JP2024174428A - Eel larvae feed, eel production method, and agent for inhibiting morphological abnormalities in eel larvae - Google Patents

Abstract

[Problem] To provide feed for eel larvae that can reduce the frequency of appearance of individuals exhibiting morphological abnormalities during the metamorphosis from eel larvae to glass eels by feeding the eels.
[Solution] Nucleic acid-containing feed for eel larvae. The content of the nucleic acid in the total dry matter weight of the feed for eel larvae is preferably 0.25% by dry matter weight or more. The feed for eel larvae may contain a yeast extract, and the yeast extract may contain the nucleic acid.
[Selection diagram] None

Description

The present invention relates to feed for eel larvae, a method for producing eels, and an agent for inhibiting morphological abnormalities in eel larvae.

Currently, glass eels are mainly used as seedlings for eel farming, and farming is carried out using wild-caught glass eels.
However, the catch of wild glass eels has drastically decreased, and there is a need to develop technology for artificially producing glass eels in order to enable stable eel farming while minimizing the impact on wild eels.

The Fisheries Research and Education Agency (formerly the Fisheries Research Agency), to which the inventors belong, became the first in the world to succeed in artificially rearing glass eels in 2002. The journey to this point was extremely difficult, and it took more than 40 years of research to achieve this.
Because the environment and ecology of wild eel larvae were largely unknown, much trial and error was required to develop feed that would enable the growth of eels at the larval stage. In particular, the most difficult hurdle was the process of transforming the larvae (leptocephali) that hatched from the eggs into young fish (glass eels).

Shark egg powder (Patent Document 1) has been proposed as an initial feed for eel larvae, but despite many attempts, no other feed than shark egg powder has been found that has feeding attraction or growth properties for eel larvae. Moreover, even with the aforementioned shark eggs, it was impossible to grow and metamorphose the leptocephali into glass eels.

However, it was later confirmed that eels could metamorphose into glass eels for the first time by feeding them a feed that contained shark eggs as well as krill hydrolysate and/or soybean peptides that had been treated to reduce phytic acid (Patent Document 2). In this way, shark eggs (especially dogfish eggs) have become the main ingredient in feed for eel larvae.

However, it is difficult to obtain a large, stable supply of feed made from natural resources, and there are concerns about the sustainable and stable supply of spiny dogfish eggs in the future due to resource depletion.

The feed that was developed for this purpose is eel larvae feed containing milk protein and chicken egg yolk powder (Patent Document 3). By feeding this feed to eel larvae and raising them, it is possible to metamorphose them into glass eels, making this a promising feed that can be supplied stably and replaces the conventional dogfish egg feed.

Japanese Patent Application Publication No. 11-253111 JP 2005-13116 A JP 2018-153147 A

Eels have a very unique developmental process. Figure 1 explains the developmental process of eels from fertilized eggs to glass eels. The larvae that hatch from fertilized eggs go through a preleptocephalus stage and then take on a form called a leptocephalus. Leptocephali are transparent and have a unique shape resembling a willow leaf. The leptocephali then metamorphose into glass eels (young eels).

However, when eels are reared using the milk protein-containing feed described in Patent Document 3, approximately half of the eels exhibit morphological abnormalities during the metamorphosis process in which they change from larval eels to glass eels (young eels).

The present invention has been made to solve the problems described above, and has an object to provide feed for eel larvae that, when fed to the eel larvae, can reduce the frequency of appearance of individuals exhibiting morphological abnormalities during the metamorphosis from eel larvae to glass eels.
Another object of the present invention is to provide a method for producing eels that can reduce the frequency of individuals exhibiting morphological abnormalities during the metamorphosis from eel larvae to glass eels.
Another object of the present invention is to provide an agent for suppressing morphological abnormalities in juvenile eels, which can reduce the frequency of appearance of individuals exhibiting morphological abnormalities during the metamorphosis from eel larvae to glass eels.

As a result of intensive research aimed at solving the above-mentioned problems, the inventors discovered that by feeding eel larvae feed containing nucleic acids, it is possible to reduce the frequency of appearance of individuals exhibiting the above-mentioned morphological abnormalities, and thus completed the present invention.
That is, the present invention has the following aspects.

(1) Feed for eel larvae containing nucleic acids.
(2) The feed for eel larvae according to (1) above, wherein the content of the nucleic acids relative to the total dry matter weight of the feed for eel larvae is 0.25% by dry matter weight or more.
(3) The feed for eel larvae according to (1) or (2) above, wherein the content of the nucleic acids relative to the total dry matter weight of the feed for eel larvae is 10% by dry matter weight or less.
(4) The feed for eel larvae according to any one of (1) to (3), wherein the nucleic acids include at least one selected from the group consisting of nucleosides, nucleotides, polynucleotides, and salts thereof.
(5) The nucleic acid has a structure in which a base and a sugar are bound to each other,
The feed for eel larvae according to any one of (1) to (4), wherein the base comprises at least one base selected from the group consisting of adenine, guanine, thymine, uracil, cytosine, and hypoxanthine.
(6) The nucleic acid has a structure in which a base and a sugar are bound to each other,
The feed for eel larvae according to any one of (1) to (5), wherein the base includes a purine base and a pyrimidine base.
(7) The feed for eel larvae according to any one of (1) to (6), wherein the feed for eel larvae contains a yeast extract, and the yeast extract contains the nucleic acids.
(8) The feed for eel larvae according to any one of (1) to (7), which does not contain any shark egg components.
(9) The feed for eel larvae according to any one of (1) to (8), further comprising a milk protein.
(10) The feed for eel larvae according to any one of (1) to (9), further comprising a chicken egg component.
(11) A method for producing eels, comprising feeding eel larvae the eel larvae feed according to any one of (1) to (10) above.
(12) The method for producing eels according to (11) above, wherein the feeding is satiation feeding.
(13) The method for producing eels according to (11) or (12) above, further comprising producing artificial seedlings.
(14) An agent for inhibiting morphological abnormalities in eel larvae, which contains nucleic acids as active ingredients.

The present invention can also have the following aspects.
(15) The agent for suppressing morphological abnormalities in eel larvae according to (14) above, wherein the nucleic acids include at least one selected from the group consisting of nucleosides, nucleotides, polynucleotides, and salts thereof.
(16) The nucleic acid has a structure in which a base and a sugar are bound to each other,
The agent for suppressing morphological abnormalities in eel larvae according to (14) or (15) above, wherein the base comprises at least one base selected from the group consisting of adenine, guanine, thymine, uracil, cytosine, and hypoxanthine.
(17) The nucleic acid has a structure in which a base and a sugar are bound to each other,
The agent for suppressing morphological abnormalities in eel larvae according to any one of (14) to (16), wherein the base includes a purine base and a pyrimidine base.
(18) The agent for suppressing morphological abnormalities in eel larvae according to any one of (14) to (17) above, which comprises a yeast extract, and the yeast extract contains the nucleic acids.

According to the present invention, it is possible to provide feed for eel larvae that can reduce the frequency of appearance of individuals exhibiting morphological abnormalities during the metamorphosis from eel larvae to glass eels by feeding the eel larvae.
Furthermore, according to the present invention, a method for producing eels can be provided that, by feeding the eel larvae feed, can reduce the frequency of appearance of individuals exhibiting morphological abnormalities during the metamorphosis from eel larvae to glass eels.
Furthermore, according to the present invention, it is possible to provide an agent for suppressing morphological abnormalities in juvenile eels, which can reduce the frequency of appearance of individuals exhibiting morphological abnormalities during the metamorphosis from eel larvae to glass eels.

FIG. 1 is a diagram illustrating the developmental process of eels up to glass eels. 11 is an image showing an example of an glass eel determined to have "no morphological abnormality" or "morphological abnormality" in the examples. 1 is a graph showing the incidence of morphological abnormalities for each test group in Experiment 1. 1 is a graph showing the incidence of morphological abnormalities for each test group in Experiment 2. 1 is a graph showing the relationship between the nucleic acid content of the feed and the average total length and average body height of 20-day-old eel larvae, as shown in Experiment 3.

The following describes embodiments of the eel larvae feed, eel production method, and eel larvae morphological abnormality inhibitor of the present invention.

<Feed for eel larvae>
The eel larvae feed of the embodiment contains nucleic acids. By feeding the eel larvae feed of the embodiment containing nucleic acids to eel larvae and raising them, it is possible to grow the eel larvae into glass eels (juvenile eels) and reduce the frequency of appearance of individuals exhibiting morphological abnormalities.

As used herein, the term "eel larvae" refers to eels before the completion of metamorphosis into glass eels. Eel larvae include eels in the process of metamorphosing into preleptocephali, leptocephali, and glass eels.
In this specification, the term "juvenile eel" refers to an eel that has completed metamorphosis into a glass eel, and may be, for example, an eel whose pre-anal length/total length ratio is 40% or less and whose body height/total length ratio is 7% or less. An example of a juvenile eel is glass eel.

The reduction in the frequency of occurrence of individuals exhibiting morphological abnormalities can be confirmed, for example, by comparing the frequency of occurrence of individuals exhibiting morphological abnormalities during metamorphosis between a group of eel larvae fed with a control eel feed and a group of eel larvae fed with the eel larvae feed of the embodiment.

The type of eel to be fed with the eel larvae feed of the embodiment may be any species belonging to the eel family, such as the Japanese eel (Anguilla japonica), European eel (Anguilla anguilla), American eel (Anguilla rostrata), etc., with the Japanese eel being preferred.

In this specification, the term "nucleic acids" refers to nucleosides, nucleotides, polynucleotides, salts thereof, as well as analogs, decomposition products, and fragments thereof.

A nucleoside has a structure in which a base and a sugar are linked together.
Examples of the base include adenine, guanine, thymine, uracil, cytosine, and hypoxanthine.
The base is preferably a nucleic acid base.
The sugar can be ribose or deoxyribose. The nucleoside can be a ribonucleoside, in which the sugar is ribose, or a deoxyribonucleoside, in which the sugar is deoxyribose.

Specific examples of nucleosides include adenosine, guanosine, 5-methyluridine, uridine, cytidine, inosine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, and deoxyinosine.

A nucleotide has a structure in which a nucleoside and a phosphate are bonded together.
The sugar in the nucleoside can be ribose or deoxyribose, and the nucleotide can be a ribonucleotide in which the sugar is ribose or a deoxyribonucleotide in which the sugar is deoxyribose.

Specific examples of ribonucleotides and deoxyribonucleosides include adenosine monophosphate (AMP, also called adenylic acid), adenosine diphosphate (ADP), adenosine triphosphate (ATP); deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP); guanosine monophosphate (GMP, also called guanylic acid), guanosine diphosphate (GDP), guanosine triphosphate (GTP); deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP); 5-methyluridine monophosphate (TMP), 5-methyluridine diphosphate (TDP), 5-methyluridine triphosphate (TTP); thymidine monophosphate (dTMP, also called thymidylic acid), thymidine diphosphate ( dTDP), thymidine triphosphate (dTTP); uridine monophosphate (UMP, also called uridylic acid), uridine diphosphate (UDP), uridine triphosphate (UTP); deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP); cytidine monophosphate (CMP, also called cytidylic acid), cytidine diphosphate (CDP), cytidine triphosphate (CTP); deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP); inosine monophosphate (IMP, also called inosinic acid), inosine diphosphate (IDP), inosine triphosphate (ITP); deoxyinosine monophosphate (dIMP), deoxyinosine diphosphate (dIDP), deoxyinosine triphosphate (dITP), etc.

In this specification, the term "polynucleotide" refers to a concept that includes nucleotide polymers and oligomers. Examples of polynucleotides include polymers in which two or more of the ribonucleotides listed above are linked together, and polymers in which two or more of the deoxyribonucleotides listed above are linked together.

Specific examples of polynucleotides include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

The salt of a nucleoside, nucleotide, or polynucleotide is not particularly limited, but examples thereof include alkali metal salts or hydrochloride salts, with sodium salts being preferred.

The nucleoside, nucleotide, or polynucleotide may contain modified bases as known in the art.

Nucleoside, nucleotide, or polynucleotide analogues have functions equivalent to those of natural nucleic acids, and include those in which any group or atom in the molecule of a natural nucleic acid has been replaced with any substituent, or those to which any modification has been imparted.
Further examples of the analogues include peptide nucleic acid (PNA) and locked nucleic acid (LNA).

As the decomposition product or fragment of a nucleoside, nucleotide, or polynucleotide, a decomposition product or fragment of a polynucleotide is preferable, and may be a hydrolysate.

The nucleic acids may be of the type found in natural eels, or may be salts, decomposition products or fragments thereof.

The nucleic acids preferably include at least one selected from the group consisting of nucleosides, nucleotides, polynucleotides, and salts thereof.

The nucleic acids preferably have a structure in which a base is bound to a sugar, and the base preferably includes at least one base selected from the group consisting of adenine, guanine, thymine, uracil, cytosine, and hypoxanthine.

In this specification, nucleic acids refer to those that have a structure in which bases and sugars are bound together, and may be expressed simply as "containing bases."

The nucleic acids have a structure in which a base and a sugar are bound, and more preferably, the base includes at least one selected from the group consisting of nucleosides, nucleotides, and polynucleotides, including at least one base selected from the group consisting of adenine, guanine, thymine, uracil, cytosine, and hypoxanthine, and salts thereof. It is even more preferable that the nucleotide includes ribonucleotides or deoxyribonucleotides, and particularly preferably includes ribonucleotides.

Among nucleic acids, nucleic acids containing purine bases can be converted into each other or can be decomposed and used as a common raw material for nucleic acids in biosynthesis.
Furthermore, among nucleic acids, nucleic acids containing pyrimidine bases can be converted into each other or can be decomposed and used in biosynthesis as a common raw material for nucleic acids.

From this perspective, it is preferable that the nucleic acids contained in the eel larvae feed of the embodiment have a structure in which a base and a sugar are bonded, and that the base includes a purine base and a pyrimidine base.

The types of bases contained in the nucleic acids are taken into consideration with respect to the entire nucleic acids contained in the eel larvae feed of the embodiment.
For example, when the nucleic acids are nucleosides or nucleotides, the purine bases and pyrimidine bases in the nucleic acids contained in the eel larval feed of the embodiment may each be contained in a different nucleic acid molecule.
For example, when the nucleic acids are polynucleotides, in the nucleic acids contained in the eel larvae feed of the embodiment, the purine bases and pyrimidine bases may be contained in different nucleic acid molecules, or may be contained in the same nucleic acid molecule as in conventional DNA and RNA.

Among the above-listed bases, examples of purine bases include adenine, guanine, and hypoxanthine.
Among the above-listed bases, examples of pyrimidine bases include thymine, uracil, and cytosine.

The nucleic acids contained in the eel larvae feed of the embodiment have a structure in which a base and a sugar are bonded, and it is further preferable that the base includes at least one base selected from the group consisting of adenine, guanine, and hypoxanthine, and at least one base selected from the group consisting of thymine, uracil, and cytosine.

The nucleic acids contained in the eel larvae feed of the embodiment have a structure in which a base and a sugar are bound together, and it is even more preferable that the base contains at least one selected from the group consisting of ribonucleosides, deoxyribonucleosides, ribonucleotides, deoxyribonucleotides, ribonucleic acids, deoxyribonucleic acids, and salts thereof, including at least one base selected from the group consisting of adenine, guanine, and hypoxanthine, and at least one base selected from the group consisting of thymine, uracil, and cytosine.

From the viewpoint of being more efficiently utilized in eel larvae, it is preferable that the nucleic acids contain a variety of bases.
The nucleic acids have a structure in which a base is bound to a sugar, and the base preferably includes adenine and/or hypoxanthine, guanine, thymine and/or uracil, and cytosine.
The nucleic acids have a structure in which a base is bound to a sugar, and more preferably, the base includes adenine and/or hypoxanthine, guanine, uracil, and cytosine.

It is further preferred that the nucleic acids have a structure in which a base is bound to a sugar, and the base comprises at least one selected from the group consisting of ribonucleosides, ribonucleotides, and ribonucleic acids, including adenine and/or hypoxanthine, guanine, uracil, and cytosine, and salts thereof.

The details of why the inclusion of nucleic acids in eel larvae feed reduces the frequency of individuals exhibiting the above-mentioned morphological abnormalities are unclear, but the following reasons are thought to be the cause. Normally, fish larvae are able to synthesize nucleic acids in their bodies. However, eel larvae probably synthesize only small amounts of nucleic acids, and while they need to ingest nucleic acids from their feed, it is possible that conventional feed does not adequately meet their requirements. In particular, it is believed that many gene expressions and cell divisions occur in conjunction with morphological changes during the metamorphosis from leptocephalus to glass eels, and feeding them feed containing nucleic acids contributes to the smooth progression of these processes.

The content of the nucleic acids in the eel larvae feed of the embodiment relative to the total dry matter weight (100% by weight) of the eel larvae feed may be, in terms of dry matter weight, more than 0.04% by weight, 0.05% by weight or more, 0.07% by weight or more, 0.1% by weight or more, 0.15% by weight or more, 0.2% by weight or more, 0.25% by weight or more, 0.5% by weight or more, 0.8% by weight or more, 0.9% by weight or more, 1.0% by weight or more, 1.5% by weight or more, 2.0% by weight or more, 2.5% by weight or more, 3.0% by weight or more, 3.5% by weight or more, 3.8% by weight or more, or 3.9% by weight or more.

By using eel larvae feed that contains the nucleic acids in an amount greater than or equal to the lower limit value above, calculated on a dry matter basis, relative to the total dry matter weight (100% by weight), it is possible to more effectively reduce the frequency of occurrence of individuals exhibiting morphological abnormalities during the metamorphosis of eel larvae into glass eels.

In the eel larvae feed of the embodiment, the content of the nucleic acids relative to the total dry matter weight (100% by weight) of the eel larvae feed may be 20% by weight or less, 15% by weight or less, 10% by weight or less, 9% by weight or less, 8% by weight or less, 7% by weight or less, 6% by weight or less, or 5% by weight or less, by dry matter weight.

By using eel larvae feed containing the nucleic acids in an amount equal to or less than the upper limit as calculated on a dry matter basis relative to the total dry matter weight (100% by weight), it is possible to grow the eel larvae even better.

As an example of the above-mentioned numerical range of the nucleic acids contained in the eel larvae feed of the embodiment, the content of the nucleic acids in the eel larvae feed of the embodiment relative to the total dry matter weight (100% by weight) of the eel larvae feed may be, by dry matter weight, more than 0.04% by weight and not more than 20% by weight, may be 0.05% by weight or more and not more than 20% by weight, may be 0.07% by weight or more and not more than 15% by weight, may be 0.1% by weight or more and not more than 15% by weight, may be 0.15% by weight or more and not more than 10% by weight, may be 0.2% by weight or more and not more than 10% by weight, may be 0.25 ... % or less by weight, may be 0.5% to 9% by weight, may be 0.8% to 9% by weight, may be 0.9% to 8% by weight, may be 1.0% to 8% by weight, may be 1.5% to 7% by weight, may be 2.0% to 7% by weight, may be 2.5% to 6% by weight, may be 3.0% to 6% by weight, may be 3.5% to 5% by weight, may be 3.8% to 5% by weight, or may be 3.9% to 5% by weight.

The dry weight of eel larvae feed or feed ingredients can be determined by subjecting the eel larvae feed or feed ingredients to a drying process in which they are heated at 105°C for 6 hours.

The content of nucleic acids in the eel larvae feed of the embodiment can be measured by a known analytical method. As the analytical method, high performance liquid chromatography can be used. For example, various nucleic acids can be measured and their content calculated using feed or feed ingredients, or processed products thereof, as a measurement sample. The measurement sample may be feed or feed ingredients that have been treated according to the type of nucleic acid to be measured. The measurement sample may be feed or feed ingredients that have been subjected to perchloric acid extraction or nuclease treatment, for example.

The eel larvae feed of the embodiment preferably contains a yeast extract. The yeast extract may contain the nucleic acids. The yeast extract is also called yeast extract. The eel larvae feed of the embodiment may contain nucleic acids and/or yeast extract.

The content of the yeast extract relative to the total dry matter weight (100% by weight) of the eel larvae feed may be 1% by weight or more, 1% to 50% by weight, 5% to 40% by weight, or 10% to 30% by weight, in terms of dry matter weight.

The numerical values of the content of nucleic acids given as examples of the content of nucleic acids in the eel larvae feed of the embodiment above (the numerical values given as examples in the range of more than 0.04% by weight and in the range of 20% by weight or less, based on the total dry weight (100% by weight) of the eel larvae feed) may be interpreted as the numerical values of the content of nucleic acids derived from the yeast extract.

The eel larvae feed of the embodiment preferably further contains milk protein, as this allows for better rearing of the eel larvae.

In this specification, "milk protein" refers to a protein derived from milk. A wide variety of milk protein products are available on the market using milk as a raw material, and milk protein is one of the proteins that is inexpensive and stably available. Specific examples include skim milk powder and various caseins, which are made from milk.

The content of the milk protein relative to the total dry matter weight (100% by weight) of the eel larvae feed may be 10% by weight or more, 10% by weight or more and 80% by weight or less, 20% by weight or more and 60% by weight or less, or 30% by weight or more and 50% by weight or less, by dry matter weight.

The eel larvae feed of the embodiment may further contain a protein source other than milk protein. The protein source is preferably one that is stably available and easily digestible by eel larvae, and may be bird egg components or chicken egg components. Chicken egg yolk powder, for example, is preferred because it is more readily available.

The content of the egg components in the eel larvae feed relative to the total dry matter weight (100% by weight) may be 5% by weight or more, 5% by weight or more and 30% by weight or less, 7% by weight or more and 25% by weight or less, or 10% by weight or more and 20% by weight or less, by dry matter weight.

An example of the eel larvae feed of the embodiment is an eel larvae feed that contains nucleic acids, milk protein, and egg components (e.g., egg yolk powder), and in which, based on the total dry matter weight of the eel larvae feed, the nucleic acid content is 0.25% by weight or more and 10% by weight or less, the milk protein content is 10% by weight or more and 80% by weight or less, and the egg component content is 5% by weight or more and 30% by weight or less.

An example of an eel larvae feed according to an embodiment is an eel larvae feed that contains nucleic acids, yeast extract, milk protein, and egg components (e.g., egg yolk powder), the yeast extract contains the nucleic acids, and the yeast extract has a dry weight content of 1% to 50% by weight, milk protein content of 10% to 80% by weight, and egg components content of 5% to 30% by weight, relative to the total dry weight of the eel larvae feed.

Another protein source other than milk protein is fish meal, and from the viewpoint of improving digestibility and absorption, enzyme-treated fish meal is preferred. The enzyme-treated fish meal may be made from the meat of any fish species, and may be, for example, fish meal treated with peptidase, protease, or proteinase.

It is preferable to mix milk protein and other proteins in such a ratio that the amount of lactose in the total dry matter weight of the eel larvae feed is 10% or less by dry matter weight of the total feed. The amount of milk protein can be increased by using low-lactose milk protein or casein.

An example of an embodiment of eel larvae feed is eel larvae feed containing nucleic acids and/or yeast extract and milk protein.

Another embodiment of the eel larvae feed is an eel larvae feed containing nucleic acids and/or yeast extract, milk protein, and chicken egg yolk powder.

Another embodiment of the eel larvae feed is an eel larvae feed containing nucleic acids and/or yeast extract, milk protein, chicken egg yolk powder, and fish meal.

In addition, since it is possible to improve the growth and survival rate of eel larvae, it is preferable that the eel larvae feed of the embodiment further contains vitamins. Examples of vitamins include vitamin C, vitamin A, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin D, vitamin K, niacin, pantothenic acid, folic acid, biotin, etc., as well as choline, inositol, etc.

In addition, it is preferable that the eel larvae feed of the embodiment contains taurine, as this can improve growth and survival rate.

Another embodiment of the eel larvae feed is an eel larvae feed containing nucleic acids and/or yeast extract, milk protein, chicken egg yolk powder, fish meal, vitamins, and taurine.

In addition, since it is generally preferable to feed larval eels n-3 highly unsaturated fatty acids such as EPA and DHA, the eel larval feed of the embodiment preferably contains fish oil such as liver oil that contains the above-mentioned n-3 highly unsaturated fatty acids.

The eel larvae feed of the embodiment may contain soybean peptides (preferably soybean peptides that have been treated to reduce phytic acid) as other optional ingredients.

The eel larvae feed of the embodiment may be, for example, any basic eel larvae feed to which nucleic acids and/or yeast extract have been further added. Here, the basic eel larvae feed refers to a feed that can be fed to eel larvae to allow them to grow and metamorphose into fry, and examples of such feed include the eel larvae feed described in Patent Document 2 and the eel larvae feed described in Patent Document 3. The amount of nucleic acids and/or yeast extract added may be an amount that satisfies the content of the nucleic acids relative to the total dry matter weight of the eel larvae feed exemplified above.

On the other hand, the eel larvae feed of this embodiment is useful as an alternative feed to feed containing shark egg components. Therefore, it is preferable that the eel larvae feed of this embodiment does not contain shark egg components. In this specification, "shark egg components" are components derived from shark eggs. An example of shark eggs is the eggs of the spiny dogfish.

The eel larvae feed of the embodiment may be in the form of a powder, and may be in the form of a dry powder since it is suitable for distribution and storage. In addition, since it is easy for the larvae to swallow, when feeding the eel larvae, a liquid such as water or seawater can be appropriately added to the powdered eel larvae feed to obtain a paste-like eel larvae feed, which can then be fed to the eel larvae.

By feeding eel larvae with the eel larvae feed of the embodiment, it is possible to reduce the frequency of occurrence of individuals exhibiting morphological abnormalities during the metamorphosis from eel larvae to glass eels.

In this specification, morphological abnormalities refer to severe deformations of the spine, including curvature, undulation, bending, and broken necks (see Figure 2). Severe deformations are those that are difficult for an observer to distinguish when observed under an optical microscope.

The rate of morphological abnormalities in the eel fry produced may be 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less, based on the results of observing 16 or more individuals.

<Method of producing feed for eel larvae>
The method for producing eel larval feed of the embodiment includes mixing various raw materials and raw material components all at once or in an appropriate order to obtain the eel larval feed of the embodiment.

According to the method for producing eel larvae feed of the embodiment, it is possible to produce the eel larvae feed of the embodiment.

The various raw materials and raw material components, and their contents, include those exemplified in the above <<Feed for eel larvae>>. In addition to nucleic acids, the various raw materials and raw material components include the above-mentioned yeast extract, milk protein, chicken egg yolk powder, fish meal, vitamins, taurine, fish-liver oil, etc., and other optional components. Eel larvae feed may contain liquids such as water or seawater as appropriate.

As an example of a method for producing eel larvae feed according to an embodiment, a method for producing eel larvae feed is illustrated, which includes blending the nucleic acids and/or yeast extract so that the eel larvae feed contains 0.25% by dry matter weight or more of nucleic acids relative to the total dry matter weight (100% by weight) of the eel larvae feed.

As an example of a method for producing eel larvae feed according to an embodiment, a method for producing eel larvae feed is illustrated, which includes blending the nucleic acids and/or yeast extract so that the eel larvae feed contains 10% or less by dry matter weight of nucleic acids relative to the total dry matter weight (100% by weight) of the eel larvae feed.

As an example of a method for producing eel larvae feed according to an embodiment, a method for producing eel larvae feed is illustrated, which includes blending the nucleic acids and/or yeast extract so that the eel larvae feed contains 0.25% by weight to 10% by weight of nucleic acids by dry matter weight relative to the total dry matter weight (100% by weight) of the eel larvae feed.

The eel larvae feed of the embodiment can also be produced by, for example, adding nucleic acids and/or yeast extract to any basic eel larvae feed. Here, the basic eel larvae feed refers to feed that can be fed to eel larvae to transform them into fry.
The amount of nucleic acids and/or yeast extract to be added may be an amount that satisfies the content of the nucleic acids relative to the total dry matter weight of the feed for eel larvae exemplified above.

The eel larvae feed of the embodiment can be produced by blending, for example, 0.25 to 20 parts by weight of nucleic acids and/or yeast extract, 10 to 50 parts by weight of milk protein, 5 to 30 parts by weight of chicken egg yolk powder, and 10 to 50 parts by weight of fish meal. The weight parts of these raw materials may be the weight parts in the state used in the production of the feed (for example, wet weight ratio).

In one embodiment, the present invention provides the use of nucleic acids for producing feed for eel larvae.
In one embodiment, the present invention provides the use of a yeast extract for producing feed for eel larvae.

<Eel production method>
The method for producing eels of this embodiment includes feeding the eel larvae feed of the embodiment to eel larvae.

In one embodiment, a method for reducing the frequency of appearance of glass eels exhibiting morphological abnormalities is provided, which includes feeding eel larvae with the eel larvae feed of the embodiment.

Examples of eel larvae feed in this embodiment include those exemplified in the above "eel larvae feed".

The method for rearing eel larvae is not particularly limited, and can be carried out according to known methods for rearing eel larvae. As an example, eel larvae can be reared in water with a temperature of 23 to 25°C and a salt concentration of 16 to 34 psu (practical salinity units).

The method of feeding the eel larvae feed is not particularly limited, and the feed can be provided in accordance with the usual breeding method for eel larvae. The number of times the eel larvae feed of the embodiment is fed may be once or more per day, for example, 3 to 5 times per day (for example, it is preferable to provide a feeding interval of at least 2 hours). The amount of the eel larvae feed of the embodiment can be appropriately adjusted taking into account the feeding state of the eel larvae, and satiation feeding is preferable.

The feeding period for feeding the eel larvae feed of the embodiment to the eel larvae is not particularly limited as long as it is during the larval stage, but from the viewpoint of more effectively suppressing morphological abnormalities during the metamorphosis process, it is preferable to at least include the period before the start of metamorphosis into glass eels. As an example, the eel larvae feed of the embodiment is fed at least once a day to eel larvae that are in the period from 10 days before the start of metamorphosis to the completion of metamorphosis, more preferably from 30 days before the start of metamorphosis to the completion of metamorphosis, and even more preferably from 60 days before the start of metamorphosis to the completion of metamorphosis. The feeding period for the eel larvae feed of the embodiment may be continuous or discontinuous, and may be, for example, 10 days or more, 50 days or more, 100 days or more, or 150 days or more.

In addition, the start of metamorphosis can be induced by raising eel larvae without feeding them for an appropriate period of time before the start of metamorphosis.

The method for producing eels of the embodiment is preferably a method for producing artificial seedlings, which includes feeding the eel larvae feed of the embodiment to eel larvae.
The production of artificial seedlings here means artificially spawning and hatching eel larvae, and then raising them to glass eels.

The eel production method of the embodiment can be full-scale aquaculture. Full-scale aquaculture involves artificially spawning and hatching eels, raising them until they become adults, and then raising the next generation of adult eels from the eggs laid by the adult eels.

<Eel larvae morphological abnormality inhibitor>
The agent for suppressing morphological abnormalities in juvenile eels according to the embodiment contains nucleic acids as an active ingredient.
The agent for suppressing morphological abnormalities in juvenile eels according to the embodiment may contain a yeast extract as an active ingredient.

The nucleic acids and yeast extracts contained in the eel larvae morphological abnormality inhibitor include those exemplified in the "eel larvae feed" above.

The eel larvae morphological abnormality inhibitor of the embodiment can be administered to eel larvae, for example by being added to any eel feed, preferably eel larvae feed, to reduce the frequency of appearance of glass eels exhibiting morphological abnormalities.

In one embodiment, the present invention provides use of nucleic acids as an agent for suppressing morphological abnormalities in eel larvae.
In one embodiment, the present invention provides use of a yeast extract as an agent for suppressing morphological abnormalities in eel larvae.

The eel larvae feed, eel production method, and eel larvae morphological abnormality inhibitor of the above-described embodiments make it possible to artificially raise eel larvae using stable raw materials, and to produce high-quality glass eels. The eel larvae feed, eel production method, and eel larvae morphological abnormality inhibitor of the present embodiments are extremely useful in contributing to the realization of the long-desired complete eel farming and commercialization of mass production.

The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experiment 1] Evaluation of the effect of adding nucleic acids to feed on the frequency of morphological abnormalities (feed ingredients)
・Dried egg yolk powder (Kewpie Egg Corporation, Dried Egg Yolk No. 1)
・Enzyme-treated fish meal (manufactured by Scientific Feed Research Institute Co., Ltd., CPSP SPECIAL G)
・Sodium caseinate (manufactured by Nippon Shinyaku Co., Ltd., sodium caseinate LW)
・Skimmed milk powder (Yotsuba Milk Industry Co., Ltd., Hokkaido skimmed milk powder)
Soybean peptide (Nihon Pharmaceutical Co., Ltd., Hypopolypeptone N)
Yeast extract (high IMP) (Kojin Life Sciences Co., Ltd., Aromild (registered trademark))
Nucleic acid mix (an equal weight mixture of disodium salts of AMP, IMP, GMP, CMP, and UMP)
AMP: Adenosine 5'-monophosphate disodium salt (Fujifilm Wako QB-7150)
IMP: Inosine 5'-monophosphate disodium salt (Nacalai Tesque 06400-22)
GMP: Guanine 5'-monophosphate disodium salt (Fujifilm Wako QB-3304)
CMP: Cytidine 5'-monophosphate disodium salt (Tokyo Chemical Industry Co., Ltd. C0524)
UMP: Uridine 5'-monophosphate disodium salt (Fujifilm Wako QR-1512)
- Taurine (Nacalai Tesque, Inc., Taurine Reagent)
・Vitamin Mix (manufactured by Japan Nutrition Co., Ltd., Fisheries Research and Education Agency Vitamin)
・Cod liver oil (Hi-calor E, manufactured by Kanematsu Shintoa Foods Co., Ltd.)

method)
Five-day-old eel larvae were housed in 12 tanks (acrylic bowl-shaped tank, water volume approximately 10 L), with 500 fish per tank. The six-day-old eel larvae were fed a shark egg substitute diet FSD (see Table 1, a diet obtained by partially modifying the fish meal feed described in Table 1 of Patent Document 3) and were pre-bred until they were 80 days old. They were fed five times a day (at two-hour intervals), with a feed amount of 7-10 mL per time, water temperature of 23°C, water injection rate of 0.5-0.7 L/min, and the tank was changed after the final feeding each day.
Larvae were collected at 80 days of age, and redistributed to a total of 12 rearing tanks with 80 larvae per tank. Each test feed was prepared by mixing each feed ingredient in the ratio (g) shown in Table 1. From 81 days of age, test groups were set up to feed each test feed shown in Table 1. Six test groups were set up in total: a control group (FSD group) fed with FSD, a group (FSD+NM_ group) fed with a feed in which 0.5g, 1.0g, 2.0g, or 4.0g of nucleic acid mix was added to the FSD feed, and a group (FSY group) fed with a feed in which the soybean peptides in the FSD feed were replaced with yeast extract and the mixing ratio of some of the raw materials was modified. The number of tanks per test group was two. The basic rearing conditions were the same as those for the first 80 days of age, and the fish were fed until they reached 340 days of age. From 341 days of age onwards, the fish were kept without food to induce the initiation of metamorphosis.
Individuals showing signs of metamorphosis (such as anal position moving forward, body height decreasing, etc.) were removed from the breeding tank and individually housed in 250 mL polycarbonate containers, filled with breeding water, and maintained without feeding to observe the progression of metamorphosis. The point at which the body shape completely changed to that of an glass eel was defined as the completion of metamorphosis, and photographs were taken after anesthesia, and the body shape at the completion of metamorphosis was recorded as image data.
Morphological abnormalities were determined by defining individuals with severe spinal deformity as abnormal, and the judgments were made independently by two experienced judges. The judgment results were collated, and for individuals where the judges' judgments differed, a reconciliation was carried out. An example of an individual judged as "no morphological abnormality" or "with morphological abnormality" is shown in Figure 2.
In order to verify the effect of the nucleic acid mix or yeast extract added to the feed on the frequency of morphological abnormalities, binary data regarding the presence or absence of morphological abnormalities at the completion of metamorphosis was used as the objective variable, and the type of feed (test group) was used as the explanatory variable. Logistic regression analysis was used to calculate the odds ratio and 95% confidence interval for the occurrence of morphological abnormalities between feeds using JMP16.0 software.

In Experiments 1 to 3, the notation "-" in Tables 1, 3 and 6 indicates that the corresponding raw material was not blended.
In Experiments 1 to 3, the total dry matter weight was calculated by subjecting the feed raw materials to a drying treatment of heating at 105° C. for 6 hours, and then calculating the sum of the values after the drying treatment of the feed raw materials.
When nucleic acids in the form of disodium salts were used, the amount of nucleic acids was shown as 0.88 times the amount used, which is the weight of the nucleic acids themselves excluding the disodium content.

The "Nucleic acid content (wt%)" column in the table shows the percentage (wt%) of nucleic acid dry matter content relative to the total dry matter weight of the feed.

result)
Individuals that began to metamorphose appeared from 138 days of age, and the cumulative number of individuals that had begun to metamorphose by 340 days of age was 308 (FSD: 29, FSD+NM0.5: 48, FSD+NM1.0: 52, FSD+NM2.0: 65, FSD+NM4.0: 54, FSY: 60), and the number of individuals that had completed metamorphosis was 283 (FSD: 27, FSD+NM0.5: 43, FSD+NM1.0: 50, FSD+NM2.0: 57, FSD+NM4.0: 49, FSY: 57).
The incidence and odds ratio of morphological abnormalities for each test group are shown in FIG. 3 and Table 2. In the four test groups to which the nucleic acid mix was added, the higher the concentration of the nucleic acid mix was, the lower the odds ratio for morphological abnormality was, and the lowest in the FSY group. When the incidence in the FSD group estimated from the odds ratio was taken as 100%, the reduction rates were 50.0% in the FSD + NM 0.5 group, 63.6% in the FSD + NM 1.0 group, 69.1% in the FSD + NM 2.0 group, 75.8% in the FSD + NM 4.0 group, and 94.7% in the FSY group, indicating that the incidence of morphological abnormalities can be reduced by adding the nucleic acid mix or yeast extract. The proportion of individuals exhibiting morphological abnormalities decreased depending on the content of nucleic acids in the feed.

[Experiment 2] Examination of various yeast extracts Feed ingredients) Items not common to Experiment 1 above Yeast extract (high AMP) (Kohjin Life Sciences Co., Ltd., Nucleamine (registered trademark))
- Yeast extract (reagent) (Sigma-Aldrich, Yeast Extract 09182)

method)
Six-day-old eel larvae were housed in a breeding tank (a polycarbonate half-pipe tank with a water volume of approximately 30 L) with 1,000-1,500 fish per tank. The six-day-old eel larvae were fed a shark egg substitute diet FSD and were pre-bred until they reached 39 days of age. They were fed five times a day (at two and a half hour intervals), with a feed volume of 30-40 mL per feeding, water temperature of 23°C, water injection volume of 1.6-1.7 L/min, and the tank was changed after the final feeding each day.
Larvae were collected at 40 days of age, and 400 larvae were redistributed to 14 sides of a 30L half-pipe tank. Each test feed was prepared by mixing each feed ingredient in the ratio (g) shown in Table 3. After 40 days of age, test areas were set up to give each of the four types of test feed shown in Table 3. The number of tank sides per test area was 3 or 4 (FSD: 4 sides, FSY2: 4 sides, FSX: 3 sides, FSZ: 3 sides). The basic rearing conditions were the same as up to 39 days of age, and feeding was continued until 304 days of age. From 305 days to 330 days of age, the animals were reared without feeding to induce the start of metamorphosis.
The definitions of the onset and completion of metamorphosis, the method of recording phenotypes, the discrimination of morphological abnormalities, and statistical analysis were the same as in Experiment 1.

result)
The nucleotide content of the FSD feed and the three types of yeast extracts added was analyzed. The analysis was performed on samples extracted with 5% perchloric acid to measure the content of various nucleic acids.
For the feed FSD and the reagent yeast extract (Sigma-Aldrich), samples that had been enzymatically hydrolyzed with nuclease P1 were also analyzed to measure the content of various nucleic acids (for the analysis of 5'-inosinic acid, samples extracted with 5% perchloric acid were analyzed and the content was measured).
The main source of nucleic acids in yeast extract (Aromild and Nucleamine, manufactured by Kohjin Life Sciences Co., Ltd.) is RNA, and since RNA is decomposed during the manufacturing process, the nucleic acids in the yeast extract are present in the form of nucleotides. As for the reagent yeast extract (manufactured by Sigma-Aldrich), no nucleotides were detected in the feed that was not enzymatically decomposed with nuclease P1, so the nucleic acids in the reagent yeast extract were contained as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).

The analysis results of the nucleotides contained in the feed FSD and the three types of yeast extracts used are shown in Table 4 as the content (wt%) per dry matter weight of the feed FSD or yeast extract. The sum of the total nucleotides and total nucleosides in the analysis values was taken as the nucleic acid content.
Feed FSD contained almost no nucleic acids, and all analytical items other than GMP were below the detection limit (0.01g/100g). Aromild contained IMP, UMP, GMP, and CMP, with a total nucleotide content of 23.05% by weight and a total nucleoside content of 1.44% by weight. Nucleamine contained AMP, UMP, GMP, and CMP, with a total nucleotide content of 24.86% by weight and a total nucleoside content of 1.74% by weight. Reagent yeast extract contained AMP, UMP, GMP, and CMP, with a total nucleotide content of 4.24% by weight and a total nucleoside content of 1.22% by weight.
In addition, in the plots where 5'-thymidylic acid was below the detection limit, the contents of deoxynucleotides, deoxynucleosides, and DNA were presumed to be below the detection limit.

By the end of the rearing period, a total of 163 fish had begun metamorphosis (FSD: 18, FSY2: 59, FSX: 64, FSZ: 22), and a total of 138 fish had completed metamorphosis (FSD: 16, FSY2: 46, FSX: 59, FSZ: 17).
The incidence and odds ratio of morphological abnormalities for each test group are shown in Figure 4 and Table 5. Compared to the FSD group, the odds ratio for the incidence of morphological abnormalities was lower in the three test groups to which yeast extract was added. When the incidence frequency in the FSD group estimated from the odds ratio was taken as 100%, the reduction rate was 68.6% in the FSY2 group, 96.5% in the FSX group, and 58.3% in the FSZ group, demonstrating that the addition of yeast extract containing nucleic acids can reduce the incidence frequency of morphological abnormalities.

[Experiment 3] Examination of the content of nucleic acids in feed Feed ingredients) Items not common to Experiments 1 and 2 above Nucleic acid mix (manufactured by Margo Corporation, a mixture of sodium 5'-ribonucleotide, nucleic acid, IMP and GMP)
- Vitamin mix (see Furuita et al. (2014) Fisheries Science, 80, 581-587)
・Taurine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)

method)
Six-day-old eel larvae were housed in seven aquariums (acrylic bowl-shaped aquariums, water volume approximately 10 L) with 250 fish per aquarium. Each test feed was prepared by mixing each feed ingredient in the ratio (g) shown in Table 6. Eel larvae were fed and reared with each test feed shown in Table 6 from 7-day-old to 19-day-old. The number of feedings was five times a day (2-hour intervals), the amount of feeding was 7 mL/time, the water temperature was 23°C, the amount of water poured was 0.4-0.6 L/min, and the aquarium was changed after the last feeding every day. There were seven test zones in which a feed containing 1 g, 2 g, 5 g, 10 g, 15 g, or 20 g of nucleic acid mix (a mixture of IMP and GMP) was added to the feed FSD, and the number of aquariums per test zone was one.
At 20 days of age, 31-33 fish per tank were randomly taken and photographed under a stereomicroscope after anesthesia, and total length and body height were measured by image analysis using ImageJ. The relationship between the average total length and average body height and nucleic acid content (proportion of nucleic acid content in dry weight to the total dry weight of the feed (wt%)) for each test group was fitted to a sigmoid curve, and the inflection point at 50% of the maximum response was estimated.

result)
The relationship between the average total length and average body height at 20 days of age and the nucleic acid content of the feed is shown in Figure 5. The inflection point and 95% confidence interval for each parameter were estimated to be 9.99 (7.7-12.3) and 9.79 (7.8-11.8), respectively. This shows that even better growth of eel larvae can be achieved by feeding a feed containing 10% or less by weight of nucleic acids per dry matter weight, for example.

The configurations and combinations thereof in each embodiment are merely examples, and additions, omissions, substitutions, and other modifications of the configurations are possible without departing from the spirit of the present invention. Furthermore, the present invention is not limited to each embodiment, but is limited only by the scope of the claims.

Claims (14)

Feed for eel larvae containing nucleic acids. The eel larvae feed according to claim 1, wherein the content of the nucleic acids relative to the total dry matter weight of the eel larvae feed is 0.25% by dry matter weight or more. The eel larvae feed according to claim 1 or 2, wherein the content of the nucleic acids relative to the total dry matter weight of the eel larvae feed is 10% by dry matter weight or less. The eel larvae feed according to claim 1 or 2, wherein the nucleic acids include at least one selected from the group consisting of nucleosides, nucleotides, polynucleotides, and salts thereof. The nucleic acids have a structure in which a base and a sugar are bound to each other,
3. The feed for eel larvae according to claim 1 or 2, wherein the base comprises at least one base selected from the group consisting of adenine, guanine, thymine, uracil, cytosine, and hypoxanthine. The nucleic acids have a structure in which a base and a sugar are bound to each other,
The feed for eel larvae according to claim 1 or 2, wherein the base comprises a purine base and a pyrimidine base. The eel larvae feed according to claim 1 or 2, wherein the eel larvae feed contains a yeast extract, and the yeast extract contains the nucleic acids. The eel larvae feed according to claim 1 or 2, wherein the eel larvae feed does not contain any shark egg components. The eel larvae feed according to claim 1 or 2, further comprising milk protein. The eel larvae feed according to claim 1 or 2, further comprising a chicken egg component. A method for producing eels, comprising feeding the eel larvae feed according to claim 1 or 2 to eel larvae. The method for producing eels according to claim 11, wherein the feeding is satiation feeding. The method for producing eels according to claim 11, which produces artificial seedlings. An agent that inhibits morphological abnormalities in eel larvae, containing nucleic acids as the active ingredient.

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