming the substantially homogeneous mixture into a desired shape may involve providing the mixture to a mould. In a particularly

preferred form, the substantially homogenous mixture is added (poured) into a cavity in a housing (120) manufactured at step 810.

[000235] Alternatively, the mould may be a separate component which receives the

substantially homogenous mixture. In these embodiments, once the desired shape has been formed, the core can subsequently be provided to a cavity in a housing (120).

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[000236] The method also includes the step of allowing the substantially homogenous

mixture to cool. As it cools, the carrier material hardens and assumes a shape according to the

shape of the mould or housing into which it has been provided.

Exampleformulations

[000237] The following cores were formulated for use in the bolus of the present invention.

Amount (w/w %)

Example 1 2 3 4 5 6 7 8 9 10 11 12

Bromoform 20 20 20 25 12.5 8.3 25 12.5 8.3 25 12.5 8.3

Paraffin 80 30 30 50 50 50 - - - - -

Beeswax 50 - - - - 50 50 50 - -

PEG4000 - 50 - - - - - - 50 50 50

PEG400 - - - - - - - - - -

AC - - 25- - 25 - - 25 -

Kaolin - - - 37.5 - - 37.5 - - 37.5

Zeolite - - - - 41.7 - - 41.7 - - 41.7

Amount (w/w %)

Example 13 14 15

Bromoform 20 33 33

Paraffin - 66

Beeswax - - 66

PEG4000 50 -

PEG 400 30 -

AC - - -

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Kaolin - -

Zeolite - -

The following additional high bromoform content cores were also formulated for use in the bolus of the present invention.

Amount (w/w %)

Example 16 17 18 19 20 21 22 23 24 25 26 27

Bromoform 33 50 67 75 33 50 67 75 33 50 67 75

Beeswax 67 50 33 25 - - - - - - -

Paraffin - - - - 67 50 33 25 - - -

wax

Carnauba - - - - - - - - 67 50 33 25

wax

CastorWax - - - - - - - - - - -

Activated - - - - - - - - - - -

Carbon

Bentonite - - - - - - - - - - -

ZincOxide - - - - - - - - - - -

Amount (w/w %)

Example 28 29 30 31 32 33 34 35 36

Bromoform 33 50 67 75 50 50 50 50 50

Beeswax - - - - - - - 25 25

Paraffinwax - - - - - - - -

Carnauba wax - - - - - - - - 25

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Castor Wax 67 50 33 25 - - - 25

Activated Carbon - - - - 50 - - -

Bentonite - - - - - 50 - -

ZincOxide - - - - - - 50 -

Validation

Example 1: Release/Diffusion study

[000238] Trials with 2mm thick 3D printed large capped boluses (LCB2) filled with 66.7%

bromoform and 33.3% beeswax in the RME (RME trial 2) were conducted to determine the diffusion rate of bromoform from the bolus.

Bolus Design

[000239] A reinforced bolus as shown in Figure 15 was used for this study. It includes an

internal reinforcing structure as well as ribs spread apart to support the wall, an upper part was adapted for the attaching a cap. The bolus with reinforcing was found to be more robust and

held its shape better than without reinforcing when the molten bromoform/beeswax mixture was poured in and cooled, as well as a more physically robust bolus for the trial.

Method

Materials

[000240] Bromoform (reagent grade, Sigma Aldrich, 96% bromoform, 4% ethanol), beeswax (food grade, NZ Beeswax, MP 65 °C and zinc oxide from Native Ingredients NZ.

Bolus Manufacture

[000241] The boluses were drawn in Solidworks, coverted to .stl files, opened in FlashPrint

to create the print jobs. The boluses were printed in three parts (case, internal structure and cap) on FlashForge Creator Pro 3D printers using E-Sun PLA+ at 100% fill, standard resolution,

first layer height 0.27 mm, layer height 0.18 mm, 2 perimeter shells, 3 top solid layers, 3 bottom

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solid layers, fill pattern hexagon, print speed 60 mm/s, extruder temperature 200°C and plate temperature 50°C.

[000242] Eight LRB boluses were prepared at 67% bromoform, eight LRB boluses were prepared at 75% bromoform, and six LCB2 boluses with no bromoform (controls). Ingredients

are listed below (Table 1). All ingredients were weighed in beakers on a calibrated 4dp electronic balance. Bromoform solutions were covered with parafilm to prevent evaporation.

Ingredients were prepared by melting pre-weighed beeswax and zinc oxide in beakers at 100°C (Thermoprism Oven), letting the mixture cool to 80°C, adding the bromoform and the mixture

kept well mixed to prevent the zinc oxide from settling out, before pouring into the boluses. Caps were press fitted and soldered to seal the bolus.

Table 1. Formulationfor the shortened reinforced bolusesfor the diffusion test Per bolus Total

Zinc Zinc Oxide Beeswax Bromoform Oxide Bromoform Type Quantity (g) (g) (g) (g) Beeswax (g) (g) LCB2 6 28.0 80.4 0.0 168.0 482.7 0.0 LRB1 8 28.0 47.3 96.1 224.0 378.8 769.0 LRB1 8 28.0 39.7 119.0 224.0 317.3 952.0 Total 616.0 1178.7 1721.0

[000243] The boluses were placed in 500 ml polypropylene bottles with approximately 380 ml 0.02M phosphate buffer (Merck) in distilled water, prepared in 2L or greater batches,

adjusted to pH 6.5 using 1M HCI (Merck) and a pre-calibrated pH meter (using pH 4, 7, and 10 pH buffers). The bottles were sealed and placed in the incubator at 400 C. 10 ml samples were

collected and the entire solution changed every 24 hours.

[000244] 10 ml samples was collected using a 10 ml autopipette in 15 ml Falcon tubes. 1 g

of sodium chloride was added to each Falcon tube. For GC-MS analysis, 1 ml of ethyl acetate (analytical grade, Merck) was added to each Falcon tube. When GC-FID was used 2 ml of ethyl

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acetate was added to each Falcon tube. The Falcon tubes were capped, well mixed using a Vortex, and centrifuged at 4000 rpm for 15 minutes. For GC-MS analysis, all the ethyl acetate

was recovered using a graduated glass syringe and the volumes noted.

[000245] For GC-FID analysis, 0.5 ml of ethyl acetate was recovered. For GC-FID analysis, 200 ul of sample was injected using an autosampler, and analysed using a ZB5HT 30 m capillary column using a temperature ramp of 30-300oC over 20 minutes, at 5 ml/min nitrogen gas flow,

in splitless mode. Bromoform had a retention time of 7.5 minutes. Peak areas were compared to calibration standards made up in ethylene acetate to determine the mass of bromoform

(mg). This was divided by the volume injected to obtain the concentration of bromoform in the ethyl acetate (mg/L). The concentration in ethyl acetate was multiplied by the total volume of

ethyl acetate added to the sample and divided by the recovery to obtain mass of bromoform in

the sample. This was then divided by the volume of sample collected to obtain a concentration in the solution, which was then multiplied by the volume of solution in the Shott bottle to

obtain mass transferred from the bolus to the solution. Bromoform recovery from solution was checked using standard solutions made up to different concentrations of bromoform and was typically 43%. GC-FID performance was checked for each run of ten samples using a calibration

sample as a reference.

Results

[000246] A lower diffusion rate followed by a rapid increase in diffusion rate was observed for both boluses (Figure 8). The 67% bolus had a lag time of 4-5 days before reaching its

maximum diffusion rate, whereas the 75% reached maximum diffusion rate with 3 days.

[000247] The rate of diffusion was higher for the 75% bolus at 1010 mg/day when compared to 66.7% which was 730 mg/day. This was a surprising, but also good result (as it

means that a single bolus could be used to dose 700 kg bulls and achieve the required methane reduction), as the predicted diffusion rates for an LCB1 bolus for 67% bromoform was 300

mg/day and 462 mg/day for an LCB1 bolus with 75% bromoform. The expectation for the LRB boluses was a lower diffusion rate because it had a reduced surface area at 1mm thick (about

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71% that of a LCB1 bolus) (Table 2). In theory the LRB bolus should only be delivering 220 mg/day for 67% bromoform and 344 mg/day for 75%.

Table2. Expected diffusion ratefor an LRB bolusfrom the different parts of the bolus.

Expected rates Exposed wax (mg/cm2/day) (mg/cm2/day) Total (mg/day) Contribution(%) Length Width Diameter Area Thickness Bitsofthebolus Quantity (cm) (cm) (cm) (cm2) (mm) 0.67 0.75 0.67 0.75 0.67 0.75 0.67 0.75 Cap 1 1.7 3.4 27.2 2 0.357 0.49 85.0 116.2 9.7 13.3 4.4 3.9 Ribs 4 0.3 3.4 12.8 3 0.082 0.086 85.0 116.2 1.1 1.1 0.5 0.3 Active diffusion area 3 3 3.4 96.1 1 1.939 3.042 85.0 116.2 186.4 292.4 84.3 84.9 Eye 1 3.0 1.2 3.6 3 0.082 0.086 85.0 116.2 0.3 0.3 0.1 0.1 Curved bit 12.2 1 1.939 3.042 85.0 116.2 23.7 37.3 10.7 10.8 Total (mg/day) 221.2 344.4 Tot, Actual (mg/day) 731 1064 Grand tot; Factorout 3.30 3.09

Table 1. Calculation of the porous area to achieve the same diffusion rate as what was measuredfrom the LRB boluses using previously determined diffusion rates. 67% bromoform 75% bromoform mg/day mg/day mg/day mg/day through through through through Proportion area open closed Proportion open closed open area area area open area area

0.01 23.2 9.6 0.01 31.6 13.2 0 0.0 1.1 0 0.0 1.1 0.06 449.4 176.1 0.06 614.1 276.4 0 0.0 0.3 0 0.0 0.3 0.06 57.3 22.4 0.06 78.2 35.2 Total (mg/day) 529.8 209.6 724.0 326.2 Grand total (mg/day) 739.4 1050.2

[000248] Variability in diffusion data was high initially with a coefficient of variation of around 1, and this decreased to between 0.05-0.22, as the boluses reached their maximum diffusion rates (Figure 9). The 75% bolus settled within 2 days, while the 67% bolus settled within 4 days.

[000249] A zero-order release was observed for both boluses indicating the rate of release was independent of concentration of bromoform in the bolus (Figure 11).

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Conclusion

[000250] The rate of diffusion for LRB boluses was 1010 mg/day for the 75% bolus, and 730 mg/day for the 66.7% bolus which was higher than predicted from the previous diffusion

studies.

[000251] The concentration of bromoform in the media for the 75% bolus, is close to the solubility limit of bromoform in water (3.2 g/L), therefore diffusion rates may be higher than

measured in this study.

Example 2: Release testing of carriers

[000252] Release testing of various carriers was undertaken for this study.

Method

Materials

[000253] Bromoform (reagent grade, Sigma Aldrich, 96% bromoform, 4% ethanol), ruminal fluid (Dairy NZ Trial), paraffin waxes (MPs 46-48, 55 and 65°C, Sigma Aldrich), castor

wax (Lotus Oils), carnauba wax (PureNature NZ), zinc oxide (PureNature NZ).

pH and buffer capacity of ruminalfluid

[000254] The rumen fluid collected from Dairy NZ was thawed and centrifuged before

analysing for pH and buffer capacity. A volume of 10 ml of Rumen fluid received from each cow was taken and titrated against 0.05 N NaOH with continuous pH monitoring. Volume of NaOH

to change the pH by a unit was recorded.

Release and testing of various carriers

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[000255] Small capped boluses were prepared as described in example 1 above.

[000256] Paraffin waxes, beeswax, carnauba wax and castor wax were mixed with

bromoform to 33%, 50%, 67% and 75% by weight bromoform. The mixes were placed in the

following: a. Paraffin waxes: 2 mm thick small capped boluses and 15 ml falcon tube;

b. Castor, carnauba and beeswaxes: 1, 2, and 3 mm small capped boluses and 15 ml falcon tubes.

[000257] These were placed in 500 ml polypropylene bottles with 400 ml 0.02M

phosphate buffer (Merck) in distilled water, prepared in 2L or greater batches, adjusted to pH 6.5 using 1M HCI (Merck) and a pre-calibrated pH meter (using pH 4, 7, and 10 pH buffers). The

bottles were sealed and placed in the incubator at 40°C. 10 ml samples were collected and the

entire solution changed every 2 days (Monday, Wednesday, Friday), except for the weekend hours.

[000258] Samples were analysed by GC-MS and GC-FID as described in example 1 above.

Results

pH and buffer capacity

[000259] The mean pH and the buffer capacity were 6.9±0.2 (n=4) and 7.47±1.4 mMol/L/delta pH (n=4) respectively. While there has been published literatures for pH values

for ruminal fluid, no data for buffer capacity is available. The buffer capacities obtained for ruminal fluid indicates that the rumen environment is resilient as it is 5-6-fold higher than that

of phosphate buffer saline. We found the pH of phosphate buffer in diffusion experiment remained stable even around 3 mg/ml of Bromoform concentration (Report No BR 2021-01,

Figure 4). Given the volume of rumen fluid 91 L, the maximum concentration of bromoform at extreme condition of complete bolus rupture would reach around 1.09 mg/ml, which is lower

than observed earlier in PBS. Therefore, with this concentration and given the strong buffer

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capacity of Rumen fluid, there is a less possibility of pH drop in the event of abrupt bolus rupture.

Release testing of carriers

[000260] Paraffin wax had the highest release rate at 190 mg/cm2/day, followed by beeswax, carnauba and castor wax (Figure 12). Carnauba and castor wax would be better

options for the carrier in case of bolus failure as the release rate is 50 to 40% less compared to beeswax. The lower release rates correspond to the differences in chemical make-up of the

waxes, paraffin wax has no ester linkages or polar groups, beeswax and carnauba is a mixture of ester waxes, paraffins, fatty alcohols with some hydroxyl groups, while the major component of

castor wax is tri-ester of glycerol and recinoloeic acid.

[000261] Bromoform had the greatest release rate in boluses made with paraffin waxes at

3.5 to 5.4 mg/cm2/day in the 2 mm thick small capped boluses (Figures 13A-C).

[000262] Boluses made with carnauba wax had release rates up to 5.5 mg/cm2/day in the

1 mm thick bolus and 1.66 mg/cm2/day in the 3 mm thick bolus. Trials with the 2 mm thick

bolus are still underway (Figure 13B).

[000263] In comparison, boluses made with beeswax had a release rate of 3 mg/cm2/day

at 75% bromoform (Figure 13C).

[000264] The bromoform had dissolved the castor wax and it had diffused through the bolus and pooled on the bottom of the container, dissolving the container, and no release rates

were able to be determined as bromoform was not detected in the water for the samples that had been collected. The trials with castor wax will need to be repeated in glass bottles.

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Release ratesfrom reinforced bolus

[000265] Average release rates for large reinforced boluses with 67% and 75%

bromoform, prepared as described previously in example 1 above, from another trial are shown in Figure 14 and compared to release rates from the same boluses measured in the lab. Half of

the boluses were in 20 L buckets with 1 kg of sand filled with buffer at pH 6.5, and the other half were in 20 L buckets with 400 g of wood shavings and 1 kg of sand. Release rates are

comparable at day 28 to those observed in the lab. Little difference in bromoform concentration was observed between buckets containing wood shavings and buckets without

wood shavings. Boluses have largely remained intact, with some compression due to sand, and some have had their lids opened.

Example 3: Animal study

[000266] An animal study was conducted to determine methane emissions from an animal implanted with a bolus of the present invention. The experiment was designed as an unbalanced, completely randomized design with three treatments and three repeated

measurements over time in three periods 8 to 12 weeks apart.

[000267] Nineteen dairy beef heifers (312 ±14 kg live weight), including three spare

animals, were selected from a mob of 50 based on behaviour traits and liveweight from a

research farm in the Manawatu, New Zealand. They were assigned to one of three treatments: a bolus containing no bromoform (CONTROL; n = 4); a bolus releasing bromoform at a rate of

300 mg/day (LOW, n = 6); or a bolus releasing 450 mg/day (HIGH, n = 6). SmaXtec boluses were administered at the same time to monitor rumen temperature as an animal health monitor and

to complement the weekly blood samples.

[000268] The heifers were transported from research farm to a testing centre for diet adaptation and gas measurements using respiration chambers. The heifers were adapted to the

environment of the cattle yards and the fresh cut pasture for 7 days before receiving their allocated treatment bolus. Gas measurements started 13 days after the boluses were

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administrated. Each heifer was in the respiration chambers for 48 hours during the period of gas measurements, which took two weeks for four measurement groups. At the end of the

measurements in respiration chambers, the animals were transported back to research farm.

Bolus Preparation

[000269] The bonuses were manufactured in accordance with the procedure described in

example 1 above. The following formulations used in this trial are shown table 4 below.

Table4. Formulation for the shortened reinforced boluses for the Research Trial

Perbolus Total

Bromoform mass fraction Zinc Oxide Bromoform ZincOxide Bromoform Type inwax Quantity (g) Beeswax (g) (g) (g) Beeswax (g) (g) LCB2 0 6 28.0 80.4 0.0 168.0 482.7 0.0 LRB1 0.67 8 12.1 21.3 43.2 93.4 164.0 332.9 LRB1 0.75 8 12.1 17.8 53.5 93.4 137.4 412.1 Bolus Administration

[000270] The three versions of boluses were made within the first 10 days of the experiment. The first version was a short bolus which was regurgitated by all animals within the

days after the boluses were administered. Because the control boluses were longer than the

treatment boluses and these had not been regurgitated during the first 3 days, it was assumed that the bolus size was the major factor for regurgitation. All first-version treatment boluses

were replaced with second-version boluses on day 5 after administration. However, the longer boluses of the second version were also regurgitated. Therefore, these boluses were then

replaced with a third version treatment bolus, which was a significantly heavier bolus of the same size as the second version bolus. The third-version boluses have not been regurgitated to

date. Currently almost all heifers have been dosed with third-version boluses, except for three of the LOW treatment heifers. Details of boluses regurgitation and re-administration are in

Table 5.

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[000271] Two control boluses were regurgitated, but only one was identified because the bolus ID was illegible. None of control boluses were re-administered because it was not

possible to identify the heifer-bolus match.

Table 5. Bolus administration events of the different bolus versions during thefirst three weeks

after initial administration.

V1I V2 V2 bolus V3 V3 bolus Animal ID Treatment bolusID bolusID administration bolusID administration 780 CONTROL 1 782 CONTROL 2 789 CONTROL 3 796 CONTROL 5 797 CONTROL 4 783 LOW 1 1 30/07/2021 1 13/08/2021 787 LOW 3 3 30/07/2021 Not regurgitated 788 LOW 2 2 31/07/2021 Not regurgitated 790 LOW 5 5 30/07/2021 5 7/08/2021 791 LOW 5 4 30/07/2021 Not regurgitated 793 LOW 6 6 30/07/2021 6 13/08/2021 794 LOW 7 7 30/07/2021 7 10/08/2021 784 HIGH 9 9 1/08/2021 9 10/08/2021 785 HIGH 10 10 30/07/2021 10 10/08/2021 786 HIGH 11 14 30/07/2021 8 70812021 792 HIGH 12 12 31/07/2021 12 13/08/2021 795 HIGH 13 13 30/07/2021 13 12/0812021 798 HIGH 14 11 1/08/2021 11 9/8/2021 781 HIGH 15 8 30/07/2021 14 13/08/2021 V1: all boluses administrated on 270721

Feed intake and liveweight

[000272] The heifers were fed cut ryegrass-based pasture offered adlibitum. The forage

was harvested daily at approximately 10:00 at research farm and transported to the testing

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centre. The harvested forage was divided into two allocations, the first allocation was fed in the afternoon at 15:30 and the second allocation was stored at 4°C until the next morning feeding

at 08:30. Samples were collected from each pasture delivery for dry matter determination and

feed analysis. Dry matter (DM) was determined from triplicate subsamples by oven drying at 105°C for 24 h. A separate subsample was oven dried at 65°C for 48 h for chemical nutrient

analyses. Both drying ovens used were forced-air ovens (Avantgarde FED 720, Binder GmbH, Germany).

[000273] Two days prior to entering respiration chambers for methane measurements, the cows were put into metabolic crates to adapt them to confined spaces and being tied. When the animals were in metabolic crates or respiration chambers, feed refusals were

collected twice daily, and refusal DM was determined as described above. Daily dry matter

intake of the heifers was then determined from the difference of the dry matter offered and refused.

[000274] Liveweight was recorded pre-trial when animals were grazing at the research farm on two occasions (13/7/2021 and 16/7/2021). The animals were weighed again on

19/07/2021 on arrival at testing farm and every 7-10 days while on site. Initial liveweight was

measured on 23/07/2021 before bolus administration and final liveweight was once animals left the respiration chambers. Final liveweight dates are different for some animals because

measurements were undertaken over two weeks.

Gas measurements

[000275] Fermentation gases methane (CH 4 ), carbon dioxide (C0 2 ) and hydrogen (H 2 )

were quantified in four open-circuit respiration chambers at the New Zealand Ruminant Methane Measurement Centre (AgResearch, Palmerston North, New Zealand). Each chamber is

15.4 m3 (3.5 m long x 2 m wide x 2.2 m high) with an air flow rate of around 1.0m 3 /min, which was continuously monitored by measuring differential pressure using a Venturi flowmeter.

Temperature inside respiration chambers was approximately 20°C and the relative humidity was on average approximately 79%. All gases were measured at ~2.8-min intervals using a

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4900C Continuous Emission analyser (Servomex Group Ltd, East Sussex, UK) and daily production of each gas was calculated from the difference between concentration flowing in

and out of the chamber (Pinares-Patino et al., 2012). Respiration chambers were opened twice

daily (~20 min each time) for cleaning, feeding, faecal sampling and feed refusal collection. No measurements were performed during the period when chambers were opened, and missing

data were interpolated by taking the average of the last 12 values (~45 min) before the doors were opened.

Statistical analyses

[000276] Data from the first period of gas measurements was analysed using the 'predictmeans' and 'Ime4' packages in the statistical software R 4.0.3 (R Core Team, 2020). Data

for dry matter intake and gas emissions for each heifer were averaged across the two measurement days. Heifer served as the experimental unit. The mixed model included

treatment as fixed effect and respiration chamber nested in measurement group as random effect.

[000277] Liveweight analyses included treatment as a fixed effect and time as a repeated

measurement, with heifer as a subject for the repeated measurements. Only initial and final liveweight were included in this analysis.

Results

Dry matter intake and gas emissions

[000278] Dosing heifers with bromoform at 300 mg/day (LOW) or 450 mg/day (HIGH) did

not affect the dry matter intake measured over the two days the animals were in respiration chambers compared with the control group (p = 0.42). Both. CH 4 production (g/day) and CH 4

yield (g/kg unit of dry matter intake) decreased by more than 99% in LOW and HIGH compared with CONTROL (p < 0.01). The decrease in CH 4 emissions at LOW and HIGH treatments was

accompanied by an increase in H 2 emissions per day (Table 7). As both treatments decreased methane emissions completely, a lower dose needs to be determined to achieve levels of

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methane reduction between 30 and 90%. A reduction in the daily dose would ensure that not more bromoform than necessary is used to increase the lifetime of the bolus and would

decrease the risk of negative effects on the animal and potential contamination of animal

products. Given that methane emissions are fully inhibited, it is noteworthy that dry matter intake was not negatively affected as has been observed when bromoform containing

Asparagopsis is fed (Roque et al. 2019).

Table 7: Dry matter intake (DMI) methane (CH4) and hydrogen (H2) emissions measured in respiration chambers over two days in heifers dosed boluses releasing no bromoform

(CONTROL), 300 mg/d (LOW) or 450 mg/d (HIGH) of bromoform

CONTROL LOW HIGH SED p-value

DMI [kg/d] 5.20 4.98 4.50 0-79 0.420 CH4 [g/d] 120.25a 0-34b 0.77b 2-74 < 0.01 CH 4 [g/kg DMI] 23.32a 0.14b 0.11b 0.33 < 0.01 H 2 [gid] D.15P 20.60a 20.08a 3.46 < 0.01

Conclusion

[000279] As observed, the results above indicate treatment using a bolus with the present

invention is highly effective a few weeks after the boluses were administered, as demonstrated by the ~99% reduction in methane.

[000280] Unless the context clearly requires otherwise, throughout the description and

the claims, the words "comprise", "comprising", and the like, are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense, that is to say, in the sense of "including,

but not limited to".

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[000281] The entire disclosures of all applications, patents and publications cited above and below, if any, are herein incorporated by reference.

[000282] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that that prior art forms part of the common

general knowledge in the field of endeavour in any country in the world.

[000283] The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or

collectively, in any or all combinations of two or more of said parts, elements or features.

[000284] Where in the foregoing description reference has been made to integers or

components having known equivalents thereof, those integers are herein incorporated as if individually set forth.

[000285] It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such

changes and modifications may be made without departing from the spirit and scope of the

invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications be included within the present invention.

Claims (5)

-50/50 Claims What we claim is:

1. A bolus for administration to a ruminant animal, wherein said bolus contains at least one methane inhibitor and is configured to

release the methane inhibitor into an animal's rumen after it is administered thereto, wherein the at least one methane inhibitor is a haloform selected from the list of

chloroform, bromoform, iodoform, or combinations thereof.

2. The bolus as claimed in claim 1, wherein the bolus includes a core that is formed from a carrier material and further wherein the methane inhibitor is admixed with the

carrier material.

3. The bolus as claimed in claim 2, wherein the carrier material comprises at least one

wax.

4. The bolus as claimed in claim 3, wherein the wax is selected from the group consisting of myristic acid, stearic acid, steryl alcohol, cetyl alcohol, cetosteryl alcohol, castor

wax, bee's wax, paraffin wax, PEG4000, Carnauba, Candellila, Jojoba, Lanolin, and a combination thereof.

5. The bolus as claimed in any one of claims 2-4, wherein the bolus comprises a housing that contains the core, and further wherein the housing is made from one or more

plastic materials.