WO1999048358A1

WO1999048358A1 - Method for constructing pseudo-hibernant fishes and shellfishes, method for storing fresh fishes and shellfishes and method for measuring degree of hibernation of pseudo-hibernant fisches and shellfishes

Info

Publication number: WO1999048358A1
Application number: PCT/JP1999/001127
Authority: WO
Inventors: Yoichi Kadokami; Yoshinori Taniguchi; Mitsuyuki Horiuchi
Original assignee: Nagase & Co., Ltd.; Jifas Corporation
Priority date: 1998-03-20
Filing date: 1999-03-08
Publication date: 1999-09-30
Also published as: AU3275899A

Abstract

A method for constructing pseudo-hibernant fishes and shellfishes which comprises administering one or more substances selected from the serum of a hibernant mammal under hibernation, delta-opioid peptide and delta-opioid peptide receptor ligand to the fishes and shellfishes. Thus establishment is made of a method for inducing fishes and shellfishes which would not hibernate in nature into a state physiologically close to hibernation (hereinafter called psuedo-hibernation), maintaining this state and awaking them. Consequently, a method is established for safely storing and transporting fresh fishes at a high survival ratio, thus enabling transportation of fresh fishes at an elevated efficiency.

Description

Description Method for producing pseudo-hibernating seafood, method for preserving live seafood, and method for measuring the degree of hibernation of pseudo-hibernating seafood

The present invention relates to a method for producing a pseudo-hibernating fish and shellfish using a substance that induces hibernation and a method for awakening the same. The present invention relates to a method for producing pseudo-hibernating seafood by administering serum from a pseudo-hibernating animal to seafood, and a method for awakening the same. Background art

The transportation of live fish and shellfish, particularly cultured fish and shellfish, is carried out from hatchery to farm or from farm to farm.

Conventionally, live fish and shellfish have been transported mainly by the method using the icon. In this case, water occupies about 95% by weight, and transport efficiency was extremely poor. In addition, since transportation is carried out while awake, the consumption of oxygen by metabolism and the accumulation of excreta, especially harmful ammonia, become a problem, and water quality management during transportation is required.In addition, the survival rate after transportation is about 20%. It was poor in sex. In addition, the stress of seafood during transportation is large, which may cause serious damage to fish eyes, body surface, fins, etc.

When the temperature of the live fish and shellfish declines, physiological functions decline within the range where body fluids do not freeze, and many of them fall into a so-called dormant state. By taking advantage of this property, a technology was introduced to measure the forced decrease in body temperature of seafood and transport it in hibernation, but in this case, the physiological changes in seafood caused by dormancy are normal hibernation Unlike the hibernating state of mammals, the lack of appetite and complete cessation of excretion, which are the characteristics of hibernating animals, necessitated continuous water quality management for prolonged treatment. In addition, prolonged dormancy often leads to death. Also many raw seafood The optimal temperature is low (2 to 20 ° C). In particular, seafood that Japanese people prefer to eat is often inhabited at low temperatures. The temperature difference from C) was small, so slight fluctuations in the aquarium temperature often broke hibernation and caused fish and shellfish to freeze to death, resulting in poor yields due to transportation.

It is also an important issue for commercial farmed fish to obtain healthy farmed fish, and there is a need to safely transport fry that is free of bacteria and viruses to farms.

At present, there is no known storage or transportation method that takes advantage of the phenomenon of “hibernation” of live fish and shellfish.

An object of the present invention is to establish a method of inducing, maintaining, or awakening from a physiologically close to hibernating state (hereinafter, referred to as pseudo-hibernation) for seafood that does not naturally hibernate, and thus surviving Establish safe and efficient methods for storing and transporting live fish> _.

In general, as a factor inducing hibernation, a physiological change is caused by a decrease in environmental temperature or a change in photoperiod as a signal, and a decrease in body temperature in response to a decrease in external temperature results in a hibernation. It is thought that it will be done.

Hibernation is associated with hypothermia, extreme loss of appetite, and consequent cessation of digestive activity and elimination, a decrease in heart rate and blood pressure due to reduced cardiac function, and a complete cessation of urine output due to reduced renal function. Etc. are characteristic.

Hibernation is, to be precise, a complex state in which unit cycles of 1) isothermal period, 2) induction period, 3) deep hibernation period, and 4) arousal period are repeated, and mammals regularly spontaneously during hibernation. Arousal is taking place.

In the past, it has been confirmed that the introduction of serum from a mammal in deep hibernation into a non-hibernating animal of the same type induces hibernation even in summer. It has also been confirmed that administration of a substance contained in the albumin fraction in the serum of another mammal in deep hibernation to a monkey, a non-hibernating animal, results in a state close to hibernation.

Therefore, the applicant of the present application has proposed a patent for the suspicion of seafood using a hibernation-inducing substance. Hibernation Inducing Trigger, a substance present in the serum of mammals in deep hibernation or the albumin fraction in the serum, in “Transportation method of live fish by induction of similar hibernation” (Japanese Patent Application No. 9-193387). (HIT) and the administration of endogenous oboioid peptides and ligands of the oboioid peptide receptors to the fish and shellfish, the fish and shellfish become physiologically close to hibernation (hereinafter referred to as pseudo-hibernation) ) Was confirmed to be able to be guided and maintained. In addition, it has been confirmed that seafood induced in the pseudo-hibernation state awakens from the pseudo-hibernation state by administering a substance that awakens the pseudo-hibernation state.

Thus, for example, if fish and shellfish can be induced and maintained in pseudo-hibernation, oxygen consumption and excretion due to biological metabolism can be reduced in the stored state, and complicated water quality management and temperature management This method has the advantage that live fish can be stored and transported efficiently and economically in a relatively high-density state without having to do so.

The method described in the earlier application patent (Japanese Patent Application No. 9-193857) is to directly administer HIT, delubiooid peptide or delta oboioid peptide receptor ligand to animals. And induces pseudo-hibernation.

Since HIT was collected from mammals in deep hibernation and prepared as HIT, the preparation was complicated, and the amount of preparation was limited. Commercially available ligands of delta oboioid peptide or delta oboioid peptide receptor, for example, methioenkephalin, are expensive, and when these hibernation inducers are administered to the majority of individuals, the cost is low. There was a risk of soaring.

Accordingly, the present invention provides a method for inducing a pseudo-hibernation of an animal to which a hibernation-inducing substance is administered, the serum of the animal in which the pseudo-hibernation is induced is used, and the other serum is secondarily induced in the pseudo-hibernation using the serum. The purpose is to provide. Disclosure of the invention

As a result of intensive studies, the present inventors have found that hibernation or a state similar to hibernation is induced by hibernation or a hibernation-inducing substance synergistic obioid peptide not only in fish and shellfish, but also in vertebrate animals including vertebrate and vertebrate animals. And even animals that induced hibernation artificially Over time, we found that hibernation or a substance that induces hibernation appeared in the serum, which caused secondary hibernation.

The present inventor has applied this finding to fish and shellfish, and originally used a hibernation-inducing substance and a hibernation-awakening substance, in particular, a serum of a hibernating mammal, an obioid peptide or a ligand of an obioid peptide receptor, to thereby obtain a natural product. While hibernating, it succeeded in inducing pseudo-hibernation for seafood. Furthermore, the present inventors succeeded in maintaining the hibernation state and awakening from the hibernation state, and subsequently established a method for measuring the pseudo-hibernation state of fish and shellfish, thereby completing the present invention.

That is, the present invention includes the embodiments described in the following items.

(1) A method for producing pseudo-hibernating seafood, which comprises administering one or more substances selected from the serum of a hibernating animal, a delta-obioid peptide, and a ligand of a delta-obioid peptide receptor to live fish and shellfish. .

(2) The method for producing pseudo-hibernating seafood according to (1), wherein the hibernating animal has been subjected to pseudo-hibernation processing.

(3) The method for producing pseudo-hibernating seafood according to (1) or (2), wherein the hibernating animal is a mammal.

(4) The method for producing a pseudo-hibernating seafood according to the above (1) or (2), wherein the hibernating animal is a seafood.

(5) The method for producing a pseudo-hibernating seafood according to (1), wherein the delta oboi peptide is methionine enkephalin.

(6) The pseudo-hibernating seafood described in any of paragraphs (1) to (5) is a method in which administration to live fish and shellfish is performed by introduction from gills or lateral lines using osmotic shock. Production method.

(7) The method according to any of (1) to (5), wherein the method of administration to live fish and shellfish is a method performed by injection into blood vessels, peritoneal cavity, or cerebrospinal fluid. Manufacturing method for hibernating seafood.

(8) A method for preserving live fish and shellfish, which comprises administering an awakening substance after a certain period of time to the fish and shellfish pseudo-hibernated by the production method described in (1). (9) The substance that wakes up is one or more substances selected from kappa-bioide peptides, myoebioide peptides, ligands for force-tuvoide peptide receptors, and ligands for mu-bioioid peptide receptors. 8. The method for storing live fish and shellfish according to item 8.

(10) Serum of simulated hibernating fish and shellfish glutamic acid-oxacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), serum urea nitrogen (BUN) and A method for measuring the degree of hibernation of a quasi-hibernating seafood, wherein the activity or quantitative measurement of a substance selected from the group consisting of serum uric acid (UA) is performed. According to a first aspect of the present invention, there is provided a method for inducing pseudo-hibernation of fish and shellfish, comprising administering to the animal a substance that induces spontaneous hibernation or a substance that induces pseudo-hibernation, and collecting serum of the animal during pseudo-hibernation, By administering this serum to fish and shellfish, pseudo-hibernation of the fish and shellfish is induced to produce pseudo-hibernating fish and shellfish.

The mechanism of hibernation is still largely unknown, and it is certain that the serum of hibernating animals contains a substance that induces hibernation, but the substance is not clear.

It is thought that this hibernation inducer acts on the receptor for the brain's affer-like substance, causing a series of protective responses such as a slight decrease in temperature, a decrease in hepatic and renal functions, and a marked decrease in appetite. BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 shows the relationship between the respiratory rate and the elapsed time after administration of a hibernation inducer in goldfish. Fig. 2 shows the respiratory volume after administration of a hibernation inducer to Brook Trout by intraperitoneal injection. FIG. 3 is a diagram showing the relationship between respiratory volume and elapsed time after administration of a hibernation-inducing substance to block trauma by intracerebrospinal fluid injection, and FIG. Fig. 5 is a graph showing the relationship between the respiratory volume and the elapsed time after administration of a hibernation inducer to a juvenile squid population, Fig. 5 is a graph showing physiological changes caused by pseudo-hibernation induction of the Japanese turtle, Fig. 6 Fig. 7 is a graph showing physiological changes caused by pseudo-hibernation induction of Japanese brown squid, and Fig. 7 is a graph showing the relationship between the respiratory volume of cut throat trout and the elapsed time after administration of a hibernation inducer. Is a substance that induces respiration and hibernation of red sea bream FIG. 9 (a) is a diagram showing the relationship between the elapsed time after administration and FIG. 9 (a) is a diagram showing the relationship between the movement of the tail fin of the cut throat trawl and the elapsed time after administration of the hibernation inducer. (b) is a diagram showing the relationship between the number of gill respirations of cut throat trout and the elapsed time after administration of the hibernation inducer. FIG. 11 is a graph showing the relationship between the ratio of the `` average diameter of the hibernation inducer '' and the elapsed time after administration of the hibernation inducer. FIG. 12 is a diagram showing the relationship between the oxygen respiratory volume of a aviation and the elapsed time after administration of the hibernation inducer, and FIG. Fig. 14 (a) and Fig. 14 (b) show the relationship between the elapsed time and the elapsed time. Fig. 15 (a) and Fig. 15 (b) are diagrams showing the effect of temperature on the hibernation effect of goldfish, and Fig. 16 is a diagram showing the respiration volume of Troughfish. is there. The symbols in FIG. 16 indicate the respiratory volume of troughs administered with physiological saline, and the respiratory volume of troughs administered with rainbow trout serum induced by pseudo-hibernation by DADLE administration. BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have found that fish, which is a thermophilic vertebrate, are not known to hibernate, but when a hibernation inducer is administered, respiratory volume decreases, hepatic renal function decreases, and it is observed that Similarly, it was confirmed that it was physiologically controlled to be in a state close to hibernation (hereinafter referred to as pseudo-hibernation).

Animals that have been induced to simulate hibernation by administration of a hibernation-inducing substance are in a state in which the existing hibernation mechanism has been activated, and by administering the serum of this animal to other animals such as fish and shellfish, However, it was assumed that the hibernation mechanism also acts on the fish and shellfish. Therefore, the present invention provides a method for administering pseudo-hibernation to another animal by administering the serum of an animal to which pseudo-hibernation has been induced by administering a hibernation-inducing substance to another animal. It manufactures pseudo-hibernating seafood.

Thus, if pseudo-hibernation of another animal can be induced from the serum of the animal in which pseudo-hibernation has been induced, HIT and deltaopio collected from the serum of the hibernating mammal can be obtained. Since there is no need to use a hibernation-inducing substance such as an id peptide for each individual, pseudo-hibernation induction can be performed at low cost.

Therefore, according to the pseudo-hibernation induction method of the present invention, a pseudo-hibernation is induced by administering a hibernation-inducing substance to an animal, and the pseudo-hibernation of another animal is gradually induced from the serum of the pseudo-hibernation-induced animal. This makes it possible to efficiently store and transport animals (eg, fish and shellfish) at low cost.

Hibernation generally means that animals spend most of their life in winter with little or no activity. Hibernation animals sense seasonal changes and take sufficient nutrients before winter to store fat in brown adipose tissue. A decrease in the temperature of the environment or a change in photoperiod is a signal that causes physiological changes and induces hibernation, but results in a decrease in body temperature in response to a decrease in the external temperature. During hibernation, extreme loss of appetite and consequent cessation of digestive activity and elimination, a decrease in heart rate and blood pressure due to decreased cardiac function, and a complete cessation of urinary excretion due to reduced renal function Etc. occur.

As described above, hibernation is considered to be a complex state in which unit cycles of 1) isothermal period, 2) induction period, 3) deep hibernation period, and 4) arousal period are repeated. The isothermal period is when the body temperature recovers, but the animal does not wake up during hibernation, but resembles a sleep state, consisting of REM (REM) and non-REM (NREM) sleep. It is. The induction period consists of so-called wakefulness (WAK) and REM (REM) sleep, and is a period in which rapid declines in body temperature and physiological activity are observed. Deep hibernation refers to the period when the body temperature of a hibernating animal falls and reaches a temperature close to the ambient temperature, and non-REM (NREM) sleep is dominant. The awakening period is the period when energy is obtained by metabolizing fat and the cycle is returned to the constant temperature period. In normal sleep, REM sleep (REM) and non-REM sleep (NREM) are periodically repeated, and the body, including the brain, is breathed. In hibernation, this activity is interrupted. It is believed that the debt of the interruption has been resolved by awakening (B. L. Krilowicz et al., (1988) Am. J. Physiol. 256, R1008-R1019).

Due to differences in the ratio of body surface area to body weight, the same mammal has a large mammal like a bear (smaller ratio) and a small mammal like a squirrel hedgehog. Have different hibernation mechanisms. For example, in large mammals such as bears, body temperature drops at most to 3 ° C (winter storm), and even during winter storms, there is complete arousal and closer to sleep. Also in bears, the metabolism of brown adipose tissue associated with awakening in bears is common to small mammals (RA Nelson et al., (1984) Science 226, 841-842, WL Davie et al. , (1990) Riochimica et Bioptiysica Acta. 1051, 276-278).

It has been reported that hibernation is induced even in summer by introducing serum from animals in deep hibernation into the same animal in non-hibernation (Dawe. AR et al., (1969) Science 163, 298) -299), and a substance present in the albumin fraction has been reported as a substance involved in the induction of hibernation contained in this serum, and is called Hibernation Inducing Trigger (HIT) (Dawe. AR et al. al., (1970) Science 168, 497-498, Deltgen, PR et al., (1987) Life Sciences 41, 2155-2120).

Injection of HIT into cerebrospinal fluid in non-hibernating monkeys observed a decrease in body temperature of 1 to 8 ° C, a decrease in heart rate of 43 to 50%, and a maximum of 7 anorexia-like states that are close to hibernation Was done. Since these changes, i.e., morphine-like effects, are also known as the effects of endogenous obioid peptides, including the pseudo-hibernation state caused by administration of HIT, hibernation state involves the involvement of the obioid peptide receptor. Is suggested. In addition, it is considered that the opioid peptide is not contained in HIT (Oeltgen, PR et al., (1988) Life Sciences 43, 1565-1574). Or a role for the Obioid peptide receptor is predicted (Margules, DL el al., (1979) Rain Res, Bull. 4, 721-724. Knowledge on rats: Swan, H. et. al., (1977) Science 195, 84-85, Findings about monkeys: Myers, RD, et al., (1981) Brain Res, Bull, 7, 691-695).

Mu (), delta (<5), kappa (K) monoreceptors and the like are known as the obioid peptide receptors. Methionin enkephalin, a kind of delta-obioid peptide, is found in the brains of deep hibernating animals about 10 times higher than in the awake state, and the serum of animals in deeper hibernating states Hibernation induced in summer The state was interrupted by the naloxone, an antagonist of the opioid peptide receptor, and the induction of hibernation by HIT was also inhibited. These results suggested that delta-obioid peptide and delta-obioid peptide receptor ligands are effective in inducing hibernation in mammals. On the other hand, mu- and bioactive peptide receptors are thought to be involved in arousal (Oeltgen, PR et al., (1988), Ueltgen, PR et al., (1988)). ).

The hibernation-inducing substance and the hibernation-awakening substance used in the present invention are not particularly limited as long as they are substances that induce and wake up a pseudo-hibernation state in fish and shellfish, but more preferably HIT, Delta opioid peptide or delta opioid peptide receptor ligands can be used. On the other hand, as a substance that wakes up hibernation, muobioid peptide, kappabiooid peptide, a ligand of muobioid peptide receptor, or a ligand of a potato biooid peptide receptor can be used.

In the present invention, the administration of the substance for inducing hibernation induces pseudo-hibernation of live fish and shellfish, but can be naturally recovered and awakened after an appropriate time. The time until awakening depends on the type and size of the target fish and shellfish, and the type of substance to be administered. In addition, it is possible to maintain a pseudo-hibernation state by repeatedly administering the substance over time. In addition, administration of a substance that wakes up from hibernation can arbitrarily wake up fish and shellfish from pseudo-hibernation.

In the present invention, HIΤ used to induce pseudo-hibernation in fish and shellfish can be obtained, for example, as an albumin fraction of serum of a mammal in deep hibernation. More specifically, for example, it can be obtained by the following method. That is, an affinity using a chromatographic carrier (eg, Affi ge 1b 1 ue, manufactured by Bio-Rad, etc.) that selectively adsorbs the albumin fraction contained in the serum of a hibernating animal. Two-column chromatography is effective. The affinity chromatography method can be performed according to a method generally used for isolating and purifying a physiologically active substance, and the following method is exemplified. Blood collected from the hibernating animal is centrifuged at about 1,000 X g for 20 minutes at 4 ° C to obtain a supernatant (serum). This Of the serum is removed using an ultrafiltration device (eg, Bio-Rad) to remove substances with a molecular weight of 5,000 or less, and then freeze-dried. Separately prepare a column of about 5 OmL, fill it with a chromatographic carrier that selectively adsorbs the above-mentioned albumin fraction, and wash the column with 0.02M phosphate buffer (pH 6.8). Dissolve the lyophilized serum sample in an appropriate volume of 0.02M phosphate buffer (pH 6.8) and place on the column. The non-albumin fraction elutes with 0.02M phosphate buffer at pH 6.8, followed by complete elution with 0.02M phosphate buffer at pH 5.7. The albumin fraction bound to the carrier in the column is eluted with a 0.2 M phosphate buffer (pH 5.7) containing 1.4 M sodium chloride, and immediately subjected to ultrafiltration (molecular weight cut: 5,000 or less) to salt. Is removed. The albumin fraction obtained here can be stored until use by freeze-drying.

As the HIT used in the present invention, in addition to those used as the albumin fraction as described above, serum of a mammal during deep hibernation, which contains a large amount of HIT, is preferably used. The target mammals include squirrels such as ground squirrels, chipmunks and squirrels in winter hibernation, and those collected from bears.

In addition, an extract of an organ or the like that produces HIT (for example, ligens / liver) can also be used.

To induce and maintain quasi-hibernation of fish and shellfish, Derby Obioid peptide can be used. Specific examples include methionine enkephalin, leucine enkephalin, / 3-endorphin and the like, and preferably, methionine enkephalin (Tyr-Gly-Gly-Phe-Met) is used. Further, as a ligand of the Derma obiooid peptide receptor used for inducing and maintaining pseudo-hibernation of fish and shellfish, DAD LE (Tyr-D-Ala-Gly-Phe-D-Leu) and the like can be mentioned.

To wake pseudo-hibernation of fish and shellfish, mu- and kappa-bioiod peptides, which are a kind of obioid peptides, can be used. The miu and potato bioiodide peptide receptor ligands used to wake up pseudo-hibernation in seafood include dynorphin 8, morphiceptin and the like.

As a ligand for these obioid peptides and obioid peptide receptors Alternatively, as long as it exists in nature, it is also possible to use a mammalian tissue that produces the substance, particularly one extracted as a composition from the brain, but preferably the composition is further used. Purified products are used, more preferably, synthesized products, which are commercially available. If they do not exist in nature, they can be synthesized by known techniques, and these are also commercially available. Methods of administering a hibernation-inducing substance or a hibernation-awakening substance to fish and shellfish include osmotic shock-induced introduction from the gill or lateral line, injection into the rffl tube, into the peritoneal cavity or into the cerebrospinal fluid. The osmotic shock is performed, for example, by the following method. First, the target fish and shellfish are transferred from the breeding aquarium to an aquarium containing 800 to 1,800 milliosmols, preferably 1,100 milliosmols of saline, and are added to the saline for 0.5 to 5 minutes, preferably 2 minutes. Soak. Return the submerged fish and shellfish to their original breeding aquarium, and then immediately immerse them in the aquarium containing the hibernation inducer or hibernating substance for 2 to 4 minutes. After immersion, return to the original breeding aquarium. The concentration of the hibernation-inducing substance or hibernation-inducing substance used in the aquarium used here varies depending on the substance used.For example, in the case of serum of a mammal in deep hibernation, 50 to 200 // LZmL, preferably 100 / zLZmL, 10 to 200 gZmL for HIT, preferably 50 g / mL. 5 to 50〃 gZmL for methionine enkephalin or DADLE, preferably 10 g / mL. 2 to 50 n for potato bioioid peptide / mL; preferably 15 gZmL, 2 ~ 50 gZmL for muobioid peptide, preferably 2 ~ 50 ng / mL for 1 S gZml ^ dynorphin A or morphiseptin, preferably at a concentration of 15 gZmL, respectively. Used. The saline, hibernation-inducing substance solution, and hibernation-awakening substance solution used in this treatment can be used repeatedly for fry up to a cumulative total weight of fry of 50-100 g without replacement. Dosage via osmotic shock or gill or shunt introduction is suitable for small or juvenile fish and is effective for transport from hatchery to aquaculture.

On the other hand, when administering using a syringe (needle gauge is appropriately selected according to the type, length or weight of the target fish), for example, in the case of intravascular administration, tail fins

One In the case of intra-peritoneal administration, it is easy to insert between the anus and the thoracic fin, and in cerebrospinal fluid, it is administered in the soft part just behind the skull. can do. When using these administration methods, the inducer should be prepared concentrated using a buffer solution that does not affect fish and shellfish, such as physiological saline, and if administered intravascularly or in the cerebrospinal fluid, the inducer should be expressed as the volume per g of body weight. It is preferable that the amount does not exceed 2.5 / L, and in the case of intraperitoneal administration, the amount does not exceed 1 per gram of body weight.

In the case of intravascular administration or intracerebrospinal fluid administration, the dosage may be, for example, 0.5 to 2 / L, preferably 1 μL for serum of a mammal in deep hibernation, preferably 1 μL, for HIT, per 1 g of body weight. 1 to 20 βg, preferably 5 g, and 0.5 to 10 g, preferably 1.5 g, for methionine enkephalin, which is a delta oboioid peptide, or a ligand for its receptor (for example, DADLE), kappa obioide peptide or 0.2-10 g, preferably 0.8 g, for the ligand of the receptor (eg, dynorphin A), 0.2-10 g, preferably 0.8 g for the muobioid peptide or the ligand of the receptor (eg, morphoiseptin) Or a dose of 0.8 ag.

In the case of intraperitoneal administration, the dose may be, for example, 2 to 10 / L, preferably 5 L for sera of mammals in deep hibernation, and 5 to 45 g for HIT per gram of body weight. 15 g, preferably 2 to 10 / g, preferably 5 g for methionine enkephalin or its receptor ligand (eg, DADLE), which is a delta-obide peptide, and its receptor or its receptor. 2 to 10 8, preferably 5 g, for the ligand of the body (for example, dynorphin A), 2 to 10 ag for the ligand of the mu-opioid peptide or its receptor (for example, mo-fuseptin), preferably 5 g. It is used in each dose.

The method of administration by injection is suitable for adult, medium and large fish, and is effective for transport from fishing grounds or farms to markets.

In the present invention, a pseudo-hibernation state is induced and maintained for the fish and shellfish, or By utilizing the method of awakening from that state, it becomes possible to store and transport live fish efficiently. Moreover, the hibernation-inducing substance and the hibernation-awake substance are derived from biological components or have properties very close to biological components, so that they are excellent in safety.

As a method of storing and transporting live fish, for example, specifically, administering a hibernation inducer according to the method of inducing pseudo-hibernation in fish and shellfish, storing the product in a pseudo-hibernation state, and in a pseudo-hibernation state Awakening the fry transported according to the method of transporting the preserved live fish to the market, transporting the fry preserved in a pseudo-hibernation state to the aquaculture farm, and after transport, awakening from the pseudo-hibernation state, A method of continuing aquaculture and the like can be mentioned. Conventional containers such as aquariums used for storage and transportation can be used, but as described above, seafood in a quasi-hibernating state has a low physiological activity, and therefore has a low oxygen consumption and waste products. Since it can be kept low, the decrease in pH and the accumulation of harmful ammonia due to the increase in carbon dioxide gas are also small, and the change in water quality can be extremely suppressed. Therefore, more fish and shellfish can be stored at once, and ventilation and water exchange are not required.

The present invention further provides a measurement method for more accurately and quantitatively grasping the pseudo-hibernation state of fish and shellfish. In order to measure the induction of pseudo-hibernation of fish and shellfish in the present invention, it is necessary to determine the amount of glutamate monooxacetate transaminase (GOT), glutamate monopyruvate transaminase (GPT), serum urea nitrogen (BUN) or serum uric acid (UA). For the measurement, a method usually used in a human serum test can be used. These are also commercially available and available as kits. The measurement can be easily performed by using the kit.

The method of collecting the serum of the fish and shellfish used as the sample is preferably a method that can collect the amount required for each test, but if only a small amount can be collected, increase the number of target seafood and increase the serum. It can be measured by mixing. Preferably, there is a method in which the artery is cut off by laparotomy and collected with a syringe or the like.

Example

In order to explain the present invention in more detail, the following examples are given. They are not limited in any way.

Example 1

Examination of introduction of hibernation inducer by osmotic shock

We investigated the induction of pseudo-hibernation based on the introduction of a hibernation inducer into fish and shellfish.

Considering the effect of the hibernation-inducing substance itself on fish and shellfish, and the practical use of providing a method for transporting live fish, as a method of administering the hibernation-inducing substance, an external solution caused by osmotic shock with less trauma and less stress The method of introduction through the gills or side wires was used.

As the target fish and shellfish, a small (approximately 5.6 cm long, approximately 0.5 g body weight) goldfish (Carassius auratus) suitable for this method was used in a mixed sex. As a hibernation inducer, DADLE (manufactured by Sigma), which is a ligand of the delta-obioid peptide receptor, was used. In the case of a goldfish, the effect of inducing hibernation was apparent in respiration, so the effect of inducing hibernation was measured by measuring the respiratory rate.

The specific method is as follows.

In a 200 mL beaker, place the salted gold (1,100 mOsmol) and then the DADLE (10〃 g / mL) aqueous solution in a 200 mL beaker. After immersion for a minute, they were released into non-aerated distilled water at room temperature, and the effect of inducing hibernation was immediately examined. Controls used were those that were immersed in water after the osmotic shock. Fig. 1 shows the results.

Following osmotic shock, uptake of DADLE, and its effects, appeared within 1 minute, with up to a 50% reduction in respiratory rate. The duration of hibernation was about 10 minutes. Based on these results, the amount taken up by one osmotic treatment may be limited, or the hibernation state may be caused by changing the treatment time and dosage depending on the type and size of the target fish and shellfish. Possible adjustment of duration is suggested. Even during the recovery period, the movement of Era was smaller than usual, and the respiratory volume probably decreased. In addition to respiratory rate and respiratory volume, changes such as decreased motility and decreased responsiveness to foreign body stimulation were observed. Furthermore, a 100% survival rate was shown after recovery from the treatment. Example 2.

Examination of induction of hibernation inducer by injection into peritoneal cavity or cerebrospinal fluid

In order to quantitatively introduce the hibernation-inducing substance from outside the body in a more generalized manner and with less experimental bias, the substance was introduced by injection into the peritoneal cavity or cerebrospinal fluid. As a target fish and shellfish, Brook Traut (Salvelius fontinalis) was used differently from Example 1 in order to examine effects on various fish and shellfishes. Serum collected from ground squirrels (ground squirrel-Citellus tridcceralineatus captured in the suburbs of Laramie, Wyoming, USA) at 5 weeks of hibernation, methionine enkephalin (manufactured by Sigma) and DAD LE was used. Bunolek trout 2-3 years old, weighing 10-12 g, and 3 cm in length were collected from a mountain stream and used by mixing male and female. To measure the hibernation state more quantitatively, the respiration rate was measured with oxygen consumption. The specific method is as follows. Each of the various hibernating inducers was administered to the belly about 2 cm above the anus of Brooktraut using a 24 G needle using a syringe. The dose of each hibernation inducer is 5.0 / L for hibernating ground squirrel serum, 5 g for methionine enkephalin, and 5 βg for DAD LE per lg of body weight of the target seafood. did. Physiological saline was used as a control instead of each hibernation inducer.

Quickly take out the Brook Trout from the breeding aquarium, inject various hibernation inducers into the peritoneal cavity of another Brook Trait, and immediately release them into the aerated water in a 10 L beaker. did. Periodically stopping the ventilation, stop immediately was dissolved acid elementary charge (0 _{2 i ni t),} amount of dissolved oxygen after standing for 15 minutes the (0 ₂ art) were measured in addition unaerated state. The difference [Δ = (0 ₂ , nit)-(0 ₂ a, t)] is calculated per minute,

The value converted per 1 g body weight was defined as the oxygen consumption at that time (ppmZminZg). The dissolved oxygen amount was measured by inserting a probe of a dissolved oxygen meter (DO meter) into water. In order to minimize the stress on the fish, open containers such as beakers were used. The results are shown in FIG.

Intraperitoneal administration had only a partial effect with ground squirrel serum and methionine enkephalin, whereas DAD LE significantly reduced respiratory Down to. Wild Brook Trout collected from a mountain stream was sensitive to external stimuli, but was insensitive to external stimuli as it could be lifted by hand while respiratory rate was low. The effect appeared quickly and lasted for about 2 hours. These observations suggest that the stability of the hibernation-inducing substance reflects the degree of induction. Immediately, the serum of local squirrel and methionine enkephalin were considered to be degraded while passing through the lungs in the bloodstream, making it difficult to reach specific parts of the brain. On the other hand, DADLE, which is substituted with two D-type amino acids, functions as a ligand for the obioid peptide receptor, but is unlikely to be a major degrading enzyme substrate.

Therefore, in order to avoid a decrease in the effect due to the degradation, hibernation-inducing substances were introduced into fish and shellfish by injection into the cerebrospinal fluid. The target fish and shellfish, and the substance to induce hibernation to be administered were the same as those used for intraperitoneal administration described above. The cerebrospinal fluid was administered using a 26G x 1/2 "needle using a syringe. The soft part immediately behind the fish skull was gently injected into the cerebrospinal fluid so as not to damage the cerebral spinal cord. Per gram of body weight, do not exceed 1 L ^ 11 for serum of hibernating ground squirrel, 5 ^ for 11 sera, 1.5 Hg for methionine enkephalin and DADLE, and not more than 2.5 L for 1 g of body weight For comparison, physiological saline was used as a substitute for each of the hibernation-inducing substances, and the results are shown in Fig. 3. As is clear from Fig. 3, All three of the ground squirrel sera, methionine enkephalin and DADLE reduced respiratory rates to 25% within 20 minutes after administration, with hibernation lasting about 60 minutes. However, the duration can be shortened or prolonged by adjusting the dose. Was possible. Further time by repeating the administration of winter sleep inducer recovery, the duration of the hibernation state is further extended ο

Example 3

Examination of pseudo hibernation induction effect of DADLE by precise measurement of oxygen consumption

We investigated the effect of DADLE on the induction of pseudo-hibernation by avoiding the effects of temperature such as air temperature and water temperature, and the effect of oxygen in the atmosphere, and measuring oxygen consumption more precisely. The measurement method was based on the commonly used Winkler method (Sojiro Oka et al., “Experimental Chemistry Course” 15 (under Analytical Chemistry), p.348 (1958)). Closed plastic container fitted with a water sampling tube to further reduce the effects of atmospheric oxygen

(350x230x130mm) and a stainless steel basket (with a lid for the net) was put in, and fish was put in this basket. Large fish were susceptible to stress due to sealing, and it seemed that there was a difference in individual response.Therefore, 150 fish of Amemas fry (mean length 1.8 cra, mean weight 0.15 g) were placed in a net basket. Oxygen consumption was measured for this population. Because fry usually tends to form baby boomers, it is unlikely that any stress will occur due to crowding.

Except for processing the whole basket containing the fry group, the same treatment as in Example 1 was performed with DADLE treatment and control treatment. After the treatment, the whole basket was placed in a closed container, and a small amount bottle (lOOraL) was placed through the water sampling port. The water sampled in) was used as a 0-minute sample, and after 15 minutes, water was sampled again and used as a 15-minute sample. A set of a 0-minute sample and a 15-minute sample was prepared before processing, and at 20, 40, 60, and 80 minutes after processing, and the dissolved oxygen content was determined by the Winkler method described above for 15 minutes. The difference between the dissolved oxygen amount of the sample and the 0-minute sample was defined as the oxygen consumption at that time. The results are shown in FIG. As is clear from Fig. 4,

A marked decrease in oxygen consumption was observed in the DADLE-treated group, and the effect of inducing pseudo-hibernation by DADLE treatment was confirmed by more precise measurement of oxygen consumption. Example 4

Examination of evaluation method of pseudo-hibernation state of seafood

A method for assessing whether a decrease in physiological activity characteristic of hibernation occurs in addition to measurement of respiratory rate and measurement of oxygen consumption in the simulated hibernation state of fish and shellfish caused by administration of a hibernation inducer It was investigated.

For the serum of fish and shellfish to which a hibernation-inducing substance was administered, items that were usually tested in human serum tests were examined. That is, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) are used as indicators of liver function, amylase (AMY) is used as an indicator of liver function, and kidney function. Serum urea nitrogen (BUN), creatinine (CRE) and The activity or amount of serum uric acid (UA), blood glucose (GLU), and inorganic phosphate (IP) were measured. The measurement was carried out using Ultra Liquid, Daikara Liquid, Pericolor Liquid (all manufactured by Toyobo Co., Ltd.) according to the attached instruction manual. The serum used as the sample was prepared as follows.

The DADLE treatment and the control treatment were performed in accordance with the intraperitoneal administration of Example 2 except that the target fish and shellfish were cut throat trout (average body length 12 cm, average body weight 3.9 g). Subsequently, the cutthroat trout that induced pseudo-hibernation by DADLE was removed from the aquarium immediately after induction, and at 20, 40, and 60 minutes after induction, and the laparotomy was performed quickly. The artery between the heart and gill was cut with scissors, and the blood was collected with a 1 mL syringe, collected in a microcentrifuge tube, and centrifuged (6,000 xg, 3 minutes) to obtain serum as a supernatant. In this example, the sera of the three turtles subjected to the treatment were mixed and used as a sample at the time of removal. The results are shown in FIGS. 5 and 6. As is clear from FIG. 4, a remarkable decrease in oxygen consumption is observed 20 minutes after DADLE treatment. Among the items examined in this example, GOT, GPT, BUN and UA rapidly increased 20 minutes after DADLE treatment, and recovered to the original level after 40 minutes. Other GLU, AMY, CRE and IP showed no significant difference compared with the control. From these results, it was clarified that the quasi-hibernation of fish and shellfish causes a decrease in physiological activity similar to that of higher mammals. Furthermore, these results indicate that GOT, GPT, BUN, and UA measurements can be used as a powerful method to evaluate the effect of seafood on inducing pseudo-hibernation.

Example 5

Pseudo-hibernation was administered to freshwater fish by hibernating squirrel serum, and respiratory volume during pseudo-hibernation was measured.

Fig. 7 shows the measurement results.

Example 6

We conducted a hibernation induction experiment on red sea bream, which is transported by marine fish, as in freshwater fish. 1.5 to 2 kg of red sea bream were administered with the hibernation-inducing substance methionine enkephalin (DADLE) (about l // g per lg of fish), and the effect was examined. The administration was performed by intraperitoneal injection, but the stress on the injected fish seemed to be quite large. The effect of the hibernation inducer was determined by respiratory volume, but unlike small fish, conventional closed containers cannot be used, so a plastic aquarium was used to fill 60 L of seawater, and a vinyl sheet was placed on the water surface. The semi-closed state was ensured by spreading and breaking the equilibrium with the atmosphere. Respiration was tracked up to 2 hours post-dose as a 15 minute respiration rate. This was determined from the difference between the dissolved oxygen amount at the start of measurement and the end of measurement (after 15 minutes), and the dissolved oxygen amount was directly measured by a DO meter. The unit is mg / milligram of oxygen consumption per lg of fish weight per hour. As a control, a fish to which physiological saline was administered was used. The number of samples was n == 8. The water temperature was 23.7 degrees Celsius.

Fig. 8 shows the measurement results.

According to FIG. 8, in the Thai group administered with saline (Salline), the respiratory volume increased about three times after one hour. Since this is transient, it is considered an injection stress. This is similar to other fish. One hour after administration, the volume returned to normal. In contrast, in the Thai group to which DADLE was administered, no change in respiratory volume due to stress was observed, and fish behavior was also suppressed.

Therefore, it was confirmed that the hibernation inducer works effectively in Thailand as well as other fish.

Example 7

The animal's hibernation status was assessed by its behavior rather than by physiological methods.

Behavior reflects not only physiological changes but also global changes in animals.

However, there is no standardized method for evaluating behavior, and it is difficult to evaluate in practice. In this case, the movement was performed using muscle movement (tail fin movement) and respiratory movement (gill movement).

The results are shown in FIGS. 9 (a) and 9 (b).

Fig. 9 (a) and Fig. 9 (b) both measure the movement of the tail fin and the movement of the gills using a cut throat trout. The movement of the tail fin is due to muscular activity, and various physiological changes intervene as factors. One trout in diameter The water was placed in a 90 cm tub, and dechlorinated water was poured to prevent water from staying inside the tub. As a result, the trout is washed away unless the tail fin is moved, and the trout is forced to move constantly. Under these conditions, the behavior was evaluated by counting the number of exercises of the tail fins of the control fish fed with saline and the sample fish fed with methionine enkephalin. The control that gave saline to the movement of the tail fin was the one that gave methionine enkephalin, a force that made a remarkable movement from the stress of injection after 10 minutes, and this stress was avoided. Exactly the same behavior pattern is reflected in breathing. Here, evaluation was made by observing the movement of gills, not respiratory volume.

From the results of both experiments, it was clear that the whole behavior of the fish was suppressed by methionine enkephalin, which proved to be effective in avoiding stress. In addition, obvious effects were found in non-fish animals. Murasaki Puji has a hard shell, and mobile spines are evenly distributed around it. The spines serve movement and protection, and also trap drifting drift. Numerous soft tube feet are arranged in the gaps between the spines, which have the effect of moving and sticking, as well as catching food. When methionine enkephalin was administered to this No. 2, the ducts began to shrink all at once, and remained stationary in a fixed place and could not move. It took about 20 minutes during this time. This situation continued for three words and gradually recovered. In order to evaluate this quantitatively, after administration to Murasaki Puji, take a picture over time, measure the length of the longest 10 of the tube feet on the picture, and compare the average length to The value divided by the average diameter of the shell was used as an index. This is because there is a difference in size between the solids. This operation was performed on eight independent solids, and the average and standard error were plotted in Fig. 10.

According to the results, hibernation was almost achieved 10 minutes after administration, and did not recover even after 40 minutes. In fact, this situation lasted two days.

This experiment showed for the first time that methionine enkephalin was effective in inducing hibernation in invertebrates.

Example 8

Pseudo-hibernation experiments are also performed on shrimp (button shrimp) used for live fish transport. Was. The results are shown in FIG.

The shrimp treated with DADLE clearly had a lower respiratory rate than the control shrimp, and the effect lasted from 30 minutes to 2 hours after administration. On the other hand, shrimp given DAD LE and naloxone at the same time did not show such effects and showed the same changes as the control shrimp. Therefore, it was confirmed that methionine 'enkephalin acts specifically on crustaceans and induces hibernation. The fluctuation of respiratory rate in shrimp tends to gradually increase with time in this experiment, but this may be considered as stress when measuring the respiratory rate.

Example 9

We conducted an experiment on induction of dormancy in a mollusk, abiabi. Figure 12 shows the results.

No change in respiratory rate due to stress or the like was observed in the control (administered saline) in the abalone. The abdomen administered DADLE showed a marked decrease in respiratory rate after 30 minutes and recovered in about 1 hour. Since the effects of DADLE were completely antagonized by simultaneous administration of naloxone, it was found that DADLE also acts specifically on mollusk abalone and induces a pseudo-hibernation state.

The reason why the respiratory rate of the naloxone-administered abi at 30 minutes increased specifically is probably because the amount of naloxone to the abi was too large.

Example 10

As described above, hibernation induction was remarkable in No. 2, but the change in respiratory rate was examined as in other animals. The results are shown in FIG.

In this example, the control penis, which received saline, showed a tendency for the respiratory rate to rise temporarily 30 minutes after administration and then to gradually decrease.

When DADLE was administered, the increase in respiratory rate due to such stress was suppressed, and the effect did not recover even after 3 hours. When naloxone was administered with DADLE, the change in respiratory rate was almost the same as that in the control, indicating that the drug was DADLE-specific. Example 11

DAD LE was administered to rainbow trout to induce pseudo-hibernation, and serum prepared from the rainbow trout was again administered to a power throat trout to examine whether secondary pseudo-hibernation was induced. The evaluation was performed by testing serum. The activity or amount of GOT, GPT, UA, and BUN in the serum of a pittle-mouth trout 30 minutes after administration was measured in the same manner as in Example 4, and the results were shown in FIG. 14 (a) and FIG. (b) It is shown in the figure.

In each case, pitted throat trout treated with secondary serum showed the same effect as DADLE treatment compared to control (treated with saline), and rainbow trout in which pseudo-hibernation was induced once. It was found that the serum contained a substance that induces hibernation.

Example 12

The effect of temperature on the extension of hibernation time was tested using goldfish at 6 ° C and 24 ° C. The respiratory rate with respect to the elapsed time after DADLE administration was examined and compared. The results are shown in FIGS. 15 (a) and 15 (b).

This experiment showed that at 24 ° C, the hibernation state of the goldfish lasts at most 40 minutes, but when this temperature is lowered to 6 ° C, the hibernation state can be extended to 6 hours.

From the results of the examples, it was proved that methionine enkephalinka, which is a kind of an obioid peptide recognized as a brain hormone, induces hibernation in seafood. Methionin enkephalin is captured in hibernating mammals as a key substance that induces hibernation in winter, and can induce hibernation in mammals even in summer.

It was found that this substance can be used to induce a state similar to hibernation in fish and shellfish, that is, a pseudo-hibernation state, and the effect can be used to transport or preserve fish and shellfish. In addition, it was found that administration of hibernating squirrel serum, methionine enkephalin, and the like to the body of the fish caused a temporary decrease in the respiratory rate of the fish. In particular, administration by intraperitoneal injection is most effective for goldfish, carp, trout, etc., in which case the serum components of the fish are specifically changed, and the liver and kidney functions may be significantly changed. Sharing Was. Since the effect of each fish recovers in about one hour, it was found that methionine kefarin has the same effect on fish as mammals, that is, it has the effect of inducing hibernation. This indicates that opioid peptides, which have an effect on higher mammals, work in the same way on thermothermic animals.

It can be presumed that the recovery of the effect was due to the induction of the activity of enkephalinase, a degrading enzyme, and the degradation of the administered methionine enkephalin.

This estimate is a conventional study of pain, and it is ineffective when administered to mammals unless administered into the cerebrospinal fluid, and methionine enkephalin in the bloodstream is an enkephalinase abundant in the lungs. This is well supported by the fact that it is immediately degraded by the method and that the presence of enkephalinase has been confirmed in salmonids.

Example 13

A substance that induces pseudo-hibernation is administered to rainbow trout to induce a state of pseudo-hibernation (primary pseudo-hibernation), serum is removed from the rainbow trout to which the pseudo-hibernation-inducing substance is administered, and administered to another animal (Trafugu). A method for producing a pseudo-hibernating tortoise was introduced by introducing the aforementioned torchfish into a pseudo-hibernation state (secondary pseudo-hibernation).

As a substance that induces pseudo-hibernation temporarily, DADLE (manufactured by Osaka Peptide Co., Ltd.), which is a ligand of the Deruba Obioid peptide receptor, was used.

A rainbow trout (scientific name: Salmo gair dneri), a body weight of about 250 g, and a body length of 25 cm to 35 cm was used as a test object to induce primary pseudo-hibernation.

The hibernation inducer was administered intraperitoneally to DADLE with a syringe using a 24 G needle in the abdomen 5 cm above the rainbow trout anus.

The dose of DADLE was 5 g per gram of body weight of each target fish.

DADLE was administered to 10 rainbow trout, including males and females. The rainbow trout was raised in a breeding aquarium through which groundwater flows freely to avoid physiological errors and to give no food for 24 hours before the test and to avoid physiological stress. The rainbow trout was taken out of the breeding aquarium, DADLE was injected intraperitoneally with a syringe, and then released into another aquarium. Thereafter, serum of rainbow trout in a pseudo-hibernation state 20 minutes after administration of the hibernation inducer was collected.

That is, 1 mL to 2 mL of blood was collected from a blood vessel near the tail fin of a rainbow trout in a pseudo-hibernation state using a syringe. The collected blood was collected in a centrifuge tube, centrifuged (12,000 xg, 5 minutes), and the supernatant was obtained as a serum sample.

The tiger puffer (scientific name: Fugu rubripes) used in this example was 10 months old, weighed about 120 g, and was 15 cm to 2 Ocm in length.

The rainbow trout serum was administered to the abdomen 5 cm above the anus of 10 male pufferfish, including males and females, using a syringe with a 24 G needle at 0.2 L / g body weight of the target fish.

As a comparative test, physiological saline was administered in the same manner to 10 tiger puffer fish.

After that, the oxygen dissolved amount (DO) was measured by the Winkler method [Sojiro Oka et al., “Experimental Chemistry Lecture” 15 (Analytical Chemistry), p. 48 (1958)], and the change in oxygen dissolved amount (DO) per unit time The respiratory volume was calculated from the results, and the effect of inducing pseudo-hibernation was evaluated.

In other words, in order to reduce the effect of oxygen in the atmosphere, put troughfish in a sealed plastic container equipped with a water sampling tube, prepare 10 sealed containers, and measure the dissolved oxygen (DO) for each. did.

The closed container containing the trough was covered with a lid, ventilated into the atmosphere, and placed in a large water tank through which seawater flows. Seawater in the sealed container in this state was collected as the first sample. In addition, seawater 20 minutes after the container was sealed was sampled as the second sample.

The oxygen dissolved amount (DO) of each of the first sample and the second sample was measured, and the difference was converted into lg of fish body weight and per volume of closed container (5.7 L), and the absolute oxygen consumption of fish was calculated for 1 hour. Respiratory volume per unit (mgOzZgrZhr).

The results are shown in FIG. The T bar on the graph indicates the standard error. In the figure, 1 is the average respiratory volume of the fish (n = 10) to which physiological saline was administered as a comparative test, and 2 in the figure is the rainbow trout serum induced by pseudo-hibernation by DADLE in this example. The mean respiratory volume of the fish (n = 10) that was administered and secondary induced pseudo-hibernation was shown.

As shown in the results of Fig. 16, rainbow trout induced temporarily by pseudo-hibernation in DAD LE. The respiratory volume (2 in the figure) of the fish secondary to pseudo-hibernation induced by trout serum administration was lower than the respiratory volume (1 in the figure) of the fish administered physiological saline. It was confirmed that it was reduced to 30% to 50%.

In other words, it can be confirmed that by administering the serum of an animal (rainbow trout) to which pseudo-hibernation was primarily induced by DADLE, another animal (Trafugu) is secondarily induced to be pseudo-hibernating.

As described above, pseudo-hibernation is induced by a hibernation-inducing substance, the serum of the animal is collected, and administered to another animal, whereby the other animal can be secondarily induced by pseudo-hibernation. Without using a hibernation-inducing substance, animals can be induced into pseudo-hibernation, and the cost of pseudo-hibernation induction can be reduced. In addition, pseudo-hibernation can be induced by administration of serum to allogeneic individuals, which is excellent in terms of safety. Industrial applicability

Provide more efficient and safer transport of live fish by establishing a method for preserving and transporting live fish based on methods that induce and maintain a pseudo-hibernation state for fish and shellfish that do not naturally hibernate or awaken from that state be able to. Further, by appropriately selecting the administration method depending on the type of the fish and shellfish to be administered and the type of the hibernation inducer to be administered, and by adjusting the dose and the number of administrations, a more excellent method of transporting live fish can be obtained.

In addition, administering a substance that induces pseudo-hibernation to an animal to induce pseudo-hibernation temporarily, collecting serum of the animal in which the pseudo-hibernation is induced, and administering this serum to another animal. Thus, a method for producing pseudo-hibernating fish and shellfish can be achieved efficiently and economically by a method for inducing pseudo-hibernation of fish and shellfish secondary.

Unlike drugs, pseudo-hibernation can be induced by using serum containing substances that animals naturally produce for their survival, and therefore they are also excellent in terms of safety.

According to the method for producing pseudo-hibernating seafood of the present invention, for example, a large amount of seafood can be safely stored and transported at low cost, which is industrially useful.

Claims

The scope of the claims

1. A method for producing pseudo-hibernating seafood, which comprises administering one or more substances selected from the serum of a hibernating animal, a delta-obioid peptide, and a ligand of a delta-obioid peptide receptor to a live fish and shellfish.

2. The method according to claim 1, wherein the hibernating animal has been subjected to a pseudo-hibernation treatment.

3. The method according to claim 1, wherein the hibernating animal is a mammal.

4. The method according to claim 1, wherein the hibernating animal is a seafood.

5. The method for producing a pseudo-hibernating seafood according to claim 1, wherein the delta obioide peptide is methionine enkephalin.

6. The method for producing a pseudo-hibernating seafood according to any one of claims 1 to 5, wherein the method of administration to the live seafood is a method performed by introduction from an gill or a lateral line using osmotic shock.

7. The method for producing a pseudo-hibernating seafood according to any one of claims 1 to 5, wherein the method of administration to a live fish is a method performed by injection into a blood vessel, peritoneal cavity, or cerebrospinal fluid.

8. A method for preserving live fish and shellfish, comprising administering an awakening substance after a certain period of time of the seafood that has been pseudo-hibernated by the production method according to claim 1.

9. The live fish and shellfish according to claim 8, wherein the substance that wakes up is one or two or more substances selected from a kappa pioid peptide, a mu pioid peptide, a ligand of a force paopioid peptide receptor, and a ligand of a muobioid peptide receptor. How to save.

10 0. It is selected from the group consisting of glutamate monoxacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), serum urea nitrogen (BUN) and serum uric acid (UA) in serum of pseudo-hibernating seafood A method for measuring the degree of hibernation of pseudo-hibernating fish and shellfish, which comprises measuring a substance in a quantitative manner.