WO2025029158A1

WO2025029158A1 - Improved bolus designs for administration to a ruminant animal and uses thereof

Info

Publication number: WO2025029158A1
Application number: PCT/NZ2024/050084
Authority: WO
Inventors: Prabhat BHUSAL; Geoffrey Earle Corbett; Bishal Raj ADHIKARI; David Siegel
Original assignee: Ruminant Biotech Corp Ltd
Current assignee: Ruminant Biotech Corp Ltd
Priority date: 2023-08-01
Filing date: 2024-08-01
Publication date: 2025-02-06
Anticipated expiration: 2026-02-01

Abstract

The disclosure provides an improved bolus configured for administration to an animal, wherein said bolus is configured to release a methane inhibiting agent or another active agent to the animal. Preferably, the methane inhibiting agent is a haloform but the bolus can be adapted as described herein to house other active agents such as other methane inhibiting agents. Uses of the bolus and methods of production are also provided.

Description

Improved bolus designs for administration to a ruminant animal and uses thereof

Field of the disclosure

The present disclosure relates to improvements in devices and methods for delivery of substances to animals, and in particular to devices and methods for administering at least one substance to the rumen of a ruminant animal, and methods of manufacturing of the devices.

Background of the disclosure

In farming it is often necessary to deliver substances to animals. This can be for any of various purposes, including but not limited to treatment or prevention of disease and to increase animal production.

There are various devices (e.g. dosage forms) and methods to deliver substances such as medicament to animals. Some substances are for administration to the rumen of ruminant animals. Some dosage forms are for extended release (i.e. sustained release) to the rumen of ruminant animals. Some dosage forms are for administration of multiple active ingredients to the rumen of ruminant animals. However, administration of substances to the animals without the need for a farmer or animal keeper to repeat administration is still needed. Extended release dosage forms present challenges relating to extended control of the quantity of the dosage released, and reliability of such control over time and across batches. Similarly, control of release of multiple active ingredients also poses challenges in release profile and reliability.

Extended release and/or release of multiple active ingredients within the rumen presents further challenges owing to the local environment, which is less well studied, particularly with respect to extended release dosage forms, than the digestive tract of single-stomached organisms (such as humans). Controlled extended release of the substance in the rumen can increase the efficacy and reduce the side effects of administered dosage forms by reducing the maximum concentration of the active substance to a concentration that is more consistent with the effective concentration of the active substance, and maintaining such an effective concentration over an extended period. Release of multiple active ingredients from a single dosage form reduces the number of administrations required. Administration of dosage forms to large animals such as ruminants can require significant investment of time and infrastructure. However, the possibility that the multiple active ingredients may affect release profile makes such a formulation challenging, particularly in extended release formulations.

There is a need for improved dosage forms for extended release and/or release of multiple active ingredients to the rumen. Preferably, these dosage forms provide controlled extended release to the rumen, more preferably, the control is maintained until the majority of the substance is released from the dosage form to the rumen.

A sustained release may be particularly desired wherein a low dosage of active substance for an extended period may provide best efficacy of treatment.

The challenges associated with extended release of a substance increase with the amount of extension desired. Extending release for hours, days, weeks and months is progressively more difficult, as is controlling release to deliver a biologically effective and non-toxic dosage across the extended timeframe. There is a need for improved dosage forms for delivering substances with an extended release profile to the rumen of a ruminant animal for increased periods of time. Preferably, the extended release is suitably controlled over that time period.

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

It is an aspect of the present invention to provide improved devices and methods to deliver substances to an animal, e.g. methane inhibiting agents.

It is an aspect of the invention to provide devices and methods to reduce emission of greenhouse gas (“GHGs”).

It is an aspect of the invention to provide devices and methods to improve animal production gains e.g. through reduction of methane production.

It is an aspect of the invention to provide a formulation to reduce emission of GHGs by one or more animals e.g. a ruminant animal.

It is an aspect of the invention to provide devices and methods that can release substances at different rates over a period of time, for example sustained release dosage forms comprising a methane inhibiting agents. It is also an aspect of the invention to provide devices and methods adapted to the specific properties of certain GHG inhibiting agents.

Alternatively, it is an aspect of the invention to overcome some of the disadvantages of the prior art.

Alternatively, it is an aspect of the present invention to provide the public with a useful choice of methane inhibiting agents and according device and method adaptions for administering these methane inhibiting agents.

Summary of the disclosure

The present disclosure relates to devices and methods to deliver substances to animals. In preferred forms further outlined herein, the substance is a methane inhibiting agent. The present disclosure is exemplified with reference to preferred embodiments, which however, are not to be seen as limiting on the scope of the disclosure. All documents cited herein are incorporated by reference. All embodiments disclosed herein can be combined unless stated otherwise.

In a first aspect the disclosure provides a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which surrounds (or covers at least a portion of or fully encases) the core, wherein the housing is configured such that its permeability to said methane inhibiting agent is increased when exposing the housing to the rumen of a living animal; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to the rumen of a living animal, allowing an increased release of the methane inhibiting agent from within the bolus through said opening or openings.

In a further aspect the disclosure provides a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which covers at least a portion of the core, wherein the housing is configured such that its permeability to said methane inhibiting agent is increased by at least 5 % when exposing the housing to phosphate buffer (pH: 6.5, 0.02 M) at 40 °C without agitation, relative to the permeability on the same day after exposure of the housing to phosphate buffer (pH: 6.5, 0.02 M) at a reference temperature without agitation for the same length of time, wherein permeability is assessed based on release rate of the methane inhibiting agent.

The disclosure provides in a further aspect a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent in microencapsulated particles and wherein the microencapsulated particles are dispersed in a composition comprising a carrier and optionally also comprising a dispersing agent; and a housing covering at least part of the core.

In a further aspect the disclosure provides a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a carrier and microencapsulated particles, wherein the microencapsulated particles comprise the methane inhibiting agent and wherein the microencapsulated particles are dispersed in the carrier, preferably wherein the carrier has a porous structure such as mesoporous silica.

In a further aspect, the disclosure provides a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises a core, wherein the core comprises the methane inhibiting agent and a carrier, and wherein the bolus does not comprise a housing.

In a further aspect the disclosure relates to a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent and a carrier; and a housing which comprises the core; wherein the methane inhibiting agent is selected from the group consisting of monensin, lauric acid, myristic acid and linoleic acid; and wherein the housing comprises at least one opening exposing the core to the environment surrounding the bolus.

In a further aspect the disclosure relates to a bolus of the disclosure, wherein in addition or instead of said methane inhibiting agent the bolus comprises an active agent, wherein said active agent is selected from the group consisting of an anti-inflammatory agent, an analgesic, an antibiotic and an anthelmintic.

In a further aspect, there is provided a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core; a housing that covers at least a portion of the core; wherein the core includes at least one methane inhibiting agent and at least one further active agent.

In a further aspect, there is provided a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core comprising the methane inhibiting agent dispersed in or forming part of one or more of a hydrogel, oleogel or organogel; and a housing which covers at least a portion of the core.

In another aspect the disclosure relates to a bolus of the disclosure for use in the treatment of an animal and preferably a ruminant animal. In another aspect, the disclosure relates to a bolus of the disclosure for use in reducing and/or inhibiting methane emission in a ruminant animal. The disclosure also provides a method of treating an animal comprising administering a bolus of the disclosure to said animal.

In a further aspect the disclosure relates to a method of manufacturing a bolus of the disclosure.

In a further aspect the disclosure relates to a methane inhibitor for use in reducing methane emission from a ruminant animal, wherein the methane inhibitor is selected from the group consisting of bromoform, monensin, lecithin, lauric acid, myristic acid, linoleic acid and phospholipid.

In a further aspect the disclosure relates to a method of treating a ruminant animal to reduce methane emission from said ruminant animal comprising administering to said animal a methane inhibitor selected from the group consisting of bromoform, monensin, lecithin, lauric acid, myristic acid, linoleic acid and phospholipid.

In another aspect, the present disclosure provides a method of administering a methane inhibiting agent to a ruminant animal, the method including administering to the rumen of the ruminant animal a bolus according to the present disclosure.

In another aspect, the present disclosure provides a method of reducing methane production in the rumen of a ruminant animal, the method including administering to the rumen of the ruminant animal a bolus according to the present disclosure.

In yet another aspect, the present disclosure provides a method of making a bolus, the method including: selecting a core and a housing inserting the core into the housing, optionally closing the housing to surround or substantially surround the core in the housing; wherein the core comprises a methane inhibiting agent. Optionally, the core is poured into the housing melted and solidifies in the housing. Optionally, the housing is enclosed around the core following solidification of the core.

In some embodiments, the housing is configured such that its permeability to said methane inhibiting agent is increased when exposing the housing to the rumen of a living animal.

In a further aspect there is provided a material comprising an acrylate-based polymer when used in packing a bolus of the disclosure. Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1. Release profile of bolus loaded with matrix composed of 60% tribromomethane, 20% EC and 20% HPMC when placed in buffer at room temperature (RT: 25 °C), 30 °C, and 40 °C.

Figure 2. Release profile of bolus loaded with matrix composed of 58.4% tribromomethane, 27.3% HPMC and 14.3% EC when placed in buffer at room temperature (RT: 25 °C), 30 °C, and 40 °C.

Figure 3. Release profile of bolus with 0.9, 1.2, and 1.5 mm housing thicknesses loaded matrix composed of 58.4 % tribromomethane, 27.3% HPMC and 14.3 % EC when placed in buffer at 40 °C.

Figure 4. Inverted vial containing lecithin-TBM gel.

Figure 5. Release profile of bolus loaded with matrix composed of Lecithin 55 %/ tribromomethane 45%, Lecithin 40 %/ tribromomethane 60%, and Lecithin 30 %/ tribromomethane 70% over 30 days.

Figure 6. Release profile of bolus loaded with matrix composed of Lecithin 55 %/ tribromomethane 45%, over 120 days.

Figure 7. Inverted vial containing PMMA-TBM gel.

Figure 8. Release profile of bolus loaded with matrix composed of PMMA 55 %/ tribromomethane 45%, and PMMA 30 %/ tribromomethane 70% over 45 days.

Figure 9. Release profile of bolus loaded with matrix composed PMMA 55 %/ tribromomethane 45% over 90 days.

Figure 10. Release profile of bolus loaded with matrix composed of MCW 30 %/ tribromomethane 70%, and MCW 35 %/ tribromomethane 65 % over 30 days. Figure 11. Release profile of bolus loaded with matrix composed of MCW 35 %/ tribromomethane 65 % over 80 days.

Figure 12. Release profile of bolus loaded with 17, 34, 52, and 70 g of MCW 35 %/ tribromomethane 65 % matrix.

Figure 13. Release profile of bolus loaded with matrix composed of stearic acid 30 %/ tribromomethane 70%, and stearic acid 35 %/ tribromomethane 65 %.

Figure 14. Change in release profile of bolus loaded with eicosane 35 %/ tribromomethane 65 % when moved from room temperature to 40 °C.

Figure 15. Release profile of MCW 35 %/ tribromomethane 65% with or without housing (PLA/PBAT= 9/1 ).

Figure 16. Release profile of MCW 55 %/ tribromomethane 45% with or without housing (PLA/PBAT= 9/1 ).

Figure 17. Effect of inclusion of lecithin (5 % w/w) on release profile of stearic acid matrix loaded with 65% TBM. The increase in concentration of lecithin (5%) was compensated with corresponding decrease in stearic acid from 35 to 30% w/w.

Figure 18. Effect of inclusion of monensin (2% and 5 % w/w) on release profile of TBM loaded EC/HPMC matrices. The increase in concentration of monensin (0, 2 and 5%) was compensated with corresponding decrease in HPMC content (20, 18, and 15%). The concentration of EC and TMB were consistent at 20 and 60% w/w respectively.

Figure 19. Effect of inclusion of phloroglucinol (2% and 5 % w/w) on release profile of TBM loaded EC/HPMC matrices. The increase in concentration of phloroglucinol (0, 2 and 5%) was compensated with corresponding decrease in HPMC content (20, 18, and 15%). The concentration of EC and TMB were consistent at 20 and 60% w/w respectively.

Figure 20. Effect of inclusion of albendazole (2% and 5 % w/w) on release profile of TBM loaded EC/HPMC matrices. The increase in concentration of albendazole (0, 2 and 5%) was compensated with corresponding decrease in HPMC content (20, 18, and 15%). The concentration of EC and TMB were consistent at 20 and 60% w/w respectively. Figure 21 . Effect of inclusion of ketoprofen (2% and 5 % w/w) on release profile of TBM loaded EC/HPMC matrices. The increase in concentration of ketoprofen (0, 2 and 5%) was compensated with corresponding decrease in HPMC content (20, 18, and 15%). The concentration of EC and TMB were consistent at 20 and 60% w/w respectively.

Figure 22. A bolus of the disclosure (100), which includes a housing (101 ), a core (102), a closed region (103) and a densifier (104).

Figure 23. Release profile from boluses with casings of high amorphous or high crystalline PLA content loaded with 60 g of 65%TBM/35% MCW over 12 days.

Detailed description of the embodiments

There are specific substances that pose particular difficulty in the context of extended release delivery to the rumen. One class of compounds that are difficult to deliver to animals are hydrophobic compounds. A further class of compounds that are difficult to deliver to animals, particularly in a sustained release, are volatile or somewhatvolatile compounds. The properties of these compounds present challenges to developing technology for the sustained release of these hydrophobic and/or volatile/somewhat-volatile substances, particularly via an animal’s stomach. Haloforms such as bromoform are such substances. Surprisingly, the inventors have developed an extremely extended release (months) dosage form for delivering hydrophobic and/or volatile/somewhat-volatile substances to the rumen of ruminants, a relatively little studied environment relative to the gastrointestinal tract of single stomached animals. Surprisingly, the inventors have improved the release kinetics and duration of controlled release of the dosage form through development of the components of the core.

One specific purpose to administer substances to animals is to reduce the adverse effects of agriculture. For instance, various methane and nitrification inhibitors are known to be administered to animals to reduce or mitigate the adverse effects of the methane and nitrogen-containing compounds produced by the animals.

However, despite current efforts, climate change is creating a wide range of environmental and social impacts globally. It is widely understood that these impacts will only continue to increase over time. As a result, there has been a global push to reduce harmful greenhouse gas (GHG) emissions in an effort to avoid the worst effects of climate change. The agricultural sector is considered to be a major source of GHG emissions. Total emissions of methane from global livestock accounts for an estimated 7.1 gigatons of CO2-equivalent per year, representing 14.5% of all anthropogenic GHG emissions. Therefore, this sector will play a key role in reducing overall GHG emissions.

The main GHGs released by agriculture are methane (CH4) and nitrous oxide (N2O), with the main source of methane emission attributed to livestock. Most methane is emitted when cattle, or other ruminant animals, burp. The amount of methane produced for each farm is directly related to the total animal feed intake, commonly measured as dry matter intake (DMI).

Countries which have a strong agricultural sector, such as New Zealand and other countries, face challenging goals in reducing agricultural emissions. For instance, the New Zealand government has introduced policies aimed to reduce methane emission by 24-50% before 2050. In New Zealand livestock methane production is estimated to comprise as much as half of the country's total GHG emissions. The reduction of methane is a critical component of meeting targets for emissions of GHGs and reducing the effects of global warming.

Release of GHGs by animals also has adverse effects on animal productivity. Any feed that is converted to a compound which is subsequently expired or released by the animal is an energy source that has not been converted to a productive use. Accordingly, for efficiency, it is important to optimise conversion of feeds into animal productivity, including in the form of weight gain or milk production.

Prior art devices for administering a methane inhibiting agent or other active ingredients to an animal known from the literature can still be further improved for example in terms of durability, control of the release rate of the active agent, the versatility of administration of the drug form such as the bolus, and the reduction of size and manufacturing costs for making the draft form, or preferably a bolus.

Definitions

Unless otherwise herein defined, the following terms will be understood to have the general meanings which follow. A “carboxylate glass” as used herein refers to a glass formed when one or a mixture of metal carboxylates are heated to their melting temperature or above and allowed to cool.

A “seaweed extract enriched in bromoform” as used herein refers to an extract that comprises non-trace seaweed components other than bromoform. Bromoform of at least 90% (w/w) purity, at least 95% (w/w) purity, at least 96% (w/w) purity, and at least 99% (w/w) purity is not a “seaweed extract enriched in bromoform”.

An “active agent” may be any substance which provides benefits to the animal e.g. a drug for treatment or prevention of disease, which improves animal productivity, or mitigates at least one adverse effect of agriculture. For example, an active agent may modulate an animal’s metabolism, for example, impact the amount or quality of methanogenesis.

A veterinary acceptable excipient is an excipient which upon administration to an animal subject is typically not deleterious to the subject. The skilled person will appreciate that in general veterinary acceptable excipients include pharmaceutically acceptable (i.e. acceptable for humans) excipients.

As used herein “haloform” is CHX3 where X is a halogen and each X atom may be a different halogen. Thus, “haloform” includes CHCIBr2 and the like. As used herein, “mixed haloform” refers to haloforms where not every X attached to the carbon atom is the same. In some embodiments, each X atom is the same. The terms “bromoform” and “tribromomethane” are used interchangeably herein.

As used herein, “degrade” and “degradation” do not require full break down of the bolus into other matter that is fully absorbed by the rumen fluid, but that instead only require that the bolus breaks sufficiently such that it may leave the rumen, for instance by passing through the digestive tract of the animal or being regurgitated.

As used herein, “feeds” refers to dry matter intake (DMI), supplements, grazing pasture, grains, or other feedstock.

As used herein, the term "effective amount" means that amount of an active ingredient that will elicit the biological or medical response of a tissue, system, or animal that is being sought, for instance, by a researcher or veterinarian. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function. For instance, a therapeutically effective amount of methane inhibiting agent such as a haloform reduces the methane output of an animal, preferably a ruminant.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a polymer” may include a plurality of polymers and a reference to “at least one carrier” may include one or more carriers, and so forth.

The term “and/or” can mean “and” or “or”.

The term “(s)” following a noun contemplates the singular or plural form, or both.

A “methane inhibiting agent” as used herein is an active agent, such as a compound or compound mixture, which is capable of inhibiting or reducing the production of methane gas in the rumen of a ruminant animal. A methane inhibiting agent may inhibit methanogenesis. A “methane inhibiting agent” as used herein is preferably a haloform, more preferably bromoform.

The term “surrounds the core” may mean fully encases the core. The disclosure also contemplated partially encasing the core.

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

Various features of the disclosure are described with reference to a certain value, or range of values. These values are intended to relate to the results of the various appropriate measurement techniques, and therefore should be interpreted as including a margin of error inherent in any particular measurement technique. Some of the values referred to herein are denoted by the term “about” to at least in part account for this variability. The term “about”, when used to describe a value, may mean an amount within ±25%, ±10%, ±5%, ±1% or ±0.1 % of that value. Increased permeability and/or openings

In some embodiments, the reference temperature is 20 °C, 25 °C or 30 °C. In some embodiments, the difference in permeability of the housing is assessed based on the release rate for the seventh day, fourteenth day, thirtieth day or sixtieth day. In some embodiments, the difference in permeability is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%.

Further provided is a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which surrounds (or covers at least a portion of or fully encases) the core, wherein the housing is configured such that its permeability to said methane inhibiting agent is increased when exposing the housing to a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C, allowing the methane inhibiting agent to exit the bolus through said opening or openings.

In some embodiments, the housing surrounds the core.

The release behavior of an active agent being released from the bolus to the exterior of the bolus can be determined by submerging a bolus according to the disclosure that comprises said active agent in a tank that is filled with 25 liters of phosphate buffer (pH:6.5, 0.02M) having constant temperature of 40°C and wherein the liquid buffer surrounding said bolus can be continuously stirred using a magnetic stirrer. After a given time the concentration of the active agent in said phosphate buffer is quantified (for example using GC-FID (gas chromatography in connection with flame ionization detector)). The quantification can be repeated in a certain interval, for example once daily.

In a preferred embodiment the disclosure provides a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which surrounds(or covers at least a portion of or fully encases) the core, wherein the housing is configured such that its permeability to said methane inhibiting agent is increased when exposing the housing to a temperature of at least 38°C; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to a temperature of at least 38°C, allowing the methane inhibiting agent to exit the bolus through said opening or openings.

Certain embodiments disclosed herein relate to a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which surrounds (or covers at least a portion of or fully encases) the core, wherein the housing becomes permeable to said methane inhibiting agent when exposing the housing to a temperature of at least physiological or ruminal temperature; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to a temperature of at least physiological or ruminal temperature, allowing the methane inhibiting agent to exit the bolus through said opening or openings. In preferred embodiments, the housing surrounds the core.

In a further aspect, the disclosure provides a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which surrounds(or covers at least a portion of or fully encases) the core, wherein the permeability of the housing to said methane inhibiting agent is increased when exposing the housing to a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C, allowing an increased release of the methane inhibiting agent from within the bolus through said opening or openings.

In a preferred embodiment the disclosure provides a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which surrounds (or covers at least a portion of or fully encases) the core, wherein the permeability of the housing to said methane inhibiting agent is increased when exposing the housing to a temperature of at least 38°C; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to a temperature of at least 38°C, allowing increased exit of the methane inhibiting agent from the bolus through said opening or openings.

In preferred embodiments of the aforementioned bolus type, the housing comprises a plurality of openings each opening having an average diameter of for example between 1 micrometer and 0.5 mm and wherein each opening is filled with a substance that melts at a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C, for example a wax or hydrocarbon composition. In an alternative or additional preferred embodiment of the bolus, the housing comprising said openings is surrounded by a film of wax or hydrocarbon that preferably melts at a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C, for example a wax or hydrocarbon composition.

Increased permeability

In some embodiments, permeability of the housing to said methane inhibiting agent is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when exposing the housing to the rumen of a living animal, relative to the permeability on the same day after exposure of the housing to phosphate buffer (pH: 6.5, 0.02 M) at 20 °C without agitation for the same length of time, wherein permeability is assessed based on release rate of the methane inhibiting agent.

In some embodiments, permeability of the housing to said methane inhibiting agent is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when exposing the housing to the rumen of a living animal, relative to the permeability on the same day after exposure of the housing to phosphate buffer (pH: 6.5, 0.02 M) at 25 °C without agitation for the same length of time, wherein permeability is assessed based on release rate of the methane inhibiting agent.

In some embodiments, permeability of the housing to said methane inhibiting agent is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when exposing the housing to the rumen of a living animal, relative to the permeability on the same day after exposure of the housing to phosphate buffer (pH: 6.5, 0.02 M) at 28 °C without agitation for the same length of time, wherein permeability is assessed based on release rate of the methane inhibiting agent. In some embodiments, permeability of the housing to said methane inhibiting agent is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when exposing the housing to the rumen of a living animal, relative to the permeability on the same day after exposure of the housing to phosphate buffer (pH: 6.5, 0.02 M) at 30 °C without agitation for the same length of time, wherein permeability is assessed based on release rate of the methane inhibiting agent.

In some embodiments, permeability of the housing to said methane inhibiting agent is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when exposing the housing to the rumen of a living animal, relative to the permeability on the same day after exposure of the housing to phosphate buffer (pH: 6.5, 0.02 M) at 38 °C without agitation for the same length of time, wherein permeability is assessed based on release rate of the methane inhibiting agent.

In some embodiments, the difference in permeability of the housing is assessed based on the release rate on the seventh day, fourteenth day, thirtieth day or sixtieth day after exposing the bolus to the rumen of a living animal, relative to the release rate on the same day after exposure of the bolus to phosphate buffer (pH: 6.5, 0.02 M) at the defined temperature without agitation for the same length of time.

In some embodiments, permeability of the housing to said methane inhibiting agent is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when exposing the bolus to phosphate buffer (pH: 6.5, 0.02 M) at 40 °C without agitation, relative to the permeability on the same day after exposure of the bolus to phosphate buffer (pH: 6.5, 0.02 M) at 20 °C without agitation for the same length of time.

In some embodiments, permeability of the housing to said methane inhibiting agent is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when exposing the bolus to phosphate buffer (pH: 6.5, 0.02 M) at 40 °C without agitation, relative to the permeability on the same day after exposure of the bolus to phosphate buffer (pH: 6.5, 0.02 M) at 25 °C without agitation for the same length of time.

In some embodiments, permeability of the housing to said methane inhibiting agent is increased by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% when exposing the bolus to phosphate buffer (pH: 6.5, 0.02 M) at 40 °C without agitation, relative to the permeability on the same day after exposure of the bolus to phosphate buffer (pH: 6.5, 0.02 M) at 30 °C without agitation for the same length of time.

In some embodiments, the difference in permeability of the housing is assessed based on the release rate for the seventh day, fourteenth day, thirtieth day or sixtieth day after exposing the bolus to phosphate buffer (pH: 6.5, 0.02 M) at 40 °C without agitation, relative to the permeability on the same day after exposure of the bolus to phosphate buffer (pH: 6.5, 0.02 M) at the defined temperature for the same length of time without agitation for the same length of time.

Increased permeability of the housing to said methane inhibiting agent leads to diffusion of the methane inhibiting agent out of the bolus when the concentration of methane inhibiting agent inside the bolus that is not bound by carrier is greater than it is outside the bolus. Permeability of the housing to said methane inhibiting agent may be assessed by any suitable means, for instance diffusion of said methane inhibiting agent out of the bolus. Diffusion out of the bolus can be measured by any suitable means, for instance GC-FID analysis of the surrounding mixture. GC-FID is suitable for quantifying bromoform, and thus quantifying permeability of a housing to bromoform. Samples from the rumen of living animals may be collected from fistulated animals.

Housing

The term “housing” as used herein is generally understood to refer to a casing that surrounds, covers at least a portion of or fully encases a core that comprises at least one methane inhibiting agent and possibly further active agent. A housing can include a cap.

In some embodiments, the housing surrounds the core. In some embodiments, the housing substantially surrounds the core. A substantially surrounded core has about 70 to about 99%, about 80 to about 99%, about 85 to about 99% or about 90 to about 99% of the surface area of the core covered by the housing. A housing can be made for example of a composition that comprises a biodegradable plastic. In some embodiments, the housing includes one or more biodegradable polymers. In some embodiments, the housing consists of one or more biodegradable polymers. In some embodiments, the housing does not include a non- biodegradable polymer. In some embodiments, the housing includes one or more non- biodegradable polymers, including Polyvinyl chloride (PVC), polyethylene terephthalate (PET), Buna-S, nylon, polyvinyl butyral, poly ethylene ( low, medium, high or ultra high density), polypropylene (PP) and combinations thereof. In some embodiments, the housing includes one or more non-biodegradable polymers, including high-density polyethylene (HDPE), polypropylene (PP) and combinations thereof. Optionally, non- biodegradable polymers in the housing are combined with a biodegradable polymer or are degradable in the presence of a haloform and/or the core.

In some embodiments a material such as a plastic is considered biodegradable, if the material is considered to be biodegradable under the standard set out in ISO 14855- 1 :2012 (biodegradability of plastic materials under controlled composting conditions). According to this method the percentage of biodegradation is given by the ratio of the CO2 produced from the test material to the maximum theoretical amount of CO2 that can be produced from the test material (not including the amount of carbon converted to new cell biomass, i.e. not metabolized to CO2). The maximum theoretical amount of CO2 produced is calculated from the total organic carbon content of the test material. The threshold for industrial composting biodegradability is a biodegradation for least 90% by mass of the total mass of the test material in less than 6 months. Thus, for instance, 90% of the carbon of the test material should be converted to CO2 within less than 6 months for the testing material to be considered biodegradable.

Preferably, the housing material is compatible with waste disposal regulations that apply to slaughter facilities. The housing material can generally include any material that is non-toxic when administered to the rumen of an animal. It is of particular relevance that any food animals will result in non-toxic foods (meat or milk) following exposure to the materials in the bolus. The housing material is further preferably sufficiently thick (wall thickness) so that it resists the mechanical stress and abrasive forces in a rumen allowing it to remain intact and prevent fracture or collapse for at least several weeks inside the rumen. The housing is shaped to fit with the core and any other components in the bolus such that there are no air pockets in the bolus.

The housing can be made from a material through which the methane inhibiting agent can migrate e.g. by a mass diffusion process. In a preferred embodiment, the housing may be made from at least one plastic material, e.g. a degradable plastic or material that degrades over time in the rumen. In one embodiment the housing may be made from a material selected from one or more of poly lactic acid (PLA), poly glycolic acid (PGA), poly lactic glycolic acid (PLGA), polypropylene, Polycaprolactone (PCL), poly(d-lactic acid) (PDLA), Polybutylene succinate (PBS), Polybutylene adipate terephthalate (PBAT), SLA polymer, ABS, or a combination thereof.

In any embodiment herein, a bolus comprising a housing described herein may comprise a housing which or part of which comprises at least one compound selected from the group consisting of polylactic acid (PLA), poly-butylene succinate co-adipate (PBSA), poly-butylene succinate (PBS), poly-hydroxybutyrate-co-hydroxy valerate, poly vinyl acetate (PVA), Polybutylene adipate terephthalate (PBAT), polycaprolactone (PCL), wood flour and cellulosic materials, ethyl cellulose and hydroxypropyl methyl cellulose and mixtures of two or more of the aforementioned.

In alternate embodiments, the housing includes one or more hydrophobic polymers. Optionally, the housing includes one or more hydrophobic biodegradable polymers. In alternate embodiments, the housing consists of one or more hydrophobic polymers. Optionally, the housing consists of one or more hydrophobic biodegradable polymers.

In some embodiments, the housing includes one or more ester-based polymers.

In some embodiments, the housing includes one or more polymers selected from the list consisting of high-density polyethylene (HDPE), polypropylene (PP), polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co- adipate (PBSA), polylactic acid (PLA), poly-D,L-lactic acid (PDLLA), polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D- lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), combinations thereof, and co-polymers thereof. In some embodiments, the housing consists of one or more polymers selected from the list consisting of polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co- adipate (PBSA), polylactic acid (PLA), poly-D,L-lactic acid (PDLLA), polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D- lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), combinations thereof, and co-polymers thereof. In some embodiments, the housing includes one or more polymers selected from the list consisting of high- density polyethylene (HDPE), polypropylene (PP), combinations thereof, and copolymers thereof. In some embodiments, the housing includes one or more polymers selected from the list consisting of polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polylactic acid (PLA), poly-D,L-lactic acid (PDLLA), polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D-lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), combinations thereof, and copolymers thereof. In some embodiments, the housing consists of one or more polymers selected from the list consisting of polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polylactic acid (PLA), poly-D,L-lactic acid (PDLLA), polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D-lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), combinations thereof, and copolymers thereof.

In some embodiments, the housing includes one or more of poly lactic acid (PLA), poly glycolic acid (PGA), poly lactic glycolic acid (PLGA), polypropylene, Polycaprolactone (PCL), poly(d-lactic acid) (PDLA), Polybutylene succinate (PBS), Polybutylene adipate terephthalate (PBAT), SLA polymer or one or more thermoset polymers and/or resins, ABS, combinations thereof, and co-polymers thereof. In some embodiments, the housing includes one or more of polylactic acid (PLA), poly-butylene succinate co-adipate (PBSA), poly-butylene succinate (PBS), polyhydroxybutyrate-co- hydroxy valerate, poly vinyl acetate (PVA), Polybutylene adipate terephthalate (PBAT), polycaprolactone (PCL), wood flour and cellulosic materials, ethyl cellulose and hydroxypropyl methyl cellulose, combinations thereof, and co-polymers thereof.

In some embodiments, the housing includes one or more polymers selected from the list consisting of polylactic acid, polybutylene adipate terephthalate, combinations thereof, and co-polymers thereof. In some embodiments, the housing consists of one or more polymers selected from the list consisting of polylactic acid, polybutylene adipate terephthalate, combinations thereof, and co-polymers thereof.

In some embodiments, the PLA:PBAT ratio is from about 95:5 to about 70:30 wt/wt, from about 95:5 to about 80:20 wt/wt, or from about 95:5 to about 85:15 wt/wt. In some embodiments, the PLA:PBAT ratio is about 90:10 wt/wt.

Blends of such substances can be particularly advantageous. For instance, mixing/blending a polybutylene polymer such as PBAT with PLA increases the plasticity and strength of the housing compared to a housing made of PLA alone, while preserving the biodegradability of the housing material. This stability improving effect is particularly beneficial when using for instance haloforms as methane inhibiting agent, because such compounds can otherwise promote brittleness of the housing material. Furthermore, the use of a polybutylene polymer/PLA blend compared to PLA alone, improves the durability of the housing and reduces the risk of fracturing under mechanical stress such as when placed into the rumen of an animal.

The components used or mixed to form the housing material may be selected according to their suitability regarding the use for forming the bolus housing. Upon heating for shaping the bolus housing the composition should not become too viscous for 3D printing or injection moulding and blending of two or more polymer should result in a homogeneous mixture without extensive bubbles formation. 3D printing includes stereolithography (SLA) and digital light processing (DLP).

The housing of the bolus may for instance comprise biodegradable and/or non- biodegradable materials, but preferably comprises biodegradable polymers. Such materials may be synthetic or naturally or essentially naturally derived. It is preferred that materials are selected from biodegradable polymers. Examples of such polymers include, without limitation, poly lactic acid (PLA), polybutylene terephthalate (PBT), polybutylene adipate terephthalate (PBAT), polybutylene succinate (PBS) and polybutylene succinate adipate (PBSA). Biodegradability allows repeated administration of boluses, while preventing the accumulation of bolus materials in the ruminant animal’s body, since the bolus components can be at least partially or even fully degraded in the rumen milieu. Nevertheless, it is understood that even if a bolus housing is biodegradable, it will not fully degrade to a degree that the bolus breaks down for the duration of at least 7 days when kept in the rumen for this time. Suitable non-biodegradable polymers include high-density polyethylene (HDPE), polypropylene (PP), combinations thereof, and co-polymers thereof.

Accordingly, the housing for any bolus comprising a housing described herein may be configured to have sufficient structural integrity to remain intact for a predetermined period of time. In a preferred embodiment, the housing may be configured to degrade over a predetermined period of time. A predetermined period of time may mean the period of time over which the methane inhibiting agent is to be released to the animal. In a particularly preferred embodiment, the predetermined period of time may be at least two months, preferably six months, and more preferably 12 months. In some embodiments the period of time is at least 2 weeks, 3 weeks, 4 weeks or 6 weeks.

In one embodiment the material of the housing may comprise poly lactic acid (PLA) and polybutylene adipate-terephthalate (PBAT) preferably in a PLA:PBAT weight ratio of between 95:5 to 70:30 or in a weight ratio of about 90:10. Unless otherwise defined, a ratio as used herein refers to a ratio by weight (or “weight ratio”, “w/w”), wherein the ratio is calculated with reference to the total weight of the housing components used.

The material for the housing may also comprise PLA, PBAT, PBSA and/or PBS in different ratios as shown in the table below:

Table 1 . Material for the housing.

Optionally, the housing is 5 to 100% or 10 to 100% PLA w/w. Optionally, the housing is 20 to 90 or 30 to 80% PBS w/w. Optionally, the housing is 20 to 100% or 30 to 90% PBAT w/w. Optionally, the housing is 20 to 100 or 30 to 90% PBSA w/w. Optionally, the housing is 5 to 100% or 10 to 100% PLA w/w, and either (i) 20 to 90 or 30 to 80% PBS w/w, (ii) 20 to 100% or 30 to 90% PBAT w/w, or (iii) 20 to 100 or 30 to 90% PBSA w/w.

As outlined above, the housing of the bolus of the disclosure may be designed in a way to allow the active ingredient, i.e. the methane inhibiting agent, to pass through the housing. This may provide a sustainable controlled release. In one embodiment, the methane inhibiting agent can perfuse through the housing material of the bolus of the disclosure. Optionally, the methane inhibiting agent diffuses through the housing material.

Alternatively, the housing may be made from one or more non-adsorbent materials, i.e. materials into which, or through which, the methane inhibiting agent does not migrate. Using a non-absorbent material for the housing can assist with controlling the rate of release of the methane inhibiting agent(s), for instance in a bolus comprising one or more openings, in a bolus with a housing which is able to form one or more openings or in an open-ended bolus. For instance, in these embodiments, the concentration of the methane inhibiting agent(s) in the core is not decreased by their absorption into the housing material.

The housing of the bolus of the disclosure may be given further functional features, for example, but not limited to, by adding further components to the housing material or by modifying housing’s dimensions and nature. In one embodiment the housing material of the bolus of the disclosure comprises one or more excipients. In a preferred embodiment the one or more excipients includes plasticizers, hardeners and/or colorants.

In one embodiment, the housing further comprises a compound selected from nucleating agents or stabilizers. In one embodiment, the housing does not comprise a nucleating agent and/or a stabilizer. The thickness of the housing may be selected to contribute to the rate of release of the methane inhibiting agent, i.e. a relatively thicker housing will have a relatively slower release rate than a relatively thinner housing. This is particularly the case if the housing material is permeable for the methane inhibiting agent. In one embodiment the housing may have a material thickness of below about 2 mm, preferably a material thickness in the range of about 0.3-1.8 mm, and more preferably a material thickness in the range of about 0.3-1 .5 mm. In some embodiments, the housing has a material thickness less than about 1.5 mm, less than about 1.3 mm, or less than about 1 mm. In some embodiments, the housing has a material thickness greater than about 0.8 mm, greater than about 1 mm or greater than about 1.1 mm. In some embodiments, the housing has a material thickness of about 0.9 mm. In some embodiments, the housing has a material thickness of about 1.2 mm. In some embodiments, the housing has a material thickness of about 1.5 mm. For boluses comprising a housing with one or more openings, also thicker housings may be applicable, such as up to about 5 mm of housing wall thickness.

Optionally, the core and housing have a ratio of about 3 to about 6 : 1 , about 4 to about 5 : 1 or about 4.6 : 1 by weight.

In one embodiment the housing is configured to degrade over a predetermined period of time. The predetermined period time may for instance be adjusted via the material thickness of the housing, the selection of housing materials or the manufacturing process of the housing.

In one embodiment the housing includes a cavity in which at least a portion of a core is located, wherein the core comprises the methane inhibiting agent, such as the methane inhibiting agent. In another embodiment the housing comprises no openings and completely surrounds the core. In another embodiment the housing completely covers and surrounds the core. In one embodiment the housing includes one or more openings, as described above. The housings of the boluses of the present disclosure may assist with a controlled release of the methane inhibiting agent. For instance, the housing is able to withstand the conditions in the rumen for the predetermined period of time. During this time, the housing protects the core from fluid in the rumen, yet can facilitate or contribute to the controlled release of the methane inhibiting agent. However, the design of the housing may allow the housing to disintegrate or degrade over the predetermined period of time. This can contribute to mitigating adverse effects of device administration to an animal, and could also ensure that an animal can be treated with multiple bolus e.g. a second bolus is administered at or towards, or after, the end of the predetermined period of time.

In the described aspect, the housing becomes permeable to said methane inhibiting agent and/or at least part of the housing is configured to form one or more openings when exposing the housing to a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C, allowing the methane inhibiting agent to exit the bolus through said opening or openings. Alternatively, the permeability of the housing to said methane inhibiting agent may be increased when exposing the housing to a critical minimal temperature or the exit through said formed openings may be increased. In the latter case the permeability and/or exit of the methane inhibiting agent is increased in comparison the permeability/exit from the bolus, when it is not exposed to at least said temperature, e.g. a bolus exposed to a temperature below 28 °C.

A bolus providing a temperature responsive release of the methane inhibiting agent allows a release of the methane inhibiting agent or an increase of its release rate to a substantial amount with an effect on the bolus’ environment only upon exposure of the bolus to the minimally required temperature. This means, that a bolus will release the methane inhibiting agent in an effective amount upon administration of the bolus to an animal but not before. In particular, the bolus may be kept at room temperature or be kept in a cool environment prior to administration, in order to ensure that no or no substantial amount of methane inhibiting agent is released from the bolus. Preventing a premature release of the methane inhibiting agent from the bolus in this manner may provide several benefits, including preventing or reducing any loss of methane inhibiting agent before the actual administration, preventing or reducing contamination by the potentially aggressive methane inhibiting agent of the environment/surroundings the bolus is kept in, and protecting farmers, staff, or other people handling the bolus from coming into contact with a possibly harmful amount of the methane inhibiting agent.

In one embodiment the housing comprises one or more openings and said openings are filled with and/or covered with a material that melts, dissolves or disintegrates in the rumen of a living animal. Preferably, said housing is made of a material that does not melt, does not dissolve and does not disintegrate in the rumen of a living animal, and preferably does not melt, dissolve or disintegrate in the rumen of a living animal over the course of 1 day.

To enable a temperature dependent release, at least part of the bolus, where openings may form or where permeability for the methane inhibiting agent may be increased, may comprise or consist of certain suitable compounds or mixtures of compounds with properties that may change upon reaching a certain temperature tipping point. In one embodiment a part of the housing, and preferably the portion of the housing, where the opening or openings form, is made of a material that melts, dissolves or disintegrates in the rumen of a living animal.

In one embodiment, the material that melts is a compound selected from the group consisting of a hydrogel, an oleogel, an organogel, a phase changing material (PCM), a fatty acid, an alkane, an alkene, a gelator, a wax, an L-alanine amino acid, an L-alanine amino acid derivative, poly(methyl methacrylate) (PMMA), (1 ,3:2,4) dibenzylidene sorbitol (DBS), hydroxy stearic acid, paraffin wax, gelatin, 1 -tetradecanol, polyethylene glycol, octadecane, nonadecane, eicosane, a pluronic polymer or a mixture of pluronics, an emulsifier, sucrose acetate isobutyrate (SAIB), derivatives of the aforementioned and combinations of one or more of the aforementioned compounds and wherein said compound or combination of compounds has a melting temperature of between 28°C and 42°C, more preferably of between 28 and 35 °C.

Said melting temperature is the temperature at which the substance changes from substantially solid to liquid state at atmospheric pressure.

In another embodiment the material that dissolves is a water-soluble material preferably selected from the group consisting of a polymer, a polyol, a sugar, a polyamide, a salt, cellulose acetate, polyethylene glycol, methyl cellulose, CMC, polyvinyl alcohol, alginic acid salt, polyacrylic acid or its salts, polyacrylamide, cellulose ether, carrageenan, guar and pectin. “Water-soluble” means that the substance can dissolve in distilled water having a temperature of 20°C.

In another embodiment the material that disintegrates is a compound selected from the group consisting of cellulose, polyhydroxyalkanoate (PHA), poly(butylene succinate- co-adipate) (PBSA) and a mixture of two or more of the aforementioned. The rumen environment comprises enzymes which can disintegrate or assist in disintegrating the aforementioned compounds. When said material disintegrates the release of the active agent from within the bolus to the exterior of the bolus will increase which can be tested as outlined above in the context of quantifying the release of an active agent from within the bolus.

In another aspect, the disclosure relates to a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which covers at least a portion of the core, wherein at least part of the housing is configured to form one or more openings allowing the methane inhibiting agent to exit the bolus through said opening or openings, wherein the portion of the housing, where the opening or openings form, comprises or consists of a compound selected from the group consisting of a temperature responsive hydrogel, an oleogel, an organogel, a phase changing material (PCM), a fatty acid, an alkane, an alkene, a gelator and a wax, an L-alanine amino acid or L-alanine amino acid derivative, poly(methyl methacrylate) (PMMA), (1 ,3:2,4) dibenzylidene sorbitol (DBS), hydroxy stearic acid, paraffin wax, gelatin, 1 -tetradecanol, polyethylene glycol, octadecane, nonadecane, eicosane, a pluronic polymer or a mixture of pluronics, an emulsifier, sucrose acetate isobutyrate (SAIB), derivatives of the aforementioned and combinations of one or more of the aforementioned compounds. In some embodiments, the housing surrounds the core.

In another aspect, the disclosure relates to a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which covers at least a portion of the core, wherein at least part of the housing is configured to form one or more openings, allowing an increased release of the methane inhibiting agent from within the bolus through said opening or openings, wherein the portion of the housing, where the opening or openings form, comprises or consists of a compound selected from the group consisting of a temperature responsive hydrogel, an oleogel, an organogel, a phase changing material (PCM), a fatty acid, an alkane, an alkene, a gelator and a wax, an L-alanine amino acid or L-alanine amino acid derivative, poly(methyl methacrylate) (PMMA), (1 ,3:2,4) dibenzylidene sorbitol (DBS), hydroxy stearic acid, paraffin wax, gelatin, 1 -tetradecanol, polyethylene glycol, octadecane, nonadecane, eicosane, a pluronic polymer or a mixture of pluronics, an emulsifier, sucrose acetate isobutyrate (SAIB), derivatives of the aforementioned and combinations of one or more of the aforementioned compounds. In some embodiments, the housing surrounds the core.

In another embodiment the portion of the housing, where the opening or openings form, comprises or consists of a compound selected from the group consisting of a temperature responsive hydrogel, an oleogel, an organogel, a phase changing material (PCM), a fatty acid, an alkane, an alkene, a gelator and a wax, preferably wherein the compound has a melting temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C.

In another embodiment the portion of the housing, where the opening or openings form, comprises or consists of a compound selected from the group consisting of a temperature responsive hydrogel, an oleogel, an organogel, a phase changing material (PCM), a fatty acid, an alkane, an alkene, a gelator and a wax, preferably wherein the compound is solid at 30°C and is liquid at 42°C.

In a preferred embodiment the compound is selected from the group consisting of L-alanine amino acid derivatives, poly(methyl methacrylate) (PMMA), (1 ,3:2, 4) dibenzylidene sorbitol (DBS), hydroxy stearic acid, paraffin wax, gelatin, 1 -tetradecanol, polyethylene glycol (PEG), octadecane, nonadecane, eicosane, a pluronic polymers or a mixture of pluronics, an emulsifier, sucrose acetate isobutyrate (SAIB) derivatives of the aforementioned and combinations of one or more of the aforementioned compounds.

The material of the housing portion, where said opening or openings do not form, comprises a compound selected from the group consisting of PLA, PCL, PBS, PBAT, PHB, PBSA, wood flour and combinations of one or more of these compounds. Such material has the property of having a melting temperature that is higher than the temperature in the rumen of an animal. This means that the portion, where the opening or openings form will be allowed to melt in the rumen, whereas the remaining portion of the housing stays intact and solid when exposed to the same rumen temperature.

Organogel

In a further aspect, there is provided a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core comprising the methane inhibiting agent dispersed in or forming part of one or more of a hydrogel, oleogel or organogel; and a housing covers at least a portion of the core.

Hydrogels, oleogels and organogels offer favourable release profiles (controlled and/or extended) and are relatively easy to handle, simplifying manufacture. Furthermore, at least in preferred embodiments, hydrogels, oleogels and organogels can provide relatively high levels of release at lower loadings of active ingredient relative to comparable boluses with other carriers (particularly polymer-based carriers), potentially decreasing the amount of active agent required per bolus.

A hydrogel as used herein may be a temperature responsive hydrogel. A temperature responsive hydrogel is understood as a gel comprising cross-linked polymer networks in which the swelling agent is water or an aqueous solution. The hydrogels may for instance be based on cross-linked polymers including but not be limited to natural polymers, N-isopropylacrylamide polymers, polyethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) polymers as well as polyethylene glycol)-biodegradable polyester copolymers.

Organogel is understood as preparations comprising an organic liquid and gelator components, which form gels filled with the organic liquids, including but not limited to polar organic solvents and their aqueous mixtures, ionic liquids, fats, and oils. The thermal stability of the gel can be adapted by selecting the solvent according to its boiling point and in general by selecting components according to their critical phase change temperatures. An oleogel, classified as a type of organogel is understood as preparations, the basis of which may consists of paraffin oils, fats or natural oils usually with addition of polyethylene, forming so called isogels, or consist of oils gelled by various additives, forming so called heterogels. Gelling agents used in heterogels include zinc stearate, aluminum stearate, highly dispersed silicon dioxide and ethyl cellulose.

In some embodiments, the core is a hydrogel. In some embodiments, the core is an oleogel. In some embodiments, the core is an organogel.

In some embodiments wherein the core is a hydrogel, oleogel or organogel, the core comprises a further active agent. In some embodiments wherein the core is a hydrogel, oleogel or organogel, the core comprises a further methane inhibiting agent (preferably, lecithin). In some embodiments wherein the core is a hydrogel, oleogel or organogel, the combination of the methane inhibiting agent and a further active agent (preferably lecithin), optionally further including water, forms a hydrogel, oleogel or organogel. In some embodiments wherein the core is a hydrogel, oleogel or organogel, the core consists of a methane inhibiting agent, a further methane inhibiting agent (preferably lecithin), and optionally water.

In some embodiments wherein the core is a hydrogel, oleogel or organogel, the core comprises a phospholipid (preferably lecithin) and/or an acrylate-based polymer (preferably poly(methyl methacrylate) [PMMA]). In some embodiments wherein the core is a hydrogel, oleogel or organogel, the housing surrounds the core. Optionally,

Pluronics, also known as poloxamers, are a class of synthetic block copolymers which consist of hydrophilic polyethylene oxide) (PEO) and hydrophobic polypropylene oxide) (PPO), arranged in an A-B-A triblock structure, thus giving PEO-PPO-PEO. When mixed with water, concentrated solutions of poloxamers can also form hydrogels, which may also be suitable compounds as described. An increase in temperature to a certain critical level can influence the hydrogen bonding between polyoxyethylene and water molecules and change the cohesion of the pluronic/poloxamer components, possibly increasing permeability.

Sucrose acetate isobutyrate (SAIB), which is prepared by esterification of sucrose with acetic and isobutyric anhydride, is known to be an emulsifier acceptable in food preparation and may therefor present especially tolerable properties when used in an animal.

In some embodiments, the portion of the housing, where the opening or openings form, comprises or consists of a compound, the properties of which change to allow increased permeability at a critical temperature, which is the compound’s melting point. For instance, this is the case when oils and/or waxes are comprised by a portion of the housing, where an opening or openings form and/or where the housing becomes permeable to the methane inhibiting agent.

The skilled person is aware that when the portion of the housing, where the opening or openings form, comprises or consists of polyethylene glycol, the critical temperature to allow increased permeability depends on the PEG or PEG mixture used. The skilled person is able to select suitable PEG components to achieve a desired critical release temperature. PEG may also suitably be used in the form of a PEG gel, i.e. a gel comprising PEG as a functional phase.

Phase changing material (PCM) are materials or compounds which may melt or solidify at a certain temperature. Heat energy is absorbed or released by changing the state of the material. The phase change material may be solid at room temperature and may softens when it reaches a specific temperature, which may encompass the material becoming more permeable. Biobased PCMs include glycols, alcohols, esters and fatty acids. Particularly fatty acids, and especially saturated fatty acids, such as palmitic acid and stearic acid, are suitable PCMs, which may be derived from vegetable oils and animal fats, such as from palm oil or coconut oil. Further biomedically and thus also veterinary applicable phase change materials are known in the art, for instance including but not being limited to 1 -tridecanol, 1 -tetradecanol, 1 -pentadecanol, decanoic acid or lauric acid.

The largest opening that forms in the housing when exposing the housing to the rumen of a living animal and/or to a temperature of at least ruminal or physiological temperature may suitably have a diameter of maximally 1 mm to ensure that a release of the methane inhibiting agent through said opening is still controlled and sustained.

In one embodiment the largest opening that forms in the housing when exposing the housing to the rumen of a living animal has a diameter of maximally 2 mm. In one embodiment the largest opening that forms in the housing when exposing the housing to the rumen of a living animal has a diameter of maximally 1 mm.

In one embodiment the largest opening that forms in the housing when exposing the housing to the temperature present in the rumen of a living animal and preferably to a temperature of 42°C has a diameter of maximally 2 mm. In one embodiment the largest opening that forms in the housing when exposing the housing to a temperature of present in the rumen of a living animal and preferably to a temperature of 42°C has a diameter of maximally 1 mm.

In another embodiment the largest opening that forms in the housing when exposing the housing to a temperature of at least 38°C has a diameter of maximally 2 mm. In another embodiment the largest opening that forms in the housing when exposing the housing to a temperature of at least 38°C has a diameter of maximally 1 mm. The size of formed pore may be determining for the release rate of the methane inhibiting agent from the bolus, wherein the methane inhibiting agent is released through the formed pore.

The dispensing mechanism provided by the bolus including temperature enhanced or temperature dependent release of the methane inhibiting agent comprised by the bolus is useful when a delayed release is sought. Delayed as used in this context is to be understood as delaying or substantially slowing down methane inhibiting agent release from the bolus until after administration of the bolus to the ruminant animal, i.e. until after placing the bolus in the animal’s rumen. This may be advantageous in the handling of the bolus, wherein the bolus when handled outside of the ruminal environment releases much less or even none of the methane inhibiting agent comprised by the bolus and only begins releasing the methane inhibiting agent or begins releasing the methane inhibiting agent at a higher, e.g. an effective rate after administration to an animal’s rumen. Advantages provided by this bolus modification include protecting farmers or other staff handling the bolus from methane inhibiting agents, such as bromoform, that may for instance have a health hazardous or irritating effect on humans when coming directly into contact with humans.

The release rate of the methane inhibiting agent from the bolus through the one or more openings may be increased when exposing the housing to a temperature of at least physiological or ruminal temperature, which allows an increased exit of the methane inhibiting agent from the bolus through the opening or openings.

In one embodiment the bolus of the disclosure comprises the methane inhibiting agent bromoform and is adapted to reach a maximum release rate of approximately 0.1 - approximately 0.5 g per day, and more preferably approximately 0.2 g per day. Such release rates may provide a sustained release of haloforms, such as bromoform. A bolus with such release rate is for instance suitable for use in cattle and sheep.

To reach a preferred release rate, also in smaller farm animals, the concentration of the methane inhibiting agent, such as a haloform, or the housing material thickness may for instance be adjusted. Furthermore, to reach a preferred release rate, also in smaller farm animals, the size of forming openings in a bolus of the disclosure upon administration into the rumen may for instance be adjusted. Furthermore, to achieve a desired release rate for the methane inhibiting agent, the overall polarity of a carrier material, which can be admixed with the methane inhibiting agent, may be adjusted to achieve the desired affinity for the methane inhibiting agent admixed therewith. However, in alternative embodiments, the methane inhibiting agent may be provided in a substantially pure form e.g. is not mixed with a carrier.

In one embodiment of any bolus comprising a housing, which surrounds the methane inhibiting agent, the bolus is adapted to release the methane inhibiting agent over a period of at least two months. In a preferred embodiment, the bolus is adapted to release the substance over a period of at least six months, such as at least seven, eight, nine or at least ten months and more.

In one embodiment of any bolus described herein the methane inhibiting agent is selected from a haloform.

In one embodiment the release rate of the methane inhibiting agent from the bolus through the one or more openings of the housing is increased when exposing the housing to the rumen of a living animal and/or a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C, as compared to the release rate of a bolus that is exposed to a temperature of 20°C. In other words, an increased exit of the methane inhibiting agent from the bolus through said opening or openings is present when the bolus is inside the rumen of a living animal. In one embodiment the release rate of the methane inhibiting agent from the bolus through the one or more openings is increased when exposing the housing to a temperature of at least 38°C, allowing increased exit of the methane inhibiting agent from the bolus through said opening or openings.

Said increase of the release rate of the methane inhibiting agent from the bolus through the one or more openings is influenced by the diameter of the opening or pore forming, which as described above is for instance up to 1 mm or less in order to maintain a controlled and sustained release.

A preferred embodiment of the first aspect of the disclosure relates the bolus of the first aspect producible by carrying out the following steps:

(i) provision of a housing wherein the housing material comprises PLA and PBAT, preferably in a wt% ratio of about 90:10 PLA:PBAT and preferably with a wall thickness of about 0.5 to 2 mm;

(ii) filling into the housing at least 20 g of steel balls;

(iii) mixing ethyl cellulose with tribromomethane;

(iv) mixing the composition obtained from (iii) with hydroxypropyl methylcellulose, whereby preferably the weight ratio of the resulting mixture comprising tribromomethane, ethyl cellulose and hydroxypropyl methylcellulose is about 3:1 :1 ;

(v) closing the housing by spin-welding a cap onto it; and wherein the housing is configured such that its permeability to said methane inhibiting agent is increased when exposing the housing to the rumen of a living animal; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to the rumen of a living animal, allowing the methane inhibiting agent to exit the bolus through said opening or openings.

The disclosure provides in a further aspect a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent in microencapsulated particles and wherein the microencapsulated particles are dispersed in a carrier; and optionally a dispersing agent, and a housing covering at least part of the core.

The disclosure provides in a further embodiment a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent in microencapsulated particles and wherein the microencapsulated particles are dispersed in a carrier; and a dispersing agent, and a housing covering at least part of the core.

Encapsulating the methane inhibiting agent as microencapsulated particles can provide several benefits which will become clear in the following. Microencapsulation promotes an even and sustained release of the methane inhibiting agent from the microcapsules contained in the bolus, for instance because the microcapsules can be evenly dispersed in the bolus. Furthermore, encapsulating the methane inhibiting agent prevents or reduces its direct contact with other bolus components and protects these components from the possibly aggressive activity of the methane inhibiting substance.

The “dispersing agent” as used herein is a substance added to a mixture or suspension of solid or even liquid particles in a carrier to improve separation of the particles and to prevent their settling or clumping. Suitable dispersing agents include ionic (such as anionic) and non-ionic surfactants, poly ethylene glycol and derivatives thereof, glucosides and others and the skilled person will be aware of suitable compounds and polymers to be used as dispersing agents.

In one embodiment the microencapsulated particles are produced by microencapsulation in at least one encapsulating agent, preferably wherein the encapsulating agent is selected from the group consisting of polymers, surface-active agents, emulsifiers, gelatin-sorbitol mixture and gelatin-starch syrup and mixtures thereof, more preferably wherein the encapsulating agent is selected from the group consisting of PLA, PBSA, PBS, PHBV, PVA, PBAT, PCL, PDLA, gelatin-sorbitol mixture and gelatin- starch syrup. Due to the small size of microcapsules, a wide range of, for instance, polymeric compounds may be used for microencapsulation, particularly since a certain degree of brittleness of the encapsulating compound is not detrimental for stability at a small microcapsule size. Microencapsulation methods are known in the art and will be clear to the skilled person. For instance, a method as described by Aida et al. (1989) may be applied to microencapsulate a methane inhibiting agent, such as a haloform (Aida et al., “Practical Application of Microcapsulation for Toxicity Studies Using Bromodichloromethane as a Model Compound”, JOURNAL OF THE AMERICAN COLLEGE OF TOXICOLOGY, Volume 8, Number 6, 1989). In one embodiment the microencapsulated particles are produced by microencapsulation in gelatin-starch syrup.

When an oil phase is used, into which the substances to be encapsulated are admixed, the oil phase may suitably contain further stabilizing agents, such as emulsifiers, to assist in particle formation. Thus, in one embodiment, an oil phase used in the process of forming microencapsulated particles may comprise at least one stabilizer, preferably at least one emulsifier, and more preferably the oil phase comprises lecithin.

In one embodiment, the microencapsulated particles may be microencapsulated using interfacial polymerization. During interfacial polymerization the polymerization occurs at the interface between two immiscible phases, such as two liquids, resulting in a polymer that is located at the interfacial layer. Suitable process modifications of interfacial polymerization for capsule formation are known in the art, for instance as described by Song et al. (2017) (Song et al., "Recent progress in interfacial polymerization." Materials Chemistry Frontiers 1.6 (2017): 1028-1040) and will be clear to the skilled person. In one embodiment the microencapsulated particles may be microencapsulated without using interfacial polymerization.

Porous carrier substances may be suitable to disperse the microcapsules. For instance, while bromoform as an exemplary methane inhibiting agent may not be directly compatible with hydrophilic carrier substances, microcapsules comprising bromoform may also be dispersed in a hydrophilic carrier, provided that the bromoform may still exit the bolus once released from the microcapsules, e.g. via a porous structure of the carrier.

In a further aspect the disclosure provides a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a carrier and microencapsulated particles, wherein the microencapsulated particles comprise the methane inhibiting agent and wherein the microencapsulated particles are dispersed in the carrier, preferably wherein the carrier has a porous structure. Such a porous structure can comprise small or minute spaces or holes through which air and liquid, such as rumen liquid, may pass. Thus, microencapsulated particles dispersed in the carrier having a porous structure may be contacted by rumen liquid passing through the pores, facilitating the delivery of the methane inhibiting agent from the microencapsulated particles to the rumen fluid.

In one embodiment the microencapsulated particles are microencapsulated using a compound selected from the group consisting of a hydrophilic material, gelatin, zein, methyl cellulose and poly(N-isopropylacrylamide) (PNIPAM) microgel, starch, cyclodextrins and a combination of two or more of the aforementioned compounds.

For instance, cyclodextrins may be used particularly for their beneficial capacity to hold hydrophobic compounds in an encapsulated core. For instance, poly(N- isopropylacrylamide) (PNIPAM) microgel is a substance widely used in biomedical application, which comprises colloidal particles forming said microgel.

Microencapsulating the methane inhibiting substance allows to use a variety of different methane inhibiting agents with a variety of different bolus carrier components the microcapsules are dispersed in, since no particular compatibility of the carrier with the methane inhibiting agent is required to load as much of the methane inhibiting agent as possible, as the methane inhibiting agent is not loaded directly into the carrier but loaded as content of the microcapsules. In one embodiment the carrier comprises a compound selected from the group consisting of silica, cellulose and activated carbon, gelatin, chitosan, poly(lactic-co-glycolic acid) (PLGA), cyclodextrins , collagen, poly alphahydroxy esters, hydroxy alkanoates and dioxanes, starch, gluten, zein, polyethylene, polypropylene, polyamide, polyethylene terephthalate and ethylene-vinyl acetate.

Like the exemplary housing materials described herein, also the microencapsulating compounds and the carrier compounds used in the boluses of the disclosure may be biodegradable, however, this is not a prerequisite. It is rather preferred that the ingredients used for a bolus as described herein should not be detrimental to the animal’s health or the environment, particularly when accumulating in larger quantities. Thus, also non-biodegradable bolus components are acceptable for the boluses of the disclosure.

Furthermore, the production mechanism of the microparticles ensures a substantially homogenous structure of all particles, e.g. they have about the same size and are enclosed by about the same layer thickness of encapsulating agent. Thus, the release rate for a sustained long-time release from all microcapsules will be about the same. In one embodiment the microencapsulated particles have an average diameter of 50 nm to 2 mm, preferably an average diameter of 1 to 1000 pm. The diameter of microencapsulated particles can for instance be determined by sieving analysis, e.g. by using different standard sieve sizes. Furthermore, scanning electron microscopy of the particles can be applied, which further allows to determine particle shape and surface morphology.

In one embodiment the bolus is configured to release the methane inhibiting agent over a period of at least 6 months. In one embodiment the bolus is configured to release the methane inhibiting agent over a period of up to 6 months. In one embodiment the bolus is configured to release the methane inhibiting agent over a period of at least 4 months. In one embodiment the bolus is configured to release the methane inhibiting agent over a period of at least 2 months.

No housing

In some embodiments, preferably embodiments where the bolus does not comprise a housing, the carrier is hydrophobic. In some embodiments, preferably embodiments where the bolus does not comprise a housing, the carrier consists of one or more hydrophobic materials. In some embodiments, preferably embodiments where the bolus does not comprise a housing, the carrier has a melting point of at least 60 °C, preferably at least 75 °C.

In some embodiments, preferably embodiments where the bolus does not comprise a housing, the bolus does not comprise a carboxylate glass. In some embodiments, preferably embodiments where the bolus does not comprise a housing, the bolus does not comprise a glass.

In some embodiments, preferably embodiments where the bolus does not comprise a housing, the bolus does not comprise a seaweed extract enriched in bromoform. In some embodiments, preferably embodiments where the bolus does not comprise a housing, the bolus does not comprise a seaweed extract.

In some embodiments where the bolus does not comprise a housing, the bolus does not comprise hydrophobic fumed silica. In some embodiments where the bolus does not comprise a housing, the bolus does not comprise silica.

In some embodiments where the bolus does not comprise a housing, the carrier comprises one or more materials selected from the list consisting of polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polylactic acid (PLA), poly-lactic acid, poly-d-lactic acid, poly-L-lactic acid, poly-D,L-lactic acid (PDLLA), poly-lactide-co-glycolide, lignin, polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D-lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), epoxy-based chain extenders, magnesium silicate, cellulosic materials, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), fumed silica, gelatin, wax, castor wax, paraffin wax, silica, microcrystalline wax, methyl cellulose, starch, polyethylene glycol, polyvinyl alcohol, hydroxyethyl cellulose, carboxymethyl cellulose, soluplus, Poly(acrylic acid) , poly(vinylpyrrolidone), poly(vinyl alcohol), poly(acrylamide), poly(2-hydroxypropyl methacrylamide), poly(N,N-dimethylacrylamide), poly([2-

(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), poly(2- (methacryloyloxy)ethyl phosphorylcholine), poly(carboxybetaine methacrylamide), polyethylene glycol), polyethylene imine), poly(sarcosine), poly(2-methyl-2-oxazoline), polyamino esters, polyester amides, polyphosphoesters, poly(l-lysine), poly(l-proline) , polyphosphazenes, dextran, sodium alginate, gelatin, agarose, carrageenan, gellan, xantham gum, urea, sucrose, derivatives thereof, combinations thereof, and co-polymers thereof. In some embodiments where the bolus does not comprise a housing, the carrier consists of one or more materials selected from the list consisting of polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polylactic acid (PLA), poly-lactic acid, poly-d-lactic acid, poly-L-lactic acid , poly-D,L-lactic acid (PDLLA), poly-lactide-co-glycolide, lignin, polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D-lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), epoxy-based chain extenders, magnesium silicate, cellulosic materials, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), fumed silica, gelatin, wax, castor wax, paraffin wax, silica, microcrystalline wax, methyl cellulose, starch, polyethylene glycol, polyvinyl alcohol, hydroxyethyl cellulose, carboxymethyl cellulose, soluplus, Poly(acrylic acid) , poly(vinylpyrrolidone), poly(vinyl alcohol), poly(acrylamide), poly(2-hydroxypropyl methacrylamide), poly(N,N-dimethylacrylamide), poly([2-

(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), poly(2- (methacryloyloxy)ethyl phosphorylcholine), poly(carboxybetaine methacrylamide) , polyethylene glycol), polyethylene imine), polyearcosine), poly(2-methyl-2-oxazoline), polyamino esters, polyester amides, polyphosphoesters, poly(l-lysine), poly(l-proline) , polyphosphazenes, dextran, sodium alginate, gelatin, agarose, carrageenan, gellan, xantham gum, urea, sucrose, derivatives thereof, combinations thereof, and co-polymers thereof. In some embodiments where the bolus does not comprise a housing, the carrier consists of PLA, PBSA, PBS, PHBV, PVA, PBAT, PCL, PDLA, epoxy-based chain extenders, magnesium silicate, cellulosic materials, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), fumed silica, gelatin, wax, castor wax, paraffin wax, silica and combinations thereof. In some embodiments where the bolus does not comprise a housing, the carrier comprises microcrystalline wax. In some embodiments where the bolus does not comprise a housing, the carrier consists of microcrystalline wax.

A “carrier” as used herein is a compound that can be mixed with a methane inhibiting agent and/or other active agent without changing the chemical structure of the methane inhibiting agent and/or other active agent. Preferably, the carrier when used in a bolus of the disclosure delays the release of the methane inhibiting agent and/or other active agent from the bolus.

For instance, the carrier may comprise at least one polar functional group.

A functional group covalently linked to the carrier can be selected from the group consisting of an ester, a fatty acid, a fatty alcohol, a carbonyl and a fatty amine. Without wishing to being bound by theory, such modified carriers may interact via the polar functional group with said methane inhibiting agent, in part possibly via hydrogen bonds.

A range of substances may be suitable for use as a carrier in the boluses of the present disclosure and the following examples are not limiting. For instance, the carrier may be selected from the list of waxes, myristic acid, stearic acid, steryl alcohol, cetyl alcohol, cetosteryl alcohol or a combination thereof. The carrier may be a waxy substance, for example, the carrier may be selected from the list of bee’s wax, paraffin wax, PEG4000, Carnauba, castor wax, Candellila, Jojoba, or Lanolin was or a combination thereof. The carrier may comprise a mixture two or more components, such as of at least one relatively polar substance with a relatively non-polar substance. As a result, the overall polarity of the carrier may be adjusted to achieve the desired affinity for the methane inhibiting agent. This can be used to achieve a desired release rate for the methane inhibiting agent. For instance, in some forms the carrier may include a mixture of paraffin wax (a mixture of alkanes with no polar functional groups) and castor wax and/or carnauba wax (which have a relatively high amount of polar functional groups).

In one embodiment, the bolus may be adapted to exhibit a release rate of between 0.02g and 2 g per day into the rumen, preferably a release rate of approximately 0.1 to 0.5g of bromoform per day. When a bolus exhibits such release rates for the methane inhibiting agent (e.g. a haloform, such as bromoform), this can reduce methane production. The rate of release of the methane inhibiting agent into the rumen may increase overtime, i.e. the rate of release starts from zero on administration to the animal and increases to a maximum due to several factors. However, the foregoing should not be seen as limiting, and other release rates are envisaged as within the scope of the present disclosure.

The carrier of a bolus described herein may have a melting point which is less than the boiling point of the methane inhibiting agent. This may be useful as the carrier can be melted and mixed with the methane inhibiting agent without substantial loss of the methane inhibiting agent due to evaporation. Furthermore, having a melting point above 37°C, and more preferably above 40°C, can assist the carrier in stabilizing the methane inhibiting agent when the bolus is in the rumen. This means, that in the rumen, which may have temperatures of up to around 40 °C in some cases, the bolus core does not melt. This may be beneficial to control release of the methane inhibiting agent e.g. for boluses comprising a housing including movement of the methane inhibiting agent through the material forming the housing.

Additionally, carriers may comprise powdered activated carbon, zeolite or bentonite, elemental zinc or zinc oxide. Preferably, a high-density material, such as a piece of metal (preferably steel) may be comprised in the carrier. The additional components may be used to achieve a desired density for the core and / or bolus. It should be appreciated by a person skilled in the art that other carriers and/or core components may be selected or used depending on the application. It is envisioned that certain carriers can be selected in order to provide a desired release profile for the methane inhibiting agent, or alternatively provide the desired physical properties of the core material -density or volume etc. In one embodiment, the carrier comprised by a bolus also comprising a housing may have a relatively higher affinity for the methane inhibiting agent compared to the affinity of the housing for the methane inhibiting agent. This may for instance be achieved by the relative polarity of the substances forming the carrier and the housing, and matching these materials appropriately to the methane inhibiting agent.

In one embodiment, the core comprises one or more materials selected from the list consisting of polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polylactic acid (PLA), poly-lactic acid, poly-d-lactic acid, poly-L-lactic acid , poly-D,L-lactic acid (PDLLA), poly-lactide-co-glycolide, lignin, polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D-lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), epoxy-based chain extenders, magnesium silicate, cellulosic materials, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), fumed silica, gelatin, wax, castor wax, paraffin wax, silica, hydrophilic silica, microcrystalline wax, methyl cellulose, starch, polyethylene glycol, polyvinyl alcohol, hydroxyethyl cellulose, carboxymethyl cellulose, soluplus, Poly(acrylic acid) , poly(vinylpyrrolidone) , poly(vinyl alcohol) , poly(acrylamide) , poly(2-hydroxypropyl methacrylamide), poly(N,N- dimethylacrylamide) , poly([2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), poly(2-(methacryloyloxy)ethyl phosphorylcholine) , poly(carboxybetaine methacrylamide) , polyethylene glycol), polyethylene imine) , poly(sarcosine), poly(2- methyl-2-oxazoline), polyamino esters, polyester amides, polyphosphoesters, poly(l- lysine) , poly(l-proline) , polyphosphazenes, dextran , sodium alginate, gelatin, agarose, carrageenan, gellan, xantham gum, urea, sucrose, beeswax, polyethylene glycol (PEG), sodium starch glycolate, croscarmellose sodium, crospovidone, carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), carrageenan, guar gum, xanthan gum, sodium alginate, locust bean gum, polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate co-polymer (PVP/VA), a polyacrylic acid and/or their co-polymer variants, polyisobutylene, ethyl vinyl acetate (EVA), a functional wax with a melting point less than about 120 °C, derivatives thereof, combinations thereof, and co-polymers thereof. In one embodiment, the core consists of one or more materials selected from the list consisting of polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co- adipate (PBSA), polylactic acid (PLA), poly-lactic acid, poly-d-lactic acid, poly-L-lactic acid , poly-D,L-lactic acid (PDLLA), poly-lactide-co-glycolide, lignin, polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D- lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), epoxy-based chain extenders, magnesium silicate, cellulosic materials, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), fumed silica, gelatin, wax, castor wax, paraffin wax, silica, microcrystalline wax, methyl cellulose, starch, polyethylene glycol, polyvinyl alcohol, hydroxyethyl cellulose, carboxymethyl cellulose, soluplus, Poly(acrylic acid) , poly(vinylpyrrolidone) , poly(vinyl alcohol) , poly(acrylamide) , poly(2- hydroxypropyl methacrylamide), poly(N,N-dimethylacrylamide) , poly([2- (methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), poly(2- (methacryloyloxy)ethyl phosphorylcholine) , poly(carboxybetaine methacrylamide) , polyethylene glycol), polyethylene imine) , poly(sarcosine), poly(2-methyl-2-oxazoline), polyamino esters, polyester amides, polyphosphoesters, poly(l-lysine) , poly(l-proline) , polyphosphazenes, dextran , sodium alginate, gelatin, agarose, carrageenan, gellan, xantham gum, urea, sucrose, beeswax, polyethylene glycol (PEG), sodium starch glycolate, croscarmellose sodium, crospovidone, carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), carrageenan, guar gum, xanthan gum, sodium alginate, locust bean gum, polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate copolymer (PVP/VA), a polyacrylic acid and/or their co-polymer variants, polyisobutylene, ethyl vinyl acetate (EVA), a functional wax with a melting point less than about 120 °C, derivatives thereof, combinations thereof, and co-polymers thereof.

In one embodiment, the core comprises a compound selected from the group consisting of Polylactic acid (PLA), Poly(butylene succinate-co-butylene adipate (PBSA), Polybutylene succinate (PBS), Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), Polyvinyl alcohol (PVA), Polybutylene adipate terephthalate (PBAT), Polycaprolactone (PCL), Poly(D,L-lactic acid) (PDLA), epoxy-based chain extenders, magnesium silicate, cellulosic materials, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), fumed silica, gelatin, wax, castor wax, paraffin wax, silica and combinations thereof. The skilled person will be aware of further compounds and particularly of further polymers, preferably biodegradable polymers, that may be suitably used as core components of the bolus of this embodiment.

In one embodiment, a bolus of the disclosure as described comprises hydrophobic fumed silica. Preferably, such fumed silica is amorphous or consists of or comprises hydrophobic fumed silica particles (HFSPs). In one preferred embodiment the average particle diameter of said hydrophobic fumed silica is between 5 nm and 15 nm. For instance, the bolus may comprise at most 10 wt%, at most 8 wt %, or at most 5 wt% of said hydrophobic fumed silica. Preferably, the bolus comprises at most 5 wt% of said hydrophobic fumed silica, wherein the methane inhibiting agent is bromoform. In one embodiment the hydrophobic fumed silica is silica producible by contacting silica with a hydrophobic silane and preferably contacting said silica with a compound selected from the group consisting of dimethyldichlorosilane (DDS), methyl acrylic silane, octyl silane, octamethylcyclotetrasiloxane, hexadecyl silane, octylsilane, methylacrylsilane, polydimethylsiloxane, hexamethyldisilazane (HMDS), silicone oil, silicone oil plus aminosilane, HMDS plus aminosilane, an organic phosphate, HMDS (hexamethyldisilazane), and combinations of the aforementioned compounds.

In one embodiment, a bolus of the disclosure as described comprises hydrophilic silica.

The core of a bolus not comprising a housing as described herein may be selected to provide sufficient sustainability of said uncased bolus in the rumen environment. In one embodiment the bolus has a Shore D hardness of at least 20. In one embodiment the bolus may have a Shore D hardness of at least 40. Shore D hardness may for instance be adjusted via the selection of core materials or the manufacturing process of the core. It will be clear to an average skilled person that the bolus hardness may be selected so that the bolus is able to persist in the environment of the rumen, withstanding the physical and chemical influences. In such an embodiment, it is believed that a bolus (without a housing) having a Shore D hardness of less than 20 may result in a bolus that is too soft, which could hinder administration of the bolus to an animal or lead to it being otherwise damaged or prematurely degraded before the full amount of methane inhibiting agent is administered. Methods to determine Shore D hardness are known in the art and will be clear to the skilled person. For instance, this may be done by use of a durometer, which determines Shore D hardness by the penetration of the Durometer indenter foot into the sample under a defined spring force.

While the described can function without a housing, the core of a bolus without a housing can be partly or fully coated. Thus, in a further embodiment, the bolus may comprise a core, wherein the core comprises a methane inhibiting agent (preferably a haloform such as bromoform); and a coating which covers art least a portion of the core or preferably the entire core; wherein the bolus is configured to release the methane inhibiting agent. A coating layer thickness of less than 2 mm is preferred to allow the methane inhibiting agent, such as a haloform, to permeate from the core material outwardly in an optimal rate.

In one embodiment the bolus is in the form of a pellet, pill, lozenge or tablet. The size and shape of a pellet, pill, lozenge or tablet may be suitably selected by an average skilled person to match the dose to be administered, the time period for which administration is intended and the size of the subject animal. For example, pellet, pill, lozenge or tablet size may be selected larger in a large animal such as cattle, while for smaller ruminants, like sheep, smaller pellet, pill, lozenge or tablet size may be suitable.

The bolus in the form of a pellet, pill, lozenge or tablet may be a small bolus, such as a bolus with a length of about 1 to 5 cm. An animal can also be administered multiple of such small boluses at the same time or subsequently. For instance, an animal may be administered multiple of such boluses, which are admixed with the animal’s feed, i.e. are used as a feed additive.

In one embodiment the bolus is configured to dissolve in the rumen of a ruminant animal over a period of time of less than 48 h. In a preferred embodiment the bolus is configured to dissolve in the rumen of a ruminant animal over a period of time of less than 12 h, more preferably of less than 6 h, even more preferably of less than 2 h. The skilled person is aware how to select the size and possibly a coating for a bolus of the disclosure in order to obtain the aforementioned dissolution time periods.

Whether and how quickly a bolus is dissolved in an animal’s rumen can be tested for example by in vitro testing, e.g. by placing the bolus in a solution with conditions that simulate the rumen environment and determining whether and when the bolus is dissolved, i.e. partially or entirely disintegrates over time. For in vitro testing a bolus can for example be placed in a tank or vessel containing a known volume (e.g. 1 liter) phosphate buffer (pH: 6.5, 0.02 M) at 40°C. Optionally, the solution containing the bolus can be agitated to simulate agitation in the rumen.

Further active agent

In some embodiments, the housing surrounds all of the core.

In some embodiments, the further active agent is selected from the group consisting of a methane inhibiting agent, a hydrogen sequester, an anti-inflammatory agent, an analgesic, an anthelmintic, a nonsteroidal anti-inflammatory drug (NSAID), an antibiotic, a growth promoter, a lactation promoter, a sustainability improver, an antimicrobial, a ketosis prevention agent, a mineral/element/vitamin supplement and combinations thereof. In some embodiments, the further active agent is selected from the group consisting of a methane inhibiting agent, a hydrogen sequester, a nonsteroidal antiinflammatory drug (NSAID), an anthelmintic and a ketosis prevention agent. In some embodiments, the further active ingredient is selected from a methane inhibiting agent, an antibiotic, and a ketosis prevention agent.

In some embodiments, the further methane inhibiting agent is selected from the group consisting of a haloform (for instance a haloform other than bromoform if the first methane inhibiting agent is bromoform), monensin, a phospholipid (such as lecithin), and a fatty acid (such as lauric acid, myristic acid and linoleic acid); a plant extract or derivative including tannins, oils, essential oil; a fumarate (such as fumaric acid and sodium fumarate), an acrylate (such as sodium acrylate), a statin (such as atorvastatin and simvastatin), a sulfur-containing salt (such as sulfate and sodium sulfate), nitrate (such as potassium nitrate, calcium nitrate, calcium ammonium nitrate and sodium nitrate), malate, a Ce-ufatty acid (preferably a Ce-i2fatty acid such as aproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, undecylic acid, lauric acid), an unsaturated fatty acid (such as a-Linolenic acid, Stearidonic acid, Eicosapentaenoic acid, Cervonic acid, Linoleic acid, Linolelaidic acid, y-Linolenic acid, Dihomo-y-linolenic acid, Arachidonic acid, Docosatetraenoic acid , Palmitoleic acid, Vaccenic acid, Pau llinic acid, Oleic acid, Elaidic acid, Gondoic acid, Erucic acid, Nervonic acid, Mead acid), and a lipid (such as a fatty acyl, a glycerolipid, a glycerophospholipid, a sphingolipid, a sterol, a prenol, and a saccharolipid). In some embodiments, the further methane inhibiting agent is selected from the group consisting of a haloform (for instance a haloform other than bromoform if the first methane inhibiting agent is bromoform), monensin, a phospholipid (such as lecithin), a fatty acid (such as lauric acid, myristic acid and linoleic acid. In some embodiments, the further methane inhibiting agent is not 3-nitrooxypropanol (3-NOP). In some embodiments, the hydrogen sequester is selected from the group consisting of fumaric acid, sodium fumarate, a phenolic compound, phloroglucinol, gallic acid, resorcinol, catechols, hydroquinone and pyrogallol. In some embodiments, the antiinflammatory agent/analgesic/NSAID is selected from Nonsteroidal Anti-inflammatory Drugs (such as aspirin, ibuprofen, ketoprofen, carprofen, meloxicam, robenacoxib, firocoxib, mavacoxib, and flunixin), a corticosteroid, an alpha-2 antagonist, ketamine, and an opioid receptor agonist (such as tramadol). In some embodiments, the antiinflammatory agent/analgesic/NSAID is selected from meloxicam and ketoprofen. In some embodiments, the anthelmintic is selected from the group consisting of a benximidazole (such as mebendazole, flubendazole, fenbendazole, oxfendazole, oxibendazole, albendazole, albendazole sulfoxide, thiabendazole, thiophanate, febantel, netobimin, and triclabendazole, netobimin, albendazole, and triclabendazole), an imidazothiazole (such as levamisole), a tetrahydropyrimidines (such as pyrantel tartarate or embonate, and oxantel), a macrocylic lactone (such as ivermectin, abamectin, doramectin, eprinomectin, selamectin, milbemycin oxime and moxidectin), a salicylanilide (brotianide, clioxanide, closantel, niclosamide, oxyclozanide, and rafoxanide), a substituted phenol (such as bithionol, disophenol, hexachlorophene, niclofolan, menichlopholan, and nitroxynil), an aromatic amide diamfenetide (such as diamphenethide), praziquantel, epsiprantel, an amino-acetonitrile derivative, a cyclic octadepsipeptide (such as emodepside), a spiroindoles (such as derquantel), piperazine, clorsulon, bunamidine, and nitroscanate. In some embodiments, the anthelmintic is albendazole. In some embodiments, the antibiotic is selected from the group consisting of meloxicam, ketoprofen, penicillin, a tetracycline, macrolides, monensin, ceftiofur, florfenicol, tilmicosin, enrofloxacin, and tulathromycin. In some embodiments, the antimicrobial is selected from the group consisting of a tetracycline (such as chlortetracycline, oxytetracycline, doxycycline, tetracycline), an amphenicol (such as florfenicol, thiamphenicol), a penicillin and clavulanic acid (such as amoxicillin, ampicillin, cioxacillin, pennthamate, procaine benzylpenicillin, phenoxymethyphenicillin), a cephalosporin (such as cefalonium, cefalexin, cefaprin, cefoperazone, cefquinome, and ceftiofur), a lincoamide (such as lincomycin), a sulfonamide, trimethoprim, a macrolide (such as gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin, and tylvalosin), a aminoglycoside (such as dihydrostreptomycin, apramycin sulfate, framycetin, neomycin, paromomycin, streptomycin, and spectinomycin), a fluoroquinolone (such as enrofloxacin, marbofloxacin, and danofloxacin), a polymyxins (such as colistin), a pleuromutilin (such as tiamulin), and chloramphenicol. In some embodiments, the ketosis prevention agent is monensin. In some embodiments, the mineral/element/vitamin supplement is selected from the group consisting copper, cobalt, selenium, manganese, magnesium, sodium and chloride, potassium, zinc, iodine, sulphur, chromium, vitamin A, vitamin E, vitamin D3, and combinations thereof. In some embodiments, the further active agent is selected from the group consisting of monensin, phloroglucinol, albendazole, ketoconazole, lecithin and combinations thereof.

The inclusion of multiple methane inhibiting agents may be beneficial, particularly if they act upon one or more of different pathways, enzymes and organisms of the methanogenic organisms of the rumen. The inclusion of a further active agent that ameliorates side-effects of the methane inhibiting agent may be beneficial, for instance if the methane inhibiting agent can lead to ketosis then inclusion of a ketosis prevention agent in the bolus may be particularly useful. The inclusion of a further active ingredient that improves the release profile of the methane inhibiting agent may be beneficial. Generally, a more extended release profile is preferred. The inclusion of a further active agent that results in reduced need for administration, for instance through administration of at least 2 actives in a single instance is desirable. Besides methane, also hydrogen gas may be produced in the rumen. Although hydrogen is a weaker greenhouse gas than methane, it would be ideal if also at least part of the hydrogen gas emission could be reduced. For this purpose, it would be desirable to bind or remove at least part of the hydrogen gas that is generated in the rumen. Phloroglucinol degradation in the rumen was found to promote the sequestration of excess hydrogen, which would otherwise be used for methane production (see: Martinez- Fernandez G, et al., Front Microbiol. 2017 Oct 5;8:1871 . doi: 10.3389/fmicb.2017.01871], Also, other compounds can be used to promote the growth of microbes in the rumen which utilize hydrogen and therefore will reduce the partial pressure of hydrogen in the rumen and the amount of this gas eructated by the animals. Accordingly, in one embodiment the bolus of the disclosure comprises a hydrogen sequester, preferably selected from the group of hydrogen-sequesters consisting of fumaric acid, sodium fumarate, a phenolic compound, phloroglucinol, gallic acid, resorcinol, catechols, hydroquinone and pyrogallol.

A bolus as described herein, and particularly a bolus comprising hydrogensequestering compounds, may in some embodiments also be a small bolus such as a pill, tablet or pellet. Such small boluses can be administered as such or can be administered by addition of the boluses, for instance in the form of tablets, pills or pellets, to an animal’s feed, whereby the boluses are consumed by the animal. For instance, such small boluses may be added to the feed of the consuming animal in an amount, so that the amount of consumed feed comprises about 20 g of hydrogen-sequestering compounds per kg of dry matter of feed, wherein said compounds are comprised by the admixed boluses. For instance, an animal such as cattle consuming from about 15 kg to about 25 kg of dry matter of feed per day may consume around 300-500 g of hydrogensequestering compounds per day comprised by the boluses as described herein, for instance as part of the described pills, tablets or pellets.

In one embodiment in any bolus described herein, the methane inhibiting agent is selected from the group consisting of bromoform, monensin, nisin, lecithin, lauric acid, myristic acid, linoleic acid and phospholipid, and combinations of the aforementioned. Preferably, the phospholipid comprises one or more polyunsaturated fatty acids, more preferably wherein the phospholipid comprises one or more fatty acids selected from 18- carbon polyunsaturated fatty acids (C18 PUFAs), palmitic acid, stearic acid and oleic acid. In one embodiment the methane inhibiting agent is contained in the bolus admixed with a carrier.

It is understood that a controlled release of a methane inhibiting agent through the housing can be influenced by a number of factors. For example, the controlled release may be influenced by the affinity of the methane inhibiting agent for a carrier comprised by the bolus of the disclosure, in which the carrier may play a role in the diffusion of the methane inhibiting agent through the housing of a bolus comprising said housing. It is understood that more polar carriers or carriers containing a high degree of polar functional groups will have a higher affinity with polar inhibiting agents than less polar carriers or carriers with a lower degree of functional groups.

The relative affinity for the methane inhibiting agent of the compounds forming a housing (in a bolus comprising said housing) and of a core of a bolus described herein may also affect controlled release of the methane inhibiting agent from the core. For example, having a housing with a relatively lower affinity for the methane inhibiting agent compared to the affinity of the carrier for the methane inhibiting agent, could be a factor in controlling the rate of release of the methane inhibiting agent from the core.

In one embodiment the methane inhibiting agent comprised in the core comprised in the bolus is a haloform, preferably selected from the list of bromoform, chloroform, iodoform, and combinations thereof. In a particularly preferred form, the haloform may be bromoform (CHBr3). Bromoform is reactive and has a short half-life in animals (0.8 h in rats, 1 .2 hours in mice, US Dept of Health, 2003). It is a liquid at room temperature and is denser than water. Previous trials demonstrated no residues in meat and tissue from slaughtered steers, after 48 hour with holding period (Kinley et al. Mitigating the carbon footprint and improving productivity of ruminant livestock agriculture using a red seaweed, Journal of Cleaner Production 259 (2020) 120836), and no significant increase in the level in milk (Roque et al. Inclusion of Asparagopsis armata in lactating dairy cows’ diet reduces enteric methane emission by over 50 percent; Journal of Cleaner Production 234 (2019) 132-138).

The use of bromoform may provide a number of advantages. For instance, it has a high efficacy for a relatively small dose, which enables one device to deliver sufficient amounts of the methane inhibiting agent over an extended period of time. In addition, bromoform also has a relatively high density which adds to the overall weight of the bolus and allows for the bolus to be retained in the rumen i.e. it sinks to the ventral part of the rumen rather than floats reducing regurgitation. In one embodiment, the bolus may comprise the haloform, preferably bromoform, in an amount of 10% (by weight) to 80% (by weight), preferably in an amount of 20% (by weight) to 50% (by weight).

The methane inhibiting agent may also be synthetic or derived from a naturally occurring source such as from a plant such as from algae. In one embodiment the methane inhibiting agent is Asparagopsis or a derivative thereof. The methane inhibiting agent, may for instance be obtained from Asparagopsis by extraction. For example, lysing of the algae can be achieved by breaking the algal cell wall or membrane to separate the methane inhibiting substances from the rest of the algae biomass. The algae such as Asparagopsis or parts and derivatives thereof may also be directly included in the bolus, as a source that releases the methane inhibiting agent, such as bromoform.

In another embodiment the methane inhibiting agent is monensin. Monensin is a carboxylic polyether ionophore which may modify rumen fermentation dynamics by selectively inhibiting growth of gram-positive bacteria, which produce most of the acetate, lactate, and hydrogen in the rumen, which can contribute to methane formation. Advantageously, monensin is also known to prevent ketosis in ruminants. Administration of a ketosis prevention agent can be beneficial when administering a methane inhibiting agent such as a haloform. Another methane inhibiting agent, which may be used in addition or alternatively to monensin, is the bacteriocin nisin. Both nisin and monensin inhibit methanogenic bacteria by primarily increasing the permeability of their cell membrane.

Lecithin is known have an effect on ruminal fermentation and digestion and may therefore contribute to methane inhibition. For instance, soybean lecithin may be suitable in this context.

Certain saturated and unsaturated fatty acids may also be used for their ability to influence ruminal fermentation and microbial composition in the rumen, thus influencing the methanogenic potential in the rumen. In one embodiment, the methane inhibiting agent is selected from lauric acid, myristic acid and linoleic acid.

Phospholipids are known to typically comprise a glycerol molecule, the carbon atoms of which are connected to two fatty acids and a phosphate group, wherein the fatty acids and phosphate group are attached to the glycerol molecule through an ester bond. In one embodiment a bolus described herein comprises a methane inhibiting agent selected from the group consisting of phospholipids comprising a glycerol molecule linked via ester bonds to a phosphate group and to two fatty acids, preferably wherein the phospholipid comprises one or more polyunsaturated fatty acids, more preferably wherein the phospholipid comprises one or more fatty acids selected from 18-carbon polyunsaturated fatty acids (C18 PUFAs), palmitic acid, stearic acid and oleic acid.

In view of possible phospholipids suitably used, phospholipids are preferred, which comprise one or more fatty acids with a methane mitigating effect. For instance, saturated fatty acids (SFAs) are known to suppress ruminal methanogenesis, including for instance lauric (C12), myristic (C14), or palmitic (C16) and stearic acid (C18). Furthermore, polyunsaturated fatty acids (PUFA) such as C12 and C18 PUFAs are potent against methanogenesis. Without wishing to be limited to this supposed effect of these fatty acids, it is currently believed that the presence of these fatty acids in the rumen impacts the microbiome in the intestinal tract of ruminant animals. In particular, it is believed that such fatty acids can act against rumen methanogens colonizing the rumen, which otherwise scavenge H2 and CO2 produced by other fermentative members of the ruminal microbiome and produce methane (CH4). In a preferred embodiment, the phospholipid is selected from lecithin, phosphatidylcholine and derivatives of the aforementioned compounds.

In one embodiment at least 50wt% of the bolus of the disclosure comprises the methane inhibiting agent. In another embodiment at least 60wt%, at least 70wt% of the bolus of the disclosure comprises the methane inhibiting agent.

It should be appreciated that the ratio of methane inhibiting agent to carrier or to full bolus weight may be selected to optimize the function of the bolus e.g. to suit the desired release profile for the respective inhibiting agent(s). In one embodiment a haloform, preferably bromoform, is comprised in the core of the bolus of the disclosure in an amount of between 10 wt% to 80 wt% and preferably in an amount of between 15 wt% and 70 wt%. The term “wt%” as used herein in the context of a methane inhibiting agent, such as a haloform or bromoform, comprised in a bolus as described herein refers to the weight percent of said methane inhibiting agent based on the total weight of said bolus. In connection with the above methane inhibiting substances, suitable carrier materials may advantageously be used in the bolus of the disclosure, which have a high capacity to hold the methane inhibiting agent. Due to the volatility of some methane inhibiting agents such as bromoform and its reactivity with many compounds including organic compounds, it is inherently difficult to contain such methane inhibiting compound in a stable way and at a high concentration (to be able to reduce the size of the formulation) in a delivery device such as a bolus. One example of especially suitable carrier material is fumed silica, preferably hydrophobic fumed silica, which can consist of particles of amorphous silica that can be fused into branched particles. Fumed silica, available for instance as a powder, provides a low bulk density and high surface area. Using fumed silica as carrier in a bolus will stabilize the formulation, improve the stability and increase the loading capacity of the drug formulation for the methane inhibiting compound and particularly for haloforms such as bromoform. Thus, when bromoform is used as a methane inhibiting agent, it can be suitable for the bolus to comprises fumed silica.

For some methane inhibiting agent, it may be suitable to incorporate in the housing of the bolus one or more openings in order to facilitate release of the methane inhibiting agent from the bolus. In a further aspect the disclosure relates to a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent and a carrier; and a housing which comprises said core; wherein the methane inhibiting agent is selected from the group consisting of monensin, lauric acid, myristic acid and linoleic acid; and wherein the housing comprises at least one opening exposing the core to the environment surrounding the bolus. Preferably, the housing material comprises PLA and PBAT.

This embodiment may be particularly useful in cases where the as methane inhibiting agent other compounds are used than a haloform.

In one embodiment the bolus according to any aspect or embodiment comprises a housing, wherein said housing comprises a stabilizer. In a preferred embodiment the stabilizer is selected from the group consisting of a surfactant, a plasticizer, a phthalate ester and a triglyceride. In a more preferred embodiment the stabilizer is selected from the group consisting of lecithin, nitrile and triacetin.

The stabilizer may have an effect on the stability of the housing by for instance introducing more flexibility into the housing material or material blend or by reducing the brittleness or tendency to become brittle of said housing material or material blend. In the case of a housing comprising a blend of different materials, a stabilizer may also be beneficial by promoting the mixing efficiency of the blend components, thereby producing a more homogenous housing, which may therefore be more stable against forces acting through the digestive system of the ruminant. Lecithin for instance, is known to be a suitable surfactant and stabilizer used in foods and pharma. The average skilled person is aware of further compounds that may suitable be used as stabilizers forming part of the housing.

In one embodiment the core comprised by the bolus comprises at least one filling agent. This embodiment pertains to any bolus described herein. Using a filling agent may provide additional internal substance of the bolus, providing sufficient stability from the inside of the bolus to counteract forces acting on the bolus from the outside. In addition, the filling agent may provide an additional bulking substance to evenly distribute the methane inhibiting agent therein (inside of the bolus) without the filling agent promoting any significant undesirable interactions and while at the same time being well tolerable to the ruminant animal.

In a preferred embodiment the at least one filling agent is a stabilizer. In another preferred embodiment the at least one filling agent is selected from the group consisting of gelatin, milk, milk derivatives, infant formula, milk powder, triglycerides, medium chain triglycerides and oil thereof, ethanol, lecithin, tween, xanthum gum, cellulose derivatives, alkyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), zein and surfactant.

In another preferred embodiment the core comprised by any bolus described herein comprises gelatin. In yet another preferred embodiment the core comprised by any bolus described herein comprises milk, milk derivatives, infant formula or milk powder. In yet another preferred embodiment the core comprised by any bolus described herein comprises milk powder. In yet another preferred embodiment the core comprised by any bolus described herein comprises at least one stabilizing and/or filling protein. In yet another preferred embodiment the core comprised by any bolus described herein comprises casein and/or zein. In yet another preferred embodiment the core comprised by any bolus described herein comprises cellulose derivatives and preferably alkyl cellulose, ethyl cellulose, and/or hydroxypropyl methyl cellulose (HPMC). In yet another preferred embodiment the core comprised by any bolus described herein comprises cellulose derivatives and preferably alkyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), combinations thereof, and co-polymers thereof.

In one embodiment the core comprised by the bolus according to any aspect or embodiment comprises at least one PEG glyceride composed of mono-, di- and triglycerides and mono- and diesters of PEG, and preferably comprises PEG esters of palmitic, stearic and/or lauric acids.

In one embodiment the core comprised by the bolus according to any aspect or embodiment described herein comprises at least one surfactant and preferably a nonionic water-dispersible surfactant. In a preferred embodiment, the surfactant comprises a mixture of glycerides and fatty acid esters, preferably of a mixture of mono-, di- and triglycerides and PEG.

In another embodiment the surfactant comprises polyoxylglycerides, more preferably oleoyl polyoxyl-6 glycerides. In one embodiment the surfactant comprises mono-, di- and triglycerides and PEG-6 (MW 300) and mono- and diesters of oleic (C18:1 ) acid.

In another embodiment the surfactant comprises mono- and diesters of caprylic (C8) and capric (C10) acids.

In another embodiment the surfactant comprises mono-, di- and triglycerides and PEG-6 (MW 300) mono- and diesters of lauric (C12) and stearic (C18) acids

In another embodiment the surfactant comprises mono, di- and triglycerides and PEG-32 (MW 1500) mono- and diesters of lauric acid (C12).

In one embodiment the bolus core comprises zein. Zein is a composition comprising at least one protein and prolamine, which can typically be found for example in protein bodies in the endosperm of the corn kernel. Due to the proteins amphiphilic character zein is also useful in forming protective coatings. Products may be encapsulated based on zein’s ability to associate or self-assemble when solution polarity changes toward a more hydrophilic environment. Therefore, zein may not only be used as a core component of the bolus of the disclosure but may also suitably be applied as a coating of a bolus. The bolus of the disclosure may also be used as a delivery platform, for instance to allow the sustained local release of other methane inhibiting and non-anti- methanogenic molecules. In one embodiment, in addition to or instead of said methane inhibiting agent, the bolus of the disclosure comprises an active agent, wherein said active agent is not a methane inhibiting agent. In one embodiment, in addition to said methane inhibiting agent the bolus of the disclosure comprises an active agent, wherein said active agent is not a methane inhibiting agent. In one embodiment, instead of said methane inhibiting agent the bolus of the disclosure comprises an active agent, wherein said active agent is not a methane inhibiting agent.

In one embodiment, in addition to or instead of said methane inhibiting agent the bolus of the disclosure comprises an active agent, wherein said active agent is not a methane inhibiting agent, wherein the release of said active agent is a local and/or a sustained release in a ruminant intestinal system. Preferably, the release of said active agent is a local and a sustained release in a ruminant intestinal system.

In one embodiment the active agent, which is not a methane inhibiting agent, is selected from the group consisting of anti-inflammatory agent, analgesic, and anthelmintic. The active agent may, however, be any health and/or growth promoting and/or sustainability improving agent known in the art or combinations of such agents. For instance, the anti-inflammatory agent and/or analgesic may be selected from nonsteroidal anti-inflammatory drugs (NSAID). In one embodiment the active agent is selected from meloxicam and ketoprofen. For instance, the anthelmintic may be albendazole. Thus, in one embodiment the active agent is albendazole. The further active agent may also be an antibiotic, preferably selected from the group consisting of penicillin, tetracyclines, macrolides, monensin, ceftiofur, florfenicol, tilmicosin, enrofloxacin, and tulathromycin. Thus, in a preferred embodiment the active agent is selected from the group consisting of anti-inflammatory agent, analgesic, antibiotic and anthelmintic. In a more preferred embodiment the active agent is selected from the group consisting of meloxicam, ketoprofen, penicillin, tetracyclines, macrolides, monensin, ceftiofur, florfenicol, tilmicosin, enrofloxacin, tulathromycin and albendazole. Other compounds routinely and commonly administrated to ruminants and substances with one or more advantageous effects on ruminant animals are known in the art and the average skilled person is aware how to suitably implement such substances in the bolus of the disclosure. Such compounds also include, but are not limited to, growth promoters, lactation promoters and sustainability improvers. A sustainability improver is an agent that improves the sustainability of husbandry of the ruminant. This improvement can a reduction in pollution, for instance a reduction in greenhouse gas emissions.

In one embodiment the core of the bolus described herein may comprise one or more metal particles (preferably steel particles), wherein the particles are preferably rounded. For instance, the total of all particles per bolus may have a mass of at least 100 g. Incorporating metal particles in the bolus adds weight to the bolus and allows the bolus inserted into an animal’s rumen to be retained more effectively, preventing the regurgitation of the bolus after administration.

A bolus of the disclosure may be used in the treatment of an animal. In one aspect the disclosure relates to a bolus of the disclosure for use in the treatment of a ruminant animal. In a preferred embodiment the disclosure relates to a bolus of the disclosure for use in the treatment of cattle or sheep.

In another aspect, the disclosure relates to a bolus of the disclosure for use in reducing methane emission in a ruminant animal. In a preferred embodiment the ruminant animal is cattle or sheep. In a further embodiment the disclosure provides a method for administering a methane inhibiting agent to an animal, the method comprising the step of administering to said animal the bolus of the disclosure. In a further embodiment the disclosure provides a method for reducing methane production in the rumen of a ruminant animal, the method comprising the step of administering to said ruminant animal the bolus of the disclosure.

In a preferred embodiment, the bolus may be configured to be administered to a ruminant, the ruminant may include beef or dairy cows, sheep, goats, buffalo, deer, elk, giraffes or camels.

In one embodiment, the bolus may be adapted to reduce the release of one or more greenhouse gases (“GHGs”) from the ruminant.

In one embodiment the ruminant animal may also be a goat or deer.

Reduced methane emission and/or greenhouse gas emission from a ruminant animal is considered to be reduced in comparison to a ruminant animal not treated with a bolus or methane inhibitor of the disclosure. In one embodiment methane emission in a ruminant animal may be reduced by at least 30 %, preferably by as at least 50 %, more preferably by at least 70%, 80% most preferably by at least 90%. In another embodiment methane emission in a ruminant animal may be reduced at least 99%. In a preferred embodiment the bolus is administered orally. In another preferred embodiment the bolus is configured to remain in the rumen after administration.

The bolus is delivered orally into the rumen of the ruminant animal to be treated, entering the rumen via the oesophagus. In the rumen, stomach fluids (and other matter such as plant fibers) can act to erode or dissolve the core to release the methane inhibiting agent over time or the contents of the bolus, such as the methane inhibiting agent, can diffuse or seep out of the bolus into the rumen.

If the bolus comprises a housing, for the duration of the treatment period said housing is substantially intact. In case the bolus housing comprises one or more opening(s), these one or more opening(s) allow stomach fluids and possibly fibrous matter to come into contact with the core

Core and housing may be designed to facilitate release of the methane inhibiting agent over a period of time for which an animal is to be treated. The bolus can be adapted to release the methane inhibiting agent over a period of at least three months, preferably at least six months, more preferably 12 months, and potentially up to two years. Preferably, the release rates of the methane inhibiting agent may be calculated based on the weight of the ruminant animal to be treated and the type of inhibiting agent used. As such, it will be appreciated that the desired release rates may vary from animal to animal. Typically, the desired release rates may be calculated on an amount of inhibiting agent/weight of animal. Alternatively, the desired release rates may also be calculated based on the amount of feed consumed by the animal. Particularly preferred release rates for bromoform as an exemplary methane inhibiting agent include from approximately 0.1 - approximately 0.5 g/day, and more preferably approximately 0.2 g/day.

A ruminant animal can also be treated by multiple boluses according to the present disclosure in order to achieve a preferred dosage of the methane inhibiting agent. This can allow a bolus to be manufactured which has a concentration and total load of the methane inhibiting agent. Multiple of those boluses can be administered to an animal concurrently or sequentially. This will allow the desired dosage to be provided to the animal. This can be particularly beneficial to allow the bolus to be used with animals requiring different doses of inhibiting agent e.g. larger or smaller animals, or to compensate for natural growth over time.

A ruminant animal can also be treated by multiple boluses according to the present disclosure in order to achieve a preferred substance combination, wherein different substances may be administered at the same time. For instance, a bolus combination may be suitable, wherein one bolus provides a methane inhibiting agent and a further bolus provides a different active agent, such as an antibiotic or other ruminant’s health improving compound.

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

It should be appreciated by the person skilled in the art that the size, thickness and/or dimensions of the bolus, including the core and a housing if provided can be adjusted depending on the dose of inhibiting agent to be delivered to the ruminant, without departing from the spirit and scope of the disclosure. For example, a smaller size bolus can be adapted for use in smaller ruminant animals such as sheep or goats, while a larger sized bolus can be used in larger ruminant animals such as cattle. Preferably, the bolus has a weight of less than 180g. For example, each cattle could be administered two boluses, each having a dimension of about 75 mm in length and about 34 mm in width, whereby each of these boluses may have a weight of about 80 g.

In addition, reducing production of methane may provide animal production benefits, the bolus may improve the ruminant’s conversion of feed for animal production. For example, by reducing methane production during digestion, it is believed that this may lead to more efficient utilization of ingested feed, and result in improved growth and weight gain, or other production such as milk or meat production. As a result, farmers may be able to improve efficiency by either securing greater productivity for a given feed volume or reduce feed accordingly. In addition, the compositions for the core and synergistic effects arising from the combination of carrier and inhibiting agent(s) may enable the provision of a slow-release, long term delivery device to improve animal productivity and / or reduce emission of greenhouse gases. In a further aspect the disclosure relates to a method of manufacturing a bolus as defined herein. The method of manufacturing may comprising the steps: (1 ) providing a housing, preferably a housing made of a polymer material, more preferably a biodegradable polymer or preferably a housing of a material as disclosed herein; and (2) filling a core, preferably comprising a material as disclosed herein, into said housing; wherein the bolus comprises: a core, wherein the core comprises a methane inhibiting agent that inhibits the production of methane in the rumen of a ruminant animal and a carrier and a housing which houses the core. Providing the housing in step (1 ) may for instance and without limitation be performed by 3D printing or injection moulding. Filling a core into said housing in step (2) may for instance and without limitation be performed by melting or mashing and mixing the core materials and filling the core material components or mixtures into the housing while the components are flowable or at least flexible or malleable.

It is preferred that the method of manufacturing a bolus may further comprise the step (3) closing the housing that contains the core with a cap. The housing may be closed with a cap by friction-welding the cap to the housing. This is advantageous compared to a screwed-on or glued-on cap, which could become lose or be pushed out of the housing when the bolus is exposed to the chemical and mechanical stress and turbulent motion in the rumen of an animal.

Providing the housing in step (1 ) may occur using any technique as should be known to one skilled in the art. For instance, a suitable material may be extruded into a desired shape defining a cavity. Alternatively, an additive layering manufacturing process could also be used to build the housing shape defining a cavity or a moulding process could be used, such as injection moulding, 3D printing or hot melt extrusion processes.

Step (2) of filling the core may for instance include one or more of the following steps: melting and/or mashing a carrier material to provide a melted and/or mashed carrier material, adding the methane inhibiting agent(s) to the melted and/or mashed carrier material, mixing the methane inhibiting agent and the melted and/or mashed carrier material to create a substantially homogenous mixture, filling the substantially homogeneous mixture into the prepared housing.

For a bolus without a housing, step (1 ) of forming the housing may be omitted and step (2) may be a step of forming the core, which may for instance include one or more of the following steps: melting and/or mashing a carrier material to provide a melted and/or mashed carrier material, adding the methane inhibiting agent(s) to the melted and/or mashed carrier material, mixing the methane inhibiting agent and the melted and/or mashed carrier material to create a substantially homogenous mixture, forming the substantially homogeneous mixture into a desired shape. It should be understood that the step of forming the substantially homogeneous mixture into a desired shape may involve providing the mixture to a mould.

It should be understood that the substantially homogenous mixture contains the methane inhibiting agent(s) at a concentration sufficient to achieve the desired release profile for the methane inhibiting agent on administration of the device to a ruminant animal. The concentration can be varied according to the type of ruminant animal to be treated, the shape and dimensions of the device, or the desired release profile to be achieved.

The method also includes the step of allowing the substantially homogenous mixture to cool, particularly if it has previously been heated and melted. As it cools, the carrier material hardens and assumes a shape according to the shape of the mould or housing into which it has been provided.

The disclosure also provides a bolus obtainable or obtained by carrying out a method of the disclosure of manufacturing a bolus.

In another aspect the disclosure relates to a methane inhibitor for use in reducing methane emission from a ruminant animal, wherein the methane inhibitor is selected from the group consisting of bromoform, monensin, lecithin, lauric acid, myristic acid, linoleic acid and phospholipid, preferably wherein the phospholipid comprises one or more polyunsaturated fatty acids, more preferably wherein the phospholipid comprises one or more fatty acids selected from 18-carbon polyunsaturated fatty acids (C18 PUFAs), palmitic acid, stearic acid and oleic acid. In a preferred embodiment the ruminant animal is cattle.

In another aspect the disclosure relates to a method of treating a ruminant animal to reduce methane emission from said ruminant animal comprising administering to said animal a methane inhibitor selected from the group consisting of bromoform, monensin, lecithin, lauric acid, myristic acid, linoleic acid and phospholipid, preferably wherein the phospholipid comprises one or more polyunsaturated fatty acids, more preferably wherein the phospholipid comprises one or more fatty acids selected from 18-carbon polyunsaturated fatty acids (C18 PUFAs), palmitic acid, stearic acid and oleic acid. In a preferred embodiment the ruminant animal is cattle. The inventors deem the technology described herein may provide a number of benefits. These benefits may be the result of the unique synergistic interactions between different aspects and embodiments of the technology. The technology of the present disclosure is therefore described based on the inventor’s current understanding of such possible interactions. It should be appreciated any aspect or embodiment described herein, or the interaction of two or more aspects/embodiments, may form a distinct disclosure.

Modified release dosage forms

Modified release dosage forms are dosage forms that change the timing, rate or site of release of an active ingredient to achieve a clinical outcome not achievable by a non-modified release dosage form.

A form of modified release is temperature dependent release, where release of an active ingredient changes in response to differences in temperature. This can lead to release only under suitable temperatures, potentially controlling the site of release. Temperature dependent release offers advantages in administration but also storage, potentially allowing storage under harsher temperatures, particularly beneficial in the harsher storage conditions often encountered when dealing with livestock.

Another form of modified release is sustained, prolonged or extended release, which slows down the release of an active ingredient so that one dosage form can provide release of an active ingredient over a longer time. This has the advantage of reducing the frequency of dosing. Reducing dosing frequency in humans is usually for a matter of hours as once-daily oral dosing is usually considered acceptable. The benefits of extended release dosage forms are even more pronounced in the treatment of livestock. Daily or even weekly dosing may be prohibitive for many livestock, particularly those needing to be herded for treatment. A treatment may only become viable with weekly, fortnightly, monthly, six-weekly, eight-weekly, two-monthly, or 10-weekly dosing. This is an extraordinary extension of release for a dosage form to achieve. Extended release can be primarily due to the core. Extended release can be primarily due to the housing. The amounts of extension can vary from small to large such as a day to many months or a year depending on the composition of the core and housing.

Extended release can be a release profile where the bolus is releasing methane inhibiting agent at least 20%, at least 30%, at least 40%, at least 50% of the maximal rate 24 h after the maximal rate is achieved. Extended release can be a release profile where the bolus is releasing methane inhibiting agent at least 20%, at least 30%, at least 40%, at least 50% of the maximal rate 1 week after the maximal rate is achieved. Extended release can be a release profile where the bolus is releasing methane inhibiting agent at least 20%, at least 30%, at least 40%, at least 50% of the maximal rate 1 month after the maximal rate is achieved. Extended release can be a release profile where the bolus is releasing methane inhibiting agent at least 20%, at least 30%, at least 40%, at least 50% of the maximal rate 6 months after the maximal rate is achieved.

Extended release can be a release profile where the bolus is releasing methane inhibiting agent at least 20%, at least 30%, at least 40%, at least 50% of the maximal rate 24 h before the maximal rate is achieved. Extended release can be a release profile where the bolus is releasing methane inhibiting agent at least 20%, at least 30%, at least 40%, at least 50% of the maximal rate 1 week before the maximal rate is achieved. Extended release can be a release profile where the bolus is releasing methane inhibiting agent at least 20%, at least 30%, at least 40%, at least 50% of the maximal rate 1 month before the maximal rate is achieved. Extended release can be a release profile where the bolus is releasing methane inhibiting agent at least 20%, at least 30%, at least 40%, at least 50% of the maximal rate 6 months before the maximal rate is achieved.

Matrix systems, extended release coatings and other systems such as extended release particles within a matrix can be used to extend release of an active ingredient. The housing of the present disclosure functions as an extended release coating. The core of the present disclosure also has extended release properties. In preferred embodiments, the core of the present disclosure is a matrix system in which the active ingredient (as particles or granules optionally with non-extended release carrier) is homogeneously mixed into the core excipients. Alternatives that are contemplated by the disclosure include extended release particles containing active ingredient disbursed within binder, where the binder may or may not have additional extended release properties. Preferably the active ingredient is without excipient and directly and homogenously dispersed within the other components of the core.

In preferred embodiments, the matrix system in the core is hydrophobic (or water insoluble with minimal swelling). Optionally, the core is a blend of hydrophobic and hydrophilic ingredients, however the release characteristics are largely controlled by the hydrophobic ingredients.

In preferred embodiments, the housing is hydrophobic. In some embodiments, the core is hydrophobic. Optionally both the core and the housing are hydrophobic.

In any embodiment of any aspect of the disclosure, the core comprises a methane inhibiting agent and a carrier and the methane inhibiting agent is optionally dispersed in the carrier.

Zero-order release

Zero-order release of the active ingredient, which is a consistent release of active ingredient over the duration of release, is a goal of preferred embodiments of the dosage form of this disclosure.

Active ingredient

In some embodiments, the methane inhibiting agent is a haloform including a mixed haloform. Preferably the haloform is selected from chloroform, bromoform, iodoform, or combinations thereof; more preferably, bromoform.

Optionally, the active ingredient or haloform is about 20 to about 90%, about 30 to about 80%, about 40 to about 80%, about 50 to about 70% or about 60% w/w of the core.

Hydrophobicity

In some embodiments, one or more of the housing and core are hydrophobic. The skilled person will appreciate that hydrophobicity may be measured through analysis of water contact angles using a goniometer. In some embodiments, the difference in static water contact angle between one or more of the housing and core is at least about 60°, at least about 50°, at least about 40°, at least about 30°, at least about 20°, at least about 15°, at least about 10°, or at least about 5° at 20 °C.

Ruminant

In some embodiments, the bolus is for or suitable for administration to the rumen of a ruminant animal. In some embodiments, the ruminant is bovine, ovine, caprine or cervine. In some embodiments, the ruminant is bovine. In some embodiments, the ruminant is ovine.

Bolus

Broadly, a bolus is a dosage form having a discrete dosage of a substance such as a medicine, supplement or metabolism adjuster. In the context of this disclosure, a bolus may be solid, semisolid, or a combination thereof. The bolus may also be a combination of liquid with solid, semisolid, or a combination thereof provided the liquid is encased in solid, semisolid, or a combination thereof. The semi-solid may be a blend of a liquid with a solid or semisolid substance. The bolus is usually used for oral administration to the gastrointestinal tract of the animal, preferably to the rumen of a ruminant. The bolus is swallowed but may be administered with the assistance of a bolus gun or balling gun, several versions of which are commercially available. The shape of a bolus can vary but round, oblong or capsule shapes are common. The size of the bolus can vary as is suitable for administration to the relevant animal. A bolus can be hard or be of softer more malleable consistency. The bolus may be in the form of a pill, capsule or tablet so long as the pill, capsule or tablet could be administered using a bolus or balling gun as opposed to the smaller pills, capsules or tablets sized for inclusion into animal feeds.

In a preferred embodiments as depicted in Figure 22, the bolus of the disclosure (100) includes a housing (101 ) that encapsulates or substantially encapsulates the core (102). The housing further comprises a closed region (103). The housing is optionally about 0.5 to about 2.0 mm thick, about 0.8 to about 2.0 mm thick, about 0.8 to about 1 .8 mm thick, about 0.9 to about 1 .8 mm thick, about 1 .0 to about 1 .8 mm thick, about 0.8 to about 1 .5 mm thick, about 0.9 to about 1 .5 mm thick, about 1 .0 to about 1 .5 mm thick or about 1.2 mm thick. The housing is optionally about 5 to about 15%, about 6 to about 12%, about 6 to about 10%, about 7 to about 15%, about 7 to about 12%, about 7 to about 10% or about 8% w/w of the bolus. The core is optionally about 20 to about 55%, about 25 to about 50%, about 30 to about 45%, about 35 to about 40% w/w or about 37% w/w of the bolus. The bolus further comprises a densifier (104), the densifier separates the closed region of the housing from the core. The densifier is optionally about 30 to about 75%, about 40 to about 70%, about 45 to about 65%, about 50 to about 60%, about 55% w/w of the bolus.

In some embodiments, the bolus comprises a therapeutically effective amount of a methane inhibiting agent and:

• about 5% to about 15% (w/w) housing;

• about 20 to about 55 (w/w) core; and

• about 30 to about 75 (w/w) densifier.

Release and duration

In some embodiments, the bolus of the disclosure comprises the methane inhibitor bromoform and is adapted to reach a maximum release rate of approximately 0.1 - approximately 0.5 g per day, and more preferably approximately 0.2 g per day. Such release rates may provide a sustained release of haloforms, such as bromoform. A bolus with such release rate is for instance suitable for use in cattle and sheep.

In some embodiments, the bolus may be adapted to exhibit a release rate of between 0.02 g and 2 g per day into the rumen, preferably a release rate of approximately 0.1 to 0.5g of bromoform per day. When a bolus exhibits such release rates for the methane inhibiting agent (e.g. a haloform, such as bromoform), this can reduce methane production. The rate of release of the methane inhibiting agent into the rumen may increase over time, i.e. the rate of release starts from zero on administration to the animal and increases to a maximum due to several factors. However, the foregoing should not be seen as limiting, and other release rates are envisaged as within the scope of the present disclosure. In some embodiments, the bolus is formulated to administer haloform to the rumen of the ruminant animal for at least about 8 weeks after administration. In some embodiments, the bolus is formulated to administer haloform to the rumen of the ruminant animal for at least about 20 weeks after administration.

Retention in the rumen

The length of time that the bolus is retained in the rumen can be increased by formulating the bolus to have a density greater than that of the fluid in the rumen. One way of achieving this outcome is to include a densifier in the bolus. In some embodiments, the bolus further comprises a densifier. A densifier is a component that increases the density of the bolus. The densifier can be a metal powder such as ZnO, metal balls such as steel balls or other dense material that is suitable for inclusion in a bolus. Preferably, the densifier increases the density of the bolus to a density greater than 1 .0 g/cm³. In some embodiments, the densifier is a densifier matrix comprising densifier and at least one veterinary acceptable excipient. Optionally, the densifier matrix includes a matrix material more hydrophobic than the at least one carrier. Optionally, the densifier matrix includes a wax. Optionally, the densifier is either dispersed in the core, in or on the housing, or separate to the core and housing (preferably within the housing). Optionally, the densifier is about 30 to about 75% w/w of the bolus, preferably about 45 to about 65%, or about 55% w/w of the bolus.

An alternative approach to increasing the length of time the bolus is in the rumen is to ensure the bolus, when in the rumen, is too large to pass from the rumen. Devices of this type are known to the skilled person and often involve components that are held close to the bolus during administration and expand following administration to increase the size of the bolus. They can include attaching a further component to the bolus that increases the cross-section of the bolus to at least 4 cm², at least 5 cm², at least 6 cm² in area at, at least, one point. The centre of the cross-section does not need to be solid.

Diffusion testing

Diffusion testing is a common technique for assessing the nature of a dosage form in vitro. The diffusion test results are often correlated with in vivo performance of the dosage form and used for quality control testing to ensure consistent manufacture of the dosage form. Diffusion of the haloform from boluses of the disclosure into the surrounding solution was tested in 1 L of a 0.02 M phosphate buffer at pH 6.5 (simulating rumen pH) and at 39 °C (simulating rumen temperature) over a period of months without agitation. Samples of the buffer were taken daily and analyzed for tribromomethane by GC-FID.

Diffusion testing has similarities to dissolution testing. Dissolution testing involves placing the dosage form in a liquid of specific pH and temperature, and with specific agitation and determining the time it takes for the active ingredient to release from the dosage form. There are standardised dissolution tests in the US and European Pharmacopoeias (USP & EP). See for example Chapter <71 1 > of the USP. However, these dissolution tests are not suitable for measuring diffusion of the dosage forms of the present disclosure, at least due to the size and extended for the length of the boluses.

Methods of administration

In embodiments of the methods of administration of the disclosure, the bolus administers haloform to the rumen of the ruminant animal for at least about 8 weeks after administration. Optionally, the bolus administers haloform to the rumen of the ruminant animal for at least about 20 weeks after administration. in any embodiment of the invention referring to a bolus for release of a methane inhibiting agent into and animal, the animal is preferably a ruminant animal and the bolus is preferably for release of the methane inhibiting agent into the rumen of the ruminant.

In embodiments of the methods of administration of the disclosure, following administration of the bolus the bolus sinks below the liquid surface or to the bottom of the rumen. In embodiments of the methods of administration of the disclosure, the bolus remains in the rumen following administration for at least about 8 weeks or at least about 20 weeks.

In embodiments of the methods of administration of the disclosure, following release of the active ingredient the bolus degrades in the rumen. Optionally, the degradation is until the remnants of the bolus are of a size that can safely pass through the ruminant.

In embodiments of the methods of administration of the disclosure, following administration of the bolus the methane emitted by the ruminant is reduced by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80% by g/day. Optionally, this reduction occurs by about 5, about 10, or about 15 days following administration of the bolus. Optionally, the reduction continues for about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks or about 20 weeks. Optionally, the reduction continues at about 40 to about 90%, about 40 to about 70%, about 40 to about 50%, about 60 to about 90%, or about 70 to about 90% by g/day over the about 4 weeks, about 6 weeks, about 8 weeks, about 12 weeks, about 16 weeks or about 20 weeks.

In embodiments of the methods of administration of the disclosure, a second bolus is administered to the ruminant at about 8 to about 20 weeks, about 12 to about 20 weeks, about 8 to about 16 weeks or about 12 to about 16 weeks following the initial administration. Optionally, further bolus administration occurs regularly at these intervals. Optionally, this dosage regimen results in ongoing methane reduction of about 40 to about 90%, about 40 to about 70%, about 40 to about 50%, about 60 to about 90%, or about 70 to about 90% by g/day.

In some embodiments of the methods of the disclosure, the bolus of the disclosure comprises the methane inhibiting agent bromoform and reaches a maximum release rate of approximately 0.1 - approximately 0.5 g per day, and more preferably approximately 0.2 g per day.

In some embodiments, the bolus exhibits a release rate of between 0.02 g and 2 g per day into the rumen, preferably a release rate of approximately 0.1 to 0.5 g of bromoform per day. The foregoing should not be seen as limiting, and other release rates are envisaged as within the scope of the present disclosure.

In some embodiments, the bolus exhibits near zero-order release kinetics. In some embodiments, the bolus exhibits near zero-order release kinetics 2 months, 4 months and/or 6 months following administration.

Methods of production

In yet another aspect, the present disclosure provides a method of making a bolus, the method including: selecting a core and a housing inserting the core into the housing, optionally closing the housing to surround or substantially surround the core in the housing; wherein the core comprises a methane inhibiting agent. Optionally, preparing the core and housing prior to their selection.

In some embodiments, the housing is configured such that its permeability to said methane inhibiting agent is increased when exposing the housing to the rumen of a living animal. Optionally, the bolus further includes a densifier either dispersed in the core, in or on the housing, or separate to the core and housing (preferably within the housing).

In yet another aspect, the present disclosure provides a method of making a bolus wherein the bolus does not comprise a housing, the method including: forming the core from a methane inhibiting agent and a carrier.

Optionally, forming the core comprises use of a mould. Optionally, forming the core comprises use of a housing and subsequent removal of the housing.

These methods may be used to prepare bolus dosage forms according to the disclosure. In some embodiments, inserting the core into the housing occurs prior to inserting the densifier into the housing. In some embodiments, inserting the densifier into the housing occurs prior to closing the closing region of the housing.

In some embodiments, closing the closing region comprises closing two sections of the housing together. Alternatively, the closing includes closing of a cap. For example, closing the closing region comprises attaching a cap to the closing region of the housing or closing a cap already attached to the housing over the core (optionally attaching to another portion of the closing region of the housing). In some embodiments, the closing is by sealing or stitching. Optionally, the closing includes soldering and/or spin welding.

In some embodiments, the closing region includes a means to close the housing and the housing is closed using the means to close. Optionally, the means to close the housing is a cap.

In some embodiments, following closing of the closing region a closed region is formed from previously separate portions of housing that have been melted and/or soldered together.

In some embodiments, the densifier is above room temperature when it is inserted in the housing. In some embodiments, at least a component of the densifier and/or densifier matrix is liquid when it is inserted in the housing.

In some embodiments, the housing is prepared by injection molding.

In some embodiments, the densifier and/or densifier matrix is in direct contact with the core. In some embodiments, the densifier and/or densifier matrix is in direct contact with the closed region. Preferably, the densifier is in direct contact with the core and the closed region. In some embodiments, the densifier and/or densifier matrix does not directly contact one or both of the core and the closed region (for instance, a further spacing component may be present preventing directing contact).

In some embodiments, the closing region includes a means to close the housing. In some embodiments, the means to close the housing is a cap. In some embodiments, the closing region includes previously separate portions of housing that are melted and/or soldered together. Packing material

In a further aspect there is provided a material comprising an acrylate-based polymer when used in packing a bolus of the disclosure.

In some embodiments, the acrylate-based polymer is PMMA.

In some embodiments, PMMA is the only polymer that contacts the bolus when the bolus is packed in the packing. In some embodiments, PMMA is the only material that contacts the bolus when the bolus is packed in the packing.

Unless stated otherwise herein any described embodiment disclosed herein can be freely combined with any other embodiment disclosed herein.

It will be understood that the disclosure disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

Examples

Boluses for administration to ruminant animals are known. They can therefore be made as known in the art and for example as described in WO2022124914, incorporated herein by reference. In the following the production of improved boluses of the disclosure are descripted in non-limiting examples. In view of these examples it will be a parent how to make also alternative boluses of the disclosure.

Example 1

In the following, a general description is provided how to make a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which covers the outer surface of the bolus, wherein the housing becomes permeable to said methane inhibiting agent when exposing the housing to a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to a temperature of between 28°C and the temperature present in the rumen of a living animal and preferably a temperature of between 28°C and 42°C, allowing the methane inhibiting agent to exit the bolus through said opening or openings.

In a first step a housing is made by 3D print or by injection moulding. The housing can have various bottle-like shapes and is preferably shaped like a cylinder. The housing is typically made from a biodegradable polymer and the housing material comprises, for example, PLA and PBAT. The housing can have a wall thickness of about 1 .2 mm and a dimension of for example about 35 mm (diameter) x about 73 mm (length). The housing is then perforated for example by drilling openings or holes into it. The holes can have an average diameter of, for example, from about 0.1 mm to about 1 mm. The diameter of the holes/openings can vary within a given housing.

Next, the holes/openings are closed by filling into them a compound that melts between 28°C and the temperature present in the rumen of a living animal. Exemplary compounds and compound mixtures that can be used for this purpose are disclosed herein. Alternatively, the perforated bolus can also be wrapped into a foil having the mentioned melting temperature.

Optionally, a part of the volume inside of the housing can be filled by a densifier composition comprising for example steel balls.

In the next step, tribromomethane is added to ethyl cellulose and mixed until a homogenous paste is obtained. To that paste, HPMC is added and mixed until a homogenous dough is obtained. This dough (for example 60 grams) is then pushed into the prepared housing (which optionally comprises the densifier composition).

In a further step, the housing is closed by adding a cap that is spin welded onto the housing to close it. The steps to produce the bolus can also be carried out in any alternative order, for example by perforating the housing after having filled the housing with the core material and/or by sealing the openings in the housing in a last step.

Also provided herein is a bolus of the disclosure producible by carrying out the above outlined method steps.

Example 2 In the following, a general description is provided how to make a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent in microencapsulated particles and wherein the microencapsulated particles are dispersed in a composition comprising a carrier and optionally also comprising a dispersing agent; and a housing covering at least part of the core.

Methods for producing microencapsulated substances is known for example from US6458118B1 or US7105158B1 , both incorporated herein by reference. In such system small amounts of the drug, e.g. 1 microgram, are encapsulated in an inert material, e.g. a stable polymer. Such approach can be applied to encapsulate a methane inhibiting agent, for example bromoform. Following the encapsulation, the encapsulated haloform is then filled into a bolus of the present disclosure. This embodiment can also be manufactured by including a porous carrier such as mesoporous silica in the bolus.

Example 3

A housing-free bolus can be made, for example by following the steps in Example 1 but not using the housing. Instead the core material can be further densified by adding additional filling agents such as fumed silica. The core is then compressed into for example pellets or similar.

Example 4

In the following, a general description is provided how to make a bolus for administration to a ruminant animal, a bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent and a carrier; and a housing which comprises said core; wherein the methane inhibiting agent is selected from the group consisting of monensin, lauric acid, myristic acid and linoleic acid; and wherein the housing comprises at least one opening exposing the core to the environment surrounding the bolus. Such a bolus comprising an alternative methane inhibiting compound can be produced for example by following the steps outlined above in Example 1 and substitute bromoform with one or more alternative methane inhibiting compounds.

In such embodiment it is also possible to not fill the holes/openings of the perforated bolus.

Example 5

In the following, a general description is provided how to make a bolus, wherein in addition or instead of said methane inhibiting agent the bolus comprises an active agent, wherein said active agent is selected from the group consisting of an anti-inflammatory agent, an analgesic, an antibiotic and an anthelmintic; more preferably wherein the active agent is selected from the group consisting of meloxicam, ketoprofen, penicillin, a tetracycline, a macrolide, monensin, ceftiofur, florfenicol, tilmicosin, enrofloxacin, tulathromycin and an albendazole.

Such a bolus can be made in accordance with the steps outlined above in any of Examples 1 to 4 by substituting the methane inhibiting agent is replaced by an alternative active agent.

Example 6

In the following, a general description is provided how to use a bolus as described herein in a method of treating an animal, comprising administering to said animal a bolus as described herein to said animal, wherein said animal is preferably a ruminant animal such as cattle.

A bolus as described herein, for instance any bolus of examples 1 to 5, may be administered per os. (oesophagus) into the rumen of a ruminant animal, such as of cattle. After administration of the bolus, animals can be left to graze freely, while the bolus remains in the rumen. Administering an active agent, such as a methane inhibiting agent, in a bolus providing a sustained release of said active agent has the advantage that the active agent need not be re-administered very frequently.

To determine the effectivity of the active agent released from the bolus and the tolerability of the administered bolus and active agent, such as of a methane inhibiting agent, certain parameters can be assessed. This allows to for instance improve the dosing regimen for the respective active agent and bolus type employed. For instance, feed intake of the animals, such as dry-matter intake, as well as animal liveweights may be recorded as an indicator of animal health and well-being.

If the bolus is to release a methane or greenhouse gas inhibiting agent, the reduction of these emissions may be quantified by measuring the animals’ gas emissions, such as methane, hydrogen, and/or carbon dioxide emissions, in respiration chambers, for instance using a 4900C Continuous Emission Analyser and measuring emissions every 3 min over a 48-hour period.

If the bolus is to release an antibiotic or anthelmintic, the effect on microbiome or parasites may be assessed by taking a sample from the animals intestine and assessing it for presence of microbiota or parasites. Methods to assess and quantify microbiota and parasites include microscopy and cell culture methods, antibiotic challenging of bacteria and molecular methods such as polymerase chain reaction (PCR).

The described assessments of animals may be repeated regularly during the assessment period to verify the development of effectiveness over time. When using a haloform (e.g. bromoform) as methane inhibiting agent the bolus can be configured to release an amount of 0.4 mg haloform per kg ruminant animal per day. In one embodiment an average sized large ruminant animal (i.e. having a weight of about 350- 400 kg) can be administered haloform (e.g. bromoform) in an amount of 200 mg/d.

Example 7

The effect of temperature on release behaviour of the boluses was tested by preparing boluses and putting them in 1 L Schott bottles, containing 0.02M phosphate buffer as described below. These bottles were stored at room temperature (RT, 25 °C), 30 °C (±2 °C), and 40 °C (±2 °C) for comparison.

Preparation of the bolus

Two different bromoform loadings of boluses (1 and 2) were prepared with carrier/matrix composition as shown in the Table 2. Briefly, in each case, bromoform (purity >95%, ethanol as stabilizer: 1 -3%) was added to ethyl cellulose (EC) (Ethoxyl: 48- 49.5;Chloride- <0.05%; Apparent Viscosity- 41 -49 mPa.s) to form a sticky paste in a mortar and pestle. The skilled person will appreciate that other mixing apparatus can also be used, particularly on an industrial scale. To this mass, hydroxypropyl methyl cellulose (HPMC) (Methoxyl content- 19-24%;Hydroxypropyl content- 7-12%;Apparent Viscosity- 75000-140000 mPa.s) was added in small aliquots followed by adequate fixing to form a uniform mix. This process was repeated until all the HPMC was added, and a uniform dough/matrix was formed.

Once, the bromoform/EC/HPMC matrix was prepared, -60 g (accurately measured ±1 g) of it was loaded into the body of the housing. On top of the matrix, densifier (-100 g, accurately measured ±5 g) was added. The densifier was free stainless- steel (SS) shots (0.1 -0.5 mm diameter) in an approximately 15:1 (w/w) ratio with paraffin wax. The densifier matrix was introduced as a mix of molten paraffin wax/ stainless steel shots directly poured on top of the bromoform/EC/HPMC matrix. The housing was filled to make sure that there were little to no air gaps. As an alternative densifier option, the densifier matrix may be a premanufactured tablet of stainless steel shot/paraffin wax or any other suitably dense material. The method described for the densifier should not be considered as a limiting factor for the scope of the densifier as the purpose of the densifier is to make sure the bolus has enough density so that it sinks in the buffer and does not float. From the ruminant application perspective, the skilled person will appreciate that enough density is desired to achieve an effective bolus which can retain itself in the rumen once it has been administered to a ruminant animal. Following the addition of both carrier/matrix and densifier into the body of the housing, the cap was attached to the body by spin welding. Alternatively, a soldering gun may be used for attachment.

The housing in this case was prepared using injection moulding technique from 90% polylactic acid (PLA) (average molecular weight- -145000 g/mole; D lactic acid- 1.2%) and 10% polybutylene adipate terephthalate (PBAT) (average molecular weight- -80000 g/mol) blend. The length of each bolus housing was 73 mmm, diameter was 35 mm and the thickness was 1.2 mm. The skilled person will appreciate that other techniques in addition to injection moulding are also suitable.

Table 2. Composition of the carrier/matrix in each bolus. TBM = Tribromomethane (bromoform); EC = Ethyl Cellulose, and HPMC= Hydroxypropyl Methyl Cellulose.

DSC data on the EC suggests that it is acting amorphously.

Release testing

Once the boluses were prepared, they were kept in 1 L Schott bottles, containing 0.02 M phosphate buffer (pH = 6.5) and were stored at room temperature (RT, 25 °C), 30 °C (±2 °C), and 40 °C (±2 °C) without agitation. The buffer was changed every day except over weekends. Nevertheless, minimum 4 daily release data points were collected per week in all cases. Bromoform (TBM) was quantified using GC-FID (Shimadzu, Nexus GC-2030). Briefly, in each case 10 mL sample was collected using a 10 ml autopipette in 15 ml Falcon tubes. To this, 1 mL of ethyl acetate (analytical grade, Merck) was added to each Falcon tube as extraction solvent for TBM. The Falcon tubes were capped, well mixed using a Vortex, and centrifuged at 4000 rpm for 15 minutes. 0.5 mL of ethyl acetate was recovered and loaded in GC vial. 200 pl of sample was injected using an autosampler, and analysed using a ZB5HT 30 m capillary column using a temperature ramp of 30-300 °C over 20 minutes, at 5 mL/min nitrogen gas flow, in splitless mode. TBM had a retention time of -5 minutes. Peak areas were compared to calibration standards made up in ethyl acetate to determine the mass of TBM (mg) in the solution and were correlated to quantify TBM release per day in the 1 L buffer solution.

Result

Figures 1 and 2 show the release profile of the boluses (type 1 and 2; Table 2) when placed in buffer at different temperatures. A temperature dependent release was observed. In both the cases, it was observed that boluses start to release TBM earlier and in higher rate when placed at higher temperature than at lower temperature. Nevertheless, surprisingly, a bolus (Figure 1) which releases -150 mg TBM per day at 40 °C was measured to start significantly releasing from day 14 at 30 °C. However, for a bolus (Figure 2) which releases -80 mg TBM/day at 40 °C, it was observed that this bolus did not significantly release TBM for over 35 days. This suggests multiple factors at play beyond the mere role of temperature. It is well established that increase in temperature of a system leads to higher thermal energy, which can often contribute to increased diffusion. Further increased temperature may also make polymers more permeable, contributing to an increased release rate. Permeability of polymers may be affected by their composition, as well as further factors such as crystallinity.

Without wishing to be bound by theory, it is suggested that here, surprisingly, the plasticization effect of TBM (as a solvent, apart from being the active ingredient) on the polymers of the housing appears to play an important role in release behaviour. The inventors have observed that the solvent content of the housing material increases when exposed to bromoform/water. For example: the solvent content of the PLA/PBAT housing of Example 7 was found to increase by at least 3 folds when the housing was exposed to -100 mg /L of bromoform solution for -10 days. The solvent content was evaluated using thermogravimetric analyser (TA instruments; model -TGA55). Briefly, -5 mg (accurately measured) of the sample was heated from room temperature to 200 °C at a heating rate of 20 °C/min in an inert atmosphere of nitrogen and the change in weight was recorded. Further, with the increase in solvent content, the inventors have observed that the glass transition temperature of the housing can decrease. The glass transition temperature of the PLA/PBAT housing of Example 7 was observed to drop by -10% from -55 to -50 °C. The glass transition temperature was evaluated using differential scanning calorimeter (TA instruments, model- DSC250). Briefly, -5 mg (accurately measured) of the housing material was loaded into Tzero aluminium pan and heated in modulated condition. The samples were heated from 0 °C to 195 °C at a heating rate of 1 °C/min, amplitude of 0.16 and modulation period of 60 s. The sorption of solvent further potentially causes swelling and increases the permeability. Swelling of PLA in presence of organic solvent and water is well established (Udayakumar M et al. (2020) Polymers (Basel), May 6;12(5) :1065).

Such changes in release behaviour can also potentially be realized through other changes such as degree of crystallinity of carrier and/or housing matrix. For instance, the inventors used amorphous EC in Example 7. The change in temperature and plasticization effect of TBM as a solvent can contribute to changes in crystallization, which can disrupt bonding interactions between TBM and EC in the matrix, contributing to increased free TBM in the matrix which can diffuse into the housing. Further, the solid state of the housing itself may be changed to high crystallinity by annealing using dry heat or use of solvent. Such changes in crystallinity may also be used to modulate the permeation or diffusion coefficients. For instance, the release of TBM from a bolus with a housing which had been crystallized (as measured by DSC) by exposure to high temperature (~80 °C) over a few hours showed lowered release of TBM over a week compared to a bolus with a housing which had not been treated with heat, and thus had more amorphous content.

Example 8

The effect of housing thickness on release profile was investigated using three different housing thicknesses - 0.90 mm, 1 .2 mm, and 1 .5 mm. The housing composition was the same 90% polylactic acid (PLA) and 10% polybutylene adipate terephthalate (PBAT) blend described in Example 7.

Preparation of the bolus

The boluses were assembled by loading them with matrix (bolus 2 matrix - Table 2) and the densifier matrix and then capping them as described in Example 7.

Release testing

Each bolus was placed in 1 L 0.02 M phosphate buffer as described in Example 7 and the amount of TBM released daily from the bolus was quantified.

Result

The bolus with 0.9 mm housing thickness started to significantly release from day 10 (Figure 3). The bolus with 1.2 mm housing thickness started to significantly release from day 29 and the bolus with 1 .2 mm housing thickness started to significantly release from day 35. The release rate was greater for the thinner housing. This suggests an inverse relationship between housing thickness and release rate and a direct relationship between housing thickness and TBM release lag period. Here, lag period refers to the time during which the bolus does not significantly release TBM.

Example 9

The influence of buffer (water) or TBM on housing material was tested. Bolus housings were the same as described in Example 7. A housing was kept along with a bolus in 1 L buffer solution at 40 °C. The change in volatile content and glass transition temperature of the housing was evaluated at the end of 25 days. The volatile content was analysed by heating ~5 mg (accurately measured) of the sample in thermogravometric analyser at 20 °C/min from room temperature to 200 °C. The weight loss during the process was taken as volatile content and expressed in percentage relative to the original mass.

The glass transition of the materials was evaluated during modulated differential scanning calorimetry (MDSC). Briefly, approximately 5 mg (accurately measured) of the samples was loading into Tzero aluminum pans and heated from 0 to 195 °C at 1 °C per min heating rate with an amplitude of ±0.16 and modulation period of 60 s. The glass transition temperature was taken from the reversing heat flow curve and analysed using the TRIOS software from TA instruments.

Results of the testing is shown in Table 3. Exposure of PLA/PBAT to water/ bromoform increases the total volatile content of the housing and reduces its glass transition temperature. The data suggests that the housing material has the capacity to hold solvents in it and likely itself acts as a reservoir of bromoform and modulates release. This also suggests that presence of bromoform and water plasticizes the housing. Plasticization likely promotes further release of bromoform with time.

Table 3. Influence of buffer (water) or TBM on housing material. TBM = Tribromomethane.

Example 10

Surprisingly, TBM formed organogels with lecithin (refined, acetone insoluble-98%, residual water <1 %) and polymethyl methacrylate (PMMA) (molecular weight- 450-550 kDa). With lecithin, gel was preferably formed with a TBM concentration of >40 % ^w/_w. Here, the gel could be formed even at lower concentration by employing water. Water potentially contributes to increased hydrogen bonding in the matrix and stabilizes the 3D gel structure. With PMMA, gel was preferably formed with a TBM concentration of >10% w/w.

Different boluses were prepared by loading gels of lecithin and PMMA with different TBM concentrations and their release profiles were evaluated.

Preparation of the bolus

Lecithin/TBM gels were prepared by adding TBM to lecithin in a beaker and mixing. Lecithin may dissolve and form a yellowish translucent viscous mass in the beginning of mixing which will begin to gel later. One skilled in the art may appreciate that heat may be employed. Three different gels with different bromoform loadings- 45, 60, and 70% were prepared as outlined in Table 4.

PMMA/TBM gels were prepared by adding TBM to PMMA in a beaker. One skilled in the art may appreciate that heat may be employed. The mixture may dissolve and form translucent viscous mass which changes to gel on long standing. Two different gels with 45 and 70% TBM loading were prepared (Table 4).

Table 4. Composition of the carrier/matrix in each bolus. TBM = Tribromomethane; PMMA = Polymethyl Methacrylate.

Bolus housings were the same as described in Example 7. The carrier matrix was loaded into the housing along with densifier and sealed as described in Example 7. The bolus with lecithin-based gel was loaded in slightly lower quantity due to its lower density and its tendency to occupy more space. One skilled in art would appreciate that the larger surface area for diffusion can affect the mass diffusing out of the bolus. Here, an attempt was made to minimise variation among boluses in terms of surface area contacted by the TBM-loaded matrix inside the housing.

Release testing

As described in Example 7, each bolus was placed in 1 L 0.02 M phosphate buffer and the amount of TBM released daily from the bolus was quantified.

Result

For the boluses with lecithin-based gel (Figure 4), release rate was directly proportional to the TBM loading in the gel (Figure 5). However, the release profile was first-order for gels loaded with 60% or 70% TBM loading. A near zero-order release profile was observed for gel loaded with 45% TBM. Such behaviour was observed over a long period of over 4 months (Figure 6).

Similarly, PMMA gel (Figure 7) also exhibited TBM concentration dependent release (Figure 8). It was interesting to observe that PMMA gel with 45% TBM loading released minimum TBM (<10 mg/day) over 3 months (Figure 9) suggesting PMMA could be an excellent packing material for TBM loaded boluses. Nevertheless, PMMA can be used as a stabilizer to modulate the release from other matrices, such as a stearic acid based matrix, which offer limited control over release in the absence of stabilizer as discussed later.

Example 1 1

Boluses with waxes of different melting points (MP) loaded with TBM were also prepared. Microcrystalline wax (MCW) supplier-Alchemy agency, Specific density-0.92 at 20 °C, melting point 80 °C) with a melting of ~80 °C, stearic acid (purity- 95%) with a melting point of ~70 °C, and eicosane (purity >95%) with a relatively low melting of ~40 °C were used. For the eicosane system, the aim was primarily to achieve temperature dependent release.

Preparation of the boluses Different boluses with wax/TBM matrix as described in Table 5 were prepared. Briefly, for MCW systems, the wax was first melted and maintained at ~100 °C, to this pre-melt TBM (kept at room temperature) was added which lowered the temperature to ~70 °C. Here, caution was undertaken to pour the mixture in clear liquid state into the housing body, before any solidification to avoid non-uniformity in the matrix loading, particularly as multiple boluses were prepared from the same melt. One skilled in the art would appreciate that the pouring temperature or agitation/precipitation prior to pouring of such systems can affect their release profile by affecting their solid state. Stearic acid was melted and maintained at ~90 °C to which TBM was added. Care was taken to pour in clear liquid state at discussed earlier for MCW system. Eicosane was melted and maintained at ~60 °C to which TBM was added. Here the mixture was also above 40 °C and in clear liquid state when poured into the housing. Bolus housings were the same as described in Example 7. In all cases the wax/TBM systems were allowed to solidify before adding the densifier and sealing them with a cap as described in Example 7.

Table 5. Composition of the carrier/matrix in each bolus. TBM = Tribromomethane; MCW = Microcrystalline Wax.

Release testing

As described in Example 7, each bolus was placed in 1 L 0.02 M phosphate buffer and the amount of TBM released daily from the bolus was quantified. Unique to boluses with eicosane, the boluses were kept at room temperature for ~2 weeks and then moved to 40 °C incubator. The release of TBM from the bolus during this time including the change in release pattern through the transition was recorded.

Result Although both MCW systems displayed release, it was observed that a TBM loading of <70% provided more desirable control over the release of TBM (Figure 10). The MCW system with 65% TBM loading showed a consistent release of -150 mg per day over 80 days (Figure 11). Further it was observed that the release could also be modulated based on the mass loading of MCW/TBM for the MCW-35% ^w/_w/TBM-65% ^w/_w system in the boluses (Figure 12). This is a desirable feature of such systems. Here, boluses can be designed to deliver the desired dose to animals based on their weights by simply changing the loading of the matrix in the boluses.

For stearic acid system, the release rate was much higher than for the same loadings in MCW systems, the release being in the range of -500 mg/day (Figure 13). A greater release rate may be desirable in some applications. However, at 65% TBM loading, the stearic acid system did not offer as much control over release as the MCW system (Figures 10 and 13).

For the eicosane system (Figure 14), it was observed that no significant TBM release occurred at room temperature, however, extensive release occurred when the bolus was heated to 40 °C. Without wishing to be bound by theory, it is thought that melting of the carrier was responsible for the temperature dependent release. One skilled in the art will appreciate that such systems can be advantageous as they would not release significant amounts of active during the storage but only release significant amounts of active once administered to the animal. Further, the release from such system can be modulated by including an excipient such as EC, lecithin, MCW or SAIB which can further control release of active agent, such as TBM.

It is also interesting to note that release of TBM from the wax system with the same TBM loading increased as the melting point of the carrier wax decreased. At 65% TBM loading into the wax system, MCW (MP = -80 °C) offered better control in release over stearic acid (MP = -70 °C) which in turn offered better control over release from eicosane (MP = -80 °C) as carrier.

Example 12

Housing free boluses were also prepared using MCW as carrier.

Preparation of the boluses Bolus housings (when used) were the same as described in Example 7. Here, MCW matrices loaded with 65% or 45% TBM loading were prepared and either loaded into a bolus or a housing free bolus was prepared (Table 6). Briefly, boluses with the housing were prepared as described in Example 1 1. For the housing free bolus, a 3D printed mould was used. Alternatively, the housing may be cut out and removed once the matrix solidifies. Densifier was not included in these tests as the boluses were dense enough to sink in water. However, the skilled person would be readily able to further include densifier if required in equivalent boluses for administration to animals.

Table 6. Composition of the carrier/matrix in each bolus. TBM = Tribromomethane; MCW = Microcrystalline Wax.

Release testing

As described in Example 7, each bolus was placed in 1 L 0.02 M phosphate buffer and the amount of TBM released daily from the bolus was quantified. All the housing free boluses also sank in the dissolution medium, potentially due to the high density of TBM which form a more significant proportion of the bolus in the absence of housing.

Result

Boluses without housing appeared to release TBM at around 2-3 the rate of equivalent boluses with housing (Figures 15 and 16). Despite this, housing-free boluses were stable in the dissolution medium and did not break during the trial suggesting that they had satisfactory mechanical properties, at least when MCW was employed. Based on this result, at least other waxes with a higher melt point than MCW may also be employed in housing-free boluses. Housing-free boluses can offer advantages in using reduced materials in a potentially simpler method of production. Housing-free boluses may also be advantageous when high release rates are desired. Housing may be advantageous when greater modulation of release rate is required.

Example 13

The inventors have observed that different excipients/carriers offer different levels of control over the release of TBM. Particularly, EC, lecithin, SAIB and MCW have been observed to offer significant control over release of TBM; an attribute that is desirable in extended release formulations. Thus, EC, lecithin, SAIB, and MCW act as stabilizing agents for the release of TBM. Here, lecithin was used as an example of a stabilizing agent for system to improve control over the release of TBM over a stearic acid system.

Preparation of the boluses

Matrices with stearic acid loaded with 65% TBM loaded were prepared with or without 5% lecithin (Table 7). Inclusion of lecithin was compensated by a decrease in stearic acid concentration. Bolus housings were the same as described in Example 7. Here, the boluses were assembled as described in Example 1 1 . In case of matrix with lecithin, lecithin was first dissolved in TBM and then the solution was added to molten stearic acid.

Table 7. Composition of the carrier/matrix in each bolus. TBM = Tribromomethane.

Release testing

Result

It was observed that inclusion of only 5% lecithin was able to stabilize the release profile of stearic acid system loaded with 65% TBM (Figure 17). This suggests similar control can be achieved during other stabilising agent excipients such as SAIB, EC, Lecithin, MCW, etc.

Example 14

Boluses may contain more than one active ingredient. Inclusion of more than one active agent offers an advantage at least in reduced applications of active agents. Further, they potentially may have an advantage from a formulation perspective. Here, boluses were prepared which included further active agents - monensin (sodium salt, purity >90%), phloroglucinol (dihydarate, purity >98%), ketoprofen (purity >98%), and albendazole (purity >98%). Monensin has been known to exhibit antimethanogenic activity (Cooke RF et al. (2024) Transl Anim Sci, Mar 9;8:txae032). Phloroglucinol is a hydrogen sequester that would potentially help redirect excess hydrogen to acetate in rumen where antimethanogenic actives like haloforms have inhibited methanogenesis (which used hydrogen) and also known to reduce methane production (Sarwono KA et al. (2019) Tropical Animal Science Journal, 42(2):121 -127). Ketoprofen is a common anti-inflammatory used in cattle. Albendazole is a common anthelminthic used in cattle.

Preparation of the boluses

EC/HPMC/TBM matrices loaded with 60% TBM with or without the inclusion of 2 or 5% monensin, phloroglucinol, albendazole and ketoconazole were prepared as detailed in Table 8. The inclusion of the further active agents in the formulation was compensated for by the decrease in HPMC. Here, the ratio of EC and TBM was kept constant as EC has been established to provide stability to release profile. These actives were first added to TBM. The matrices and housings were prepared as described in Example 7.

Table 8. Composition of the carrier/matrix in each bolus. TBM = Tribromomethane; EC = Ethyl Cellulose, and HPMC = Hydroxypropyl Methyl Cellulose.

Release testing

Result

Inclusion of additional active agents generated only minor changes in release profile (Figure 18 to Figure 21).

Example 15 The influence of casing crystallinity on release rate was assessed.

Preparation of the boluses

Bolus housings were the same as described in Example 7, with the exception that bolus housing with high crystalline PLA content and high amorphous crystalline content were prepared. The skilled person would be aware of techniques to influence crystalline polymer content, for instance, annealing. Here, MCW matrices loaded with 65% TBM to a total mass of 60 g loading were prepared as described in Example 1 1 .

Release testing As described in Example 7, each bolus was placed in 1 L 0.02 M phosphate buffer (pH = 6.5) and the amount of TBM released daily from the bolus was quantified.

Result

Boluses with high crystalline PLA content exhibited a greater release rate than boluses with low crystalline PLA content. This suggests that the crystallinity of the casing influences release rate.

Statements of the invention

1 . A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which surrounds the core, wherein the housing is configured such that its permeability to said methane inhibiting agent is increased when exposing the housing to the rumen of a living animal; and/or wherein at least part of the housing is configured to form one or more openings when exposing the housing to the rumen of a living animal, allowing an increased release of the methane inhibiting agent from within the bolus through said opening or openings.

2. The bolus according to statement 1 , wherein the housing comprises one or more openings and wherein said openings are filled with and/or covered with a material that melts, dissolves or disintegrates in the rumen of a living animal.

3. The bolus according to statement 1 , wherein a part of the housing, and preferably the portion of the housing, where the opening or openings form, is made of a material that melts, dissolves or disintegrates in the rumen of a living animal.

4. The bolus of statements 2 or 3, wherein said material that melts is a compound selected from the group consisting of a hydrogel, an oleogel, an organogel, a phase changing material (PCM), a fatty acid, an alkane, an alkene, a gelator, a wax, an L-alanine amino acid, an L-alanine amino acid derivative, poly(methyl methacrylate) (PMMA), (1 ,3:2, 4) dibenzylidene sorbitol (DBS), hydroxy stearic acid, paraffin wax, gelatin, 1 - tetradecanol, polyethylene glycol, octadecane, nonadecane, eicosane, a pluronic polymer or a mixture of pluronics, an emulsifier, sucrose acetate isobutyrate (SAIB), derivatives of the aforementioned and combinations of one or more of the aforementioned compounds and wherein said compound or combination of compounds has a melting temperature of between 28°C and 42°C, more preferably of between 28°C and 35°C.

5. The bolus of statements 2 or 3, wherein said material that dissolves is a water- soluble material preferably selected from the group consisting of a polymer, a polyol, a sugar, a polyamide, a salt, and cellulose acetate.

6. The bolus of statements 2 or 3, wherein said material that disintegrates is a compound selected from the group consisting of cellulose, polyhydroxyalkanoate (PHA), poly(butylene succinate-co-adipate) (PBSA) and a mixture of two or more of the aforementioned.

7. The bolus according to any one of statements 1 to 6, wherein the largest opening that forms in the housing when exposing the housing to the rumen of a living animal has a diameter of maximally 2 mm.

8. The bolus according to any one of statements 1 to 7, wherein the methane inhibiting agent is selected from a haloform.

9. A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent in microencapsulated particles and wherein the microencapsulated particles are dispersed in a composition comprising a carrier and optionally also comprising a dispersing agent; and a housing covering at least part of the core.

10. The bolus according to statement 9, wherein the microencapsulated particles are producible by microencapsulation of said particles in at least one encapsulating agent, preferably wherein the encapsulating agent is selected from the group consisting of a polymer, a surface-active agent, an emulsifier, a gelatin-sorbitol mixture, gelatin-starch syrup and mixtures thereof, more preferably wherein the encapsulating agent is selected from the group consisting of PLA, PBSA, PBS, PHBV, PVA, PBAT, PCL, PDLA, gelatin- sorbitol mixture, gelatin-starch syrup and a mixture comprising two or more of any of the aforementioned compounds.

1 1. A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a carrier and microencapsulated particles, wherein the microencapsulated particles comprise the methane inhibiting agent and wherein the microencapsulated particles are dispersed in the carrier, preferably wherein the carrier has a porous structure such as mesoporous silica.

12. The bolus according to any one of statements 9 or 1 1 , wherein the microencapsulated particles are microencapsulated using a compound selected from the group consisting of a hydrophilic material, gelatin, zein, methyl cellulose and poly(N- isopropylacrylamide) (PNIPAM) microgel, starch, cyclodextrins and a combination of two or more of the aforementioned compounds.

13. The bolus according to any one of statements 9 to 12, wherein the carrier comprises a compound selected from the group consisting of silica, cellulose and activated carbon, gelatin, chitosan, poly(lactic-co-glycolic acid) (PLGA), cyclodextrins, collagen, poly alpha-hydroxy esters, hydroxy alkanoates and dioxanes, starch, gluten, zein, polyethylene, polypropylene, polyamide, polyethylene terephthalate and ethylenevinyl acetate.

14. The bolus according to any one of statements 9 to 13, wherein the microencapsulated particles have an average diameter of 50 nm to 2 mm, preferably an average diameter of 1 pm to 1000 pm.

15. The bolus according to any one of statements 9 to 14, wherein the bolus is configured to release the methane inhibiting agent over a period of at least 3 months.

16. A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises a core, wherein the core comprises the methane inhibiting agent and a carrier, and wherein the bolus does not comprise a housing.

17. The bolus according to statement 16, wherein the core comprises a compound selected from the group consisting of PLA, PBSA, PBS, PHBV, PVA, PBAT, PCL, PDLA, epoxy-based chain extenders, magnesium silicate, cellulosic materials, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), fumed silica, gelatin, wax, castor wax, paraffin wax, silica and combinations thereof.

18. The bolus according to any one of statements 16 or 17, wherein the bolus has a Shore D hardness of at least 20.

19. The bolus according to any one of statements 16 to 18, wherein the bolus is in the form of a pellet, pill, lozenge or tablet.

20. The bolus according to any one of statements 16 to 19, wherein the bolus is configured to dissolve in the rumen of a ruminant animal over a period of time of less than 48 h. 21. The bolus according to any of the preceding statements, wherein the bolus comprises a hydrogen sequester, preferably selected from the group of hydrogensequesters consisting of fumaric acid, sodium fumarate, a phenolic compound, phloroglucinol, gallic acid, resorcinol, catechols, hydroquinone and pyrogallol.

22. The bolus according to any of the preceding statements, wherein the methane inhibiting agent is selected from the group consisting of bromoform, monensin, lecithin, lauric acid, myristic acid, linoleic acid and phospholipid, preferably wherein the phospholipid comprises one or more polyunsaturated fatty acids, more preferably wherein the phospholipid comprises one or more fatty acids selected from 18-carbon polyunsaturated fatty acids (C18 PUFAs), palmitic acid, stearic acid and oleic acid.

23. A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent and a carrier; and a housing which comprises said core; wherein the methane inhibiting agent is selected from the group consisting of monensin, lauric acid, myristic acid and linoleic acid; and wherein the housing comprises at least one opening exposing the core to the environment surrounding the bolus.

24. The bolus according to any one of the preceding statements, wherein said bolus comprises a housing, wherein said housing comprises a stabilizer, preferably a stabilizer selected from the group consisting of a surfactant, a plasticizer, a phthalate ester and a triglyceride, more preferably wherein the stabilizer is selected from the group consisting of lecithin, nitrile and triacetin.

25. The bolus according to any one of the preceding statements, wherein said core of the bolus comprises at least one filling agent, preferably wherein the at least one filling agent is a stabilizer, preferably wherein the at least one filling agent is selected from the group consisting of gelatin, milk, milk derivatives, infant formula, milk powder, triglycerides, medium chain triglycerides and oil thereof, ethanol, lecithin, tween, xanthum gum, cellulose derivatives, alkyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), zein and surfactant.

26. A bolus as defined in any one of the preceding statements, wherein in addition or instead of said methane inhibiting agent the bolus comprises an active agent, wherein said active agent is selected from the group consisting of an anti-inflammatory agent, an analgesic, an antibiotic and an anthelmintic; more preferably wherein the active agent is selected from the group consisting of meloxicam, ketoprofen, penicillin, a tetracycline, a macrolide, monensin, ceftiofur, florfenicol, tilmicosin, enrofloxacin, tulathromycin and an albendazole.

27. The bolus according to any one of statements 1 to 26 for use in the treatment of a ruminant animal.

28. The bolus according to any one of statements 1 to 26 for use in reducing methane emission in a ruminant animal.

29. Method of treating an animal comprising administering to said animal a bolus as defined in any one of the preceding claims; wherein said animal is preferably a ruminant animal such as cattle.

30. A method of manufacturing a bolus according to any one of statements 1 to 28 comprising the steps of

(a) providing a housing;

(b) creating in the wall of said housing multiple openings, where each opening has a diameter of maximally 3 mm;

(c) closing said openings of said housing with said material that melts, dissolves and/or disintegrates in the rumen of a living animal; and

(d) filling said housing with said inhibiting agent.

31. A methane inhibitor for use in reducing methane emission from a ruminant animal, wherein the methane inhibitor is selected from the group consisting of bromoform, monensin, lecithin, lauric acid, myristic acid, linoleic acid and phospholipid, preferably wherein the phospholipid comprises one or more polyunsaturated fatty acids, more preferably wherein the phospholipid comprises one or more fatty acids selected from 18- carbon polyunsaturated fatty acids (C18 PUFAs), palmitic acid, stearic acid and oleic acid.

32. Method of treating a ruminant animal to reduce methane emission from said ruminant animal comprising administering to said animal a methane inhibitor selected from the group consisting of bromoform, monensin, lecithin, lauric acid, myristic acid, linoleic acid and phospholipid, preferably wherein the phospholipid comprises one or more polyunsaturated fatty acids, more preferably wherein the phospholipid comprises one or more fatty acids selected from 18-carbon polyunsaturated fatty acids (C18 PUFAs), palmitic acid, stearic acid and oleic acid.

Claims

1 . A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core; a housing that covers at least a portion of the core; wherein the core includes at least one methane inhibiting agent and at least one further active agent.

2. The bolus of claim 1 , wherein the further active agent is selected from the group consisting of a methane inhibiting agent, a hydrogen sequester, an anti-inflammatory agent, an analgesic, an anthelmintic, a nonsteroidal anti-inflammatory drug (NSAID), an antibiotic, a growth promoter, a lactation promoter, a sustainability improver, an antimicrobial, a ketosis prevention agent and combinations thereof.

3. The bolus of claim 1 or 2, wherein the further active agent is selected from the group consisting of monensin, phloroglucinol, albendazole, ketoconazole, lecithin and combinations thereof.

4. The bolus of any one of the preceding claims, wherein the further active agent is monensin.

5. A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent; and a housing which covers at least a portion of the core, wherein the housing is configured such that its permeability to said methane inhibiting agent is increased by at least 50 % when exposing the housing to phosphate buffer (pH: 6.5, 0.02 M) at 40 °C without agitation, relative to the permeability on the same day after exposure of the housing to phosphate buffer (pH: 6.5, 0.02 M) at a reference temperature without agitation for the same length of time, wherein permeability is assessed based on release rate of the methane inhibiting agent on the fourteenth day; and wherein the reference temperature is 20 °C..

6. The bolus of claim 5, wherein the reference temperature is 25 °C.

7. A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core comprising the methane inhibiting agent dispersed in or forming part of one or more of a hydrogel, oleogel or organogel; and a housing which covers at least a portion of the core.

8. The bolus of claim 7, wherein the core comprises a phospholipid and/or an acrylate-based polymer.

9. The bolus of claim 7 or 8, wherein the core comprises lecithin and/or an poly(methyl methacrylate) (PMMA).

10. The bolus of any one of the preceding claims, wherein the housing surrounds the core.

1 1. The bolus of any one of the preceding claims, wherein the housing comprises one or more polymers selected from the list consisting of high-density polyethylene (HDPE), polypropylene (PP), polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polylactic acid (PLA), poly-D,L-lactic acid (PDLLA), polybutylene adipate terephthalate (PBAT), styrene-acrylic copolymer (such as Joncryl®), talc-filled poly(D-lactide) (TALC PDLA), Poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), combinations thereof, and copolymers thereof.

12. The bolus of any one of the preceding claims, wherein the housing comprises one or more biodegradable polymers.

13. The bolus of any one of the preceding claims, wherein the housing comprises one or more polymers selected from the list consisting of polylactic acid, polybutylene adipate terephthalate, combinations thereof, and co-polymers thereof.

14. The bolus of any one of claims 1 to 13, wherein the housing fully encases the core.

15. The bolus of any one of claims 1 to 14, wherein the housing is 5 to 100% or 10 to 100% PLA w/w.

16. The bolus of any one of claims 1 to 15, wherein the housing is 20 to 100% or 30 to 90% PBAT w/w.

17. The bolus of any one of claims 1 to 16, wherein the housing has a material thickness of below about 2 mm, preferably a material thickness in the range of about 0.3- 1 .8 mm, and more preferably a material thickness in the range of about 0.3-1 .5 mm.

18. The bolus of any one of claims 1 to 17, wherein the core and housing have a ratio of about 3 to about 6 : 1 , about 4 to about 5 : 1 or about 4.6 : 1 by weight.

19. The bolus of any one of claims 1 to 18, wherein the bolus has a Shore D hardness of at least 20.

20. A bolus for administration to a ruminant animal, wherein said bolus is configured to release a methane inhibiting agent in the animal, wherein said bolus comprises: a core, wherein the core comprises the methane inhibiting agent and a carrier, and wherein the bolus does not comprise a housing.

21 . The bolus of any one of the preceding claims, wherein the core comprises one or more materials selected from the group consisting of polycaprolactone (PCL), polybutylene succinate (PBS), polybutylene succinate-co-adipate (PBSA), polylactic acid (PLA), poly-lactic acid, poly-d-lactic acid, poly-L-lactic acid , poly-D,L-lactic acid (PDLLA), poly-lactide-co-glycolide, lignin, polybutylene adipate terephthalate (PBAT), styrene- acrylic copolymer (such as Joncryl®), talc-filled poly(D-lactide) (TALC PDLA), Poly(3- hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), polyvinyl alcohol (PVA), epoxy-based chain extenders, magnesium silicate, cellulosic materials, ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), fumed silica, gelatin, wax, castor wax, paraffin wax, silica, hydrophilic silica, microcrystalline wax, methyl cellulose, starch, polyethylene glycol, polyvinyl alcohol, hydroxyethyl cellulose, carboxymethyl cellulose, soluplus, Poly(acrylic acid) , poly(vinylpyrrolidone) , poly(vinyl alcohol) , poly(acrylamide) , poly(2-hydroxypropyl methacrylamide), poly(N,N-dimethylacrylamide) , poly([2-

(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide), poly(2- (methacryloyloxy)ethyl phosphorylcholine) , poly(carboxybetaine methacrylamide) , polyethylene glycol), polyethylene imine) , poly(sarcosine), poly(2-methyl-2-oxazoline), polyamino esters, polyester amides, polyphosphoesters, poly(l-lysine) , poly(l-proline) , polyphosphazenes, dextran , sodium alginate, gelatin, agarose, carrageenan, gellan, xantham gum, urea, sucrose, beeswax, polyethylene glycol (PEG), sodium starch glycolate, croscarmellose sodium, crospovidone, carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), carrageenan, guar gum, xanthan gum, sodium alginate, locust bean gum, polyvinylpyrrolidone (PVP), polyvinylpyrrolidone-vinyl acetate copolymer (PVP/VA), a polyacrylic acid and/or their co-polymer variants, polyisobutylene, ethyl vinyl acetate (EVA), a functional wax with a melting point less than about 120 °C, derivatives thereof, combinations thereof, and co-polymers thereof.

22. The bolus of any one of the preceding claims, wherein the core comprises one or more materials selected from the group consisting of ethyl cellulose, hydroxypropyl methyl cellulose (HPMC), combinations thereof, and co-polymers thereof.

23. The bolus of any one of claims 1 -22, wherein the core comprises microcrystalline wax.

24. The bolus of any one of the preceding claims, wherein the methane inhibiting agent is a haloform, preferably bromoform.

25. The bolus of claim 24, wherein the haloform, preferably bromoform, is comprised in the core of the bolus of the disclosure in an amount of between 10 wt% to 80 wt% and preferably in an amount of between 15 wt% and 70 wt%.

26. The bolus of claim 25, wherein the bromoform is not a seaweed extract enriched in bromoform.

27. The bolus of any one of the preceding claims, wherein the bolus further comprises a densifier.

28. The bolus of any one of the preceding claims, wherein the bolus comprises the methane inhibitor bromoform and is adapted to reach a maximum release rate of approximately 0.1 - approximately 0.5 g per day, and more preferably approximately 0.2 g per day.

29. The bolus of any one of the preceding claims, wherein the bolus comprises a therapeutically effective amount of a methane inhibiting agent and:

• about 5% to about 15% (w/w) housing;

• about 20 to about 55 (w/w) core; and

• about 30 to about 75 (w/w) densifier.

30. A method of administering a methane inhibiting agent to a ruminant animal, the method including administering to the rumen of the ruminant animal a bolus according to any one of the preceding claims.

31 . A method of reducing methane production in the rumen of a ruminant animal, the method including administering to the rumen of the ruminant animal a bolus according to any one of claims 1 -29.

32. A method of making a bolus, the method including: selecting a core and a housing inserting the core into the housing, optionally closing the housing to surround or substantially surround the core in the housing; wherein the core comprises a methane inhibiting agent.

33. The method of claim 32, wherein the melted core is poured into the housing and solidifies in the housing.

34. The method of claim 33, wherein the housing is enclosed around the core following solidification of the core.

35. A packaged bolus according to any one of claims 1 to 29 or a bolus prepared according to claims 32 and 33, wherein the bolus is packaged in a material comprising an acrylate-based polymer.