WO1999048358A1 - Procedes de creation, de stockage et de mesure du degre d'hibernation, de poissons et coquillages pseudo-hibernant - Google Patents

Procedes de creation, de stockage et de mesure du degre d'hibernation, de poissons et coquillages pseudo-hibernant Download PDF

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Publication number
WO1999048358A1
WO1999048358A1 PCT/JP1999/001127 JP9901127W WO9948358A1 WO 1999048358 A1 WO1999048358 A1 WO 1999048358A1 JP 9901127 W JP9901127 W JP 9901127W WO 9948358 A1 WO9948358 A1 WO 9948358A1
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WIPO (PCT)
Prior art keywords
hibernation
pseudo
fish
hibernating
serum
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PCT/JP1999/001127
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English (en)
Japanese (ja)
Inventor
Yoichi Kadokami
Yoshinori Taniguchi
Mitsuyuki Horiuchi
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Nagase & Co., Ltd.
Jifas Corporation
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Priority claimed from JP14610298A external-priority patent/JPH11318273A/ja
Priority claimed from JP28704798A external-priority patent/JP2000106787A/ja
Application filed by Nagase & Co., Ltd., Jifas Corporation filed Critical Nagase & Co., Ltd.
Priority to AU32758/99A priority Critical patent/AU3275899A/en
Publication of WO1999048358A1 publication Critical patent/WO1999048358A1/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/10—Culture of aquatic animals of fish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/50—Culture of aquatic animals of shellfish
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K63/00—Receptacles for live fish, e.g. aquaria; Terraria
    • A01K63/02—Receptacles specially adapted for transporting live fish
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Definitions

  • 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.
  • the transportation of live fish and shellfish, particularly cultured fish and shellfish, is carried out from hatchery to farm or from farm to farm.
  • 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> _.
  • pseudo-hibernation a physiologically close to hibernating state
  • 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.
  • 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 Hibernation Inducing Trigger
  • pseudo-hibernation the fish and shellfish become physiologically close to hibernation
  • HIT was collected from mammals in deep hibernation and prepared as HIT, the preparation was complicated, and the amount of preparation was limited.
  • 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
  • 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.
  • the present invention includes the embodiments described in the following items.
  • 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. .
  • 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.
  • 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).
  • 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.
  • GAT glutamic acid-oxacetate transaminase
  • GPT glutamate-pyruvate transaminase
  • BUN serum urea nitrogen
  • 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.
  • 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
  • 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.
  • FIG. 9 (a) is a diagram showing the relationship between the elapsed time after administration
  • 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.
  • FIG. 15 (a) and Fig. 15 (b) are 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. 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.
  • 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).
  • 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.
  • 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.
  • a 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.
  • animals eg, fish and shellfish
  • 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.
  • 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.
  • 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).
  • the same mammal 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).
  • Hibernation Inducing Trigger HIT
  • 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.
  • delta-obioid peptide and delta-obioid peptide receptor ligands are effective in inducing hibernation in mammals.
  • 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.
  • 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.
  • 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.
  • administration of a substance that wakes up from hibernation can arbitrarily wake up fish and shellfish from pseudo-hibernation.
  • HI ⁇ 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.
  • an ultrafiltration device eg, Bio-Rad
  • 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.
  • 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.
  • an extract of an organ or the like that produces HIT for example, ligens / liver
  • HIT for example, ligens / liver
  • 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.
  • 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.
  • 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.
  • 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.
  • the concentration of the hibernation-inducing substance or hibernation-inducing substance used in the aquarium used here varies depending on the substance used.
  • 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.
  • 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.
  • tail fins 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
  • 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.
  • the dosage may be, for example, 0.5 to 2 / L, preferably 1 ⁇ L for serum of a mammal in deep hibernation, preferably 1 ⁇ L, for HIT, per 1 g of body weight.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • GAT glutamate monooxacetate transaminase
  • GPT glutamate monopyruvate transaminase
  • BUN serum urea nitrogen
  • U serum uric acid
  • 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.
  • 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.
  • DADLE manufactured by Sigma
  • the effect of inducing hibernation was apparent in respiration, so the effect of inducing hibernation was measured by measuring the respiratory rate.
  • the substance was introduced by injection into the peritoneal cavity or cerebrospinal fluid.
  • Brook Traut Salvelius fontinalis
  • Example 1 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.
  • 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 ⁇ g 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.
  • 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.
  • 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.
  • Example 2 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,
  • GAT glutamate-oxaloacetate transaminase
  • GPT glutamate-pyruvate transaminase
  • AY amylase
  • 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.
  • 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.
  • Fig. 8 shows the measurement results.
  • 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.
  • 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.
  • 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.
  • 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.
  • control penis which received saline, showed a tendency for the respiratory rate to rise temporarily 30 minutes after administration and then to gradually decrease.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • DADLE manufactured by Osaka Peptide Co., Ltd.
  • Osaka Peptide Co., Ltd. which is a ligand of the Deruba Obioid peptide receptor
  • 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.
  • 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.
  • physiological saline was administered in the same manner to 10 tiger puffer fish.
  • 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.
  • 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.
  • 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.
  • animals can be induced into pseudo-hibernation, and the cost of pseudo-hibernation induction can be reduced.
  • pseudo-hibernation can be induced by administration of serum to allogeneic individuals, which is excellent in terms of safety.
  • 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.
  • 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.

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  • Life Sciences & Earth Sciences (AREA)
  • Environmental Sciences (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Zoology (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention porte sur une méthode de création de poissons et coquillages pseudo-hibernants consistant à administrer à des poissons et coquillages une ou plusieurs substances sélectionnées dans le sérum d'un mammifère hibernant en cours d'hibernation, ainsi que des delta-opioïdes et des ligands du récepteur du delta-opioïde. Cette méthode met les poissons et coquillages n'hibernant pas naturellement dans un état proche de l'hibernation, dit pseudo-hibernation, où ils sont maintenus jusqu'à leur réveil. Elle permet donc de stocker et transporter avec sûreté des poissons frais avec un taux élevé de survie et une grande efficacité.
PCT/JP1999/001127 1998-03-20 1999-03-08 Procedes de creation, de stockage et de mesure du degre d'hibernation, de poissons et coquillages pseudo-hibernant WO1999048358A1 (fr)

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AU32758/99A AU3275899A (en) 1998-03-20 1999-03-08 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

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JP10/72462 1998-03-20
JP7246298 1998-03-20
JP14610298A JPH11318273A (ja) 1998-03-20 1998-05-27 動物の疑似冬眠誘導方法
JP10/146102 1998-05-27
JP28704798A JP2000106787A (ja) 1998-10-08 1998-10-08 魚介類の製造方法
JP10/287047 1998-10-08

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CN109430158A (zh) * 2018-11-05 2019-03-08 贵港市庄满园农业服务有限公司 一种便于清洗及保护乌龟的乌龟饲养盒

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EP3289864A4 (fr) * 2015-04-30 2019-02-13 University Industry Foundation, Yonsei University Wonju Campus Composition pour induire un hypométabolisme, procédé pour induire un hypométabolisme, et procédé de transport de poisson utilisant ses techniques
CN109430158A (zh) * 2018-11-05 2019-03-08 贵港市庄满园农业服务有限公司 一种便于清洗及保护乌龟的乌龟饲养盒

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