How to produce Asparagopsis

Abstract

Methane production in ruminants can be reduced by improving rumen fermentation efficiency through dietary supplementation with Asparagopsis taxiformis. This disclosure relates to a method for producing A. taxiformis and its use. In a specific form, this disclosure relates to a method for producing tetrasporophytes and gametophytes of A. taxiformis by environmental manipulation.

Description

Technical Field

[0001] The present disclosure relates to a method for producing Asparagopsis taxiformis. In certain forms, the present disclosure relates to a method for producing tetrasporophytes and gametophytes of A. taxiformis by environmental manipulation. Priority Documents

[0002] This application claims priority from Australian Provisional Patent Application No. 2023901004, filed on 5 April 2023, entitled "METHODS OF PRODUCING ASPARAGOPSIS", the entire contents of which are incorporated herein by reference in their entirety.

Background Art

[0003] In the report Climate Check 2013 / 2014 on greenhouse gas emissions by gas from 2000 to 2010 issued by the Intergovernmental Panel on Climate Change (IPCC), it was highlighted that methane is the second most abundant contributor to greenhouse gas (GHG) after carbon dioxide (CO2), accounting for approximately 16% of global emissions (IPCC, 2015). Importantly, methane can trap heat from the atmosphere much more effectively than other gases such as carbon dioxide, and even small amounts can have a significant impact on global warming (Howarth, 2014). Human-related activities such as energy production, waste management, industrial manufacturing, and agriculture have significantly contributed to the dramatic increase in atmospheric methane concentrations over the past few centuries (Baceninaite et al., 2022; Karakurt et al., 2012). Notably, methane production by enteric fermentation, a natural process of digestion in ruminants, accounted for 59.84% of agricultural emissions (Karakurt et al., 2012). Therefore, reducing intestinal CH4 emissions is urgently needed to mitigate global warming and climate change.

[0004] A proposed solution to reduce methane production in ruminants is to improve rumen fermentation efficiency (Karakurt et al., 2012). This involves supplementing feed to reduce the number of methanogenic archaea. Red algae in the form of feed additives have been shown to inhibit the growth of methane-producing methanogenic archaea (Abbott et al., 2020; Machado et al., 2015). Among red algae, species of the genus Asparagopsis show the greatest potential for reducing methane emissions because they are rich in bioactive bromoforms that can reduce the number of methanogenic bacteria in the rumen (Abbott et al., 2020; Baceninaite et al., 2022; Machado et al., 2016). Methanogenic bacteria rely on the Wolfe cycle for methane formation, in which carbon dioxide (CO2) is reduced to methane (CH4) using hydrogen (H2). Bromoform inhibits methyl transfer from methyl coenzyme M and methane release by competing with the substrates of the enzymes methyl coenzyme M reductase and methyl coenzyme M transferase. Recent studies have shown that adding small amounts of this red alga to livestock feed significantly reduces methane emissions without adversely affecting animal performance (Kinley et al., 2016; Machado et al., 2016a,b). According to Stefenoni et al. (2021), the inclusion of 1% dry matter of A. taxiformis in a growth culture medium reduced methane levels by 98% in an in vitro assay. In in vivo studies, dietary supplementation with 0.20% organic matter of A. Taxiformis reduced methane production by 98% in feedlot beef cattle without adversely affecting meat quality, and slightly improved growth rate by 42%. Bromoform levels were below detectable in the meat, fat, organs, or feces of experimental animals (Kinley et al., 2020). Stefenoni et al. (2021) reported that dairy cows supplemented with low (0.25%) or high (0.5% dry matter) levels of A. taxiformis showed methane production reduced by 65% ​​and 55%, respectively.Similarly, steers fed low (0.25%) and high (0.5%) levels of A. taxiformis for 147 days showed a 45% and 68% reduction in methane production, respectively (Roque et al., 2021). Furthermore, A. taxiformis can effectively minimize methane emissions in other ruminants such as sheep (Li et al., 2016). However, commercial aquaculture of A. taxiformis remains very immature, and little is currently known about its breeding and farming techniques.

[0005] A. taxiformis is sexual, heteromorphic, and possesses a triphasic life cycle (Bonin and Hawkes 1987; Zanolla et al., 2014). A. taxiformis grows in rocky areas and reefs in tropical and temperate regions of the Indian, Pacific, and Atlantic Oceans. The gametophyte stage is dark brown to reddish, with feathery branches reaching up to 40 cm in height, attached by rhizoids (Zanolla et al., 2014). The main stem is mainly covered with dense and irregularly branched radial lateral walls, 1-2 cm long (Zanolla et al., 2022). The carposporophyte stage is identified by the presence of cystic fruits and spermangia (Bonin and Hawkes 1987; Zanolla et al., 2014; Zanolla et al., 2022). The tetrasporophyte stage (diploid), which is a red, sphere-shaped filamentous structure, looks completely different from both the gametophyte (haploid) and carposporophyte (diploid) stages (Abbott 1999). They are often found growing attached to other algae up to a depth of 15m in the intertidal zone, or floating (Huisman et al. 2007; Zanolla et al., 2022). Recently, research has begun on each phase of the life cycle of A. taxiformis used in aquaculture.

[0006] Tetrasporophytes can be propagated vegetatively by cleaving the filaments into smaller parts and have been successfully grown in different cultivation systems (Schuenhoff et al. 2006; Mata 2008; Mata et al. 2007, 2010, 2012). Currently, tetrasporophyte starting materials are collected from the wild or induced by excising cysts to obtain carpospores and growing the carpospores into young tetrasporophytes (Mata et al., 2017; Schuenhoff et al. 2006; Paul et al., 2006). However, wild tetrasporophyte filaments contain several contaminated species and require a strong and complex washing process. Furthermore, the current method of excising cysts to release carpospores is manual, time-consuming, and labor-intensive. Furthermore, the gametophyte phase of A. taxiformis has not been successfully cultivated in experimental facilities and decomposes rapidly (Zanolla et al., 2022).

[0007] Therefore, there is a need to develop technologies that can provide commercial-scale production of Asparagopsis tetrasporophytes and gametophytes in ground cultivation or land-based aquaculture systems. [Overview of the project]

[0008] This disclosure arises from research into methods for cultivating A. taxiformis to produce tetrasporophytes and gametophytes naturally, sustainably, and commercially under controlled conditions.

[0009] According to the first aspect, a method is provided for producing at least one tetrasporophyte of A. taxiformis, the method being Obtain a carposporophyte of A. taxiformis with at least one mature cystic, To induce the release of at least one carpospore from at least one cystocarp, the carposporophyte is exposed to approximately 80–170 μmol·m over a first timed exposure period ranging from approximately 10–48 hours. -2 ·s -1The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. To induce at least one carpospore to germinate and produce at least one tetrasporophyte, at least one carpospore is exposed to a second timed exposure period ranging from approximately 10 to approximately 72 hours, at a rate of approximately 80 to approximately 170 μmol·m³. -2 ·s -1 The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. Includes.

[0010] In one embodiment, the duration of illumination during either or both of the first and second time-delayed exposure periods is in the range of about 16 to about 24 hours per 24-hour period. In another embodiment, the duration of illumination during either or both of the first and second time-delayed exposure periods is substantially continuous.

[0011] In one embodiment, the illumination intensity during either or both of the first and second time exposure periods is approximately 120 to approximately 170 μmol·m². -2 ·s -1 The range is as follows: In one embodiment, the illumination intensity during either or both of the first and second time-delay exposure periods is about 140 to about 150 μmol·m -2 ·s -1 It is within the range.

[0012] In one embodiment, the culture temperature during the first and second exposure periods is in the range of 21-25°C. In a specific embodiment, the culture temperature during the first and second exposure periods is in the range of 22-24°C.

[0013] In some embodiments, prior to the first timed exposure period, the method includes a pre-culture in which the temperature of the carposporophyte is adjusted from a first temperature (e.g., the temperature provided in a culture medium such as seawater) to the culture temperature of the first timed exposure period. Thus, in certain embodiments, the method includes a pre-culture that includes culturing the carposporophyte at a temperature (e.g., a temperature in the range of about 20 to about 26 °C) that is adjusted from the first temperature to the temperature of the first timed exposure period.

[0014] In certain embodiments, the first temperature is in the range of about 17 to about 23 °C.

[0015] In certain embodiments, the temperature during the pre-culture is adjusted in the range of about 1 to about 8 °C.

[0016] In certain embodiments, the temperature during the pre-culture is adjusted at a pace in the range of about 0.25 to about 3 °C per hour.

[0017] According to a second aspect, a method of producing at least one gametophyte of A. taxiformis is provided, the method comprising procuring a tetrasporophyte of A. taxiformis, and culturing the tetrasporophyte at a temperature in the range of about 20 to about 26 °C while exposing it to illumination having an intensity in the range of about 30 to about 100 μmol·m -2 ·s -1 for a timed exposure period in the range of about 5 to about 30 days to induce the growth of at least one gametophyte thallus, and optionally, prior to inducing the growth of at least one gametophyte thallus, first inducing the formation of tetrasporangia containing tetraspores, inducing the tetrasporangia to release the tetraspores, and inducing the tetraspores to germinate to produce at least one gametophyte thallus, and comprising.

[0018] In certain embodiments, the duration of illumination during the timed exposure period is in the range of about 6 to about 18 hours per 24 hours. In certain embodiments, the duration of illumination during the timed exposure period is in the range of about 7 to about 16 hours per 24 hours.

[0019] In one embodiment, the illumination intensity during the timed exposure period is approximately 40 to approximately 80 μmol·m -2 ·s -1 The range is as follows: In one embodiment, the illumination intensity during the timed exposure period is approximately 50 to approximately 70 μmol·m -2 ·s -1 It is within the range.

[0020] In one embodiment, the culture temperature during the timed exposure period is in the range of approximately 21 to approximately 25°C. In a specific embodiment, the culture temperature during the timed exposure period is in the range of approximately 22 to approximately 24°C.

[0021] In one embodiment, the timed exposure period is in the range of approximately 6 to approximately 21 days.

[0022] In one embodiment, the method of the second embodiment further includes first inducing the formation of a tetrasporangium containing tetraspores, inducing the tetrasporangium to release tetraspores, and inducing the tetraspores to germinate and produce at least one gametophyte thallus, before inducing the growth of at least one gametophyte thallus.

[0023] In one embodiment, the tetrasporophytes are produced by the method of the first embodiment.

[0024] In one embodiment, at least one tetrasporophyte is brought into a state capable of producing at least one gametophyte. To increase the biomass of at least one tetrasporophyte, at least one tetrasporophyte is exposed for approximately 30 to 90 days, with a third exposure period ranging from approximately 16 to 24 hours per 24 hours, at a rate of approximately 30 to 100 μmol·m³. -2 ·s -1 The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. To develop at least one tetrasporophyte to a state where it can produce at least one gametophyte, at least one tetrasporophyte is exposed to approximately 30 to 100 μmol·m³ over a fourth exposure period ranging from approximately 8 to 16 hours per 24 hours for approximately 30 to 90 days. -2 ·s -1 The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. To promote development.

[0025] According to a third embodiment, a tetrasporophyte of A. taxiformis produced by the method of the first embodiment is provided.

[0026] According to the fourth aspect, a gametophyte of A. taxiformis produced by the method of the second aspect is provided.

[0027] According to the fifth aspect, a composition is provided comprising at least one tetrasporophyte of A. taxiformis according to the third aspect or a gametophyte of A. taxiformis according to the fourth aspect, or an extract(s) of the above-mentioned tetrasporophyte or gametophyte containing one or more halogen compounds.

[0028] According to the sixth aspect, an animal nutritional supplement is provided that contains an effective amount of the composition of the fifth aspect.

[0029] Embodiments of this disclosure will be described with reference to the accompanying drawings. [Brief explanation of the drawing]

[0030] [Figure 1] This is a diagram of the life cycle of A. taxiformis. [Figure 2] This is a photograph of a branch of A. taxiformis selected to induce carpospore formation. [Figure 3] This is a photograph of a cystic fruit spontaneously releasing carpospores from an A. taxiformis plant. [Figure 4] This is a photograph of a germinating carpospore of A. taxiformis. [Figure 5] This is a photograph of a healthy tetrasporophyte of A. taxiformis under 8 hours of illumination per 24 hours. [Figure 6] This is a photograph of a pale-colored tetrasporophyte of A. taxiformis under continuous illumination conditions (i.e., 24 hours of illumination per 24-hour period). [Figure 7] This is a photograph of a young gametophyte of A. taxiformis, processed with 8 hours of illumination per 24 hours (8 light / 16 dark), and then magnified 5 times. [Figure 8] This is a photograph of a gametophyte that developed from a tetrasporophyte. [Figure 9] This is a photograph of the gametophyte in its early stages. [Modes for carrying out the invention]

[0031] As used herein, the term “approximately” means plus or minus 5% of a given value, for example, a 10-hour ± 5% exposure period is 9.5 to 10.5 hours, or a 22°C ± 5% culture temperature is 20.9 to 23.1°C. As used herein, the phrases “range of approximately” and “up to approximately” mean plus or minus 5% of a given value, for example, a range of approximately 20 to approximately 26 degrees means a range from 20°C ± 5% (21 to 23°C) to 26 degrees ± 5% (24.7 to 27.3°C). The term “approximately” may be omitted to improve precision or clarity, as will be understood by those skilled in the art.

[0032] Commercial production of A. taxiformis faces challenges due to limited resources in the wild. Current research attempts to replicate the life cycle of the gametophyte, carposporophyte, and tetrasporophyte stages in the laboratory. Currently, there are two methods for obtaining tetrasporophytes: 1) collecting mature cysts from the wild, manually excising them to obtain carpospores, germinating these carpospores, and developing them into young tetrasporophytes; and 2) collecting tetrasporophytes from the wild. These methods have many drawbacks. They are time-consuming, labor-intensive, have low hatching rates, and low germination rates. Furthermore, wild tetrasporophytes are covered with many contaminants, which are difficult to remove, making it difficult to obtain clean tetrasporophytes.

[0033] This disclosure arises from research on methods for cultivating A. taxiformis. This research involves cultivating A. taxiformis through different phases of the triphasic life cycle shown in Figure 1. The inventors have developed a method for producing tetrasporophytes and gametophytes. Broadly speaking, this method involves inducing carpospores through environmental manipulation, germinating them, and growing them into tetrasporophytes. This allows for the rapid generation of large quantities of clean biomass of tetrasporophytes. Furthermore, this method includes further environmental manipulation for gametophyte production.

[0034] According to the first aspect, a method is provided for producing at least one tetrasporophyte of A. taxiformis, the method being Obtain a carposporophyte of A. taxiformis with at least one mature cystic, To induce the release of at least one carpospore from at least one cystocarp, the carposporophyte is exposed to approximately 80–170 μmol·m over a first timed exposure period ranging from approximately 10–48 hours. -2 ·s -1 The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. To induce at least one carpospore to germinate and produce at least one tetrasporophyte, at least one carpospore is exposed to a second timed exposure period ranging from approximately 10 to approximately 72 hours, at a rate of approximately 80 to approximately 170 μmol·m³. -2 ·s -1 The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. Includes.

[0035] The method of the first embodiment is useful for the mass production of carpospores and for the germination and cultivation of carpospores to the tetrasporophyte stage. In this method, the duration of exposure, the intensity of illumination, and the culture temperature are controlled to induce at least one carpospore to be released from at least one cyst, and then to induce at least one carpospore to germinate and produce a tetrasporophyte. As will be understood by those skilled in the art, when a carpospore is released from a cyst, it is released from the cyst.

[0036] Carposporophytes of A. taxiformis with at least one mature cyst can be collected from the wild (e.g., in the marine habitat where A. taxiformis grows naturally). Those skilled in the art can easily determine the production season of the carposporophytes and when the cysts are mature and ready to produce carpospores. Those skilled in the art will understand that the timing of carposporophyte development in the wild is geographically dependent and typically occurs in the fall. For example, in the examples herein, carposporophytes of A. Taxiformis with mature cysts were collected from Abrojos and the Rat Islands in Western Australia on May 23, 2022, and on June 13 and 20, 2022. At least one mature cyst can be identified by the pink to reddish coloration of the cyst body. As will be readily apparent to those skilled in the art, the timing of cyst maturation varies based on the season and therefore on latitude. Germinating carpospores can be identified by the initiation of a germ tube (having the appearance of an arrowhead) that begins to elongate from the carpospore. As will be understood by those skilled in the art, the water temperature at which carpospores are harvested varies with the season. For example, the temperature may be around 16-19°C in the period closer to winter, and around 22-24°C in the period closer to summer. Therefore, the water temperature needs to be adjusted to the temperature of the first exposure period.

[0037] Alternatively, carposporophytes of A. taxiformis with at least one mature cystocarp can be produced in an artificial environment (e.g., a bioreactor). At least one mature cystocarp can be identified by the pink to reddish coloration of the cystocarp body.

[0038] The culture step in the first time-delayed exposure period should preferably be started within 72 hours, more preferably within 48 hours, and most preferably within 24 hours, of the collection of carposporophytes from the wild or artificial environment. As will be understood by those skilled in the art, the culture step in the first time-delayed exposure period may be started after a longer period if the conditions closely resemble the seawater in which A. taxiformis grows naturally.

[0039] In one embodiment, prior to the first time-delayed exposure period, the method includes a pre-culturing in which the temperature of the carposporophytes (e.g., the temperature applied to a culture medium such as seawater) is adjusted to the culture temperature of the first time-delayed exposure period. Thus, the method includes a pre-culturing in which the carposporophytes are cultured at a temperature adjusted from a first temperature to a temperature in the range of about 20 to about 26°C for the first time-delayed exposure period. In one embodiment, the adjustment includes raising the temperature. In one embodiment, the pre-culturing includes raising the culture temperature of the carposporophytes from a first temperature to the temperature for the first time-delayed exposure period.

[0040] The first temperature may be, for example, the temperature of the water in which the carposporophytes are harvested, or the temperature of the water in which the carposporophytes are produced. In some embodiments, the first temperature is in the range of about 17 to about 23°C. In some embodiments, the first temperature is in the range of about 17 to about 22°C, or in the range of about 18 to about 22°C, for example, 18, 19, 20, 21, or 22°C. In some embodiments, the temperature during pre-culturing is adjusted in the range of about 1 to about 8°C. That is, the change from the first temperature to the temperature during the first time-limited exposure period is 1 to 8°C. In some embodiments, the temperature during pre-culturing is adjusted in the range of about 1 to about 4°C. In certain embodiments, the temperature during pre-culturing is adjusted in the range of about 2 to about 4°C.

[0041] In one embodiment, the temperature during pre-culture is adjusted at a rate of approximately 0.25 to 3°C per hour. That is, the rate of temperature change is within the range of approximately 0.25 to 3°C per hour. In another embodiment, the temperature during pre-culture is adjusted at a rate of approximately 1 to 2°C per hour. In a specific embodiment, the temperature during pre-culture is adjusted at a rate of approximately 1°C per hour. In a specific embodiment, the temperature during pre-culture is adjusted at a rate of approximately 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, or 1.9°C per hour.

[0042] In some embodiments where the first temperature is high, for example, in summer when the water temperature in which the carposporophytes are harvested is high (e.g., 23, 24, 25, 26, or 27°C), the culture temperature may be lowered first (during pre-culture) and then raised to the temperature for the first time-limited exposure period (e.g., 20–26°C). For example, the culture temperature (e.g., water temperature or first temperature) may be lowered first from 26, 25, 24, 23, or 22°C to a lower temperature such as 24, 23, 22, 21, 20, 19, or 18°C. In some embodiments, the rate at which the temperature is lowered is less than 2°C per hour. In some embodiments, the rate at which the temperature is lowered is 0.1–1°C per hour. In some embodiments, the rate at which the temperature is lowered is 0.1–0.5°C per hour. In some embodiments, the rate at which the temperature is lowered is 0.2–0.3°C per hour. In some embodiments, the rate at which the temperature is lowered is about 0.25°C per hour. Following the decrease, the temperature may be increased during pre-culturing.

[0043] In one embodiment, the duration of illumination during the first and second exposure periods is in the range of approximately 16 to approximately 24 hours per 24-hour period. In another embodiment, the duration of illumination during the first exposure period is different from that of the second exposure period. In yet another embodiment, the duration of illumination during the first exposure period is the same as that of the second exposure period. In one embodiment, the duration of illumination during the first exposure period is approximately 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours per 24-hour period, or any range between these numbers. In another embodiment, the duration of illumination during the second exposure period is approximately 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours per 24-hour period, or any range between these numbers. In some embodiments, the duration of illumination during either or both of the first and second time-delay exposure periods is in the range of about 16 to about 18 hours per 24 hours, about 18 to about 20 hours per 24 hours, about 20 to about 22 hours per 24 hours, about 22 to about 24 hours per 24 hours, about 23 to about 24 hours per 24 hours, about 20 to about 24 hours per 24 hours, or about 21 to about 24 hours per 24 hours. In some embodiments, the duration of illumination during either or both of the first and second time-delay exposure periods is substantially continuous, for example, continuous or exceeding 23.5, 23.6, 23.7, 23.8, or 23.9 hours per 24 hours. In some embodiments, the duration of illumination during the second time-delay exposure period is sufficient to cause the carpospores to germinate and may be changed thereafter. Thus, those skilled in the art can monitor germination and adjust the duration of illumination accordingly. For example, initial long-duration lighting, such as continuous lighting or 22-23 hours per 24-hour period, may be sufficient to germinate the carpospores, after which the duration may be reduced to, for example, 16-20 hours per 24-hour period.

[0044] As described above, the illumination intensity during the first and second exposure periods was approximately 80 to 170 μmol·m². -2 ·s -1The range is as follows: In one embodiment, the illumination intensity during the first time exposure period is different from that during the second time exposure period. In another embodiment, the illumination intensity during the first time exposure period is the same as that during the second time exposure period. In one embodiment, the illumination intensity during the first time exposure period is approximately 80, approximately 90, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150, approximately 160, or approximately 170 μmol·m -2 ·s -1 , or any range between these values. In one embodiment, the illumination intensity during the second timed exposure period is about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, or about 170 μmol·m -2 ·s -1 , or any range between these values. In one embodiment, the illumination intensity during either or both of the first and second time-delay exposure periods is about 80 to about 170 μmol·m -2 ·s -1 , about 110 to about 170 μmol m -2 ·s -1 , about 120 to about 170 μmol m -2 ·s -1 , about 130 to about 160 μmol m -2 ·s -1 , about 140 to about 150 μmol m -2 ·s -1 , about 140 to about 160 μmol m -2 ·s -1 , about 140 to about 170 μmol m -2 ·s -1 , about 110 to about 160 μmol m -2 ·s -1 , or approximately 100 to 150 μmol·m -2 ·s -1 The range is as follows: In a particular embodiment, the illumination intensity during either or both of the first and second time-delay exposure periods is 140-150 μmol·m -2 ·s -1As will be understood by those skilled in the art, the illumination intensity can be measured, for example, using a full-spectrum underwater quantum meter that accurately measures photosynthetically active radiation in water, below the surface of the culture medium where A. taxiformis is growing and as close as possible to the depth of A. taxiformis.

[0045] As described above, the method includes culturing carposporophytes at a temperature in the range of approximately 20 to approximately 26°C during a first and a second exposure period. In one embodiment, the temperature during the first exposure period is different from that during the second exposure period. In another embodiment, the temperature during the first exposure period is the same as that during the second exposure period. In one embodiment, the method includes culturing carposporophytes at a temperature of 20, 21, 22, 23, 24, 25, or 26°C, or any range between these values, during the first exposure period. In one embodiment, the method includes culturing carposporophytes at a temperature of 20, 21, 22, 23, 24, 25, or 26°C, or any range between these values, during the second exposure period. In one embodiment, the temperature during the first exposure period is different from that during the second exposure period. In another embodiment, the temperature during the first exposure period is the same as that during the second exposure period. In some embodiments, the temperature during either or both of the first and second time-delay exposure periods is in the range of about 20–25°C, about 20–24°C, about 21–26°C, about 22–26°C, about 21–25°C, about 22–25°C, or about 22–24°C. In certain embodiments, the temperature is 22±0.5°C or 24±0.5°C. Preferably, the temperature should not fluctuate outside the described range, as this may adversely affect the growth and / or health of A. taxiformis. For example, temperatures below 16°C, e.g., 8°C, may result in carpospore death.

[0046] As described above, the duration of the first timed exposure period is in the range of approximately 10 to approximately 48 hours, and the duration of the second timed exposure period is in the range of approximately 10 to approximately 72 hours. In some embodiments, the duration of the first timed exposure period is different from that of the second timed exposure period. In other embodiments, the duration of the first timed exposure period is the same as that of the second timed exposure period. In some embodiments, the duration of the first timed exposure period is in the range of approximately 10 to approximately 42, approximately 10 to approximately 36, approximately 10 to approximately 30, approximately 10 to approximately 24, approximately 12 to approximately 42, approximately 12 to approximately 36, approximately 12 to approximately 30, approximately 12 to approximately 24, approximately 14 to approximately 42, approximately 14 to approximately 36, approximately 14 to approximately 30, approximately 14 to approximately 24, approximately 16 to approximately 42, approximately 16 to approximately 36, approximately 16 to approximately 30, or approximately 16 to approximately 24 hours. In certain embodiments, the duration of the first timed exposure period is 18 to about 30 hours. In some embodiments, the duration of the second timed exposure period is in the range of about 10 to about 66, about 10 to about 60, about 10 to about 54, about 10 to about 48, about 18 to about 72, about 18 to about 66, about 18 to about 60, about 18 to about 54, about 18 to about 48, about 24 to about 72, about 24 to about 66, about 24 to about 60, about 24 to about 54, or about 24 to about 48 hours. In certain embodiments, the duration of the second timed exposure period is 24 to 72 hours.

[0047] The product of the method according to the first embodiment is at least one tetrasporophyte. The produced at least one tetrasporophyte can be cultured under conditions suitable for producing a developed tetrasporophyte suitable for use in the method according to the second embodiment (e.g., in an artificial environment). These conditions may include culture temperature and lighting intensity for a second time-delay exposure period that is maintained for a sufficient period for the tetrasporophyte(s) to develop. In one embodiment, these conditions include increasing the biomass of at least one tetrasporophyte by culturing at least one tetrasporophyte over a third time-delay exposure period ranging from about 30 to about 90 days, with a biomass of about 30 to about 100 μmol·m³, ranging from about 16 to about 24 hours per 24 hours. -2 ·s -1This involves culturing at a temperature in the range of approximately 20 to approximately 26°C while exposing the plants to illumination of an intensity in the range of [specify intensity]. This also includes culturing at least one tetrasporophyte to a state where it can produce at least one gametophyte, over a fourth exposure period of approximately 30 to approximately 90 days, with exposure of approximately 30 to approximately 100 μmol·m³ for approximately 8 to approximately 16 hours per 24 hours. -2 ·s -1 This involves culturing at a temperature in the range of approximately 20 to approximately 26°C while exposing the cells to illumination of an intensity in the range of . At least one developed tetrasporophyte may be used by the method of the second embodiment.

[0048] As described above, the duration of illumination during the third timed exposure period is in the range of approximately 16 to approximately 24 hours per 24 hours. In some embodiments, the duration of illumination during the third timed exposure period is approximately 16, approximately 17, approximately 18, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, or approximately 24 hours per 24 hours, or any range between these numbers. In some embodiments, the duration of illumination during the third timed exposure period is in the range of approximately 16 to approximately 18 hours per 24 hours, approximately 18 to approximately 20 hours per 24 hours, approximately 20 to approximately 22 hours per 24 hours, approximately 22 to approximately 24 hours per 24 hours, approximately 23 to approximately 24 hours per 24 hours, approximately 20 to approximately 24 hours per 24 hours, or 21 to approximately 24 hours per 24 hours. In one embodiment, the duration of illumination during the third timed exposure period is substantially continuous, for example, continuous or exceeding 23.5, 23.6, 23.7, 23.8, or 23.9 hours per 24 hours.

[0049] As described above, the duration of illumination during the fourth exposure period is in the range of approximately 8 to approximately 16 hours per 24 hours. In some embodiments, the duration of illumination during the fourth exposure period is approximately 8, approximately 9, approximately 10, approximately 11, approximately 12, approximately 13, approximately 14, approximately 15, or approximately 16 hours per 24 hours, or any range between these numbers. In some embodiments, the duration of illumination during the fourth exposure period is approximately 8 to approximately 15 hours per 24 hours, approximately 8 to approximately 14 hours per 24 hours, approximately 8 to approximately 13 hours per 24 hours, approximately 8 to approximately 12 hours per 24 hours, approximately 8 to approximately 11 hours per 24 hours, approximately 9 to approximately 16 hours per 24 hours, approximately 9 to approximately 15 hours per 24 hours, approximately 9 to approximately 14 hours per 24 hours, approximately 9 to approximately 13 hours per 24 hours, approximately 9 to approximately 12 hours per 24 hours, The duration of illumination during the fourth exposure period is approximately 9 to 11 hours per 24 hours, approximately 10 to 16 hours per 24 hours, approximately 10 to 15 hours per 24 hours, approximately 10 to 14 hours per 24 hours, approximately 10 to 13 hours per 24 hours, approximately 10 to 12 hours per 24 hours, approximately 10 to 11 hours per 24 hours, approximately 11 to 16 hours per 24 hours, approximately 11 to 15 hours per 24 hours, approximately 11 to 14 hours per 24 hours, or approximately 12 hours per 24 hours. In one embodiment, the duration of illumination during the fourth exposure period is approximately 12 ± 1 hours per 24 hours.

[0050] As described above, the culture temperature during the third and fourth exposure periods is in the range of approximately 20–26°C. In some embodiments, the culture temperature during either or both of the third and fourth timed exposure periods is approximately 20, approximately 21, approximately 22, approximately 23, approximately 24, approximately 25, or approximately 26°C, or any range between these specified temperatures. In some embodiments, the culture temperature during either or both of the third and fourth timed exposure periods is in the range of approximately 20–approximately 25, approximately 20–approximately 24, approximately 21–approximately 25, approximately 21–approximately 26, approximately 22–approximately 26, approximately 22–approximately 25, or approximately 22–approximately 24°C. In certain embodiments, the culture temperature during either or both of the third and fourth timed exposure periods is 22±1°C or 22±0.5°C. Preferably, the temperature should not fluctuate outside the described range, as this may adversely affect the growth and / or health of A. taxiformis.

[0051] As described above, the illumination intensity during the third and fourth exposure periods was approximately 30 to 100 μmol·m². -2 ·s -1 The range is as follows: In one embodiment, the illumination intensity during either or both of the third and fourth timed exposure periods is approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, or approximately 100 μmol·m -2 ·s -1 In one embodiment, the illumination intensity during either or both of the third and fourth time-delay exposure periods is approximately 30 to approximately 90, approximately 30 to approximately 80, approximately 30 to approximately 70, approximately 30 to approximately 60, approximately 40 to approximately 90, approximately 40 to approximately 80, approximately 40 to approximately 60, approximately 50 to approximately 80, approximately 50 to approximately 70, or approximately 50 to approximately 60 μmol·m -2 ·s -1 This range is as follows: In certain embodiments, the illumination intensity during either or both of the third and fourth time-delay exposure periods is 40-80 μmol·m -2 ·s -1 or 50-70 μmol·m -2 ·s -1 The illumination intensity may be measured as described elsewhere in this specification.

[0052] As described above, the duration of the third timed exposure period is in the range of approximately 30 to approximately 90 days, and the duration of the fourth timed exposure period is in the range of approximately 30 to approximately 90 days. In some embodiments, the duration of the third exposure period is different from that of the fourth exposure period. In other embodiments, the duration of the third exposure period is the same as that of the fourth exposure period. In some embodiments, the duration of either or both of the third and fourth timed exposure periods is in the range of approximately 32 to approximately 88, 34 to approximately 86, 36 to approximately 84, 38 to approximately 82, 40 to approximately 80, 42 to approximately 78, 44 to approximately 76, 46 to approximately 74, 48 to approximately 72, 50 to approximately 70, 52 to approximately 68, 54 to approximately 66, 56 to approximately 64, or approximately 58 to approximately 62 days.

[0053] As those skilled in the art will understand, at least one tetrasporophyte is developing and, at least about two months old, is capable of producing at least one gametophyte. In the wild, A. taxiformis remains in the tetrasporophyte stage during the winter. Therefore, A. taxiformis can remain in the tetrasporophyte stage for two to four months. After that, A. taxiformis may progress to the gametophyte stage during the spring. This means that those skilled in the art can determine when the fourth time-limited exposure period should end and when at least one tetrasporophyte is capable of producing at least one gametophyte.

[0054] Alternatively, at least one A. taxiformis tetrasporophyte may be collected from the wild (e.g., in a marine location where A. taxiformis naturally grows). As described above, those skilled in the art can identify that a tetrasporophyte collected from the wild is developing and has at least one tetrasporangia, and is therefore capable of producing at least one gametophyte. The culture step in the timed exposure period of the second embodiment should preferably be started within 72 hours, more preferably within 48 hours, and most preferably within 24 hours, of the timed exposure period in which the tetrasporophyte was collected from the wild or artificial environment. As those skilled in the art will understand, the culture step in the timed exposure period may be started after a longer period if the conditions closely resemble the seawater in which A. taxiformis naturally grows.

[0055] According to a second aspect, a method for producing the gametophyte of A. taxiformis is provided, and this method is To obtain the tetrasporophytes of A. taxiformis, To induce the growth of at least one gametophyte thallus, tetrasporophytes are exposed to approximately 30–100 μmol·m over a timed exposure period ranging from approximately 5–30 days. -2 ·s -1 The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. Optionally, before inducing the growth of at least one gametophyte thallus, first induce the formation of a tetrasporangium containing tetraspores, induce the tetrasporangium to release tetraspores, and induce the tetraspores to germinate and produce at least one gametophyte thallus. Includes.

[0056] The method of the second embodiment is useful for mass production of gametophytes from the tetrasporophyte stage.

[0057] In one embodiment, the duration of illumination during the timed exposure period is in the range of about 6 to about 18 hours per 24 hours. In another embodiment, the duration of illumination during the timed exposure period is about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18 hours per 24 hours. In one embodiment, the duration of illumination during the timed exposure period is in the range of approximately 6 to 17 hours per 24 hours, 6 to 16 hours per 24 hours, 6 to 15 hours per 24 hours, 6 to 14 hours per 24 hours, 6 to 13 hours per 24 hours, 6 to 12 hours per 24 hours, 7 to 18 hours per 24 hours, 7 to 17 hours per 24 hours, 7 to 16 hours per 24 hours, 7 to 15 hours per 24 hours, 7 to 14 hours per 24 hours, 7 to 13 hours per 24 hours, 7 to 12 hours per 24 hours, 8 to 18 hours per 24 hours, 8 to 17 hours per 24 hours, 8 to 16 hours per 24 hours, 8 to 15 hours per 24 hours, 8 to 14 hours per 24 hours, 8 to 13 hours per 24 hours, or 8 to 12 hours per 24 hours. In certain embodiments, the duration of illumination during the timed exposure period is 7 to 16 hours per 24-hour period. In some embodiments, the duration of illumination during the timed exposure period is substantially continuous, for example, continuous or exceeding 23.5, 23.6, 23.7, 23.8, or 23.9 hours per 24-hour period.

[0058] As described above, the illumination intensity during the timed exposure period is approximately 30 to 100 μmol·m -2 ·s -1 The range is as follows: In one embodiment, the illumination intensity during the exposure period is about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 μmol·m -2 ·s -1 In one embodiment, the illumination intensity is approximately 30 to 90, 30 to 80, 30 to 70, 30 to 60, 40 to 90, 40 to 80, 40 to 60, 50 to 80, 50 to 70, or 50 to 60 μmol·m -2 ·s -1 In certain embodiments, the illumination intensity is 40-80 μmol·m-2 ·s -1 or 50-70 μmol·m -2 ·s -1 The illumination intensity may be measured as described elsewhere in this specification.

[0059] As described above, the method involves culturing carposporophytes at a temperature in the range of approximately 20 to approximately 26°C during a timed exposure period. In some embodiments, the method involves culturing carposporophytes at a temperature of 20, 21, 22, 23, 24, 25, or 26°C, or any range between these specified temperatures, during a timed exposure period. In some embodiments, the temperature is in the range of approximately 20 to approximately 25°C, approximately 20 to approximately 24°C, approximately 21 to approximately 25°C, approximately 21 to approximately 26°C, approximately 22 to approximately 26°C, approximately 22 to approximately 25°C, or approximately 22 to approximately 24°C. In certain embodiments, the temperature is 22 to 24°C. Preferably, the temperature should not fluctuate outside the described range, as this may adversely affect the growth and / or health of A. taxiformis.

[0060] As described above, the duration of the timed exposure period ranges from about 5 to about 30 days. As will be understood by those skilled in the art, the tetrasporophytes can be monitored for induction of growth of at least one gametophyte thallus, which typically occurs within 5 to 30 days. In some embodiments, the duration of the timed exposure period ranges from about 5 to about 28, 5 to about 26, 5 to about 24, 5 to about 22, 5 to about 20, 5 to about 18, 5 to about 16, 6 to about 14, 5 to about 12, 5 to about 10, 6 to about 28, 6 to about 26, 6 to about 24, 6 to about 22, 6 to about 20, 6 to about 18, 6 to about 16, 6 to about 14, 6 to about 12, 6 to about 10, 7 to about 20, 7 to about 18, 7 to about 16, 7 to about 14, 7 to about 12, 7 to about 10, 8 to about 12, or about 8 to about 10 days. In certain embodiments, the duration of the timed exposure period is 6 to 12 days or about 9 days.

[0061] In one embodiment, the method further includes first inducing the formation of a tetrasporangium containing tetraspores, inducing the tetrasporangium to release tetraspores, and inducing the tetraspores to germinate and produce at least one gametophyte thallus, before inducing the growth of at least one gametophyte thallus. Those skilled in the art will understand that the conditions for inducing the formation of a tetrasporangium containing tetraspores, inducing the tetrasporangium to release tetraspores, and inducing the tetraspores to germinate and produce at least one gametophyte thallus can be derived from published literature, for example, the conditions used to achieve the same results in the closely related species A. armata. In Oza (1977), tetrasporophytes of A. armata developed tetrasporangia when grown under short-day conditions (8 light:16 dark) at 15°C, when cultured in a culture medium with reduced nitrogen and phosphorus. Similarly, Guiry & Clinton (J.Exp.Mar.Biol.158.2(1992):197-217) grew tetrasporophytes of A. armata from Ireland, Italy, and Australia for 5 weeks under the following conditions: Irish plants at 15–21°C and 8–9 hours of lighting per 24 hours, Italian plants at 17–21°C and 9–10 hours of lighting per 24 hours, and Australian plants at 13–17°C and 8–9 hours of lighting per 24 hours, resulting in the development of tetrasporangia. Alternatively, the culture temperature, lighting intensity, and lighting duration during the above-mentioned timed exposure period can be maintained for a sufficient period to induce the formation of tetrasporangia containing tetraspores, to induce the tetrasporangia to release tetraspores, and to induce the tetraspores to germinate and produce at least one gametophyte thallus.

[0062] According to a third embodiment, a tetrasporophyte of A. taxiformis produced by the method of the first embodiment is provided. The tetrasporophyte of A. taxiformis is a product of the first embodiment, and therefore, a tetrasporophyte of A. taxiformis is produced by following the method of the first embodiment.

[0063] According to the fourth aspect, a gametophyte of A. taxiformis is provided, which is produced by the method of the second aspect. The gametophyte of A. taxiformis is a product of the second aspect, and therefore, the gametophyte of A. taxiformis is produced by following the method of the second aspect.

[0064] Throughout this disclosure, there are references to cultivating, farming, or producing various growth stages of A. taxiformis, such as carposporophytes, tetrasporophytes, and gametophytes. As will be understood by those skilled in the art, A. taxiformis can be farmed in any suitable liquid culture medium. The composition of the liquid culture medium may be similar to the seawater in which A. taxiformis naturally grows. In one embodiment, the liquid culture medium may be seawater collected from the site where A. taxiformis naturally grows. The liquid culture medium may be treated to remove contaminating microorganisms. Preferred treatments include, for example, pasteurization (e.g., heat treatment), sterilization (e.g., ultraviolet treatment), and / or filtration (e.g., using a filter with a pore size that removes contaminating organisms, e.g., 0.05–5 μm, typically 0.2 μm). The seawater culture medium may also be fortified to provide A. taxiformis with nutrients suitable for growth. An example of a suitable fortified culture medium is Provassoli fortified seawater (Bold, HC & Wynne, MJ Introduction to the Algae; 1978; Redmond et al., 2014). Fortified culture media may contain, for example, macronutrients, micronutrients, and / or vitamins. Other suitable culture media can be found in the literature, e.g., Andersen, RA; Jacobson, DM & Sexton, JP - Provasoli-Guillard Center for Culture of Marine Phytoplankton. Catalogue of Strains. 98pp. West Boothbay Harbor, Maine, USA, 1991; Castenholz, RW - Culturing methods for Cyanobacteria. Published in: L. Packer and AN Glazer, eds., Cyanobacteria. Methods of Enzymology 167 (1988), 68-93; Guillard, RRL - Culture of Phytoplankton for feeding marine invertebrates. Published in: WL Smith and MH Chanley, eds., Culture of marine invertebrate animals. pp.29-60, Plenum Book Publ.Corp., New York, 1975; Kuhl, A. & Lorenzen, H. - Handling and culturing of Chlorella. Included in: D.M. Prescott, ed., Methods in cell physiology. Vol.1, pp. 152-187, Academic Press, New York and London, 1964; Rippka, R. & Herdman, M. - Pasteur Culture Collection of Cyanobacterial Strains in Axenic Culture. Vol.1, Catalogue of strains. 103pp., Institut Pasteur, Paris, France, 1992; Starr, R.C. - Algal Cultures - sources and methods of cultivation. Included in: A. San Pietro, ed., Photosynthesis. Part A, pp. 29-53, Methods in Enzymology vol. 23, Academic Press, New York, 1971; Starr, R.C. & Zeikus, J.A. - UTEX - The Culture Collection of Algae at the University of Texas at Austin. J. Phycol. Suppl. 29 (1993); Stein, J.R. ed. - Handbook of phycological methods. Culture Methods and growth measurements, pp. 448, Cambridge at the University Press, London, New York, 1973; Thompson, A.S.; Rhodes, J.C. & Pettman, I. - Culture Collection of Algae and Protozoa.This can be found in: Catalogue of strains. 164pp., Natural Environment Research and Council, England, 5th edit., 1988; Watanabe, MM & Nozaki, H. - NIES-Collection. List of strains, microalgae and protozoa. 4th edit., 127pp. The National Institute for Environmental Studies, Japan, 1994; Werner, D. - Biologische Versuchobjekte. Kultivierung und Wachstum ausgewaehlter Versuchsorganismen in definierten Medien. 432pp. Fischer Verlag, Stuttgart, New York, 1982. As will be understood by those skilled in the art, the concentration of the culture medium may be changed according to the nutritional requirements of A. taxiformis, for example, the culture medium may be used at the concentrations described in the literature or diluted. Therefore, when using Provassoli-enhanced seawater (Bold, HC & Wynne, MJ Introduction to the Algae; 1978; Redmond et al., 2014), this culture medium may be used at the concentrations described, or diluted to, for example, 50% or 25% of the described concentrations. In one embodiment, the enhanced culture medium is undiluted (e.g., 100% or its original concentration). In another embodiment, the enhanced culture medium is diluted to 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5% of the described concentrations (i.e., 100% or its original concentration). In yet another embodiment, the concentration may be increased to, for example, 2x, 3x, or 4x compared to the described concentrations.

[0065] The salinity of the liquid culture medium is typically that of seawater, for example, 33,000 parts per thousand (ppt) to 38 ppt. In some embodiments, the salinity ranges from approximately 33.5 to approximately 37.5, 34 to approximately 37.5, 34.5 to approximately 37.5, 35 to approximately 37.5, 35.5 to approximately 37.5, 34 to approximately 37, 35 to approximately 37, 35.5 to approximately 37.5, and 36 to approximately 37. In certain embodiments, the salinity is 36.5 ± 1 or 36.5 ± 0.5.

[0066] The pH of the liquid culture medium is preferably 6.5 to 9. In some embodiments, the pH is typical of seawater, for example, 8 to 9. In some embodiments, the pH is in the range of about 6.5 to 8.5, about 7 to 8.5, about 7.5 to 8, about 8.1 to 8.8, about 8.1 to 8.7, about 8.1 to 8.6, about 8.1 to 8.5, about 8.2 to 8.8, about 8.2 to 8.7, about 8.2 to 8.6, about 8.2 to 8.5, about 8.3 to 8.8, about 8.3 to 8.7, about 8.3 to 8.6, about 8.3 to 8.5, or 8.4 to 8.5. In certain embodiments, the pH is 8.4 to 8.5.

[0067] A. taxiformis can be cultured in a bioreactor, such as a tank or flask containing a liquid culture medium. The bioreactor may include, for example, inlets and outlets for the flow of the liquid culture medium to a reservoir or aerator of the culture medium. In some embodiments, the liquid culture medium is aerated. Aeration may use a carbon dioxide-containing gas such as air. As will be understood by those skilled in the art, the carbon dioxide-containing gas may be introduced into the liquid culture medium, for example by a bubble diffuser, or by removing the liquid from the bioreactor, increasing the amount of gas dissolved in the liquid, and then returning the liquid to the bioreactor. The light source used to provide illumination in the first and second embodiments is positioned to supply light to the surface of the liquid in the bioreactor. In some embodiments, the light source is a light-emitting diode. In some embodiments, the light source is a fluorescent lamp. In some embodiments, the light source is an incandescent light.

[0068] According to a fifth embodiment, a composition is provided comprising at least one of the following: the tetrasporophyte of A. taxiformis according to a third embodiment, the gametophyte of A. taxiformis according to a fourth embodiment, or an extract(s) of the tetrasporophyte or gametophyte containing one or more halogen compounds. This composition can be added to animal feed and ultimately fed to ruminants to reduce the number of methanogenic bacteria in the rumen of ruminants. In one embodiment, the composition comprises A. taxiformis biomass, which is the material produced by the growth and / or proliferation of A. taxiformis cells. The biomass may include cells and / or intracellular inclusions, as well as extracellular material. Extracellular material includes, but is not limited to, compounds secreted by cells. In one embodiment, the extract(s) comprises intracellular or extracellular extracts. In a specific embodiment, the extract(s) comprises a brominated compound such as bromoform. Asparagopsis is known to produce halogenated low molecular weight compounds (Burreson BJ et al., Tetrahedron Lett. 1975:473-476; Burreson BJ et al. J. Agric. Food Chem. 1976;24:856-861; Woolard FX et al. Tetrahedron. 1976;32:2843-2846; McConnell O., and Fenical W. Phytochemistry. 1977;16:367-374; Combaut G. et al. Phytochemistry. 1978;17:1661-1663; Woolard FX et al. Phytochemistry. 1979;18:617-620; Abrahamsson K. et al. Limnol. Oceanogr.). 1995;40:1321-1326;Marshall RA et al. Limnol.Oceanogr. 1999;44:1348-1352).As those skilled in the art will understand, extracts(s) can be obtained using common extraction techniques such as solvent extraction or oil immersion (Magnusson, Marie, et al. Algal Research 51 (2020): 102065; Tan, S. et al., (2022). Shelf-life stability of Asparagopsis bromoform in oil and freeze-dried powder. Journal of Applied Phycology, 1-9).

[0069] The A. taxiformis used in the composition may be dried and / or pulverized to form a powder. Drying the A. taxiformis biomass in a mostly intact form or in a crushed form helps to facilitate further processing or use of the biomass in the composition. Drying refers to removing free or surface moisture / water from a mostly intact biomass, or removing surface water from a homogenized (e.g., by micronization) biomass slurry. In one embodiment, the A. taxiformis biomass is drum-dried to a flake form to produce A. taxiformis flakes. In another embodiment, to produce A. taxiformis powder, the A. taxiformis biomass is spray-dried or flash-dried (i.e., subjected to an air-drying process) to form a powder containing mostly intact cells. In a further embodiment, the A. taxiformis biomass is micronized (homogenized) to form a crushed material mainly of dissolved cells, which is then spray-dried or rapid-dried to produce A. taxiformis powder.

[0070] A sixth embodiment provides a veterinary supplement comprising an effective amount of the composition of the fifth embodiment, the veterinary supplement comprising at least one of the following: A. taxiformis tetrasporophytes of the third embodiment, A. taxiformis gametophytes of the fourth embodiment, or extracts thereof. As will be understood by those skilled in the art, an effective amount means the amount necessary to reduce methane emissions from an animal consuming the supplement. Studies have shown that supplementing the diet with 0.2% to 2% A. taxiformis reduces methane emissions from ruminants by 45% to 98% (Kinley et al., 2016; Li et al. 2016; Machado et al., 2016a,b; Kinley et al., 2020; Roque et al., 2021; Stefenoni et al., 2021). The amounts of the components in animal nutritional supplements can be easily determined by those skilled in the art based on these studies.

[0071] Accordingly, a seventh embodiment provides a method for reducing methane emissions in ruminants, the method comprising administering an effective amount of the animal nutritional supplement of the sixth embodiment to a ruminant, or an effective amount of the composition of the fifth embodiment to a ruminant. In some embodiments, the effective amount is 0.02% to 3% of the dry weight of the ruminant's diet. In specific embodiments, the effective amount is 0.02%, 0.05%, 0.1%, 0.5%, 1%, 1.5%, or 2% of the dry weight of the ruminant's diet. Those skilled in the art can formulate appropriate dosage forms and administration plans derived from published literature, e.g., WO2015109362A2. [Examples]

[0072] method

[0073] 1. Methods for producing and germinating carpospores

[0074] 1.1. Methods for producing carpospores

[0075] Carposporophytes of A. taxiformis with mature cystic fruits were collected by diving and snorkeling in Ablohos and the Rat Islands, Western Australia, on May 23, June 13, and June 20, 2022 (preliminary induction tests were conducted on May 23 and June 13, 2022, and the full experiment was conducted on June 20, 2022). The algae were kept in fresh seawater at the same temperature as the collection site. They were then transferred to a hatchery within 4 hours. The induction step was performed within 24 hours. Forty branches (20-25 cm) containing 30 mature cystic fruits (the cystic bodies are shown in pink to red) were carefully selected, and after removing any visible particles or organisms, they were washed with sterile seawater (Figure 2). These clean branches were distributed into six white containers (430 × 322 × 127 mm). Next, all containers were placed in the induction system to produce a large quantity of carpospores. Sterilized seawater was maintained at a harvest temperature of 22±0.5℃ throughout all of the above activities.

[0076] The induction system was constructed using a black steel rack with shelves (40cm deep x 90cm wide x 180cm high). Lighting intensity 140-150, 80 μmol·m -2 ·s -1 A 36W / 830 LumiLux white OSRAM 120cm (Apogee Instruments UnderwaterQuantumFlux) was mounted on top of the induction vessel. During the induction phase, the seawater temperature was controlled to 18±0.5℃, 22±0.5℃, or 24±0.5℃.

[0077] Three branches, each containing 30 mature cysts, were placed in six plastic petri dishes (150 mm in diameter), and the number of carpospores was estimated. The six petri dishes were placed in water at 18, 22, or 24°C. From 18 to 30 hours after induction, the algal branches from the petri dishes were periodically checked under a microscope for carpospore release. Empty cysts were counted under a microscope (5x magnification).

[0078] Three follow-up induction cycles were conducted at different points in time each year. In the first experiment, the seawater temperature at harvest was 18°C. During the induction phase, the temperature was increased at a rate of 1°C per hour to 22±0.5°C. In the second experiment, the seawater temperature at harvest was 22°C. During the induction phase, the temperature was increased at a rate of 1°C per hour to 26±0.5°C. In the third experiment, the seawater temperature at harvest was 22°C. The temperature was initially lowered to 18°C ​​over 16 hours, and then during the subsequent induction phase, the temperature was increased at a rate of 1°C per hour to 22±0.5°C.

[0079] Hatching rate of cysts (%) = (Number of hatched cysts / Number of mature cysts) × 100

[0080] The ratio of carpospores to cysts = number of carpospores / number of mature cysts

[0081] Ratio of carbospores per hatching sac = Number of carbospores / Number of hatching sacs

[0082] 1.2. Method for germinating carpospores

[0083] For germination, all branches from the above carpospore induction step were removed from the white container and petri dish. The carpospores attached to the bottom of the white container and petri dish were rinsed with sterile seawater at a water temperature of 18, 22, or 24°C, respectively. The carpospores were incubated in water at 18, 22, and 24°C at 140-150°C, yielding 80 μmol·m³. -2 ·s -1 The samples were kept under constant light intensity. Germination was observed 24 hours after the release of the first carpospores in the induction step described above, and again for 48–72 hours thereafter. The carpospores were checked under a microscope, and signs of germination were recorded. Signs of germination were indicated by the emergence of a germ tube (i.e., the tip of the arrow) from the carpospore (Figures 3 and 4). The germination rate was then calculated as follows:

[0084] Germination rate (%) = (Number of germinated carbospores / Total number of carbospores) × 100

[0085] Ratio of germinated carpospores to cysts = germinated carpospores / mature cysts

[0086] 2. Method of producing the gametophyte

[0087] 2.1. Tetrasporophyte material

[0088] The tetrasporophytes were induced from the carposporophyte stage, under 24-hour illumination, half the concentration of PES, and 60 μmol·m³. -2 ·s -1 Under the conditions of light intensity, salinity of 36.5 ppt, and pH: 8.4-8.5, UV-treated seawater was used and cultured in 5L bottles at 22°C ± 0.5 for 2 months to mass-produce the species. After 2 months, the tetrasporophytes were grown under the same conditions, but the light intensity was reduced to 12 hours of light and 12 hours of dark (12 light / 12 dark). To produce the gametophyte stage, tetrasporophytes at 4 months of age were selected.

[0089] 2.2. System design for producing gametophytes of A. taxiformis

[0090] The experiment was set up to determine the environmental conditions for photoperiod and trophic levels necessary for gametophyte production from tetrasporophytes. A 30W Philips fluorescent lamp mounted on top of the culture flask was used, with a temperature of 60 μmol·m³. -2 ·s -1 Four aquaculture cabinets (60cm wide x 120cm long x 60cm wide) were designed to maintain the required lighting intensity. The aquaculture plants were supplied with normal, standard air from an air generator.

[0091] Water temperature and air temperature were controlled to 24°C and 22±0.5°C, respectively. Before cultivation, seawater was filtered through a 0.2 μm filter and treated with ultraviolet light. The salinity was maintained at 36.5 ppt, and the pH was 8.4-8.5.

[0092] This study was conducted to investigate gametophyte production under different photoperiods and trophic levels. Photoperiods were set to 8 hours of light and 16 hours of dark (8 light / 16 dark), 12 light / 12 dark, 16 light / 8 dark, and 24 dark. Tropical levels were tested with half-concentration PES (10 ml / L) and full-concentration PES (20 ml / L) (Redmond et al., 2014).

[0093] Tetrasporophytes were harvested from a 5L glass bottle culture system. First, excess water was removed using tissue paper, and then the tetrasporophytes were weighed and recorded as initial weights. The tetrasporophytes were distributed into 24 flasks (250ml flasks) at a density of 0.4g / L each. The experiment was conducted for 9 days.

[0094] 2.3. Sample Sampling and Analysis

[0095] At the start of the experiment, the tetrasporophytes were photographed and carefully examined under a microscope to confirm that they were healthy and free from contamination.

[0096] Gametophyte production under different photoperiods and trophic levels

[0097] The presence of young gametophytes from each cultured plant was recorded, the number of young gametophyte thalli was counted, and photographs were taken under a microscope (5x magnification).

[0098] Performance of tetrasporophytes under different photoperiods and trophic levels

[0099] The weight of the tetrasporophytes was measured at the start and end (day 9) of the experiment. Performance was evaluated as follows.

[0100] Biomass increase (g) = W F -W I

[0101] SGR(% / ngay)=[(LnW F -LnW I ) / t]×100

[0102] WG(%) = [(W F - WL I ) / W I × 100

[0103] Yield / m 3 (g) = WG% × W s

[0104] In the formula, SGR: specific growth rate; WG: weight gain; W F : final weight; W I : initial weight; Ws: initial weight of the algae placed per 1 m 3 is the initial weight of the algae placed per 1 m.

[0105] 3. Statistical analysis

[0106] Statistics were calculated using the Statistical package for Windows (SPSS) (version 22, IBM Corp., Armonk, NY, USA). To ensure a normal distribution, Levene's test for equality of variances was used to evaluate the homogeneity of variances between the means, and an independent samples t-test was used to compare the cystocarp hatching rate, the ratio of carpospores to cystocarps, the ratio of carpospores to hatched cystocarps, the germination rate (%), and the ratio of germinated carpospores / cystocarps at 22 °C and 24 °C.

[0107] To evaluate the effects of nutrient levels and photoperiod on gametophyte production and tetrasporophyte performance, the data were analyzed using two-way analysis of variance. When significant main effects were observed, Tukey's HSD post hoc test was used to detect significant differences between treatment means. A significance level of p < 0.05 was used for all statistical tests. All values are presented as mean ± standard error of the mean.

[0108] Results

[0109] 3.1. Induction of carpospores

[0110] The induction method was successfully repeated three times at a water temperature of 22 °C on May 23, 2022 and June 13, 2022, and at water temperatures of 18, 22, and 24 °C on June 20, 2022. Consistent results were observed.

[0111] The cysts began releasing utricles after 18 hours at a water temperature of 24°C or after 24 hours at a water temperature of 22°C. The cysts did not release utricles at 18°C. Utricles were observed in all white containers and all Petri dishes. There were no significant differences between the two temperatures, 22°C and 24°C, in terms of cyst hatching rate, utricle-to-utricle ratio, and utricle-to-hatched cyst ratio (Table 1). The cyst hatching rate ranged from 5.92±0.74% to 6.67±0.64%. [Table 1]

[0112] The results obtained on May 23, 2022 and June 13, 2022 were consistent with the results obtained on June 20, 2022. However, temperature deviations led to spore death. In the May 23, 2022 method, spores were transported by air, causing the temperature to drop to approximately 8°C. All spores died. In the June 13, 2022 method, the temperature in the growth chamber dropped to 16°C overnight. All spores died.

[0113] Through three follow-up guidance sessions, we successfully induced the cystic carbos to release carpospores.

[0114] 3.2. Germination of carpospores

[0115] The first signs of germination were observed 18 and 22 hours after the release of carpospores, respectively, at water temperatures of 24°C and 22°C. No significant differences were observed in germination rate or the germinated carpospore / carp ratio between 22°C and 24°C water temperatures (Table 1). The germination rate ranged from 60.53±9.06% to 70.33±1.40%. [Table 2]

[0116] 3.3. Performance of tetrasporophytes under different photoperiods and trophic levels

[0117] The performance of the tetrasporophytes was photoperiod-dependent, but there were no significant differences in growth between treatments induced by different trophic levels (Table 2). No interaction was found between trophic level and photoperiod.

[0118] The biomass increase of tetrasporophytes under 24 hours of light was significantly higher than that of those cultured under 8 hours of light. However, there was no significant difference in tetrasporophyte biomass increase between 24-hour, 16-hour, and 12-hour photoperiods. Healthy A. taxiformis tetrasporophytes under 8 hours of lighting per 24 hours are shown in Figure 5. The highest growth rate of tetrasporophytes was recorded from the 24-hour lighting treatment, but the cultured plants visually lost their red color by the end of the experiment (Figure 6).

[0119] 3.4 Gametophyte Production under Different Photoperiods and Tropical Levels

[0120] Tetrasporophytes cultured under an 8-hour light / 16-hour dark cycle produced significantly more gametophytes than those obtained under other treatments, with gametophytes observed in 5 out of 6 cultured plants. The number of gametophyte thalli was also significantly higher in those grown under the 8-hour light / 16-hour dark photoperiod compared to those grown under other photoperiods (p<0.05). Figure 7 shows young gametophytes of A. taxiformis obtained from the 8L / 16D treatment under 5x magnification. Interestingly, in this study, gametophyte thalli were observed within 9 days of cultivation, but tetrasporangial development and the presence of tetraspores were not observed (Figure 8). This finding helps to significantly reduce the time required for gametophyte production. Young gametophytes are shown in Figure 9.

[0121] Consideration

[0122] Methods for inducing mass production of carpospores

[0123] The production period of carposporophytes and the maturation of cysts for carpospore production led to the production of tetrasporophytes for use in aquaculture systems. In the wild, the season for carposporophyte development is geographically dependent, but generally, they become abundant and mature in the fall. According to the inventors' records, carposporophytes were first observed in March 2022 in the Abrojos Islands, Western Australia, but the cysts were small and still developing. The second collection was made on May 19, 2022, and some cysts had turned red, indicating maturity. The third and fourth observations were made on June 13 and June 19, 2022, respectively, and more mature cysts were found. According to the aquaculture manager of the Abrojos Islands, some cysts were still observed in September 2022, but their color had faded, indicating the end of the season.

[0124] In species other than A. taxiformis, carpospores in algae have been induced by several methods, including drying mature germ thalli (Andersen 2005; Avila et al. 2011) or moving thalli to a high-light-intensity location after placing them in complete darkness (Andersen 2005). However, information on carpospore induction in A. taxiformis is limited. In this study, the induction method involved exposing mature cysticates to high-light-intensity conditions at water temperatures of 22°C and 24°C, which resulted in mass production of carpospores and ultimately provided a large quantity of tetrasporophytes.

[0125] Based on our observations, the hatching rate and the number of carpospores depended on the degree of maturation of the cystocarp. The higher the degree of maturation, the higher the hatching rate and the greater the number of carpospores. As described above, pink to red indicates maturation.

[0126] Methods for germinating cartilage spores

[0127] The carpospore germination process has been described for A. armata and other red algal species (Bonin and Hawkes, 1987; Orduna-Rojas and Robledo, 1999; Oza and Krishnamurty, 1967, Oza 1975). However, information on carpospore induction methods is limited for A. taxiformis. This study was conducted to test the effect of water temperatures of 22°C and 24°C on carpospore germination rates. Based on our preliminary trials, germination was recorded at 22°C and 24°C, but not at 18°C. Germination time was shorter at higher water temperatures.

[0128] Method for mass production of gametophytes

[0129] In this study, the source of tetrasporophytes used for gametophyte production originated from a cultivation system that continued from the previous carpospore induction and germination steps. Gametophytes are produced when the tetrasporophytes are approximately 4 months old. Generally, Asparagopsis species are expected to develop through the tetrasporangial stage, and when these are induced by a combination of photoperiod and water temperature, tetraspores are released. It takes 4-8 weeks for tetraspores to be released and germinate (Oza 1977; Luening 1981; Rojas et al. 1982, Guiry and Dawes 1992; Ni Chualain et al. 2004). However, in this study, while gametophyte thalli were observed within 9 days of cultivation, tetrasporangial development and the presence of tetraspores were not observed. Similar observations were reported by Ni Chualain et al. in A. taxiformis from Rottnest Island (Western Australia) (2004).

[0130] References

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[0133] Avila, M. et al., 2011. Cultivation of the red alga Chondracanthus chamissoi: sexual reproduction and seedling production in culture under controlled conditions. Journal of Applied Phycology 23:529-536.

[0134] Baceninaite, D. et al., 2022. Global warming and dairy cattle: How to control and reduce methane emission Animals, 12 (19), 2687.

[0135] Bonin, D. R. et al., (1987). Systematics and life histories of New Zealand Bonnemaisoniaceae (Bonnemaisoniales, Rhodophyta): I. The genus Asparagopsis. New Zealand Journal of Botany, 25(4), 577-590.

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[0151] Ni Chualain FN et al., (2004) The invasive genus Asparagopsis (Bonnemaisoniaceae, Rhodophyta): molecular systematics, morphology, and ecophysiology of Falkenbergia isolates. J Phycol 40:1112–1126

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[0163] The method disclosed herein is industrially applicable because it can enable the production and germination of carpospores, which can lead to rapid mass production of tetrasporophytes. This method can also provide high-quality and abundant carpospores, as well as high germination rates, for use in naturally induced methods. Furthermore, this method can enable the rapid production of a large number of gametophytes. Since the production equipment is not complex and labor requirements are low, the implementation of this method can be carried out with low input costs.

[0164] No reference to prior art in this specification acknowledges, or suggests in any way, that such prior art forms part of common general knowledge, and should not be construed as such.

[0165] As used herein and in the subsequent claims, the terms “comprise” and “include,” and their derivatives (e.g., “comprises,” “comprising,” “includes,” and “including”) are to be understood as encompassing the features referred to by those terms, and, unless otherwise stated or implied, do not mean to exclude the presence of additional features.

[0166] In some cases, multiple features may be combined in a single embodiment for the sake of brevity and / or to aid in understanding the scope of the disclosure. In such cases, these multiple features may be provided separately (in separate embodiments) or in any other suitable combination. Alternatively, if distinct features are described in a distinct embodiment, these distinct features may be combined in a single embodiment unless otherwise stated or implied. This also applies to claims that can be recombined in any combination; that is, a claim may be modified to include features defined in any other claim. Furthermore, the phrase "at least one" in a list of items refers to any combination of these items, including a single member. As an example, "at least one of a, b, or c" is intended to cover a, b, c, ab, ac, bc, and abc.

[0167] Those skilled in the art will understand that this disclosure is not limited to use for the specific(s) described. This disclosure is also not limited to preferred embodiments with respect to certain elements and / or features described or illustrated herein. This disclosure is not limited to the(s) disclosed embodiments and will be understood to be capable of various reconfigurations, modifications, and substitutions without departing from the scope described and defined in the following claims.

Claims

1. A method for producing at least one tetrasporophyte of Asparagopsis taxiformis, Obtain a carposporophyte of A. taxiformis with at least one mature cystic, To induce at least one carpospore to be released from the at least one cystocarp, the carposporophyte is exposed to an amount of approximately 80 to approximately 170 μmol·m over a first timed exposure period ranging from approximately 10 to approximately 48 hours. －２ ・s －１ The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. To induce the at least one carpospore to germinate and produce the at least one tetrasporophyte, the at least one carpospore is exposed to an amount of approximately 80 to approximately 170 μmol·m over a second timed exposure period ranging from approximately 10 to approximately 72 hours. －２ ・s －１ The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. The method, including the method described above.

2. The method according to claim 1, wherein the duration of the illumination during either or both of the first and second time exposure periods is approximately 16 to approximately 24 hours per 24-hour period.

3. The method according to claim 2, wherein the duration of the illumination in either or both of the first timed exposure period and the second timed exposure period is substantially continuous.

4. The illumination intensity during either or both of the first and second time-delayed exposure periods is approximately 120 to approximately 170 μmol·m －２ ・s －１ The method according to any one of claims 1 to 3, which is within the range of claims 1 to 3.

5. The illumination intensity during either or both of the first and second time-delayed exposure periods is approximately 140 to approximately 150 μmol·m －２ ・s －１ The method according to any one of claims 1 to 4, which is within the range of claims 1 to 4.

6. The method according to any one of claims 1 to 5, wherein the culture temperature during either or both of the first time-delayed exposure period and the second time-delayed exposure period is in the range of about 21 to about 25°C.

7. The method according to any one of claims 1 to 6, wherein the culture temperature during either or both of the first time-delayed exposure period and the second time-delayed exposure period is in the range of about 22 to about 24°C.

8. The method according to any one of claims 1 to 7, further comprising pre-culturing, which includes culturing the carpospore bodies at a temperature adjusted from a first temperature to the temperature of the first time-limited exposure period.

9. The method according to claim 8, wherein the first temperature is in the range of about 17 to about 23°C.

10. The method according to claim 8 or 9, wherein the temperature during the pre-culture is adjusted to a range of about 1 to about 8°C.

11. The method according to any one of claims 8 to 10, wherein the temperature during the pre-culture is adjusted at a rate of about 0.25 to about 3°C ​​per hour.

12. A method for producing at least one gametophyte of Asparagopsis taxiformis, To obtain the tetrasporophytes of A. taxiformis, To induce the growth of at least one gametophyte thallus, the tetrasporophyte is cultured at a temperature in the range of about 20 to about 26 °C while being exposed to illumination having an intensity in the range of about 30 to about 100 μmol·m －２ ·s －１ for a limited exposure period in the range of about 5 to about 30 days. Optionally, before inducing the growth of the at least one gametophyte thallus, first induce the formation of a tetrasporangium containing tetraspores, induce the tetrasporangium to release the tetraspores, and induce the tetraspores to germinate and produce the at least one gametophyte thallus. The method, including the method described above.

13. The method according to claim 12, wherein the duration of the illumination during the timed exposure period is in the range of about 6 to about 18 hours per 24 hours.

14. The method according to claim 13, wherein the duration of the illumination during the timed exposure period is in the range of about 7 to about 16 hours per 24 hours.

15. The illumination intensity during the aforementioned timed exposure period is approximately 40 to approximately 80 μmol·m －２ ・s －１ The method according to any one of claims 12 to 14, which is within the range of claims 12 to 14.

16. The illumination intensity during the aforementioned timed exposure period is approximately 50 to approximately 70 μmol·m －２ ・s －１ The method according to any one of claims 12 to 15, which is within the range of claims 12 to 15.

17. The method according to any one of claims 12 to 16, wherein the temperature during the timed exposure period is in the range of about 21 to about 25°C.

18. The method according to any one of claims 12 to 17, wherein the temperature during the timed exposure period is in the range of about 22 to about 24°C.

19. The method according to any one of claims 12 to 18, wherein the timed exposure period is in the range of about 6 to about 21 days.

20. The method according to any one of claims 12 to 19, further comprising first inducing the formation of the tetrasporangium containing the tetraspores, inducing the tetrasporangium to release the tetraspores, and inducing the tetraspores to germinate and produce the at least one gametophyte thallus, before inducing the growth of the at least one gametophyte thallus.

21. The method according to any one of claims 12 to 20, wherein the tetrasporophyte is produced by the method according to any one of claims 1 to 11.

22. The at least one tetrasporophyte is brought into a state where it can produce the at least one gametophyte. To increase the biomass of the at least one tetrasporophyte, the at least one tetrasporophyte is exposed to a third time-limited exposure period of approximately 30 to 90 days, ranging from approximately 16 to 24 hours per 24 hours, with an exposure of approximately 30 to 100 μmol·m³. －２ ・s －１ The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. To develop the at least one tetrasporophyte into a state capable of producing the at least one gametophyte, the at least one tetrasporophyte is exposed to an exposure of approximately 30 to 100 μmol·m over a fourth time-limited exposure period ranging from approximately 8 to 16 hours per 24 hours for approximately 30 to 90 days. －２ ・s －１ The culture is performed at a temperature in the range of approximately 20 to 26°C while being exposed to illumination of an intensity within the range of [specify range]. The method according to claim 21, wherein development is carried out by

23. A tetrasporophyte of Asparagopsis taxiformis produced by the method described in any one of claims 1 to 11.

24. A gametophyte of Asparagopsis taxiformis produced by the method described in any one of claims 12 to 22.

25. A composition comprising the tetrasporophyte of A. taxiformis as described in claim 23, the gametophyte of A. taxiformis as described in claim 24, or an extract(s) of the tetrasporophyte or gametophyte containing one or more halogen compounds.

26. A nutritional supplement for animals comprising an effective amount of the composition described in claim 25.

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