Method for reducing methane emissions in ruminant animals

Abstract

The present disclosure provides a non-therapeutic method for reducing the total gas production and / or methane production in a ruminant animal, comprising the step of administering an effective amount of insect frass to said ruminant animal. Another object of the present disclosure relates to food for ruminants, comprising an effective amount of insect frass, preferably derived from black soldier flies.

Description

REPLOID GROUP AG hj rt lI Patent &27090.20689WO [ intellectuaMETHOD FOR REDUCING METHANE EMISSIONS IN RUMINANTANIMALSDESCRIPTIONField of the Disclosure

[0001] The present disclosure relates to method for reducing methane emissions from ruminants while providing significant feed value to ruminants.Brief description of the related art

[0002] Methane is the second most greenhouse gas contributor to climate change following carbon dioxide. Methane’s ability to trap heat in the atmosphere is stronger than that of carbon dioxide. Ruminants produce methane as part of their digestion process. The increasing meat consumption in many developing and emerging countries is related to increased intensive animal husbandry comprising ruminants.

[0003] Because of the increasing amounts of methane output resulting from the increasing number of animals for food production, approaches are required for reducing the methane production. One approach relates to reducing the methane emission from ruminants.

[0004] Published Australian patent application AU 2011239541 Al relates to a method and system for reducing methane emissions by ruminants. The method includes providing a feed dispenser for feeding ruminants nutrient supplements, and the feed dispenser includes a gas analyzer where a ruminant places its head. The method includes determining a particular ruminant has accessed the feed dispenser such as by reading an identifier from an RFID ear tag and operating the feed dispenser to provide a ration of methane-controlling nutrient supplement. The method includes using the gas analyzer to determine levels of carbon dioxide and methane and operating a data analyzing station to determine a ratio of methane to carbon dioxide and modify the type or amount of nutrient supplement for the ruminant for a next feeding to controlREPLOID GROUP AG I F Poaterntt &m Recahtsannwndlte Tegethoff27090.20689WO [ intellectual Property Attorneys methane production or achieve an animal production goal, such as by operating a hopper with supplement compartments. The unit can be monitored remotely and controlled through an Internet connection.

[0005] Published International patent application WO 2015 / 109362 A2 provides a method for reducing total gas production and / or methane production in a ruminant animal comprising the step of administering to said ruminant animal an effective amount of at least one species of red marine macroalgae.

[0006] The publication of Seankamsorn et al. (Seankamsom, et al. (2021). Veterinary World, 14(5), 1158) discloses that the combination of crude glycerin at 21% with chitosan at 2% in the total mixed ration for 2-year-old native Thai bulls reduced CPU estimation by 5.08% compared with the other feed treatments.

[0007] The study of Harahap et al. (Harahap et al., Tropical Animal Science Journal 43.3 (2020): 233-239) aimed to perform a meta-analysis from published studies regarding the effects of chitosan on methane emission and rumen fermentation profile of in vitro batch culture experiments. A total of 41 studies from 12 articles were integrated into a database. Parameters included were gas production, methane emission, rumen fermentation characteristics, microbial population, nutrient digestibility, and fatty acid profile. Data were analyzed according to mixed model methodology in which different studies were treated as random effects and chitosan addition levels were treated as fixed effects. Results showed that chitosan addition was able to reduce enteric methane emissions (p<0.001). Such methane decrease was accompanied by a decline in the protozoa population (p<0.05) and a tendency of methanogen reduction (p<0.1). The increasing chitosan level was associated with a decrease in total VFA and ammonia concentrations (both at p<0.001). Chitosan addition decreased acetate proportion (p<0.001) while elevated propionate proportion (p<0.001). Chitosan was associated with an increase of dry matter digestibility, crude protein digestibility, and neutral detergent fiber digestibility (p<0.001). Chitosan increased concentrations of C18:3n3 (p<0.05), conjugated linoleic acid (p<0.01) and polyunsaturated fatty acids (p<0.01) while decreased concentration of saturated fatty acids (p<0.001). It can be concluded that chitosan addition can mitigate enteric methane emission and alters rumen fermentation profiles in a favorable direction.REPLOID GROUP AG hj rt lI Patent &27090.20689WO [ intellectua

[0008] The publication of Wasson et al (Wasson et al. (2022), Frontiers in Animal Science, 3, 999338) investigated Macroalgae supplementation as a promising tool to mitigate enteric methane emission in ruminants. The mode of action responsible for the mitigation effect centers around the content of volatile halogenated compounds, primarily bromoform. The sub-tropical red seaweed, Asparagopsis taxiformis, is the most well researched bromoform containing species. While several studies, both in vitro and in vivo, have demonstrated the effectiveness of A. taxiformis at reducing enteric methane emission (> 80% reduction), questions surrounding sustainability, animal productivity, animal product quality, and commercial practicality remain. These questions by no means disqualify the practice of feeding macroalgae to cattle to reduce methane emission, but they must be answered before implementing macroalgae as a feed additive commercially. Also, limiting scientific inquiry to a few species reduces the potential of discovering other compounds and modes of action that could produce the desired mitigation effect without the inherit drawbacks of the current options. Work conducted in both ruminant nutrition and human health fields have identified numerous bioactive compounds within plants that exhibit anti-microbial functions that could modify the rumen microbiome for beneficial outcomes. These compounds are also found in macroalgae. Phlorotannins, saponins, sulfonated glycans, other halocarbons and bacteriocins found within macroalgae have demonstrated antimicrobial activity in vitro. However, it is unclear what effect these compounds may have when used in vivo. Once identified, extracting these compounds for supplementation in lieu of feeding the entire plant may be a more practical solution. Dietary inclusion levels of macroalgae in ruminant diets can be limited by variation in active ingredient concentration, palatability to cattle, and excessive dietary mineral content. There are multiple in vitro studies that have demonstrated a methane reduction potential of non-bromoform containing species, but inclusion levels are often well above the effective levels of A. taxiformis (< 0.5% of dietary dry matter). In some animal studies, A. taxiformis supplementation has led to decreased dry matter intake and productivity and elevated mineral accumulation, such as iodine, in animal products. Therefore, methane mitigation by macroalgae will likely have to occur at low dietary concentrations to be practical.

[0009] The publication of Guyader et al. (Guyader et al., Journal of animal science 93.11 (2015): 5367-5377) showed that nitrate but not tea saponin feed additives decreased enteric methane emissions in nonlactating cows.REPLOID GROUP AG I F Poaterntt &m Recahtsannwndlte Tegethoff27090.20689WO [ intellectual Property Attorneys

[0010] The publication of Renna eta 1. (Journal of Animal Science and Biotechnology, 13: 138, 2022) investigated eight full-fat insect meals for ruminants as a lipid source for the ruminants.

[0011] The paper of Jayanegara et al. (J. Indonesian Trop. Anim. Agric., 42(4):247-254, 2017) evaluated the chemical composition, in vitro rumen fermentation, digestibility and methane emissions of some insects, i.e. Jamaican field cricket (JFC), mealworm (MW) and black soldier fly larvae age 1 and 2 weeks (BSF1 and BSF2). The provided results revealed that all insect meals contained high crude protein, i.e. above 40% DM. Proportions of neutral detergent insoluble CP (NDICP) and neutral detergent insoluble CP (ADICP) were high in the insect meals than that of soybean meal (SBM), and these were particularly very high in BSF2. All insect meals had lower IVDMD and IVOMD than that of SBM (P<0.05). All insect meals had lower methane emissions as compared to SBM at 12, 24 and 48 h (P<0.05).

[0012] Published International application WO 2024 / 115560 Al relates to a composition for animal feed comprising a fermented organic material, black soldier fly biomass, fermented black soldier fly frass and microbial biomass. Methods of producing the composition are also provided. The composition can be produced from food waste, and provides an environmentally- friendly alternative to existing protein sources for farmed animals, particularly fishmeal.

[0013] The publication of Renna et al. (J Animal Sci Biotechnol 13, 138, 2022) investigated the in vitro rumen fermentation characteristics and lipid biohydrogenation of eight full-fat insect meals (some of them being never tested before for ruminant or even monogastric animals), as potential protein sources for ruminant nutrition, comparing them to three different plant-based meals commonly used as protein source in ruminant nutrition and with fishmeal as animal-based reference protein source.

[0014] Disadvantages from solutions known from the state of the art relate to the fact that chitin and chitosan, or shrimp, fungi and algae only provide low feed value to ruminants. Chitin is currently only available in significant amounts from limited marine resources, low amounts from fungi, insects and algae. Pure chitin has to be extracted by expensive and non- environmentally friendly processes using chemicals or enzymes. Chitosan production requires an additional processing step from chitin with chemicals. Chitin and chitosan yields from fungi, algae and insects are low - reducing available amounts for ruminant feed - and will thus have no significant impact on methane emissions.REPLOID GROUP AG h / rt lI Patent &27090.20689WO [ intellectua

[0015] Thus, there is a need for a method reducing methane emissions from ruminants while providing significant feed value to ruminants.Summary of the Disclosure

[0016] The present disclosure provides a non-therapeutic method for reducing the total gas production and / or methane production in a ruminant animal, comprising the step of administering an effective amount of insect firass to said ruminant animal.

[0017] In an embodiment of the non-therapeutic, the insect frass is derived from black soldier fly larvae.

[0018] The non-therapeutic method comprises further in an embodiment an effective amount of insect frass which is to be administered to said ruminant animal by supplementing food intended for said ruminant animal with said effective amount of insect frass.

[0019] The non-therapeutic method comprises in an embodiment effective level of at least one selected from the group comprising insect exuviae, insect puparia, fermented insect exuviae and / or insect puparia and chitin, which are administered.

[0020] The non-therapeutic method comprises further effective levels of nutrients which are administered.

[0021] Another object of the present disclosure relates to food for ruminants, comprising an effective amount of insect frass.

[0022] The food comprises in an embodiment insect frass is derived from black soldier fly larvae.

[0023] In another embodiment, said food intended is supplemented with effective levels of at least one selected from the group comprising insect exuviae, insect puparia, fermented insect exuviae and / or insect puparia and chitin.REPLOID GROUP AG I F Poaterntt &m Recahtsannwndlte Tegethoff27090.20689WO [ intellectual Property Attorneys

[0024] The food comprises in an embodiment the use of effective levels of nutrients.

[0025] The foregoing summary is not intended to summarize each potential embodiment or every aspect of the present disclosure.Brief Summary of the Drawings

[0026] The disclosure will be described based on figures. It will be understood that the embodiments and aspects of the disclosure described in the figures are only examples and do not limit the protective scope of the claims in any way. The disclosure is defined by the claims and their equivalents. It will be understood that features of one aspect or embodiment of the disclosure can be combined with a feature of a different aspect or aspects of other embodiments of the disclosure, in which:

[0027] FIG. 1 shows the absolute methane production per fermented feed of control (no frass) versus two different firass types and frass mixed with additional chitin.

[0028] FIG. 2 shows the methane production per in vitro digested feed of control (no frass) versus two different frass types and frass mixed with additional chitin.Detailed Description of the Disclosure

[0029] The technical problem is solved by the independent claims. The dependent claims cover further specific embodiments of the disclosure.

[0030] The present disclosure relates to a non-therapeutic method for reducing the gas and / or methane production in a ruminant animal. A food that is suitable for feeding animals is another object of the present disclosure.

[0031] The term "reducing" relates to the reduction of an amount of a substance in comparison with a reference. For example, the reduction in the amount of total gas and / or methane produced by a ruminant animal or animals administered a composition comprising insect frass relative to an animal or animals not administered insect frass. The reduction can be measured in vitro withREPLOID GROUP AG I F Poaterntt &m Recahtsannwndlte Tegethoff27090.20689WO [ intellectual Property Attorneys an artificial rumen system that simulates anaerobic fermentation, or in vivo with animals confined in respiration chambers. It is within the knowledge and skill of those trained in the art to assess enteric methanogenesis by a ruminant animal.

[0032] The term 'reducing total gas production' refers to the reduction of the total amount of gas produced, for example the amount of total gas produced in the gastro-intestinal tract. The term includes the collective volume of all gases generated as a result of anaerobic fermentation, for example, in the systems described herein. Fermentation in the rumen and the gut of a ruminant gives rise to production of gas including methane. The present invention aims to reduce this process, such as to reduce the total amount of gas produced in the gastro-intestinal tract. It is within the knowledge and skill of those trained in the art to assess total gas production by a ruminant animal.

[0033] The term 'reducing methane production' refers to the reduction of methane produced in the gastro-intestinal tract. The term includes the specific volume of methane generated as a result of anaerobic fermentation, for example, in the systems described herein. Fermentation in the rumen and the gut of a ruminant gives rise to production of methane. The present invention aims to reduce this process, such as to reduce the total amount of methane produced in the gastro-intestinal tract. It is within the knowledge and skill of those trained in the art to assess methane production by a ruminant animal.

[0034] The term "effective amount", is meant a quantity of insect frass sufficient to allow improvement, e.g. reduction in the amount of methane production in comparison with a reference or control, reduction in the amount of total gas produced in comparison with a reference or control, maintenance of effective levels of desirable volatile fatty acids in comparison with a reference or control, reduction in the acetate to propionate ratio in comparison with a reference or control, maintenance of liveweight, dry matter intake and / or organic matter intake in comparison with a reference or control. Within the meaning of the present invention, the methane reductive effect can be measured in the rumen with an artificial rumen system, such as that described in T. Hano., J. Gen. Appl. Microbiol., 39, 35-45,1993 or by in vivo oral administration to ruminants.

[0035] The method according to the present disclosure is based on providing insect frass in a non-therapeutic method to ruminants. It turned out that the use of insect frass from black soldierREPLOID GROUP AG I F Poaterntt &m Recahtsannwndlte Tegethoff27090.20689WO [ intellectual Property Attorneys flies is appropriate for reducing methane and thus gas production in ruminants. The inclusion of black soldier fly larvae frass with and without the addition of chitin and / or chitosan from chitinous black soldier fly larvae byproducts is also intended for reducing methane emissions in ruminants. Finally, the inclusion of black soldier fly frass with lipids for reducing methane emissions in ruminants is also intended.

[0036] It is within the scope of the present disclosure that the insect frass is supplemented with effective levels of at least one selected from the group comprising insect exuviae, insect puparia, and chitin. Insect exuviae and puparia are the crude bark streams from the insect production, which also contain other substances in addition to chitin. The purification of insect exuviae and puparia with NaOH and HCL is time-consuming, but this may also be necessary because it increases the solubility and digestibility (especially in the last step from chitin with NaOH to chitosan).

[0037] It is an advantage that administering food to ruminants comprising insect frass reduces not only the total gas and / or methane production in ruminants but also provides a use for utilising insect frass in a method for protecting the environment from methane.

[0038] The anti-methanogenic potential of seven different side products derived from insect farming on in vitro fermentation was investigated. The method used to assess their methane (CH4) mitigation properties was the Hohenheim Gas Test (HGT), a well-established technique for in vitro fermentations under controlled laboratory conditions.

[0039] Prior to the in vitro fermentation, the control feed and frass plus 5% were freeze-dried, while frass 1 and frass 2 were fermented as fresh. All materials were analysed for their gross chemical composition. The results, along with the composition of the basal diet used in the fermentation, are presented in Table 1 below.REPLOID GROUP AG h / rt lI Patent &27090.20689WO [ intellectuaTablet : Gross nutrient composition of the insect farming side products used as feed for in vitro ruminant fermentation.

[0040] The insect-derived materials were tested as feedstuff ingredients, replacing 20% of the alfalfa hay in a basal diet. The basal diet (control), on a dry matter basis (g / kg DM), consisted of 700g alfalfa hay and 300 g concentrate meal mixture. Alfalfa hay is considered a premium feed among fiber-rich ruminant feeds. It contains more protein and important nutrients than conventional meadow hay. It is also easy to digest and supports animal health. Compared to other feeds, alfalfa offers a balanced supply of nutrients. It is therefore often used as a high- quality supplement in feed.

[0041] Table 2 summarizes diet composition of the treatments.Table 2: Summary of the diet composition per treatment. Alfalfa hay in the control was replaced by up to 20% with frass or frass and insect chitin.

[0042] FIG. 1 shows that frass materials showed a significant effect in reducing both absolute methane production (mL per 24h, -38.9% and -26.6%) and yield (per unit of fermented diet, - 38.8% and -20.6%, respectively) compared to the control diet.REPLOID GROUP AG I F Poaterntt &m Recahtsannwndlte Tegethoff27090.20689WO [ intellectual Property Attorneys

[0043] It can be taken from FIG. 2 that feed comprising firass reduces the methane production per digested feed. Thus, the present disclosure provides a novel feed for ruminants for reducing methane production.

[0044] It is to be noted that the publication of Renna et al. (J Animal Sci Biotechnol 13, 138, 2022) relates to insect meals made of larvae, subadult or adult insects from different species which is different from firass as defined above (cf. Methods: Insect and control meals). Moreover, Renna et al. mentions in the discussion with respect to methane production that the publications of Jayanegara et al. (Vet World. 2017; 10(12): 1439-46) and a lack of agreement on the effects of chitin and chitosan on methane mitigation potential is present. Thus, Renna et al. will not motivate a skilled person to regard frass and chitin or chitosan as promising compounds of a feed reducing methane production.

[0045] The foregoing description of preferred and other embodiments is not intended to limit or restrict the scope or applicability of the inventive concepts conceived of by the Applicants. It will be appreciated with the benefit of the present disclosure that features described above in accordance with any embodiment or aspect of the disclosed subject matter can be utilized, either alone or in combination, with any other described feature, in any other embodiment or aspect of the disclosed subject matter.

Claims

REPLOID GROUP AG I F Poaterntt &m Recahtsannwndlte Tegethoff27090.20689WO [ intellectual Property AttorneysCLAIMS1. A non-therapeutic method for reducing the total gas production and / or methane production in a ruminant animal, comprising the step of administering an effective amount of insect frass to said ruminant animal.

2. The non-therapeutic method of claim 1, wherein the insect frass is derived from black soldier fly larvae.

3. The non-therapeutic method of claim 1 or 2, wherein said effective amount of insect frass is to be administered to said ruminant animal by supplementing food intended for said ruminant animal with said effective amount of insect frass.

4. The non-therapeutic method of any one of claims 1 to 3, wherein effective levels of at least one selected from the group comprising insect exuviae, insect puparia, fermented insect exuviae and / or insect puparia, chitin, chitosan and / or lipids are administered.

5. The non-therapeutic method of any one of claims 1 to 4, wherein effective levels of nutrients are administered.

6. A food for ruminants, comprising an effective amount of insect frass.

7. The food of claim 6, wherein the insect frass is derived from black soldier fly larvae.

8. The food of claim 6 or 7, wherein said food intended is supplemented with effective levels of at least one selected from the group comprising insect exuviae, insect puparia, fermented insect exuviae and / or insect puparia, and chitin.

9. The food of any one of claims 6 to 8, wherein said food comprises effective levels of nutrients.

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