Improved devices and methods for delivering substances to animals

Abstract

The present invention provides delayed-release dosage forms and boluses configured for administration to an animal, wherein the dosage forms and boluses are configured to release a hydrophobic substance into the animal over a period of time. Preferably, the hydrophobic substance is haloform. Also provided is the use of the delayed-release dosage forms or boluses of the present invention to reduce methane production in ruminants. Methods for producing the boluses of the present invention are also provided.

Description

[Technical Field]

[0001] The present invention relates to improved devices and methods for animal production and delivery of substances to animals, and in particular to devices and methods for administering at least one beneficial substance to animals, and methods for making the devices. [Background technology]

[0002] In agriculture, it is often necessary to deliver substances to animals for a variety of purposes, including but not limited to, treating or preventing disease and increasing animal production.

[0003] There are various devices and methods for delivering substances such as drugs to animals. However, one class of compounds that is difficult to deliver to animals is hydrophobic compounds. The properties of these compounds pose challenges in developing controlled release technologies for these hydrophobic compounds, particularly through the animal's stomach.

[0004] One specific purpose of administering substances to animals is to reduce the negative impacts of agricultural practices. For example, various methane and nitrification inhibitors are known to be administered to animals to reduce or mitigate the negative effects of methane and nitrate-containing compounds produced by animals.

[0005] However, despite current efforts, climate change is having far-reaching environmental and social impacts around the world. It is widely understood that these impacts will continue to increase over time. As a result, there is a global drive to reduce harmful greenhouse gas (GHG) emissions to avoid the worst impacts of climate change.

[0006] The agricultural sector is considered to be a major source of GHG emissions. Total methane emissions from global livestock are estimated at 7.1 gigatonnes of CO2 equivalent per year, accounting for 14.5% of total anthropogenic GHG emissions. Therefore, this sector has a key role to play in reducing overall GHG emissions.

[0007] The main GHGs emitted by agriculture are methane (CH4) and nitrous oxide (N2O), and the main source of methane emissions can be attributed to livestock. Most of the methane is released when cows burp. The amount of methane produced on each farm is directly related to the total feed intake of the animals.

[0008] Countries with strong agricultural sectors, such as New Zealand, face challenging targets for reducing emissions from agriculture. For example, the New Zealand government has introduced policies aimed at reducing methane emissions by 24-50% by 2050. In New Zealand, methane production from livestock is estimated to account for half of the country's total greenhouse gas emissions. Reducing methane is a key component in meeting greenhouse gas emission targets and reducing the impact of global warming.

[0009] GHG emissions by animals also have a negative impact on animal productivity. Feed that is converted into compounds and then expires or is released by the animal is an energy source that is not converted into productive use. Therefore, to increase efficiency, it is important to optimize the conversion of feed into animal productivity in the form of weight gain and milk production. Summary of the Invention [Problem to be solved by the invention]

[0010] It is an object of the present invention to provide improved devices and methods for delivering substances, such as hydrophobic substances and / or methane inhibitors, to animals.

[0011] It is an object of the present invention to provide an apparatus and method for reducing GHG emissions.

[0012] It is an object of the present invention to provide an apparatus and method for improving or optimizing animal productivity.

[0013] Alternatively, it is an object of the present invention to provide an apparatus and method for improving animal production benefits, for example through reduced methane production.

[0014] It is an object of the present invention to provide a formulation for reducing GHG emissions by one or more animals, such as ruminants.

[0015] It is an object of the present invention to provide a device and method that can release substances at different rates over a period of time.

[0016] Alternatively, it is an object of the present invention to provide a method of manufacturing a device for delivering a substance to an animal, for example a substance that reduces GHG emissions.

[0017] Alternatively, it is an object of the present invention to overcome some of the shortcomings of the prior art.

[0018] Alternatively, it is an object of the present invention to provide the public with a useful choice. [Means for solving the problem]

[0019] According to one aspect of the present invention, there is provided a bolus configured for administration to an animal, said bolus configured to release a hydrophobic substance into the animal over a period of time.

[0020] According to one aspect of the present invention, there is provided a bolus for administration to a ruminant, wherein said bolus is configured to release an effective amount of a substance, preferably at least one inhibitor.

[0021] According to a further aspect of the present invention there is provided a method of reducing gas (preferably methane) emissions from a ruminant animal, the method comprising the step of administering to said ruminant animal a bolus comprising at least one inhibitor.

[0022] According to another aspect of the present invention there is provided the use of a methane inhibitor and a carrier in a bolus to reduce methane production in ruminants.

[0023] According to another aspect of the present invention there is provided the use of a methane inhibitor and a carrier in a bolus to reduce methane emissions from ruminants.

[0024] According to another aspect of the present invention, there is provided the use of a haloform in the manufacture of a bolus for reducing emissions of one or more greenhouse gases ("GHG") from ruminants.

[0025] In a preferred embodiment, the bolus may be configured to be administered to a ruminant, which may include beef or dairy cattle, sheep, goats, buffalo, deer, elk, giraffe or camel.

[0026] In one embodiment, the bolus may be adapted to reduce emissions of one or more greenhouse gases (“GHG”) from ruminants.

[0027] In another embodiment, the bolus may be a sustained release bolus configured to release the at least one inhibitor over a period of time in a ruminant animal, for example in the rumen of the animal.

[0028] According to a further aspect, there is provided a bolus for administration to a ruminant, the bolus comprising: a core comprising at least one substance to be administered to a ruminant mixed with a carrier; and a housing covering at least a portion of the core; The bolus is configured to release the substance through the housing over a period of time.

[0029] In another aspect of the invention, a core containing a substance to be administered to an animal; and A bolus is provided that includes a housing that at least partially covers a portion of the core; The housing is formed from at least one polylactic acid (PLA).

[0030] In a further aspect of the present invention, a bolus is provided comprising a core, the core comprising a mixture of at least one wax and a haloform.

[0031] The inventors have surprisingly discovered that the technology described herein may provide many advantages. These advantages may be the result of unique synergistic interactions between various aspects of the technology. Therefore, the technology of the present invention is described based on the inventors' current understanding of these interactions. It should be understood that any embodiment, or the interaction of two or more embodiments, described herein may form a separate invention.

[0032] Throughout this specification, reference is made to the term "substance" or "substance administered to an animal", which should be understood to mean any substance that provides a benefit to an animal, such as a drug for the treatment or prevention of a disease, thereby increasing the productivity of the animal or reducing at least one adverse effect of agriculture.

[0033] In a preferred embodiment, the substance may be a hydrophobic substance.

[0034] In a particularly preferred embodiment, the hydrophobic substance may be an inhibitor. Reference is made herein to the substance as an inhibitor. However, this should not be considered as limiting the scope of the invention, and alternatives are envisaged, for example, it may be a hydrophilic substance.

[0035] In one embodiment, at least one inhibitor may be a methane inhibitor.The use of a methane inhibitor may provide many benefits.For example, the methane inhibitor reduces or eliminates the production of methane by ruminants, for example, in the rumen.As a result, the amount of methane in the rumen that may be emitted by ruminants is reduced, and thus GHG emissions are effectively reduced.

[0036] Furthermore, reducing methane production could potentially benefit animal production. For example, reducing methane would allow a relatively larger proportion of ingested feed to be digested and converted into protein (milk or meat). As a result, farmers could potentially achieve higher productivity for a given amount of feed or increase efficiency by reducing feed accordingly.

[0037] In one embodiment, the methane inhibitor may be a haloform.

[0038] In a preferred embodiment, the methane inhibitor may be selected from the list of chloroform, bromoform, iodoform, or combinations thereof.

[0039] In a particularly preferred form, the haloform can be bromoform (CHBr3). The use of bromoform may offer many advantages. For example, it can be highly effective at relatively low doses, and a single device can deliver sufficient amounts of inhibitor over an extended period of time. Furthermore, bromoform's relatively high density increases the total weight of the bolus, ensuring that the bolus is retained within the lumen, i.e., sinks to the ventral portion of the lumen rather than floating, reducing reflux.

[0040] However, despite these advantages, inventors have faced many challenges and problems in developing a bolus for the controlled release of haloform, particularly bromoform, to ruminants.

[0041] In a further embodiment, the bolus may include a core.

[0042] The core may be formed by the inhibitor mixed with the carrier.

[0043] However, in alternative embodiments, the inhibitor may be provided in a substantially pure form, eg, purified form, eg, not admixed with a carrier.

[0044] In embodiments, the carrier may have a structure that promotes or enhances the affinity of the inhibitor for the carrier, for example, the carrier may have a polar functional group.

[0045] In embodiments, the carrier may be a relatively polar material, e.g., having a relatively high % w / w of polar functional groups. The inventors have surprisingly discovered that the carrier and the inhibitor can interact, and that this interaction can affect the release rate of the inhibitor from the bolus. This aspect of the invention will become more apparent from the following description.

[0046] Examples of suitable functional groups contained in the carrier include esters, fatty acids, fatty alcohols, carbonyls, and fatty amines. Without being limited to a specific mechanism, the inventors believe that the inhibitor may interact with the polar functional groups in the wax, potentially through the formation of hydrogen bonds. The amount of polar functional groups present in the carrier affects the mutual affinity between the carrier and the inhibitor.

[0047] The inventors have discovered that a range of materials may be suitable for use as carriers in the present invention, for example, the carrier may be selected from the list of waxes, myristic acid, stearic acid, steryl alcohol, cetyl alcohol, cetosteryl alcohol, or combinations thereof.

[0048] In a particularly preferred embodiment, the carrier may be a waxy substance, for example, the carrier may be selected from the list of beeswax, paraffin wax, PEG 4000, carnauba, castor wax, candelilla, jojoba, or lanolin, or a combination thereof.

[0049] In particularly preferred embodiments, the carrier may include paraffin wax and castor wax.

[0050] In a particularly preferred embodiment, the carrier may comprise paraffin wax and castor wax in a ratio of about 50:50 (parts by weight).

[0051] 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 and a relatively non-polar substance. For example, in some embodiments, the carrier may comprise a mixture of paraffin wax (a mixture of alkanes without 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 can be adjusted to achieve a desired affinity for the inhibitor. This can be used to achieve a desired release rate of the inhibitor.

[0052] Furthermore, in addition to the above, solid supports such as powdered activated carbon, zeolite, bentonite, etc. can also be used as supports. Therefore, the discussion herein should not be considered as limiting the scope of the present invention.

[0053] In further embodiments, the carrier may contain one or more additional components. For example, additional components such as zinc element or zinc oxide may be incorporated. Preferably, a high-density material such as a metal piece (preferably steel) may be included in the carrier. The additional components may be used to achieve the desired density of the core and / or bolus.

[0054] It should also be understood that additional ingredients may be added to the bolus cavity separately from the carrier and without being mixed with the carrier, which is particularly beneficial for forming a core with a desired release profile, and the density of the bolus can be adjusted to a desired amount by including additional ingredients.

[0055] Other suitable additives for incorporation into the carrier may also include colloidal silicon dioxide, charcoal, bentonite, and zeolites.

[0056] Further aspects of the carrier and its effect on the release of the inhibitor from the bolus, as well as the interaction of the carrier with the housing, will become more apparent from the description below.

[0057] In a preferred embodiment, the carrier may have a melting point of substantially 50 to 90°C.

[0058] In particularly preferred embodiments, the carrier has a melting point lower than the boiling point of the inhibitor, which can be useful because it allows the carrier to be melted and mixed with the inhibitor without substantial loss of the inhibitor due to evaporation.

[0059] In a preferred embodiment, the core may have a melting point above 37°C.

[0060] In a particularly preferred embodiment, the core may have a melting point above 40°C.

[0061] The melting point of the core can be beneficial to the function of the technology in several ways. For example, having a melting point above 37°C, more preferably above 40°C, can help the carrier stabilize the inhibitor when the bolus is in the lumen. This can be beneficial in controlling the release of the inhibitor, e.g., the migration of the inhibitor through the material forming the housing.

[0062] In one embodiment, the bolus may be adapted to reach a maximum release rate of about 0.05 g to 2 g of bromoform per day into the lumen.

[0063] In one embodiment, the bolus may be adapted to release an amount of between 0.02 g and 0.5 g of bromoform per day into the lumen.

[0064] In a particularly preferred embodiment, the bolus may be adapted to reach a maximum release rate of about 0.1 to 0.5 g of bromoform per day into the lumen.

[0065] In a preferred embodiment, the bolus is configured to release an amount of bromoform into the lumen between 0.02 g and 0.3 g per day.

[0066] In one embodiment of the present invention, the bolus core may comprise haloform, preferably bromoform, in an amount of 30% (wt) to 80% (wt), preferably in an amount of 55% (wt) to 75% (wt), more preferably in an amount of 50% (wt).

[0067] In a particularly preferred embodiment, the core comprises a haloform, preferably bromoform, at a concentration of up to 55% (by weight).

[0068] The inventors have discovered that the release rate of the inhibitor into the lumen increases over time. This may be the result of several factors. Thus, the release rate begins at zero upon administration to the animal and increases to a maximum. However, the above should not be considered limiting, and other release rates are considered within the scope of the present invention.

[0069] In a further embodiment, the bolus can include a housing.

[0070] Throughout this specification, references to the term "housing" should be understood to mean a structure capable of receiving and holding a core containing at least one inhibitor.

[0071] In a preferred embodiment, the housing comprises a body having a cavity in which the core is disposed.

[0072] However, it should be understood that the housing may take other shapes, for example, the housing may include two or more cavities, each capable of receiving and holding a separate core.

[0073] In one embodiment, the housing may include an open end.

[0074] The bolus can be open-ended for use, e.g., administered to an animal in an open-ended state. Consequently, in these embodiments, the open end provides an opening for exposing the contents of the core to fluid within the lumen during use.

[0075] In yet another preferred embodiment, the housing can completely enclose and surround the core, eg, have a sealed cavity in which the core is disposed.

[0076] For example, the bolus may include a housing with a cavity in which at least a portion of the core may be disposed and an open end to facilitate insertion of the core into the cavity. A cap may be used to cover the open end.

[0077] The cap may be formed separately from the housing and removably or permanently secured to the housing, or alternatively, the cap may be integrally formed with the housing.

[0078] In yet another embodiment, the housing may be constructed from at least two parts, each part having a cavity for receiving a respective portion of the core, the at least two parts together completely surrounding the core and defining a sealed cavity in which the core is disposed.

[0079] In yet another embodiment, the housing may be formed around the core, for example by molding. Alternatively, the housing and cap together may define a substantially closed and sealed cavity in which the core is disposed.

[0080] The inventors believe that providing a substantially or completely closed and sealed cavity is preferred, as this can aid in achieving the desired controlled release of the inhibitor from the bolus of the present invention, e.g., in such embodiments, the inhibitor can pass through the material forming the housing, e.g., by mass diffusion.

[0081] In embodiments, the housing may be configured to have sufficient structural integrity to remain intact for a predetermined period of time.

[0082] In a preferred embodiment, the housing may be configured to degrade over a predetermined period of time.

[0083] Throughout this specification, references to the term "predetermined period of time" should be understood to mean the period of time during which the inhibitor is released into the animal.

[0084] In particularly preferred embodiments, the predetermined period may be at least 2 months, preferably 6 months, more preferably 12 months.

[0085] The inventors have surprisingly discovered that the housing of the present invention may aid in the controlled release of the inhibitor. For example, the housing may be able to withstand conditions within the lumen for a predetermined period of time. During this time, the housing protects the core from fluids within the lumen, but may also facilitate or control the controlled release of the inhibitor. However, depending on the design of the housing, the housing may disintegrate or degrade over a predetermined period of time. This may contribute to reducing adverse effects from administering the device to an animal and may also allow the animal to be reliably treated with multiple boluses, for example, a second bolus may be administered at, toward, or after the end of the predetermined period of time.

[0086] In embodiments of the present invention, the thickness of the housing can be selected to contribute to the release rate of the inhibitor. For example, the inventors have determined that the thickness of the housing can affect the release rate of the inhibitor from the bolus. In these embodiments, a relatively thicker housing will have a relatively slower release rate than a relatively thinner housing.

[0087] In a preferred embodiment, the housing may have a thickness of at least 1 mm.

[0088] In yet another preferred embodiment, the housing may have a thickness of less than 3 mm.

[0089] In yet another preferred embodiment, the housing may have a thickness between 1.5 and 2 mm, or between 0.5 and 2 mm.

[0090] In a particularly preferred embodiment, the housing has a thickness of 1 mm.

[0091] The thickness of the housing may be particularly important for achieving the desired controlled release of the inhibitor in embodiments such as those in which the core is completely encapsulated by the housing, as should become apparent from the discussion below.

[0092] In one embodiment, the dimensions of the cavity may vary along the length of the housing.

[0093] In a preferred embodiment, the cavity includes at least two regions having different cross-sectional areas, for example a first region having a first cross-sectional area and a second region having a second cross-sectional area.

[0094] In a particularly preferred embodiment, the first region has a relatively small cross-sectional area and the second region has a relatively large cross-sectional area.

[0095] In yet another preferred embodiment, the first region can be located closer to the open end than the second region.

[0096] Having a cavity with regions having different cross-sectional areas facilitates a more controlled release of the inhibitor to better meet the needs of the animal. For example, a smaller cross-sectional area can be provided near the open end to deliver a smaller dose of the inhibitor, while a larger cross-sectional area can be provided near the distal end, which can be useful if the dose of the inhibitor needs to be increased over time, for example, as the animal grows.

[0097] It should also be understood that the reverse configuration may be provided, e.g., a relatively larger cross-sectional area may be provided near the open end and a relatively smaller cross-sectional area may be provided near the distal end. This arrangement may be useful when an initial higher dosage of inhibitor is desired, followed by subsequent lower dosages. For example, this arrangement may be used when the animal has a high demand for inhibitor, e.g., during periods of relatively high feed intake and energy requirements, such as during milking, followed by periods of relatively low feed intake, such as during the dry season.

[0098] It should further 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 and second regions.

[0099] In other embodiments, the housing can include a third region having a third cross-sectional area, which can be further used to control the dosage of the inhibitor to the animal. Therefore, the foregoing should not be considered as limiting the scope of the present technology.

[0100] In one embodiment, the wall thickness of the housing may vary along the length of the housing. In such an embodiment, the wall thickness at or toward one end of the housing may be thicker than the wall thickness at the distal end. For example, the wall thickness at or toward the open end may be thinner than that at the distal end.

[0101] This configuration can be particularly useful for controlling the release of the inhibitor over time. For example, a relatively thin wall will degrade relatively faster than a relatively thick wall. This configuration can be used to control the degradation rate along the length of the housing. For example, it can be used to ensure that the only site where fluid in the lumen can contact and erode the core is the open end.

[0102] In a preferred embodiment, the housing is made from a material to which the inhibitor can migrate during use, for example by a mass diffusion process.

[0103] In preferred embodiments, the housing may be made from at least one plastic material, for example, the housing may be made from a degradable plastic or material that degrades over time within the lumen.

[0104] In particularly preferred embodiments, the housing may be made from a material selected from the list of one or more of polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid (PLGA), polypropylene, polycaprolactone (PCL), poly(d-lactic acid) (PDLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), SLA polymer, ABS, or combinations thereof. In particularly preferred embodiments, the housing comprises PLA and PBS.

[0105] The housing material may contain PLA, PBAT, and / or PBS in different proportions (wt %) as shown in Examples 1-7 in the table below. [Table 1]

[0106] In a particularly preferred embodiment, the housing material comprises PLA and PBS in a weight ratio ranging from 100:0 to 40:60 PLA:PBS.

[0107] In a particularly preferred embodiment, the housing comprises PLA and PBS in a weight ratio ranging from 100:0 to 40:60 PLA:PBS, and the housing has a thickness between 0.4 and 1.5 mm.

[0108] In a further embodiment, the core of the bolus of the present invention is covered by multiple housings arranged concentrically (e.g., similar to an onion). Such multiple housings (e.g., two, three, or more housings) have the advantage that the bolus decomposes less rapidly in the lumen (e.g., due to wear). As a result, the haloform in the core persists in the lumen longer, reducing methane production over an extended period of time. In embodiments with multiple housings, the materials and thicknesses of the housings can be as described herein for other embodiments. In a preferred embodiment, the bolus of the present invention comprises at least two housing layers, one outer housing and one inner housing, each housing material comprising a biodegradable polymer, preferably selected from the group consisting of polylactic acid (PLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof.

[0109] Additionally, the housing may be made of non-biodegradable materials such as EVA, silicone, acrylate, etc. As a result, the discussion herein should not be considered as limiting the scope of the present invention.

[0110] Additionally, the housing material may include one or more other compounds, such as plasticizers, hardeners, colorants, and the like.

[0111] However, in alternative embodiments, the housing may be made from one or more non-absorbent materials, i.e., materials into or through which the inhibitor does not migrate. The use of non-absorbent materials for the housing may help control the release rate of the inhibitor in certain embodiments, such as an open-ended bolus. For example, in these embodiments, the concentration of the inhibitor within the core is not reduced by absorption into the housing material.

[0112] In some embodiments, the bolus may include a barrier layer. In these embodiments, the barrier layer may be disposed between at least a portion of the core and the housing. For example, the barrier layer may minimize or completely prevent contact between a portion of the core and the housing. This may help prevent dissolution of the inhibitor (or other compound) to better control the release of the inhibitor and improve the stability of the device. This may be particularly useful if the inhibitor has a high solubility in the material from which the housing is made.

[0113] Alternatively, in embodiments in which a barrier layer is provided only between a portion of the core and the housing, migration of the inhibitor into the housing may be reduced, but not completely prevented, and in fact, the barrier layer may reduce the contact area between the core and the housing, thereby slowing the release rate of the inhibitor compared to when no barrier layer is provided.

[0114] Alternatively, the bolus may not include a barrier layer. This configuration may be useful when the inhibitor has relatively low solubility in the material comprising the housing. It may also be useful when the composition of the housing and / or carrier is selected to control the release rate, e.g., the rate of diffusion of the inhibitor through the housing.

[0115] In another embodiment, the bolus may be adapted to have a dissolution rate of the core and housing that results in substantially uniform dissolution of both components over time within the lumen.

[0116] In one embodiment, a cavity within the housing may provide a reservoir configured to receive a quantity of the inhibitor, for example, the reservoir may be a closed cavity within the housing that can receive and hold a quantity of the inhibitor.

[0117] In one embodiment, the bolus may include a dispensing mechanism.

[0118] In one embodiment, the carrier may have a relatively high affinity for the inhibitor compared to the affinity of the housing for the inhibitor. As discussed elsewhere in this document, this may be achieved by the relative polarities of the materials forming the carrier and housing, and by appropriately matching these materials to the inhibitor.

[0119] In another embodiment, the housing may be formed from a material having a Shore D hardness of at least 40. In such embodiments, having a housing with a Shore D hardness lower than 40 is believed to result in a bolus that is too soft, which may prevent bolus administration to the animal, cause injury to the animal before the full amount of inhibitor is administered, or cause premature degradation.

[0120] In a further embodiment, the housing may be formed from a material having a Shore D hardness of less than 80.

[0121] In another embodiment, the housing may be configured to facilitate controlled release of the inhibitor from the core. Without being limited to a particular mechanism, the inventors hypothesize that the inhibitor may be released through the housing by a mechanism of mass diffusion.

[0122] In a further embodiment, the present invention provides a bolus for administration to a ruminant, said bolus comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a housing enclosing at least a portion of, or preferably the entire core, wherein the bolus is configured to release the haloform.

[0123] In a further embodiment, the present invention provides a bolus for administration to a ruminant, said bolus comprising: a core comprising haloform (preferably bromoform) mixed with a carrier, said core, and a housing enclosing at least a portion of or all of the core, wherein the bolus is configured to release the haloform; and the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof.

[0124] In a further embodiment, the present invention provides a bolus for administration to a ruminant, said bolus comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a housing covering at least a portion of, or preferably the entire core; wherein the bolus is configured to release the haloform, and the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; and the housing comprises a biodegradable polymer, preferably selected from the group consisting of polylactic acid (PLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof.

[0125] In a further embodiment, the present invention provides a bolus for administration to a ruminant, said bolus comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a housing covering at least a portion of, or preferably the entire core; wherein the bolus is configured to release the haloform; and the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; and the housing has a layer thickness of between 0.4 and 1.5 mm.

[0126] In a further embodiment, the present invention provides a bolus for administration to a ruminant, said bolus comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a housing covering at least a portion of, or preferably the entire core; wherein the bolus is configured to release haloform; and the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; and the housing comprises a biodegradable polymer, preferably selected from the group consisting of polylactic acid (PLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof; and the housing has a layer thickness of between 0.4 and 1.5 mm.

[0127] In a further embodiment, the present invention provides a bolus for administration to a ruminant, said bolus comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a housing covering at least a portion of, or preferably the entire core; wherein the bolus is configured to release the haloform; the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; the housing comprises a biodegradable polymer, preferably selected from the group consisting of polylactic acid (PLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof; and the housing layer has a thickness of less than 2 mm.

[0128] In a further embodiment, the present invention provides a bolus for administration to a ruminant, the bolus comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a housing surrounding at least a portion of or the entire core, the bolus being configured to release the haloform; the core further comprising at least one metal piece (such as a metal pellet and / or metal rod), preferably steel or zinc. An advantage of this embodiment is that the density of the bolus is increased, reducing the likelihood that the bolus will be regurgitated by the animal. Preferably, the bolus of the present invention further comprises a densifier, preferably the densifier comprises at least one metal piece, preferably the densifier is provided within the core.

[0129] In a further embodiment, the present invention provides a bolus for administration to a ruminant, said bolus comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a housing covering at least a portion of, or preferably the entire core; wherein the bolus is configured to release the haloform; the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; the housing comprises polylactic acid (PLA); and the housing layer preferably has a thickness of less than 2 mm. In a further aspect, the present invention provides a delayed release dosage form for administration to a ruminant, the delayed release dosage form comprising: a core comprising haloform (preferably bromoform); and a coating covering at least a portion of, or preferably the entire core; wherein the delayed release dosage form is configured to release the haloform.

[0130] In a further embodiment, the present invention provides a delayed release dosage form for administration to a ruminant, said delayed release dosage form comprising: a core comprising haloform (preferably bromoform); and a coating covering at least a portion of, or preferably the entire core; wherein the delayed release dosage form is configured to release the haloform; and the core further comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof.

[0131] In a further embodiment, the present invention provides a delayed release dosage form for administration to a ruminant, the delayed release dosage form comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a coating covering at least a portion of, or preferably the entire core; wherein the delayed release dosage form is configured to release the haloform; the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; and the coating comprises a biodegradable polymer, preferably selected from the group consisting of polylactic acid (PLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof.

[0132] In a further embodiment, the present invention provides a delayed release dosage form for administration to a ruminant, said delayed release dosage form comprising a core comprising haloform (preferably bromoform) mixed with a carrier; and a coating covering at least a portion of, or preferably the entire core; the delayed release dosage form is configured to release the haloform; and the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; and the coating has a layer thickness of between 0.4 and 1.5 mm.

[0133] In a further embodiment, the present invention provides a delayed release dosage form for administration to a ruminant, said delayed release dosage form comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a coating covering at least a portion of, or preferably the entire core; wherein the delayed release dosage form is configured to release the haloform; the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; the coating comprises a biodegradable polymer, preferably selected from the group consisting of polylactic acid (PLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof; and the coating has a layer thickness of between 0.4 and 1.5 mm.

[0134] In a further embodiment, the present invention provides a delayed release dosage form for administration to a ruminant, said delayed release dosage form comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a coating covering at least a portion of, or preferably the entire core; wherein the delayed release dosage form is configured to release the haloform; the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; the coating comprises a biodegradable polymer, preferably selected from the group consisting of polylactic acid (PLA), polybutylene succinate (PBS), polybutylene adipate terephthalate (PBAT), and combinations thereof; and the coating layer has a thickness of less than 2 mm.

[0135] In a further embodiment, the present invention provides a delayed release dosage form for administration to ruminants, the delayed release dosage form comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a coating over the core; wherein the delayed release dosage form is configured to release the haloform; the core further comprises at least one metal piece (such as a metal pellet and / or metal rod), preferably steel or zinc. An advantage of this embodiment is that the density of the delayed release dosage form is increased, making the delayed release dosage form less likely to be regurgitated by the animal.

[0136] In a further embodiment, the present invention provides a delayed release dosage form for administration to ruminants, the delayed release dosage form comprising: a core comprising haloform (preferably bromoform) mixed with a carrier; and a coating covering part or all of the core, wherein the delayed release dosage form is configured to release the haloform; the carrier comprises a wax, preferably castor wax, paraffin wax, or a mixture thereof; the coating comprises polylactic acid (PLA); and the coating layer thickness is preferably less than 2 mm. Experiments have shown that a coating layer thickness of less than 2 mm is preferred, as this thickness allows the haloform to permeate outward from the core material at an optimal rate.

[0137] In the delayed release dosage forms or boluses of the present invention, preferably less than 50% of the haloform contained in the core is released over a 3-month period. In preferred embodiments of the delayed release dosage forms or boluses of the present invention, the core contains at least 100 grams of haloform. The core of the bolus or delayed release dosage forms of the present invention preferably contains 30% to 70% by weight of haloform (preferably bromoform).

[0138] It is currently understood that the controlled release of the inhibitor through the housing can be affected by many factors. For example, the affinity of the inhibitor for the carrier can affect the diffusion of the inhibitor 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 for the inhibitor than less polar carriers or carriers with a lower degree of functional groups.

[0139] The relative affinities of the materials forming the housing and core for the inhibitor can also affect the controlled release of the inhibitor from the core. For example, having a housing with a relatively low affinity for the inhibitor compared to the affinity of the carrier for the inhibitor can be a factor in controlling the release rate of the inhibitor from the core. These aspects of the invention should become more apparent from the description herein.

[0140] Throughout this specification, references to the term "release mechanism" should be understood to mean a configuration for releasing a predetermined amount of inhibitor over time. For example, the release mechanism may include a valve device that can release a quantity of inhibitor through an outlet. Alternatively, the release mechanism may be a syringe-type mechanism with a plunger and actuator; over time, the actuator moves the plunger within the reservoir, expelling the inhibitor from the reservoir.

[0141] The following items are also according to the present invention: Item 1 provides a bolus for administration to a ruminant, said bolus configured to release an effective amount of at least one inhibitor. Item 2 provides a bolus of any one of items 1, wherein at least one inhibitor is a methane inhibitor. Item 3 provides the bolus of item 1 or 2, wherein at least one inhibitor is a haloform selected from chloroform, bromoform, iodoform, or a combination thereof. Item 4 relates to the bolus of any one of items 1 to 3, wherein at least one inhibitor is bromoform. Item 5 relates to the bolus of any one of items 1 to 4, wherein the bolus includes a core containing an amount of the inhibitor. Item 6 provides the bolus of item 5, wherein the core comprises a carrier mixed with the inhibitor. Item 7 relates to the bolus according to item 6, wherein the carrier is a waxy substance selected from beeswax, paraffin wax, PEG4000, carnauba, candelilla, jojoba, or lanolin, or a combination thereof. Item 8 relates to the bolus of any one of items 5 to 7, wherein the core has a melting point above 37°C. Item 9 relates to the bolus of any one of items 5 to 8, wherein the bolus includes a housing for receiving and holding the core. Item 10 relates to the bolus of item 9, wherein the housing includes a cavity capable of receiving and holding the core. Item 11 relates to the bolus of item 9 or 10, wherein the housing includes an opening to facilitate exposure of the core to fluids in the ruminant's lumen in use. Item 12 relates to the bolus of item 10 or 11, wherein the cavity includes a first region having a first cross-sectional area and a second region having a second cross-sectional area, the first cross-sectional area and the second cross-sectional area being different from one another to facilitate controlled release of the inhibitor from the core. Item 13 relates to the bolus of any one of items 9 to 12, wherein the housing is configured to decompose over a predetermined period of time. Item 14 relates to the bolus of any one of items 9 to 13, wherein the housing is made of one or more non-adsorbent materials selected from the following: polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid (PLGA), polypropylene, SLA polymer, PBS, or a combination thereof. Item 15 relates to the bolus of any one of items 9 to 14, further comprising a barrier layer between at least a portion of the housing and the core to isolate the portion of the housing and the core from contacting each other. Item 16 relates to the bolus of any one of items 1 to 15, wherein the bolus is adapted to release a dosage of about 0.1 g to 0.5 g of the inhibitor per day into the rumen of a ruminant. Item 17 relates to the bolus of any one of items 1 to 16, wherein the bolus is adapted to release the inhibitor over a period of at least six months. Item 18 relates to the bolus of any one of items 1 to 17, wherein the bolus is adapted to release the inhibitor within two years. Item 19 provides a method for reducing gas emissions from a ruminant, the method comprising administering a bolus of any one of items 1 to 18 to the ruminant. Item 20 provides a method for reducing methane production in a ruminant, the method comprising administering to the ruminant a bolus according to any one of items 1 to 18. Item 21 provides the use of a methane inhibitor and a carrier in a bolus to reduce methane production in ruminants. Item 22 provides the use of a methane inhibitor and a carrier in a bolus to reduce methane emissions from ruminants. Item 23 provides for the use of haloforms in the manufacture of boluses to reduce emissions of one or more greenhouse gases (“GHGs”) from ruminants. Item 24 provides a method for producing a bolus according to any one of items 1 to 18, the method comprising: a. forming a housing having a cavity; b. forming a core containing an inhibitor; c. Including transferring the core to the cavity. Item 25 relates to the method of item 24, wherein the step of forming the core includes mixing a carrier material and an inhibitor. Item 26 provides the method of item 25, wherein the step of forming the core includes heating the carrier material to melt the carrier material before mixing the carrier material with the inhibitor to form the mixture. Item 27 relates to the method according to any one of Items 24 to 26, wherein the step of transferring the core into the cavity includes injecting a mixture into the cavity.

[0142] Further aspects of the present invention, which should be considered in all its novel aspects, will become apparent to those skilled in the art upon reading the following description, which provides at least one example of the practical application of the invention. [Brief explanation of the drawings]

[0143] One or more embodiments of the present invention will now be described, by way of example only, and not by way of limitation, with reference to the following figures:

[0144] [Figure 1A-B] FIG. 1A is a front view of a bolus according to one embodiment of the present invention.

[0145] FIG. 1B is a perspective cross-sectional view of the bolus of FIG. 1A.

[0146] [Figure 2A-B] FIG. 2A is a front view of an alternative embodiment of a bolus in accordance with a further aspect of the present invention.

[0147] FIG. 2B is a perspective cross-sectional view of the bolus of FIG. 2A.

[0148] [Figure 3A-B] FIG. 3A is a front view of an alternative embodiment of a bolus in accordance with a further aspect of the present invention.

[0149] FIG. 3B is a perspective cross-sectional view of the bolus of FIG. 3A.

[0150] [Figure 4A-B] FIG. 4A is a front view of an alternative embodiment of a bolus in accordance with a further aspect of the present invention.

[0151] FIG. 4B is a perspective cross-sectional view of the bolus of FIG. 4A.

[0152] [Figure 5] FIG. 5 is a front view of an alternative embodiment of a bolus according to a further aspect of the present invention.

[0153] [Figure 6A-B] FIG. 6A is a front cross-sectional view of an alternative embodiment of a bolus in accordance with a further aspect of the present invention.

[0154] FIG. 6B is a perspective cross-sectional view of the bolus of FIG. 6A.

[0155] [Figure 7] FIG. 7 is a flow diagram illustrating representative steps of a method for producing a bolus according to one embodiment of the present invention.

[0156] [Figure 8-9] FIG. 8 is a graph showing the daily diffusion / release rate of bromoform from a bolus in medium.

[0157] FIG. 9 is a graph showing the variability of the diffusion results.

[0158] [Figure 10-11] FIG. 10 is a graph showing the concentration of bromoform in the diffusion medium over time.

[0159] FIG. 11 is a graph showing the mass (%) of bromoform released over time.

[0160] [Figure 12-13A] FIG. 12 is a graph showing the release rate of bromoform from different carriers in open-top falcon tubes.

[0161] FIG. 13A is a graph showing the release rate of bromoform from paraffin wax as a carrier.

[0162] [Figures 13B-13C] FIG. 13B is a graph showing the release rate of bromoform from carnauba wax as a carrier.

[0163] FIG. 13C is a graph showing the release rate of bromoform from beeswax as a carrier.

[0164] [Figure 14] FIG. 14 is a graph showing the mean release rate of bromoform for an enhanced bolus according to one embodiment of the present invention.

[0165] [Figure 15A-D] FIG. 15A is a side view illustrating an enhanced bolus design according to an alternative embodiment of the present invention.

[0166] FIG. 15B is a side cross-sectional view of an enhanced bolus design according to an alternative embodiment of the present invention.

[0167] FIG. 15C is a side cross-sectional view of an enhanced bolus design according to an alternative embodiment of the present invention.

[0168] FIG. 15D is a cross-sectional view of the internal structure of an enhanced bolus design according to an alternative embodiment of the present invention.

[0169] [Figure 16A] FIG. 16A shows the shrinkage of injection molded tensile bars.

[0170] [Figure 16B] FIG. 16B shows the absorbed bromoform versus the bromoform composition in the beeswax for different compositions of PLA mixed with PBS and PBAT.

[0171] [Figure 16C] FIG. 16C shows the relationship between absorbed bromoform versus bromoform composition in beeswax for 3D printed PLA and injection molded 2003D PLA.

[0172] [Figure 16D]FIG. 16D shows the rate of bromoform absorption versus the bromoform composition in beeswax for different compositions of PLA mixed with PBS and PBAT.

[0173] [Figure 16E] FIG. 16E shows the rate of bromoform absorption versus PLA composition in beeswax containing different concentrations of bromoform.

[0174] [Figure 17] FIG. 17 shows the hardness analysis of the PLA blends before and after exposure to bromoform.

[0175] [Figure 18A-B] FIG. 18A shows the release of bromoform from 1 mm thick boluses loaded with 67% (wt) and 55% (wt) bromoform.

[0176] FIG. 18B shows the cumulative release of bromoform from the bolus.

[0177] [Figure 18C-D] Figure 18C shows the cumulative plots for 7, 8, and 9 days of the 57-1 mm bolus.

[0178] FIG. 18D shows the release rates from different boluses.

[0179] The term "bromet" as used in the figures refers to a bromoform containing bolus. DETAILED DESCRIPTION OF THE INVENTION

[0180] The present invention relates to devices and methods for delivering substances, particularly hydrophobic substances, to animals. In a preferred form, the substance is an inhibitor, such as a methane inhibitor. The invention is illustrated with reference to preferred embodiments. However, this should not be considered as limiting the scope of the invention. Those skilled in the art will understand how to apply the teachings herein to devices for delivering other substances to animals.

[0181] 1A and 1B, a bolus (100) is provided. The bolus (100) is configured to reduce or eliminate the emission of one or more greenhouse gases ("GHGs") from ruminant animals. For example, the bolus (100) can reduce or eliminate the production of GHGs by ruminant animals, thus reducing the gases emitted by the animals.

[0182] Additionally or alternatively, the bolus (100) can improve animal production by preventing the conversion of feed from ruminant animals to one or more GHGs.

[0183] The bolus (100) includes a core (110) and a housing (120).

[0184] 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).

[0185] Housing (120) is generally cylindrical and has an open end generally designated as (60) and a rounded, closed end (170). Open end (160) allows fluid within the ruminant's lumen to contact core (110).

[0186] Further aspects of Bolus (100) should become more apparent from the following discussion.

[0187] core The core (110) contains at least one inhibitor, which may optionally be mixed with a suitable carrier, particularly preferred carriers include PEG4000, PEG400, natural and synthetic waxes, fatty acids, fatty alcohols, fatty amines, phospholipid-lecithin, and adsorbents, and combinations thereof.

[0188] Suitable waxes include beeswax, paraffin, castor wax, carnauba wax, candelilla wax, jojoba wax, and lanolin.

[0189] Additionally, minerals such as zeolite, bentonite, kaolin, activated carbon, or combinations thereof may be suitably mixed with the inhibitor. Other compounds such as zinc (i.e., powdered) or zinc oxide may also be included.

[0190] Alternatively, the core (110) may include the inhibitor in a concentrated (substantially pure) form.

[0191] In a preferred embodiment, the inhibitor is a methane inhibitor, particularly preferred forms include halomethanes such as haloforms, e.g., bromoform (CHBr3), as described in more detail below.

[0192] Those skilled in the art will appreciate that other carriers can be selected or used depending on the application. It is contemplated that a particular carrier can be selected to provide a desired release profile of the inhibitor, or alternatively, to provide a desired physical property of the core material, such as density or volume.

[0193] In a preferred embodiment, the carrier used in the present invention is a natural waxy substance having a preferred melting point of 50-90°C, more preferably 60-80°C.

[0194] The inventors have found that having a carrier with this melting point range allows the carrier to be melted and mixed with the inhibitor to form a homogenous core (110), which can then be solidified at room temperature.

[0195] A particularly preferred carrier is a mixture containing castor wax and one or more of paraffin wax, beeswax, and carnauba wax. Even more preferably, the carrier is a mixture containing castor wax and paraffin wax.

[0196] It will be appreciated that the ratio of carrier to inhibitor can be selected to optimize the performance of the bolus (100), for example to match a desired release profile of the inhibitor.

[0197] As formed, the core (including both the carrier and inhibitor) preferably has a melting point of at least 45° C. Having this minimum melting point helps ensure that the core (110) will not melt when the bolus (100) is administered to a ruminant. Furthermore, the bolus (100) is less likely to melt if exposed to inadvertent high temperatures, such as those that might reasonably be experienced during transport and / or storage.

[0198] It should be understood that the melting point range of the core (110) can be tailored by varying the ratio of inhibitor to carrier forming the core (110).

[0199] Preferred ratios of inhibitor to carrier include substantially 80:20 w / w% to substantially 50:50 w / w%, or preferably substantially 70:30 w / w% to substantially 60:40 w / w%, or more preferably substantially 66:33 w / w%.

[0200] inhibitors In a preferred embodiment, the inhibitor is one or more methane inhibiting compounds.

[0201] Suitable methane inhibitors include haloforms such as bromoform, chloroform, iodoform, and combinations thereof. It is contemplated that any methane inhibitor suitable for internal administration to ruminants may be used in the present invention.

[0202] The inventors have surprisingly discovered that bromoform is particularly well suited for use in the bolus (100) according to the present invention. Accordingly, the inhibitor will be referred to herein as bromoform. However, this should not be construed as limiting the scope of the invention, and alternatives are also considered to be within the scope of the invention.

[0203] Bromoform is reactive and has a short half-life in animals (0.8 hours in rats and 1.2 hours in mice; U.S. Department of Health, 2003). It is liquid at room temperature and is denser than water. Previous studies have demonstrated no residues in the meat or tissues of slaughtered steers after 48 hours, including storage (Kinley et al. Mitigating the carbon footprint and improving productivity of ruminant livestock agriculture using a red foam, Journal of Cleaner Production 259 (2020) 120836), and no significant increase in levels in milk (Roque et al. Inclusion of Asparagopsis armata in lactating pressures' diet is enteric methane exit by over 50%; Journal of Cleaner Production 234 (2019) 132-138).

[0204] Bromoform has a relatively high efficacy, e.g., effect per dose, which allows it to be provided in sufficient quantity within the core (110) to produce a bolus (100) that can deliver a controlled release of the inhibitor over an extended period of time.

[0205] Additionally, bromoform is also relatively dense, which can help achieve higher retention of the bolus (100) within the lumen because the density of the bolus can be optimized to promote the bolus (100) sinking in the ventral portion of the lumen rather than floating.

[0206] Despite the above, there are general concerns about the use of bromoform in animals, as the compound is believed to be carcinogenic and have other adverse effects at certain exposure levels.

[0207] Furthermore, technical challenges exist when administering bromoform to animals. These include the volatility of the substance and the ability to dissolve substances used for its delivery. Furthermore, achieving a precise (and relatively slow) administration rate over a period of time is challenging.

[0208] housing The housing (120) includes a cavity (not numbered in the figure) sized and dimensioned to receive the core (110). The housing (120) forms the outer structure of the bolus (100).

[0209] The housing (120) is configured to provide structural integrity to the bolus (100), but is also adapted to degrade over time, which may facilitate release of the inhibitor over a predetermined period of time.

[0210] Housing 120 is preferably non-toxic and resists erosion within the ruminant's rumen for a period of time sufficient to facilitate the desired rate of release of the inhibitor from core 110. It should be understood by those skilled in the art that the dissolution rates of housing 120 and core 110 can be configured to allow for controlled release of the inhibitor within the ruminant's rumen.

[0211] Preferably, the housing (120) is constructed of a biodegradable, non-absorbable material, or a material that is compatible with waste disposal in a slaughterhouse. It should be understood that any material suitable for internal administration to a ruminant with a desired dissolution rate can be used in the present invention.

[0212] In a preferred embodiment, the housing (120) is selected from biodegradable materials, particularly preferably biodegradable materials including polymers such as polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid (PLGA), polypropylene, SLA polymers, PBS, and combinations thereof. In a particularly preferred embodiment, the housing (120) is made from materials including PLA and PBAT.

[0213] In a preferred embodiment, the housing 120 is constructed of PLA. PLA is available in three forms: D-, L-, and a racemic mixture of both D and L. All three types of PLA can be used in the housing 120 of the present invention.

[0214] In its preferred form, PLA decomposes into lactic acid, which is commonly used in medical implants. Depending on the type of PLA used, PLA decomposes in the body within six months to two years.

[0215] Those skilled in the art will appreciate that other suitable biodegradable materials may be used for the housing (120).

[0216] In any embodiment, additional fillers, binders, surfactants, active agents and / or absorbents may be included in the boluses of the present invention.

[0217] 1A and 1B, the bolus 100 has a substantially cylindrical shape. The housing 120 includes a smooth outer surface that aids in the ingestion of the bolus 100 by a ruminant.

[0218] Those skilled in the art will appreciate that the size, thickness, and / or dimensions of the bolus (100), including the core (110), barrier layer (130), if provided, and housing (120), can be adjusted depending on the dose of inhibitor delivered to a ruminant without departing from the spirit and scope of the present invention. For example, a smaller sized bolus (100) can be adapted for use with smaller ruminants, such as sheep or goats, while a larger sized bolus (100) can be used with larger ruminants, such as cattle. A bolus for a larger animal, such as a cattle, may have dimensions of 13 cm in length, 3.4 cm in diameter, and weigh 257 gm (throughout this specification, "gm" refers to grams). A bolus for a smaller animal, such as a sheep, may have dimensions of 8.5 cm in length, 2 cm in diameter, and weigh 60 gm. Alternatively, a smaller bolus may be administered to larger ruminants such as cattle, and such a smaller bolus may have dimensions of 3.4-3.8 cm in length and 2.6-3.0 cm in diameter.

[0219] The paper also contemplates the use of multiple smaller boluses in combination. In a preferred embodiment, the bolus and delay dosage forms of the present invention have a length of at least 5 cm, most preferably at least 10 cm, preferably 10.3 cm. In a preferred embodiment, the bolus and delay dosage forms of the present invention have a diameter of at least 2 cm, preferably 3.4 cm, and a length of at least 10 cm, preferably 10.3 cm. Preferably, the bolus and delay dosage forms of the present invention weigh between 100 and 300 grams.

[0220] Additionally, the housing (120) may be configured to control the release rate of the core (110) and / or the degradation of the bolus (100). For example, the internal cross-sectional area of ​​the cavity may be adapted to control the amount of core (110) present within the bolus (100). In such embodiments, 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 help increase the amount of inhibitor over time. This may explain animal growth, which increases the animal's feed intake.

[0221] Additionally or alternatively, the cross-sectional thickness of the walls forming the housing (120) can increase along the length of the housing (120). For example, the walls may be thicker at one end of the housing (120) than at the other end. In such embodiments, the wall thickness at the open end (160) may be thinner than toward the closed end (170). This helps to provide controlled dissolution of the core formulation from the bolus.

[0222] Barrier layer 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) may 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 wax-like material, an epoxy, or a silicone material.

[0223] Those skilled in the art will appreciate that the barrier (130) layer can be selected depending on the desired application and / or release profile. For example, if further control over the release rate of the inhibitor is desired, the selection of the material, shape, and configuration of the barrier layer (130) can facilitate achieving the desired release profile.

[0224] Composition example In an exemplary embodiment, the bolus can include a core surrounded by a housing. The bolus can be approximately 13 cm long, approximately 3.4 cm in diameter, and weigh approximately 257 g.

[0225] The housing may be made of PLA (3052D, 3001D, 3251D, L130, etc.), for example by injection molding, and is 1 mm thick.

[0226] The core matrix may be made from a 50:50 (by weight) mixture of castor wax and paraffin wax, and may contain bromoform as an inhibitor at a concentration of about 50% (by weight).

[0227] Treatment method The bolus (100) is delivered orally into the rumen of the ruminant being treated, passing through the esophagus. Within the rumen, gastric fluids (and other substances, such as a plant fiber mat) act to eventually erode or dissolve the core (110), releasing the inhibitor over time. However, the housing remains substantially intact throughout the treatment period.

[0228] The open end (160) allows gastric fluids and fibrous material to contact the core (110), and it also helps control the release of the core (110) therefrom into the lumen.

[0229] The core (110) and housing (120) are designed to facilitate release of the inhibitor over the period of time that an animal is treated according to the methods disclosed herein.

[0230] The bolus (100) is adapted to release the inhibitor over a period of at least six months, preferably twelve months, and in some cases up to two years.

[0231] Preferably, the release rate of the inhibitor can be calculated based on the body weight of the ruminant being treated and the type of inhibitor used. Therefore, it will be understood that the desired release rate may vary from animal to animal. Typically, the desired release rate can be calculated based on the amount of inhibitor / animal body weight. Alternatively, the desired release rate can be calculated based on the amount of feed consumed by the animal. Particularly preferred release rates for bromoform include about 0.1 to about 0.5 g / day, more preferably about 0.2 g / day.

[0232] Additionally, those skilled in the art will appreciate that ruminants can be treated with multiple boluses (100) according to the present invention to achieve a desired dose of inhibitor. This allows for the creation of boluses (100) with different concentrations and total amounts of inhibitor. Multiple of these boluses (100) can be administered to the animal simultaneously or sequentially, allowing for the desired dose to be provided to the animal. This is particularly beneficial, allowing for the use of boluses (100) for animals requiring different doses of inhibitor, such as larger and smaller animals, or to supplement natural growth over time.

[0233] The bolus (100) is adapted to deliver a dose of an inhibitor directly into the animal's rumen. For example, bromoform can be released at a rate that effectively reduces or eliminates methane production during digestion, thereby reducing greenhouse gas emissions from animals and the environmental impact of agriculture.

[0234] Additionally, the bolus (100) may improve feed conversion for ruminant animal production. For example, it is believed that reducing methane production during digestion may lead to more efficient utilization of ingested feed, potentially leading to improvements in growth, weight gain, or other production, such as milk production. Furthermore, the synergistic effects resulting from the combination of the core composition and the carrier and inhibitor may provide a sustained-release, long-term delivery device to improve animal productivity and / or reduce greenhouse gas emissions.

[0235] First Alternate Housing Embodiment 2A and 2B, an alternative embodiment of a bolus (200) is shown in accordance with one embodiment of the present invention.

[0236] Aspects of bolus (200) are similar to those of bolus (100), and therefore like reference numerals refer to like components.

[0237] A series of ribs (240) are provided along the exterior surface of the housing (120). The ribs (240) can provide additional structural strength to the bolus (200) and can help prevent the bolus (200) from bursting if the core (110) expands. Additionally or alternatively, the ribs (240) can aid in the administration of the bolus (200) to the ruminant.

[0238] As shown, ribs 240 are provided as a series of concentric "hoops." However, ribs 240 may also be a series of parallel or non-parallel ribs (not shown) extending along the length of bolus 200.

[0239] Second Alternative Housing Embodiment Reference is now made to Figures 3A-3B, which illustrate an alternative embodiment of a bolus (300) according to one embodiment of the present invention.

[0240] Aspects of the bolus (300) are similar to those of the bolus (100) described above, and therefore like reference numerals refer to like components.

[0241] The bolus (300) includes additional features on the exterior surface of the housing (120), including a depression or groove (350).

[0242] Grooves (350) can facilitate the detachment of portions of housing (120) as housing (120) degrades, which can be used to further control the release profile of the inhibitor.

[0243] Third Alternative Housing Embodiment Reference is now made to Figures 4A-4B, which illustrate an alternative embodiment of a bolus (400) according to one embodiment of the present invention.

[0244] Aspects of the bolus (400) are similar to those of the bolus (100) described above, and therefore like reference numerals refer to like components.

[0245] The bolus (400) includes a housing (120) having a cavity (not shown) configured to receive and retain the core (110).

[0246] The housing (120) is tapered along its length, e.g., the distance between the distal outer surfaces of the housing (120) increases along the length of the bolus (400). For example, as shown in Figure 4A, the width (X) is less than the width (Y).

[0247] Alternatively, the bolus (400) may have sidewalls of substantially constant thickness, but structured and oriented to define a taper in the bolus (400).

[0248] This configuration allows for better control over the degradation of the core (110), thereby allowing for more control over the release of the inhibitor.

[0249] Fourth Alternative Housing Embodiment Referring now to FIG. 5A, an alternative embodiment of a bolus (500) is shown in accordance with one embodiment of the present invention.

[0250] The embodiment of the bolus (500) is similar to that described above, and therefore like reference numerals refer to like components.

[0251] The bolus (500) includes a reservoir (580) adapted to hold a relatively concentrated form of the inhibitor, for example, bromoform in a substantially pure liquid form.

[0252] The bolus (500) includes a dispensing mechanism configured to dispense a predetermined dose of the inhibitor from a reservoir (580).

[0253] 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 inhibitor through the valve (590) and releases the inhibitor into the lumen where the bolus (500) was administered.

[0254] The dispensing mechanism may be configured to be consistent, for example to release the same amount of inhibitor at defined intervals.

[0255] Alternatively, the dispensing mechanism may be configured to vary the amount of inhibitor released at different times. This may be useful to allow the bolus (500) to provide an effective amount of inhibitor that takes into account the growth of the animal. Additionally or alternatively, it may be possible to compensate for changes in other factors, such as seasonal variations in methane production, which may require larger doses of inhibitor.

[0256] In further embodiments, the bolus (500) can include sensors (not shown). For example, a temperature sensor can be included within the bolus (500). Additionally or alternatively, other sensors, such as motor activity or pH, can be included within the bolus. Adding such sensors can provide valuable information regarding the animal's feed intake and can help assess whether the amount of inhibitor is sufficient for the animal.

[0257] Fifth Alternate Housing Embodiment Reference is now made to Figures 6A and 6B, which illustrate an alternative embodiment of a bolus (600) according to one embodiment of the present invention.

[0258] The bolus (600) may be adapted to include additional features within the cavity of the housing, such as grooves or ribs (680) formed in the interior wall of the housing (120) that defines the cavity.

[0259] Embodiments of bolus (600) are similar to embodiments of bolus (100), and therefore like reference numerals refer to like components.

[0260] A series of ribs (680) are provided along the interior surface of the housing (120). The ribs (680) may provide additional structural strength to the bolus (600) and / or provide an additional means for retaining the contents of the core formulation within the cavity of the housing. Additionally or alternatively, the ribs (680) may also aid in retention of the core within the housing. Furthermore, the ribs may also provide for controlled dissolution of the core formulation from the bolus (600) into the ruminant.

[0261] In one embodiment, the exterior surface of the housing remains smooth or uniform.

[0262] Sixth Alternate Housing Embodiment Reference is now made to Figures 15A-15D, which illustrate a further embodiment of a bolus (700) according to one aspect of the present invention. The dimensions of the bolus in the figures are given in mm. Preferably, the bolus has a length of 13 cm, a diameter of 3.4 cm, and preferably weighs approximately 250 grams.

[0263] The bolus (700) can be adapted to include additional features with internal reinforcing structures on the housing.

[0264] Embodiments of bolus (700) are similar to those of bolus (100), and therefore like reference numerals refer to like components.

[0265] The bolus (700) includes at least one reinforcing rib (710) located within a cavity (not numbered) defined by the housing structure. A cap (720) may also be provided that releasably attaches to the bolus (700), for example to close the open end of the bolus (700). Attachment may be provided by a friction fit arrangement or a thread arrangement in which corresponding threads on the housing and cap engage with each other. Alternatively, the cap may be attached to the housing by adhesive or other mechanical fasteners.

[0266] The reinforcing ribs (720) can improve the structural integrity of the bolus (700) and help the bolus retain its shape.

[0267] Manufacturing method Reference is now made to FIG. 7, which is a flow chart illustrating exemplary steps in a method 800 for manufacturing boluses 100, 200, 300, and 400, for example, according to the present invention.

[0268] Generally speaking, the method includes the steps of forming (810) a housing (120) and forming (820) a core (110).

[0269] housing Forming the housing (120) can be accomplished using any technique known to those skilled in the art. For example, a suitable material can be extruded into the desired shape to define the cavity. Alternatively, an additive manufacturing process can be used to build the housing shape that defines the cavity. It is also contemplated that molding processes can be used, such as sacrificial or injection molding processes, 3D printing, or hot melt extrusion processes.

[0270] core In step 820, the core (110) is manufactured.

[0271] Step 820 may include one or more of the following steps.

[0272] Step 822 includes melting the carrier material to provide a molten carrier material.

[0273] Step 824 includes adding an inhibitor to the molten carrier material.

[0274] In step 826, the inhibitor and the molten carrier material are mixed to form a substantially homogenous mixture.

[0275] Step 828 involves forming the substantially homogeneous mixture into a desired shape.

[0276] It is understood that a substantially homogeneous mixture includes the inhibitor at a concentration sufficient to achieve a desired release profile of the inhibitor upon administration of the device to a ruminant, which concentration can vary depending on the type of ruminant being treated, the shape and size of the device, or the desired release profile to be achieved.

[0277] It should be understood that forming the substantially homogeneous mixture into the desired shape may include dispensing the mixture into a mold. In a particularly preferred form, the substantially homogeneous mixture is added (injected) into a cavity within the housing (120) produced in step 810.

[0278] Alternatively, the mold may be a separate component that receives the substantially homogeneous mixture. In these embodiments, once the desired shape is formed, the core may then be placed into the cavity within the housing (120).

[0279] The method also includes the step of allowing the substantially homogeneous mixture to cool, whereupon the carrier material hardens and assumes the shape of the mold or housing in which it is incorporated.

[0280] Example formulation The following cores were formulated for use in the boluses of the present invention. [Table 2] [Table 3] [Table 4] [Table 5]

[0281] verification Example 1: Release / Diffusion Studies A study (RME study 2) using a 2 mm thick 3D printed large capped bolus (LCB2) filled with 66.7% (by weight) bromoform and 33.3% (by weight) beeswax in a RME (rumen emulator) was conducted to determine the diffusion rate of bromoform from the bolus.

[0282] Bolus Design This study used a reinforced bolus, as shown in Figure 15. It included an internal reinforcing structure and extended ribs to support the walls, and the top was adapted to receive a cap. The reinforced bolus was found to be more rigid and hold its shape better than the unreinforced bolus when poured with a molten bromoform / beeswax mixture and cooled, resulting in a physically more rigid bolus in the tests.

[0283] method material Bromoform (reagent grade, Sigma Aldrich, 96% bromoform, 4% ethanol), beeswax (food grade, NZ beeswax, MP65°C), and zinc oxide from Native Elements NZ.

[0284] Bolus production The bolus was drawn in Solidworks, converted to an .stl file, and opened in FlashPrint to create the print job. The bolus was printed in three parts (case, inner structure, and cap) using E-Sun PLA+ at 100% fill, standard resolution, 0.27mm first layer height, 0.18mm layer height, two surrounding shell layers, three upper solid layers, three lower solid layers, hexagonal infill pattern, print speed 60mm / s, extruder temperature 200°C, and plate temperature 50°C.

[0285] Eight LRB boluses were prepared with 67% (by weight) bromoform, eight LRB boluses were prepared with 75% (by weight) bromoform, and six LCB2 boluses were prepared without bromoform (control). The ingredients are listed below (Table 1). All ingredients were weighed on a calibrated 4-dp electronic balance in a beaker. The bromoform solution was covered with parafilm to prevent evaporation. The ingredients were prepared by melting pre-weighed amounts of beeswax and zinc oxide in a beaker at 100°C (thermoprism oven), cooling the mixture to 80°C, adding bromoform, and thoroughly mixing the mixture to prevent the zinc oxide from settling before injecting into the bolus. The cap was press-fitted and soldered to seal the bolus. [Table 6]

[0286] The bolus was placed in a 500 ml polypropylene bottle with approximately 380 ml of 0.02 M phosphate buffer (Merck) in distilled water, prepared in batches of 2 L or more, and adjusted to pH 6.5 using 1 M HCl (Merck) and a pre-calibrated pH meter (using pH 4, 7, and 10 pH buffers). The bottle was sealed and placed in a 40°C incubator. 10 ml samples were collected, and the entire solution was replaced every 24 h.

[0287] A 10 ml sample was collected into a 15 ml Falcon tube using a 10 ml autopipette. 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 using GC-FID, 2 ml of ethyl acetate was added to each Falcon tube. The Falcon tubes were capped, mixed well using a Vortex, and centrifuged at 4000 rpm for 15 minutes. For GC-MS analysis, all ethyl acetate was collected using a graduated glass syringe, and the volume was recorded.

[0288] 0.5 ml of ethyl acetate was collected for GC-FID analysis. For GC-FID analysis, 200 μl of sample was injected using an autosampler and analyzed using a ZB5HT30m capillary column in splitless mode, with a temperature gradient from 30 to 300 °C over 20 min under a 5 ml / min nitrogen gas flow. The retention time of bromoform was 7.5 min. The mass of bromoform (mg) was determined by comparing the peak area with a calibration standard prepared with ethylene acetate. This was divided by the injection volume to determine the concentration of bromoform in ethyl acetate (mg / L). The concentration in ethyl acetate was multiplied by the total amount of ethyl acetate added to the sample and divided by the recovery rate to determine the mass of bromoform in the sample. This was then divided by the volume of the collected sample to determine the concentration in solution, which was then multiplied by the volume of solution in the shot bottle to determine the mass transferred from the bolus to the solution. Bromoform recovery from solution was checked using standard solutions prepared with various concentrations of bromoform and was typically 43%. GC-FID performance was checked after every run of 10 samples using a calibration sample as a reference.

[0289] result For both boluses, a low diffusion rate followed by a rapid increase in diffusion rate was observed (Figure 8). The 67% bolus had a 4-5 day delay before reaching the maximum diffusion rate, whereas the 75% bolus reached the maximum diffusion rate in 3 days.

[0290] The diffusion rate was higher for the 1010 mg / day 75% bolus compared to 66.7% for the 730 mg / day bolus. This was surprising, but also a positive result, since the predicted diffusion rate for a 67% bromoform LCB1 bolus was 300 mg / day and for a 75% bromoform LCB1 bolus was 462 mg / day (meaning a single bolus could be used to administer to a 700 kg bull and achieve methane reduction). Because the LRB bolus is 1 mm thick and has a reduced surface area (approximately 71% of the LCB1 bolus), a lower diffusion rate was expected (Table 2). Theoretically, the LRB bolus should deliver only 220 mg / day for 67% bromoform and 344 mg / day for 75% bromoform. [Table 7] [Table 8]

[0291] The variability of the diffusion data was initially high, with a coefficient of variation of approximately 1, which decreased to 0.05–0.22 as the bolus reached its maximum diffusion rate (Figure 9). The 75% bolus stabilized within 2 days, and the 67% bolus stabilized within 4 days.

[0292] Zero-order release was observed for both boluses, indicating that the release rate was independent of the concentration of bromoform in the bolus (Figure 11).

[0293] conclusion The diffusion rates of the LRB bolus were 1010 mg / day for the 75% bolus and 730 mg / day for the 66.7% bolus, which were higher than predicted from previous diffusion studies.

[0294] The concentration of bromoform in the 75% bolus medium is close to the solubility limit of bromoform in water (3.2 g / L), so the diffusion rate may be higher than that measured in this study.

[0295] Example 2: Carrier release test For this study, release tests were conducted on various carriers.

[0296] method material Bromoform (reagent grade, Sigma Aldrich, 96% bromoform, 4% ethanol), ruminal fluid (Dairy NZ test), paraffin wax (MPs 46-48, 55 and 65°C, Sigma Aldrich), castor wax (Lotus Oils), carnauba wax (PureNature NZ), zinc oxide (PureNature NZ).

[0297] Ruminal fluid pH and buffer capacity Rumen fluid collected from Dairy NZ was thawed, centrifuged, and then analyzed for pH and buffering capacity. 10 ml of rumen fluid received from each cow was sampled and titrated against 0.05 N NaOH, with pH continuously monitored. The amount of NaOH required to change the pH in units was recorded.

[0298] Release and testing of various carriers Small capped boluses were prepared as described in Example 1 above.

[0299] Paraffin wax, beeswax, carnauba wax, and castor wax were mixed with bromoform to give bromoform concentrations of 33%, 50%, 67%, and 75% by weight. The mixes were as follows: a. Paraffin wax: 2mm thick small capped bolus and 15ml Falcon tube; b. Castor, carnauba and beeswax: 1, 2 and 3mm small capped boluses and 15ml Falcon tubes.

[0300] These were placed in 500 ml polypropylene bottles with 400 ml of 0.02 M phosphate buffer (Merck) in distilled water, prepared in batches of 2 L or more, and adjusted to pH 6.5 using 1 M HCl (Merck) and a pre-calibrated pH meter (using pH 4, 7, and 10 buffers). The bottles were sealed and placed in an incubator at 40°C. 10 ml samples were collected, and the entire solution was replaced every two days (Monday, Wednesday, and Friday), excluding weekends.

[0301] Samples were analyzed by GC-MS and GC-FID as described in Example 1 above.

[0302] result pH and buffer capacity The mean pH and buffering capacity were 6.9 ± 0.2 (n = 4) and 7.47 ± 1.4 mMol / L / delta pH (n = 4), respectively. While the pH values ​​of rumen fluid have been reported in the literature, data on buffering capacity were not available. The buffering capacity obtained for rumen fluid is 5–6 times higher than that of phosphate-buffered saline, indicating a resilient rumen environment. The pH of phosphate buffer in the diffusion experiment was found to be stable even at bromoform concentrations near 3 mg / mL (Report No. BR2021-01, Figure 4). Given a rumen fluid volume of 91 L, the maximum bromoform concentration reached approximately 1.09 mg / mL under extreme conditions of complete bolus rupture, which was lower than that previously observed with PBS. Therefore, considering the strong buffering capacity of rumen fluid at this concentration, a drop in pH is unlikely even in the event of a sudden bolus rupture.

[0303] Carrier release test Paraffin wax had the highest release rate at 190 mg / cm2 / day, followed by beeswax, carnauba wax, and castor wax (Figure 12). Carnauba wax and castor wax appear to be better choices as carriers, as their release rates are 50-40% lower than beeswax.

[0304] Bromoform showed the highest release rate in boluses made from paraffin wax, at 3.5–5.4 mg / cm / day in small capped boluses with a thickness of 2 mm (Figures 13A–C).

[0305] The release rates of boluses made from carnauba wax were up to 5.5 mg / cm² / day for boluses with a thickness of 1 mm and 1.66 mg / cm² / day for boluses with a thickness of 3 mm.

[0306] In comparison, boluses made from beeswax showed a release rate of 3 mg / cm2 / day for 75% (by weight) bromoform (Figure 13C).

[0307] Bromoform dissolved the castor wax, diffused through the bolus, settled at the bottom of the container, and dissolved the container, and no bromoform was detected in the water samples taken, so a release rate could not be determined. The castor wax experiment could be repeated in a glass vial.

[0308] Enhanced bolus emission rate From a separate study, the mean release rates of large, fortified boluses containing 67% (by weight) and 75% (by weight) bromoform, prepared as described in Example 1 above, are shown in Figure 14 and compared with the release rates from the same boluses measured in the laboratory. Half of the boluses were placed in a 20 L bucket containing 1 kg of sand filled with pH 6.5 buffer, and the other half were placed in a 20 L bucket containing 400 g of wood chips and 1 kg of sand. The release rates at 28 days were comparable to those observed in the laboratory. Little difference in bromoform concentration was observed between the buckets containing wood chips and those without. The boluses remained largely intact, except for some compaction by the sand and some with open lids.

[0309] Example 3: Animal studies An animal study was conducted to measure methane emissions from animals 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 time periods 8-12 weeks apart.

[0310] Nineteen dairy heifers (live weight 312 ± 14 kg), including three reserves, were selected from a group of 50 from a research farm in Manawatu, New Zealand, based on behavioral characteristics and live weight. They were assigned to one of three treatments: a bromoform-free bolus (control; n = 4); a bromoform-releasing bolus (low, n = 6) at a rate of approximately 300–400 mg / day; or a bromoform-releasing bolus (high, n = 6) at a rate of approximately 450–580 mg / day. SmaXtec bolus was administered simultaneously to monitor rumen temperature and supplement weekly blood samples as an animal health monitor.

[0311] Heifers were transported from the research farm to the test center for dietary adaptation and gas measurements using a respiration chamber. Heifers were allowed to adapt to the barn and freshly cut pasture environment for 7 days before receiving their assigned treatment bolus. Gas measurements began 13 days after bolus administration. During the gas measurement period, each heifer was placed in a respiration chamber for 48 hours, spanning 2 weeks for each of the four measurement groups. After the respiration chamber measurements were completed, the animals were returned to the research farm.

[0312] Bolus preparation The boluses were prepared according to the procedure described above in Example 1. The following formulations used in this study are shown in Table 4 below. [Table 9]

[0313] Bolus administration Three versions of the bolus were administered within the first 10 days of the experiment. The first version was a short bolus that all animals regurgitated within 5 days of bolus administration. Because the control bolus was longer than the treatment bolus and did not regurgitate during the first 3 days, it was assumed that bolus size was the primary factor in regurgitation. All first-version treatment boluses were replaced with second-version boluses on the 5th day after administration. However, the longer second-version boluses were also regurgitated. Therefore, these boluses were replaced with third-version treatment boluses, which were significantly heavier and of the same size as the second-version boluses. No third-version boluses have been regurgitated to date. Currently, the third-version boluses have been administered to almost all heifers, except for three low-treatment heifers. Details of bolus regurgitation and readministration are shown in Table 5.

[0314] Two control boluses were regurgitated, but only one was identified because the bolus ID was illegible. None of the control boluses were re-administered because a match between heifer and bolus could not be identified. [Table 10]

[0315] Feed intake and live weight Heifers were fed a cut ryegrass-based pasture ad libitum. The forage was harvested daily at approximately 10:00 AM on the research farm and transported to the test center. The harvested forage was divided into two portions: the first portion was fed at 15:30 PM, and the second portion was stored at 4°C until feeding the following morning at 08:30 AM. Samples were collected from each pasture for dry matter measurement and ration analysis. Dry matter (DM) was determined from three subsamples by oven-drying at 105°C for 24 hours. Another subsample was oven-dried at 65°C for 48 hours for chemical nutrient analysis. Both drying ovens used were forced-air ovens (Avantgarde FED 720, Binder GmbH, Germany).

[0316] Two days before entering the respiration chamber for methane measurements, the cows were placed in metabolic crates and restrained to allow them to adapt to the confined space. While the animals were in the metabolic crates or respiration chambers, rejected feed was collected twice daily, and rejected DM was measured as described above. The heifers' daily dry matter intake was then determined from the difference between offered and rejected dry matter.

[0317] Live weights were recorded twice (July 13, 2021 and July 16, 2021) while the animals were grazing on the research farm prior to the study. Animals were weighed upon arrival at the test farm on July 19, 2021, and again every 7–10 days while on site. The first live weight was measured on July 23, 2021, before the bolus administration, and the final live weight was after the animals left the respiration chamber. Measurements were taken over a 2-week period, so the date of the final live weight varies for some animals.

[0318] Gas Measurement Fermentation gases methane (CH4), carbon dioxide (CO2), and hydrogen (H2) were quantified in four open-circuit respiration chambers at the New Zealand Ruminant Methane Measurement Centre (AgResearch, Palmerston North, New Zealand). Each chamber had a volume of 15.4 m 3 (Length 3.5m x Width 2m x Height 2.2m) and the air flow velocity is about 1.0 m 3The respiration chamber was continuously monitored by measuring differential pressure using a Venturi flowmeter at a flow rate of approximately 1 / min. The temperature in the respiration chamber was approximately 20°C, and the relative humidity averaged approximately 79%. All gases were measured at approximately 2.8-minute intervals using a 4900C Continuous Emission Analyzer (Servomex Group Ltd, East Sussex, UK). The daily production of each gas was calculated from the difference between the concentration entering the chamber and the concentration exiting the chamber (Pinares-Patino et al., 2012). The respiration chamber was opened twice daily (approximately 20 minutes each time) for cleaning, feeding, fecal sampling, and collection of feed refusals. No measurements were taken while the chamber was open, and missing data were interpolated by averaging the last 12 values ​​(approximately 45 minutes) before the door was opened.

[0319] statistical analysis Data from the first period gas measurements were analyzed using the "predictmeans" and "lme4" packages in the statistical software R4.0.3 (R Core Team, 2020). Dry matter intake and gas output data for each heifer were averaged across the two measurement days. The heifer served as the experimental unit. Mixed models included treatment as a fixed effect and the respiration chamber nested within measurement group as a random effect.

[0320] Liveweight analyses included treatment as a fixed effect and time as a repeated measure, with heifers as the subject for the repeated measure. Only initial and final liveweights were included in this analysis.

[0321] result Dry matter intake and gas output Bromoform administered to heifers at approximately 300–400 mg / day (low) or approximately 450–580 mg / day (high) did not affect dry matter intake measured over the two days in the respiration chamber compared to the control group (p=0.42). Both: CH4 production (g / day) and CH4 yield (g / kg dry matter intake) were reduced by more than 99% in the low and high treatments compared to the control (p<0.01). The reduction in CH4 emissions in the low and high treatments was accompanied by an increase in daily H2 emissions (Table 7). Because methane emissions were completely reduced in both treatments, lower doses could achieve 30–90% methane reduction levels. Reducing the daily dose ensures that no more bromoform than necessary is used to extend the life of the bolus, reducing the risk of adverse effects on the animals and potential contamination of animal products. Considering that methane emissions were completely suppressed, it is noteworthy that dry matter intake was not adversely affected, as observed when feeding bromoform containing asparagopsis (Roque et al. 2019). [Table 11]

[0322] conclusion As observed, the above results indicate that treatment using a bolus according to the present invention is highly effective several weeks after bolus administration, as evidenced by an approximately 99% reduction in methane.

[0323] Unless the context clearly requires otherwise, throughout the description and claims, the terms "comprises," "including," and the like are to be construed in an inclusive sense, i.e., "including but not limited to," rather than in an exclusive or exhaustive sense.

[0324] The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference.

[0325] The reference to prior art in this specification is not, and should not be construed as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any field of endeavor anywhere in the world.

[0326] The invention may also be broadly stated as consisting of the parts, elements and features individually or collectively referred to or indicated in the specification of this application, and any and all combinations of two or more of said parts, elements or features.

[0327] Where reference has been made in the foregoing description to components having integers or known equivalents, those integers are incorporated herein as if individually set forth.

[0328] 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 can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. Accordingly, such changes and modifications are intended to be included in the present invention.

[0329] Example 4 method material PLA (3052D), PBS (supplier Convex), and PBAT (supplier Convex) were freeze-dried in aluminum foil trays using a Labcono freeze dryer prior to use to reduce the water content in the blends.

[0330] Bolus production Blends of PLA(3052D), PBS, and PBAT were prepared by mixing the pellets in the following ratios: [Table 12]

[0331] Blends were prepared by melt blending in a LabTech co-rotating twin-screw extruder (L / D 44:1) at a screw speed of 200 rpm. The temperature profile increased across 11 barrel heating sections, from 70 °C at the feed throat to 220 °C along the main barrel and 230 °C at the die. Blends were granulated using a tri-blade granulator (Castin Machinery, New Zealand) equipped with 4 mm plates. Blends were stored in aluminum foil trays and packaged in Ziploc bags before use. All blends were oven-dried overnight at 40 °C before injection molding. Tensile specimens (ASTM D368) and impact specimens (ISO 179) were fabricated in a BOY 35A injection molding machine using a temperature profile of 70 to 220 °C from the feed to the nozzle. The mold temperature was held constant at 50 °C. Lanolin was used as a mold release agent and was sprayed into the mold before each tensile bar was fabricated.

[0332] Bolus analysis Injection molding shrinkage was determined by measuring the width and thickness of the tensile specimens, subtracting this from the width and depth of the mold, dividing by the width and depth of the mold, and multiplying by 100 to obtain a percentage. Tensile bars were cut to approximately 2 cm lengths using a band saw, and the ends were sanded smooth using 500-grit sandpaper. Flat-bottom glass Petri dishes with a diameter of 120 cm were filled with beeswax / bromoform mixtures with the following bromoform concentrations: 33, 50, 67, and 75 wt%. Three samples of each PLA blend were labeled, weighed on a 4-dp electronic balance, and measured for thickness, length, and width using digital calipers. They were then laid flat and gently pressed into each bromoform / beeswax blend to ensure good contact between the beeswax and the PLA surface. A glass lid was then placed on top of the Petri dishes, sealed using electrical tape, and placed in a 40°C incubator.

[0333] The hardness of the samples was tested using a Shore D hardness tester with a weight of 7 kg, and the structural properties were also tested using XRD.

[0334] Every 2–3 days, the samples were removed from the Petri dishes, washed using tissue paper, weighed using a 4dp electronic balance, and measured using digital calipers.

[0335] The absorption of bromoform was determined by measuring the total change in mass of the sample and dividing by the starting mass of the sample. The absorption rate was determined by dividing the change in mass of the sample between measurements by the area of ​​the sample in contact with the bromoform / beeswax mixture and dividing by the change in time between measurements.

[0336] Swelling was determined by measuring the change in volume of the sample and dividing by the original volume of the sample.

[0337] result injection molding The shrinkage for PLA was approximately 0.2%, but increased to approximately 1-1.2% with increasing PBS and PBAT blends (Figure 16). One of ordinary skill in the art would know how to adjust the shrinkage to produce a bolus of the desired size and dimensions. It should be understood that various bolus sizes are possible and, given the teachings of this patent application, are not critical to achieving delayed release of the haloform.

[0338] At bromoform concentrations below 50 wt% in beeswax, less bromoform was absorbed, suggesting limited mobility of bromoform at low bromoform concentrations in beeswax and the strong retention capacity of beeswax for bromoform (Figure 16B). As the bromoform concentration in beeswax increased and the mass fraction of PBAT and PBS in PLA increased, the mass of bromoform absorbed and the maximum absorption rate also increased (Figures 16C and D). The absorbed mass of the PLA blend was lower than that of 200 3D PLA and 3D-printed PLA (Figure 16E).

[0339] Example 5 method Unless otherwise indicated, samples were prepared and analyzed as described in Example 4.

[0340] The samples were tested for hardness using a Shore D hardness tester with a weight of 7 kg, and also for structural properties using XRD before and after exposure to the bromoform / beeswax mixture.

[0341] For XRD analysis, a PANalytica Empyrean XRD equipped with a flat sample stage holder with an adjustable beam to maintain an exposed area of ​​1 cm x 5 mm at all angles between 5 and 70 2Theta was used in the following configuration: [Table 13]

[0342] XRD data were exported to Excel, smoothed with 10-point smoothing, and baseline corrected between 5 and 60 2θ. result Figure 17 shows the hardness analysis of the PLA blend before and after exposure to bromoform. Therefore, the inclusion of PBS in the carrier may make the blend less sensitive to exposure to bromoform and promote shelf life.

[0343] Example 6: Enhanced large bolus delivery test (Rissington test) The bolus was drawn in Solidworks, converted to an .stl file, and opened in FlashPrint to create the print job. The bolus was printed in three parts (case, inner structure, and cap) using E-Sun PLA+ at 100% fill, standard resolution, 0.27mm first layer height, 0.18mm layer height, two surrounding shell layers, three upper solid layers, three lower solid layers, a hexagonal infill pattern, a print speed of 60mm / s, an extruder temperature of 200°C, and a plate temperature of 50°C.

[0344] Two separate formulations were prepared, containing 67% and 55% (by weight) bromoform in a carrier mixture of castor wax and paraffin wax (50:50 in this example). Then, after inserting a zinc rod as a densifying agent, each bromoform-wax mixture was injected into a 1 mm-thick casing. A soldering gun was used to attach and seal the cap. The release test was performed according to the method described in Example 1, with some modifications; instead, 2 L of medium was used, which was changed daily. A 10 ml sample was taken and, after appropriate extraction with ethyl acetate, injected into a GC to quantify bromoform release.

[0345] Bromoform was released at a higher rate from a 67% (by weight) bromoform (1150 mg / day) bolus, whereas the release rate was slower from a 9.5 mg / day 55% (by weight) bromoform loading bolus (Figure 18A).

[0346] Next, four different types of boluses (two of each type) containing 57% (by weight) bromoform with 1 mm and 2 mm casings, and 55% (by weight) and 67% (by weight) bromoform with 2 mm casings were administered to zinc rods with similar carrier compositions. Release tests were performed according to the method described above. The 2 mm casing showed a slow release rate, regardless of the bromoform content (Figures 18B-D). On the other hand, the 1 mm casing bolus containing 57% (by weight) bromoform showed a 7-day delay, reaching 240 mg on day 8 and 400 mg on day 9 (Figure 18D). The cumulative plots for 7, 8, and 9 days showed the best fit, indicating a release rate of 319 mg / day (Figure 18C).

[0347] Each bolus was tested in RME according to the method described in the above example. After 6 days of the study, the boluses were retrieved and visually inspected. The boluses remained intact with no signs of damage or deformation. Example 7: Design of a bromoform-containing bolus In one preferred embodiment tested in this example, the bolus comprises a housing and a core configured as defined below. [Table 14]

Claims

1. 1. A bolus for administration to a ruminant, comprising: The bolus comprises: a core containing a methane inhibitor to be administered to a ruminant mixed with a carrier; and a housing covering at least a portion of the core; the bolus is configured to release the methane inhibitor through the housing over a predetermined period of time; the methane inhibitor is a haloform, the haloform being selected from the list of bromoform, chloroform, iodoform, and combinations thereof; The bolus.

2. 10. The bolus of claim 1, wherein the carrier and the methane inhibitor have a relatively high affinity for each other compared to the affinity between the housing and the methane inhibitor for each other.

3. 3. The bolus of claim 1, wherein the haloform is contained in the core in an amount of 30% to 80% by weight.

4. A bolus according to any one of claims 1 to 3, wherein bromoform is contained in the core in an amount of up to 55% by weight.

5. 5. The bolus of any one of claims 1 to 4, wherein the carrier is a polar substance selected from the list of wax, myristic acid, stearic acid, steryl alcohol, cetyl alcohol, cetosteryl alcohol, castor wax, beeswax, paraffin wax, PEG 4000, carnauba, candelilla, jojoba, lanolin, and combinations thereof.

6. 6. The bolus of claim 5, wherein the wax is mixed with haloform.

7. A bolus as described in claim 6, wherein the haloform includes bromoform.

8. A bolus as described in claim 5, wherein the carrier comprises beeswax, paraffin wax and / or castor wax.

9. A bolus as described in claim 5, wherein the carrier comprises castor wax and paraffin wax in a weight ratio of castor wax to paraffin wax between 40:60 and 60:

40.

10. A bolus as described in either claim 5 or 6, wherein the bolus comprises at least one metal piece.

11. A bolus according to any one of claims 1 to 10, wherein the housing comprises a cavity in which at least a portion of the core is disposed.

12. 12. A bolus according to any preceding claim, wherein the housing includes an open end and the bolus includes a cap configured to close the open end.

13. 13. The bolus of any one of claims 1 to 12, wherein the housing and cap substantially or completely cover and surround the core to define the core.

14. A bolus according to any one of claims 1 to 13, wherein the housing completely covers and surrounds the core.

15. A bolus according to any one of claims 1 to 14, wherein the housing is made from a material having a Shore D hardness of at least 40 and / or less than 70.

16. A bolus according to any one of claims 1 to 15, wherein the housing is made from a plastic material through which the methane inhibitor can migrate.

17. A bolus according to any one of claims 1 to 16, wherein the housing is made from a plastic material.

18. 18. The bolus of claim 17, wherein the plastic is one or more of polylactic acid (PLA), polyglycolic acid (PGA), polylactic-glycolic acid (PLGA), polypropylene, SLA polymer, PBS, PBAT, or combinations thereof.

19. A bolus according to any one of claims 1 to 18, wherein the housing is made from materials including polylactic acid (PLA) and polybutylene adipate terephthalate (PBAT).

20. A bolus according to any one of claims 1 to 19, wherein the carrier comprises or consists of a wax.

21. 20. The bolus of claim 19, wherein the material comprises polylactic acid (PLA) and polybutylene adipate terephthalate (PBAT) in a weight ratio of polylactic acid (PLA):polybutylene adipate terephthalate (PBAT) ranging from 100:0 to 40:60, and the carrier comprises a wax.

22. A bolus according to any one of claims 1 to 21, wherein the housing is made from a material comprising one or more excipients selected from the group comprising plasticisers, hardeners and / or colourants.

23. A bolus according to any one of claims 1 to 22, wherein the housing has a material thickness of less than 2 mm.

24. A bolus according to any preceding claim, wherein the housing is configured to degrade over a predetermined period of time.

25. A bolus according to any one of claims 1 to 24, wherein the core has a melting point above 37°C.

26. 26. The bolus of any one of claims 1 to 25, further comprising a barrier layer between at least a portion of the housing and the core to isolate the portion of the housing and the core from contacting each other.

27. 27. The bolus of any one of claims 1 to 26, wherein the bolus is adapted to achieve a maximum release rate of bromoform into the lumen of about 0.05g to 2g per day.

28. 28. A bolus according to any one of claims 1 to 27, wherein the bolus is adapted to release the substance over a period of at least two months.

29. 1. Use of a methane inhibitor and a carrier in a bolus for reducing methane production in or methane emissions from a ruminant, wherein the methane inhibitor comprises a haloform selected from the list of bromoform, chloroform, iodoform, and combinations thereof.

30. A method for producing a bolus according to any one of claims 1 to 16, comprising the steps of: forming a housing having a cavity; forming a core comprising a methane inhibitor; Transferring the core into the cavity The method comprising:

31. 31. The method of claim 30, wherein the step of forming the core comprises mixing a carrier material with the substance.

32. 32. The method of claim 30 or 31, wherein the step of forming the core comprises heating the carrier material to melt the carrier material before mixing the carrier material with the substance to form the mixture.

33. The method of any one of claims 30 to 32, wherein the step of transferring the core to the cavity comprises injecting the mixture into the cavity.

34. 29. A bolus according to any one of claims 1 to 28, wherein the core of the bolus comprises one or more metal particles.

35. A bolus as described in claim 34, wherein the metal particles include steel particles.

36. A bolus as described in claim 34, wherein the metal particles include round metal particles.

37. The bolus of claim 34, wherein the bolus comprises at least 100 grams of metal particles.

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