Environmental stress tolerance enhancer for aquatic animals, feed containing the same, and method for using the same.

Abstract

The objective is to provide formulations or feeds that improve the tolerance of aquatic animals to environmental stress, and to provide methods for raising aquatic animals using these. [Solution] An environmental stress tolerance enhancer for aquatic animals is provided, containing a predetermined nucleic acid as an active ingredient. The weight-average molecular weight of the nucleic acid is in the range of 5,000 to 50,000, and the absorbance of the nucleic acid at a wavelength of 420 nm is 3.0 or less. This environmental stress tolerance enhancer is incorporated into a feed for aquatic animals. These agents or feeds are administered to raise or manage the growth of aquatic animals.

Description

Technical Field

[0001] The present disclosure relates to an environmental stress tolerance enhancer for aquatic animals and its applications. More specifically, it relates to an environmental stress tolerance enhancer for aquatic animals, a feed containing the environmental stress tolerance enhancer for aquatic animals, a method for raising aquatic animals using the agent or the feed, a method for growth management of aquatic animals using the agent or the feed, and the like.

Background Art

[0002] With the global population increase, the demand for food has been growing, and from the perspective of realizing a sustainable society, a stable supply of highly productive fishery resources is required. In order to stably supply animals that become fishery resources (hereinafter also referred to as "aquatic animals"), it is necessary to conduct aquaculture. However, when using natural water sources such as the sea or rivers during aquaculture, it is not preferable because the environmental load increases and the installation location of the aquaculture facility is restricted. Therefore, in recent years, so-called land-based aquaculture, in which aquatic animals are cultured in an artificially created breeding environment, has been carried out. Among them, closed-loop land-based aquaculture, in which the breeding water is circulated and used in a closed system, can avoid problems such as environmental pollution caused by residual feed and excrement during aquaculture, and is regarded as a promising next-generation aquaculture method. Closed-loop land-based aquaculture enables the installation of aquaculture facilities even in locations far from natural water sources, which is particularly significant in inland areas.

[0003] One of the problems in the aquaculture business of aquatic animals is various stresses imposed on individuals when raising aquatic animals. For example, in order to increase productivity, it is necessary to increase the stocking density, but at the same time, it may lead to an increase in infectious diseases or stress derived from the fact that individuals are in close proximity may be imposed on the individuals.

[0004] To address the aforementioned challenges, various anti-stress agents have been considered. The most common are antibiotics against pathogens, which are widely used in aquaculture. However, the overuse of antibiotics can lead to the emergence of antibiotic-resistant bacteria, and numerous cases of harm have been reported. Furthermore, antibiotics can remain and accumulate in the bodies of aquatic animals, and depending on the type of antibiotic, there is a possibility of adverse effects on human organs. From a reputational standpoint, it is preferable to avoid the use of antibiotics altogether, and if their use is unavoidable, it should be kept to a minimum.

[0005] Various anti-stress agents are being investigated for environmental stresses other than those caused by pathogens. For example, Patent Document 1 describes a feed for improving the environmental stress tolerance of aquatic animals, used for fish and crustaceans that live in water, with zerumbon as the active ingredient. Patent Document 2 also describes that polysaccharide components obtained by separating or removing lipid-soluble components from silkworm pupae can act as stress relievers. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 6855157 [Patent Document 2] Japanese Patent Publication No. 2022-159199 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] In aquaculture, especially land-based aquaculture, the scale is smaller compared to natural water sources, which means that aquatic animals may be subjected to unexpected or difficult-to-control stresses, such as increased nitrates due to residual feed or fluctuations in pH. Furthermore, because the system is small, the rearing environment is prone to rapid changes, and all of the above stresses can cause sudden shocks to aquatic animals, leading to more severe damage.

[0008] Furthermore, in land-based aquaculture, because the scale is smaller compared to natural water sources, there is a possibility of sudden increases or decreases in water temperature due to unexpected problems. Crustaceans such as shrimp, in particular, are more delicate than fish and are more susceptible to environmental stress such as water quality and stocking density, and may die even without any sudden problems. Among them, decapoda (shrimp order), represented by the family Penaeidae, originate from high-temperature regions and are vulnerable to fluctuations in water temperature, especially drops in water temperature, making land-based aquaculture difficult until now. In order to solve the above problems, various water quality data are currently being managed, but even with these measures, the mortality rate has not been sufficiently reduced.

[0009] As mentioned above, although feeds and stress relievers that improve tolerance to environmental stress have been developed, none of them have been able to cope with rapid environmental changes, such as a 10-degree Celsius rise in water temperature in one hour, or direct transfer from optimal temperature rearing water to cold water. For example, in order to maintain the freshness of aquatic animals, it is common practice to immerse them in cold water immediately after harvesting to induce a dormant state. However, species that are sensitive to drops in water temperature, such as shrimp, experience extraordinary stress during this procedure, and there is a risk that they may die and their freshness will deteriorate.

[0010] In light of the above circumstances, one of the challenges we aim to address is to provide formulations or feeds that improve the tolerance of aquatic animals to environmental stress. More specifically, one of the problems we aim to solve is to provide formulations or feeds that improve the tolerance of aquatic animals to cold stress. Another challenge we aim to address is providing methods for appropriately raising or managing aquatic animals even under environmental stress conditions. [Means for solving the problem]

[0011] Through repeated studies to address the above-mentioned problems, the inventors discovered that by adding a specific nucleic acid component, the effects of various environmental stresses on aquatic animals are mitigated, and the survival rate of aquatic animals is improved.

[0012] The invention presented in this disclosure can be understood in multiple aspects and forms, and may include, for example, the following embodiments as means for solving the problem. In this disclosure, the invention presented in this disclosure is also referred to simply as "the present invention," either conceptually or according to each individual form.

[0013] [1] An environmental stress tolerance enhancer for aquatic animals, comprising nucleic acid as an active ingredient, wherein the weight-average molecular weight (Mw) of the nucleic acid is in the range of 5,000 to 50,000, and the absorbance of the nucleic acid at a wavelength of 420 nm is 3.0 or less. [2] The environmental stress tolerance enhancer according to [2] above, wherein the nucleic acid is ribonucleic acid derived from yeast. [3] The environmental stress tolerance enhancer according to either item [1] or [2] above, wherein the aquatic animal is an arthropod. [4] The environmental stress tolerance enhancer described in [3] above, wherein the arthropod is an animal belonging to the crustaceans. [5] The environmental stress tolerance enhancer described in [4] above, wherein the crustacean is of the family Penaeidae. [6] A feed for aquatic animals, comprising an environmental stress tolerance enhancer as described in any one of the above items [1] to [5]. [7] A method for raising aquatic animals in a closed-circulation rearing facility, comprising providing an environmental stress tolerance enhancer described in any one of the above items [1] to [5] or the feed described in the above item [6]. [8] A method for managing the growth of aquatic animals, comprising placing aquatic animals raised with an environmental stress tolerance enhancer described in any one of the above items [1] to [5] or the feed described in [6] above under conditions of a water temperature of 15°C or lower. In this disclosure, "methods for raising aquatic animals" shall include aspects of "methods for producing aquatic animals." [Effects of the Invention]

[0014] In one or more embodiments of the inventions presented herein, formulations or feeds that improve the tolerance of aquatic animals to environmental stress can be provided. In one or more embodiments of the inventions presented herein, formulations or feeds that improve the tolerance of aquatic animals to cold stress can be provided. In one or more embodiments of the inventions presented in this disclosure, a method for appropriately rearing and / or managing the growth of aquatic animals even under environmental stress conditions can be provided. Specifically, in one embodiment, the survival rate of aquatic animals can be improved even under conditions of low-temperature stress. [Modes for carrying out the invention]

[0015] Embodiments of the present invention will be described below. In this disclosure, the term "one embodiment" in relation to the present invention refers to any one embodiment used to describe the present invention in detail, unless otherwise specified, and does not negate or limit the existence of other or more embodiments. As shown below, the present invention may have multiple embodiments that fall within its scope. These multiple embodiments may also be provided as modified forms, for example, by various combinations of the components (or technical features) shown in this disclosure. Furthermore, in this disclosure, where the term "embodiment" is used, it includes one or more embodiments unless otherwise specified.

[0016] In this disclosure, unless otherwise specified, the notation "AA~BB" in relation to a numerical range means "AA or greater and BB or less" (where "AA" and "BB" represent any numerical value). Furthermore, unless otherwise specified, the units for both the lower and upper limits are the same as the unit immediately following the latter (i.e., "BB" in this case). In this disclosure, the combination of the lower and upper limits of a numerical range can be any combination of numerical values ​​selected from the set of lower or upper limits provided as examples of preferred values. Also, the expression "X and / or Y" means both X and Y, or either one of them.

[0017] As one embodiment of the present invention, an environmental stress tolerance improver for aquatic animals is provided. In the present disclosure, the environmental stress tolerance improver refers to a preparation used to improve tolerance to environmental stress.

[0018] The environmental stress tolerance improver for aquatic animals, which is one embodiment of the present invention, contains a predetermined nucleic acid as an active ingredient.

[0019] In one embodiment of the present invention, it is desirable that the nucleic acid contained in the environmental stress tolerance improver satisfies the following requirements (A) and (B). (A) The weight-average molecular weight of the nucleic acid is within the range of 5,000 to 50,000. (B) The absorbance of the nucleic acid at a wavelength of 420 nm is 3.0 or less.

[0020] The nucleic acid that can be used in the present invention needs to be a nucleic acid as a polymer, and a molecule having a certain molecular weight is preferable. As an index of the molecular weight of a preferable nucleic acid, the weight-average molecular weight (Mw) may be used. The weight-average molecular weight (Mw) of the nucleic acid is preferably 5,000 to 50,000, and more specifically as follows. The lower limit of the weight-average molecular weight (Mw) of the nucleic acid may preferably be 5,000 or more, more preferably 7,000 or more, and still more preferably 10,000 or more. The upper limit of the weight-average molecular weight (Mw) of the nucleic acid may preferably be 100,000 or less, more preferably 70,000 or less, and still more preferably 50,000 or less. In addition, the molecular weight distribution of the nucleic acid can be determined by using a general method in the technical field of the present disclosure. For example, it can be determined by GPC (gel permeation chromatography) or the like.

[0021] Using nucleic acids with a weight-average molecular weight (Mw) within the above range contributes to improving stress tolerance in aquatic animals. It is presumed that after aquatic animals ingest a specific nucleic acid, a favorable reaction is triggered in their bodies, alleviating the stress response. Although the detailed mechanism is unknown, it is possible that nucleic acid molecules identified by the above weight-average molecular weight can stimulate organs and cells within the organism, thereby exerting effects such as alleviating the stress response.

[0022] Furthermore, in one embodiment of the present invention, it is desirable that the nucleic acid used exhibits a relatively low absorbance at wavelengths corresponding to the violet range of visible light. Absorbance (OD) may be used as an indicator of a suitable nucleic acid, and it is preferable to observe the absorbance at wavelengths of 400 nm to 450 nm, and more specifically, the absorbance at a wavelength of 420 nm.

[0023] The OD420 value of the nucleic acid that can be used in the present invention is preferably 3.0 or less. More details are as follows. The upper limit of the OD420 value of nucleic acids is preferably 3.0 or less, more preferably 2.5 or less, and even more preferably 2.0 or less. The lower limit of the OD420 value of nucleic acids does not necessarily have to be clearly specified by a numerical value, but if it were to be specified, it is preferably 0.01 or higher, more preferably 0.03 or higher, and even more preferably 0.05 or higher.

[0024] Using nucleic acids with OD420 values ​​that meet the above requirements contributes to improving the stress tolerance of aquatic animals. Among the elements that make up nucleic acids, the base components absorb the above wavelength range of visible light, but the absorption rate differs depending on the structure of the base components. Therefore, it is presumed that by having an OD420 value of nucleic acid that is below the above value, the ratio of base components constituting the nucleic acid becomes appropriate, and the effects expected of the present invention are fully realized.

[0025] Absorbance (OD) can be measured using methods common in the art of this disclosure. Specifically, the sample used for measuring absorbance (OD) can be prepared by the methods shown in the "Examples" section below.

[0026] The type of sugar constituting nucleic acid may be either deoxyribose or ribose. That is, nucleic acid may be either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The types of bases constituting nucleic acid mainly include adenine, guanine, thymine, cytosine, and uracil, and that is, the types of nucleosides constituting nucleic acid include adenosine, guanosine, cytidine, uridine, and thymidine. The phosphate constituting a nucleotide may be monophosphate or composed of multiple phosphate groups. Commercially available nucleic acids may be used. Nucleic acids may be formulated individually or as a mixture of multiple types. Preferably, ribonucleic acid can be used as the nucleic acid.

[0027] There are no particular restrictions on the origin of nucleic acids; they may be artificially synthesized or derived from natural products. For example, they may be extracted or purified from microorganisms such as yeast. The nucleic acids synthesized, extracted, or purified in this way can be made into a form that is easily absorbed when ingested by the aquatic animals to which they are administered. By cultivating microorganisms such as yeast using biological resources that have been discarded, such as wood sugars contained in discarded wood, to obtain nucleic acids, and incorporating them into feed compositions, it is possible to transform waste materials into useful substances and contribute to the formation of a sustainable circular society.

[0028] Examples of spore-forming yeasts include yeasts of the genera Shizosaccharomyces, Saccharomyces, Kluyveromyces, Hansenula, Pichia, Debaryomyces, and Lipomyces. More specifically, these include Shizosaccharomyces pombe, Shizosaccharomyces octosporus, Saccharomyces cerevisiae, Saccharomyces uvarum, and Saccharomyces lucii. Examples include *rouxii*, *Kluyveromyces fragilis*, *Kluyveromyces lactis*, *Hansenula anomala*, *Pichiamembranaefaciens*, *Debaryomyces hansenii*, and *Lipomyces starkeyi*.

[0029] Examples of non-sporeless yeasts include yeasts of the genera Torulopsis, Candida, and Rhodotorula. More specifically, these include Torulopsis versatilis, Candidatropicalis, Candida lipolytica, Candida utilis, and Rhodotorula glutinis. Yeasts of the genus Candida are also taxonomically known as torula yeast and are sometimes classified as yeasts of the genus Cyberlindnera.

[0030] Preferred yeasts that can be used include, for example, brewer's yeast, wine yeast, baker's yeast, Torula yeast, and more specifically, Saccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces rouxii; Kluyveromyces fragilis, Torulpsis versatilis, Candida tropicalis, Candida lipolytica, Candida utilis, and Rhodotorula glutinis. Furthermore, Candida utilis is sometimes classified taxonomically as a species of Torula yeast, specifically under the name Cyberlindnera jadinii.

[0031] As a method for preparing the nucleic acid, for example, yeast may be subjected to enucleation treatment to separate the nucleic acid from the enucleated yeast, recover the nucleic acid component, and prepare nucleic acid with a molecular weight of 5,000 to 100,000. The nucleic acid prepared in this way can improve the tolerance of aquatic animals to various stresses.

[0032] Nucleation can be performed, for example, by contacting the yeast with an alkaline chemical, saline solution, or cell wall-dissolving enzyme to dissolve the yeast cell wall, eluting the yeast contents into the culture medium, and separating the cell wall components from other components. The separated components may be purified and powdered as needed.

[0033] The alkaline treatment method may be the conventional method used to obtain yeast extracts. That is, an alkaline chemical is used to dissolve or damage part or all of the yeast cell wall so that components within the yeast cell are eluted. Preferred alkaline chemicals include, for example, sodium hydroxide, potassium hydroxide, barium hydroxide, calcium hydroxide, and sodium carbonate.

[0034] As an embodiment of the environmental stress tolerance enhancer for aquatic animals, other components may be added as needed. For example, in another preferred embodiment of the environmental stress tolerance enhancer, sulfites may be further added. Adding sulfites can contribute to oxidation inhibition or inhibition of bacterial growth.

[0035] Furthermore, the environmental stress tolerance enhancer may contain other optional components as needed, such as water, oil, pH adjusters, antioxidants, preservatives, colorants, fragrances, excipients, vitamins, hormones, and amino acids. One preferred embodiment in this disclosure is a form that does not contain antibiotics, antibacterial agents, etc., but in another embodiment, these components may be included as needed.

[0036] Environmental stress tolerance enhancers for aquatic animals are preferably in a form that can be administered into the tanks where the aquatic animals are kept, and can generally be in the same form as additives and feeds used for aquatic animals. Examples of environmental stress tolerance enhancers include powder, granules, mash, pellets, crumble, and flakes. The form of the environmental stress tolerance enhancer may be a single form, or a mixture of two or more of the above forms, for example, a mixture of pellets and flakes, or a mixture of mash and pellets.

[0037] In this disclosure, the term "aquatic animals" refers to animals that can be used as fishery resources. In this disclosure, aquatic animals are not particularly limited as long as they are animals that live in water, and can include various animal species such as fish and crustaceans.

[0038] Examples of fish include saltwater fish such as yellowtail, amberjack, greater amberjack, kingfish, sea bream, tuna, pufferfish, flounder, horse mackerel, mackerel, grouper, longtooth grouper, and salmon, as well as freshwater fish such as sweetfish, char, eel, carp, sea bass, crucian carp, trout, cherry salmon, smelt, goldfish, medaka, tilapia, and sturgeon.

[0039] Examples of arthropods or crustaceans include crabs, shrimp, krill, and water fleas. Examples of crabs include the Japanese mitten crab, snow crab, Japanese spider crab, red snow crab, king crab, hairy crab, Hanasaki crab, and swimming crab. Examples of shrimp include the Pacific white shrimp, kuruma prawn, tiger prawn, white-legged shrimp, spot prawn, sakura shrimp, white shrimp, northern shrimp (sweet shrimp, southern shrimp, red shrimp), and spiny lobster.

[0040] Specific examples of aquatic animals that are preferred targets for application of the environmental stress tolerance enhancer in this disclosure include crustaceans, more preferably decapoda (shrimp), and even more preferably shrimp of the family Penaeidae, and more specifically, Pacific white shrimp.

[0041] In this disclosure, "environmental stress" refers to stress that living organisms experience from the external environment. The environmental stress in this disclosure is not limited in its source, and examples of stress sources include high water temperature, low water temperature, low pH, high pH, ​​osmotic pressure, ammonia, nitrite, inappropriate light exposure, ultraviolet radiation, pathogens, overcrowding of biological populations, narrow, enclosed spaces, contact, and rapid environmental changes.

[0042] One of the unique effects that the environmental stress tolerance enhancer disclosed herein may exert is the mitigation (or reduction) of the effects of stress caused by rapid environmental changes. Examples of rapid environmental changes include rapid increases and decreases in water temperature, rapid fluctuations in pH, and rapid increases in the concentration of chemical substances such as ammonia. In this disclosure, "stress" to aquatic animals can be specifically defined as an external stimulus that reduces the survival rate of aquatic animals, and "rapid stress" can be defined as an external stimulus in which the value of the stressor factor increases or decreases by 30% within one hour.

[0043] Generally, many of the pathways used in stress responses are common, and among them, the synthesis of antioxidant enzymes to protect against oxidation, which is a major cause of stress, and the ATP synthesis pathway for supplying energy used in the overall stress response are activated in common with various types of stress. Examples of the above-mentioned stress responses include ATP synthesis (metabolic activity), unsaturated fatty acid synthesis (energy supply support, hormone regulation, antioxidant effect), and antioxidant enzyme synthesis (protection from oxidative attack by reactive oxygen species). In addition to cold water, it has been confirmed that these reactions are activated when exposed to ammonia and pH stress.

[0044] As described above, a preferred embodiment of the present disclosure is provided, which is a feed composition for aquatic animals containing the above-mentioned environmental stress tolerance enhancer.

[0045] There are no particular restrictions on the form of use of the environmental stress tolerance enhancer for aquatic animals disclosed herein. Examples include directly adding it to the water in which the aquatic animals are reared, introducing aquatic animals into rearing water containing the environmental stress tolerance enhancer to allow them to acclimate, and feeding the aquatic animals a diet containing the environmental stress tolerance enhancer. Preferably, examples include incorporating a predetermined nucleic acid into the diet of aquatic animals.

[0046] The environmental stress tolerance enhancer for aquatic animals disclosed herein may be (1) added to a basic feed and fed as a mixed feed, or (2) prepared separately from other feed components and given separately, and used in combination. In this disclosure, the term "combined use" without any particular limitation is used to encompass both forms (1) and (2) above.

[0047] In this disclosure, the term "basic feed" means feed provided to aquatic animals to provide them with their main nutritional components. Examples of basic feed components containing the main nutritional components for aquatic animals include plant-based feed and / or animal-based feed. Examples of aquatic animal feed include plant-derived feed such as wheat flour and soybean meal, live feed such as fish fillets, fish meal, fish oil, or mixtures thereof.

[0048] When supplying a predetermined nucleic acid, which is the active ingredient of an environmental stress tolerance enhancer, as feed, the amount of nucleic acid included may preferably be 0.01 to 50% by weight of the total feed. More details are as follows. The lower limit of the amount of nucleic acid in the total feed is preferably 0.01% by weight or more, more preferably 0.03% by weight or more, and even more preferably 0.05% by weight or more. By setting the amount of nucleic acid as described above, a feed suitable for increasing the stress tolerance of aquatic animals can be made. While there is no particularly clear upper limit on the amount of nucleic acids included in the overall feed from the standpoint of increasing the stress tolerance of aquatic animals, considering the balance with other components of aquatic animal feed, it is preferably 50% by weight or less, more preferably 30% by weight or less, and even more preferably 20% by weight or less.

[0049] The environmental stress tolerance enhancers for aquatic animals or feeds containing them disclosed herein can be suitably used for rearing aquatic animals under conditions that make them susceptible to environmental stress. Examples of rearing aquatic animals under conditions that make them susceptible to environmental stress include rearing in closed-loop rearing facilities, particularly closed-loop rearing facilities on land. In closed-loop rearing facilities, there is a risk that the rearing environment may change rapidly due to unintended and unexpected external factors, causing severe environmental stress to the reared aquatic animals. The environmental stress tolerance enhancers for aquatic animals or feeds containing them can be used as a preventive measure or preventive agent against such unexpected environmental stress risks.

[0050] Furthermore, the environmental stress tolerance enhancer for aquatic animals or the feed containing the same disclosed herein can also be suitably used when aquatic animals are to be transported and stored at low temperatures after they have grown to a sufficient size. In particular, when it is desired to transport and store aquatic animals alive at low temperatures in order to maintain their freshness, the environmental stress tolerance enhancer or the feed containing the same disclosed herein is suitable for raising aquatic animals with high environmental stress tolerance. In this case, low temperature can be, as one exemplary indicator, a temperature at which aquatic organisms can maintain their freshness without freezing. Such low temperatures can be, for example, higher than 0°C or 1°C, and around 15°C or lower, 13°C or lower, 10°C or lower, 8°C or lower, 5°C or lower, 4°C or 3°C or lower. In particular, when it is planned in the future that aquatic animals will be raised and stored alive at low temperatures of around 15°C to 10°C, it may also be suitable to use it as a feed for growing aquatic animals or as an anti-stress preventive agent for aquatic animals for which such plans are in place. [Examples]

[0051] The present invention will be described in more detail below with reference to examples, but the technical scope (or technical reach) of the invention presented in this disclosure is not limited to the following examples. Numerical values ​​and evaluations related to various shapes, physical properties, and performance (or effects) shown in this disclosure can be determined by the following measurement and evaluation methods.

[0052] <Example A: Preparation of nucleic acid components> A culture solution was prepared by diluting 1 liter of pulverized extract (15% w / v solids) generated from wood chips mainly composed of domestically produced broadleaf trees using sulfurous acid pulverization (150°C, 9 hours). This extract was then diluted with water to a sugar concentration of 3.0%, and a nitrogen source and a phosphorus source (0.1% ammonia, 0.1% monoammonium phosphate) were added.

[0053] Candida utilis was cultured as yeast in the above culture medium for 24 hours. The resulting yeast culture was heated and stirred in a 2% by weight sodium chloride aqueous solution for 2 hours to isolate the nucleic acid component. Hydrochloric acid was added to adjust the pH to 1.4 and stirred for 2 hours, and the resulting precipitate was collected. As a redissolution procedure, sodium hydroxide aqueous solution was added until the pH reached 4.7, and the mixture was heated and stirred for 2 hours. The resulting solution was spray-dried using a spray dryer to obtain the sodium salt of the nucleic acid. The weight-average molecular weight (Mw) of the sodium salt of the nucleic acid was 20,000, and the absorbance (OD420) at a wavelength of 420 nm of the solution dissolved in 3% by weight deionized water was 1.8. Furthermore, the ribonucleic acid content in the obtained sodium salt of nucleic acid was 80% by weight, and the deoxyribonucleic acid content was 1.0% by weight. The obtained nucleic acid component was used as a feed additive (hereinafter also referred to as nucleic acid additive 1).

[0054] <Breeding Experiment 1> <Example 1> A closed-loop land-based aquaculture system was implemented using the materials shown in Table 1. The aquatic animal used was the Pacific white shrimp (Litopenaeus vannamei). Larval Pacific white shrimp with an average weight of 0.002g were raised for one month. Individuals in good condition with minimal stress were selected and placed in three separate rearing tanks, 100 of each, designated as experimental groups 1, 2, and 3.

[0055] For cultivation, we used a mixed water prepared by diluting groundwater drawn from underground in Iwata City, Shizuoka Prefecture, with freshwater drawn from underground in the same city, and adding potassium. The water quality of each component is shown in Table 2.

[0056] For this study, the feed used in land-based aquaculture of Pacific white shrimp (Table 3) was used as the basic feed, and feeding was carried out twice a day during the study period.

[0057] Furthermore, in test plots 1 to 3, the feed was provided with a diet to which nucleic acid additive 1, prepared above, was added as a nucleic acid component, with a total content of 0.1% by weight.

[0058] <Comparative Example 1> The Pacific white shrimp selected according to the above criteria were placed in three separate rearing tanks, 100 shrimp in each tank, and designated as control groups 4-6. These control groups were fed the feed formulation described above (without nucleic acids), i.e., the basic feed described above, and the rearing experiment was conducted in the same manner as in Example 1.

[0059] <Water Quality Testing> During the trial period, water quality tests were conducted using the items and frequencies shown in Table 4. No significant fluctuations were observed in the tested water quality parameters during the trial period.

[0060] [Table 1]

[0061] [Table 2]

[0062] [Table 3]

[0063] [Table 4]

[0064] Based on the above breeding experiment, the following items were tested.

[0065] <Survival rate> The survival rate over 30 days of rearing was compared based on the difference in the number of fish at the start and end of the experiment.

[0066] <Growth rate> Growth was compared based on the average weight at the start of the experiment, after 15 days of rearing, and at the end of the experiment (after 30 days of rearing).

[0067] <Feeding efficiency (FCR)> Based on the standard satiety feeding regimen for Pacific white shrimp in this rearing system, the amount of food consumed was calculated from the amount of leftover food.

[0068] <Stress tolerance> After a 30-day rearing trial, Pacific white shrimp were directly placed into a rearing tank filled with cold water (water temperature 13°C) (water quality and other conditions were the same as in Example 1) and left to stand for 1 hour. Then, the survival rate was compared 14 hours after returning them to the original tank (water temperature 28°C).

[0069] It should be noted that Pacific white shrimp (Litopenaeus vannamei) are originally from tropical regions where water temperatures are around 27°C, and are therefore very vulnerable to cold water stress. Generally, it is believed that Pacific white shrimp begin to lose their internal homeostasis, especially at around 18°C, and that around 13°C is the limit of their stress response to cold water stress, meaning it becomes fatal.

[0070] <Result> The results are shown in Tables 5 and 6 below. Regarding survival rates, no difference was observed between the control group (without nucleic acid additive 1) and the test group (with nucleic acid additive 1). While the control group showed slightly better results in terms of growth and feeding efficiency, the difference was not statistically significant.

[0071] On the other hand, the survival rate after the cold water stress test was better in the experimental group. (Statistically significant difference (P<0.05))

[0072] [Table 5]

[0073] [Table 6]

[0074] In land-based aquaculture, cold water stress can occur due to factors such as a malfunction in the water temperature control system leading to the injection of cooling water, localized drops in water temperature due to the intrusion of outside air during winter, and drops in the controlled water temperature due to human error such as operational mistakes. Because the water temperature drops caused by these factors are generally rapid, especially since the systems are smaller in scale compared to those using natural water sources, there is a high risk of death of tropical aquatic animals such as Pacific white shrimp. However, it has been shown that the effects of death can be mitigated by administering the specified nucleic acid components mentioned above.

[0075] Furthermore, the main biological responses to cold water stress in this study are thought to include energy metabolism abnormalities, oxidative stress, and decreased immunity. Biological reactions that may be activated by these stress responses include ATP synthesis (metabolic activity), unsaturated fatty acid synthesis (energy supply support, hormone regulation, antioxidant effect), and antioxidant enzyme synthesis (protection from oxidative attack by reactive oxygen species). It is possible that the nucleic acid components administered in this study promoted the activation of these biological reactions.

[0076] Stress factors that activate the above-mentioned biological reactions include, in addition to cold water, high water temperature, low water temperature, low pH, high pH, ​​osmotic pressure, ammonia, nitrite, inappropriate light exposure, ultraviolet light, pathogens, excessive density of biological populations, narrow enclosed spaces, contact, and rapid environmental changes. The addition of the specified nucleic acid components mentioned above is expected to improve resistance to these stresses.

Claims

1. An environmental stress tolerance enhancer for aquatic animals, comprising nucleic acid as an active ingredient, wherein the weight-average molecular weight (Mw) of the nucleic acid is in the range of 5,000 to 50,000, and the absorbance of the nucleic acid at a wavelength of 420 nm is 3.0 or less.

2. The environmental stress tolerance enhancer according to claim 1, wherein the nucleic acid is yeast-derived ribonucleic acid.

3. The environmental stress tolerance enhancer according to claim 1, wherein the aquatic animal is an arthropod.

4. The environmental stress tolerance enhancer according to claim 3, wherein the arthropod is an animal belonging to the crustaceans.

5. The environmental stress tolerance enhancer according to claim 4, wherein the crustacean is of the family Penaeidae.

6. A feed for aquatic animals, comprising an environmental stress tolerance enhancer according to any one of claims 1 to 5.

7. A method for raising aquatic animals, comprising providing an environmental stress tolerance enhancer according to any one of claims 1 to 5 or a feed according to claim 6 in a closed-loop breeding facility.

8. A method for managing the growth of aquatic animals, comprising placing aquatic animals raised with an environmental stress tolerance enhancer according to any one of claims 1 to 5 or the feed according to claim 6, under conditions of a water temperature of 15°C or lower.

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