KR20120084500A - Method for prolonged hibernation-like state of non-hibernated animals - Google Patents

Abstract

PURPOSE: A method for inducing the hibernation-like state of non-hibernated animals is provided to be used for long distance traffic of animals. CONSTITUTION: A method for inducing the hibernation-like state of non-hibernated animals is as follows. Low metabolism and low hypothermia are induced by injecting a hibernation inducing agent to non-hibernation animals. A continued dormant state is maintained by injecting hibernation inducing agent one time or more. A wakening state is made by stopping the injection of the hibernation inducing agent.

Description

Method for inducing moving surface of non-hypnotic animals {Method for prolonged hibernation-like state of non-hibernated animals}

The present invention relates to a moving surface derivation method of non-hypnotic animals except humans.

Hibernating animals are semi-thermal animals that go into hibernation for more than 5 months by lowering their metabolic rate and body temperature during the low temperature and lack of food. As the hibernating season approaches, these animals are known to produce hibernating material in brain tissue that is not yet accurately identified. This material is called a hibernation induction trigger. Hibernating Squirrel (Winter) When administered to a summer squirrel, blood can induce hibernation, which explains the existence of such a substance.

Importantly, hibernating animals have arousal every few days, even in a torpor state [Epperson LE, Martin SL. 2002. Quantitative assessment of ground squirrel mRNA levels in multiple stages of hibernation. Physiol Genomics 10: 93 + 102; Park KJ, Jones G, Ransome RD. 2000. Torpor, arousal and activity of hibernating greater horseshoe bats (Rhinolophus ferrumequinuum). Funct Ecol 14: 580-588. Awakening time lengths vary from species to species but are generally reported to be less than 24 hours. During the awakening period, animals raise their metabolic rate quickly to normal temperatures, and there has been a lot of interest in why these animals, which need to conserve energy in hibernation, "wake up" without exception.

Recently, some research teams, including the lab, have found that awakening plays an important role in maintaining homeostasis in hibernated animals [Carey HV, Andrews MT, Martin SL. Mammalian hibernation: Cellular and molecular responses to depressed metabolism and low temperature. Physiol Rev 83: 1153-1181; Lee K, So H, Gwag T, Ju H, Lee JW, Yamashita M, Choi I. 2010. Molecular mechanism underlying muscle mass retention in hibernating bats: Role of periodic arousal. J Cell Physiol 222: 313-319. It is to remove the body waste accumulated in the dormant state, to increase the immune function, ingesting insufficient water, and especially to prevent atrophy of cardiopulmonary and muscle function through a brief exercise effect. This periodic recovery is of great importance for the survival of animals that have a long hibernation period.

To date, five kinds of hibernate derivatives are known internationally. Among these, 3-iodothyronamine (T ₁ AM) is a natural thyroid hormone that can be produced in the body through deiodination and decarboxylation in thyroxine (T4). Hormonal hibernate agents [Scanlan TS, Suchland KL, Hart ME, Chiellini G, Huang Y, Kruzich PJ, Frascarelli S, Crossley DA, Bunzow JR, Ronca-Testoni S, Lin ET, Hatton D, Zucchi R, Grandy DK. 2004. 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nat Med 10: 638-642; Panas HN, Lynch LJ, Vallender EJ, Xie Z, Chen GL, Lynn SK, Scanlan TS, Miller GM. 2010. Normal thermoregulatory responses to 3-iodothyronamine, trace amines and amphetamine-like psychostimulants in trace amine associated receptor 1 knockout mice.J Neurosci Res 88: 1962-1969], to date, even in the blood of mice, rats, hamsters, and even humans. The presence of the substance was confirmed. For this reason, the use of this material to induce the movable surface of non-hypnotic mammals is of great medical value.

An object of the present invention is to provide a method of inducing moving surfaces of non-hypnotic animals except humans.

The present invention as a means for solving the above problems,

A dormant step of sleeping by administering a hibernate inducer; And

Awakening step without hibernation-inducing substance

Provides a method of inducing movable surfaces of non-hypnotic animals, except humans.

The present invention also provides

A dormant first step of dormant by administering a hibernate inducer;

An awakening step without a hibernation inducing substance;

A dormant second step of dormant by administering a hibernate inducer;

Provides a method of inducing movable surfaces of non-hypnotic animals, except humans.

Movable surface induction method of the non-hypnotic animal according to the present invention can survive in a dormant state for a long time without disrupting brain cell protection activity, it can be very useful for emergency relief treatment or long-distance transport of animals.

In FIG. 1, A is a photograph of an ICR mouse male induced at 20 minutes after a single T ₁ AM administration, and B and C are graphs showing changes in body temperature for 6 hours after a single T ₁ AM administration (room temperature 23 ± 0.5 ° C., n = 4, arrow: T ₁ AM single dose time), D is a graph showing metabolic changes with body temperature change.
FIG. 2 is a graph showing body temperature measured from 1 day before administration to 10 days after administration of Comparative Example 2 (Control) 1 (A) and Example 1 (T ₁ AM multi-administration group) (BD) [indoor Temperature is 23 ± 0.5 ° C. Feed, water and light / dark cycles are controlled as A, long arrows indicate T ₁ AM intraperitoneal administration and short arrows indicate T ₁ AM subcutaneous administration.
Figure 3 shows the neural response in the continuous dormant state of the multiple dose group of Example 1.

The present invention

A dormant step of sleeping by administering a hibernate inducer; And

Awakening step without hibernation-inducing substance

It relates to a movable surface induction method of non-hypnotic animals, except for human.

Hereinafter, a method of inducing a movable surface of the non-hypnotic animal of the present invention will be described in detail.

More preferably, the present invention

A dormant first step of dormant by administering a hibernate inducer;

An awakening step without a hibernation inducing substance;

A dormant second step of dormant by administering a hibernate inducer;

It relates to a movable surface induction method of non-hypnotic animals, except for human.

In the present invention, the non-hypnotic animal means an animal that does not hibernate, and preferably a mammal that is not hibernated, specifically a mouse, rat, rabbit, cow, horse, pig, sheep, goat, monkey, elephant, tiger , Lions, giraffes, and the like, but not limited to these.

In the present invention, the hibernation-inducing substance is T ₁ AM (3-iodothyronamine), DADLE ([D-Ala2, D-Leu5] enkephalin), 5'-AMP (5'-adenosine monophosphate) or H ₂ S (hydrogen sulfide) Is possible, but is not limited thereto.

In the present invention, the dormant step refers to the step of entering the dormant state by inducing the movable surface of the low metabolic-hypothermic temperature by administering the hibernate inducer to the non-hypnotic animal.

The hibernate inducer is administered one or more times, preferably every 3 to 6 hours to maintain a continuous dormant state. The administration may be intraperitoneal administration, subcutaneous administration, intramuscular injection, etc., but subcutaneous administration is most preferred in order not to disturb the dormant state.

In addition, in the dormant phase, it is desirable to maintain a dark state so as to be as close as possible to the actual hibernating animal.

In addition, in the dormant step, it is preferable to adjust the ambient temperature to 20 to 25 ℃.

In addition, stopping the water and food supply a day before the first dormant phase (including the dormant first phase) is preferable for preventing indigestion or enteritis at low temperature.

The present invention includes an awakening step between dormant states to induce a long running surface. In other words, it is possible to induce a long-term moving surface by creating an awake state out of the dormant state by not administering the hibernation-inducing substance.

In the present invention, the awakening step is preferably carried out in 24 hours in accordance with the circadian rhythm of the animal.

In the awakening step, water is preferable for maintaining homeostasis such as weight loss relief and osmotic control. In addition, in the arousing step, it is more preferable to administer antibiotics to recover the immunity against enteritis by fasting in a dormant state. Furthermore, the light / dark cycle is adjusted to the original period before dormancy (light / dark = 8-16 hours: 16-8 hours, preferably 10-14 hours: 14-10 hours, more preferably 12 hours: 12 hours), ambient temperature is adjusted to 27-33 ° C. (preferably 28-32 ° C.) to maintain animal body temperature at normal levels (above 36 ° C.).

After the wake-up step, the hibernate-inducing substance is administered again to return to the dormant state (including the dormant second step). At this time, the dormant second step is performed in the same manner as the dormant first step.

After the second stage of dormancy, the hibernation-inducing substance may be discontinued to enter the recovery phase.

In the recovery phase, food and water are re-supplied to supplement energy consumed in the dormancy phase, and the light / dark cycle is adjusted to the original period before sleep (light / dark = 8-16 hours: 16-8 hours). , Preferably 10 to 14 hours: 14 to 10 hours, more preferably 12 hours: 12 hours). The ambient temperature is then adjusted to 27-33 ° C. (preferably 28-32 ° C.) on the first day of the recovery phase to maintain animal body temperature at normal levels (36 ° C.). It is also advisable to administer antibiotics on the first and second days of recovery to prevent enteritis.

Thus, the moving surface induction method of the present invention includes the awakening step in the middle to provide time for restoring immunity and physiological homeostasis, thereby minimizing the sequelae that may be caused by low metabolic-low body temperature and preventing abnormality in survival after the operating surface. Do it.

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to the following examples and comparative examples, but the scope of the present invention is not limited by the following examples.

Reference Example Statistical analysis

Data in this experiment are expressed as mean ± standard deviation. The weight difference between the control group (Comparative Example 2) and the experimental group (Example 1) was evaluated by an independent sample t-test at each time period. Protein amount differences between the five time zones between day 0 and day 7 were examined by Kruskal-Wallis and Langley post hoc comparative analysis. All statistical analyzes were performed using SPSS / PC + and the significance level was determined at P <0.05.

Comparative example  1: When hibernation-inducing substance is administered once

Week 10 ICR males (~ 33 g) were bred with unlimited feed of Purina chow and water at a room temperature of 23 ± 0.5 ° C and light / dark cycles of 12h: 12h. Both food and water were removed one day prior to T ₁ AM administration. In the experiment, a thermocouple having a diameter of 0.025 mm was inserted into the pectoral muscle and body temperature was measured at 10 minute intervals (4 mice). Dimethyl sulfoxide (DSO) and physiological saline (pH = 7.4) were dissolved in a ratio of 6: 4 to dissolve 50 mg kg ^-1 T ₁ AM and then intraperitoneally administered (ip).

Next, body temperature and oxygen consumption rate (VO ₂ ) were measured simultaneously to investigate the effect of T ₁ AM on the energy metabolic rate. The breeding conditions of the mice (n = 3) were kept the same as above. Since the body temperature should be measured while the mouse is placed in the metabolic chamber, a MiniMitter sensor (Series 3000 XM-FH; MiniMitter, Bend, OR, US) was inserted into the abdominal cavity and the transmitted data was input to the computer through the receiver (ER4000). .

Each mouse was weighed and placed in a metabolic chamber (23 ° C., 2000 ml) for 2 h. Subsequently, T ₁ AM (50 mg kg ⁻¹ ) was administered to the mouse abdominal cavity and placed in the metabolic chamber again. 500 ml of air samples were taken every minute and sent to an oxygen consumption meter (Columbus Instruments, Columbus, OH, USA). After that, the oxygen consumption was measured every two minutes through the oxygen sensor, and the oxygen consumption rate was converted according to the Hill formula and standard pressure (760 Torr) and temperature (℃).

M of mice in a single dose of T ₁ AM 50 mg / kg by intraperitoneal injection in A of FIG. 1 showed a typical pattern.

The subjects were bent for 10 minutes after the substance was administered and their tails and bodies were bent to enter the dormant phase.

During the first hour, the body temperature dropped rapidly to 29 ± 0.5 ° C, about 8 ° C below the 1.7-2 hour body temperature. Body temperature increased to 35 ° C. after 4-6 hours after administration [FIG. 1B]. This transient pattern of body temperature is similar to the metabolic rate (VO ₂ ) pattern for 6 hours [C, D of FIG. 1]. At 0 h average VO ₂ is ^{_{1.93 ± 0.05 LO 2 kg -1 h}} - ¹ was, after treatment was reduced by half to 1.8 hours. Changes in VO _{2 in the} temperature riser were shown by the recovery of the dormant state by T ₁ AM [FIG. 1D]. These total body temperature and transient changes in VO ₂ are shown similarly in previous papers [Scanlan TS, Suchland KL, Hart ME, ChielliniG, Huang Y, Kruzich PJ, Frascarelli S, Crossley DA, Bunzow JR, Ronca-Testoni S , Lin ET, Hatton D, Zucchi R, Grandy DK. 2004. 3-Iodothyronamine is an endogenous and rapid-acting derivative of thyroid hormone. Nat Med 10: 638-642; Braulke LJ, Klingenspor M, DeBarber A, Tobias SC, Grandy DK, Scanlan TS, Heldmaier G. 2008. 3-Iodothyronamine: A novel hormone controlling the balance between glucose and lipid utilisation. J Comp Physiol B 178: 167-177.

In the case of single administration of T ₁ AM, the dormant state cannot be maintained for more than 6 hours, and thus, long-term induction is impossible.

Example  1: multi-dose hibernation-inducing substance with arousal stage

10 week-old males (ICR mice) were bred at a light / dark cycle (L / D) cycle 12h: 12h at 23 ± 0.5 ° C. Water and high-fat diets (60% lipid, 20% carbohydrates, and 20% protein (Research Diets Inc., New Brunswick, NJ, USA) were fed in an unlimited supply for 4 weeks.Random control (Comparative Example 2) And multi-dose experiment group and body temperature was measured by inserting the MiniMitter sensor into the abdominal cavity as in the Comparative Example 1 experiment.

In this experiment, the temperature range (28-32 ° C.) of the bear's hibernation (denning) was applied to the mouse. Thus, a 5-day running surface experiment proceeded to the first two days of dormant (torpor-I), followed by one day of arousal and then two days of dormant (torpor-II). A dormant phase was initiated at 19:00 on time, intraperitoneally injected with 50 mg kg ⁻¹ of T ₁ AM, and the cancer was maintained by blocking the lighting of the feeding room. When raised to 32 ℃ again after the body temperature of an object by intraperitoneal administration fall each case 50 mg kg ^- it was continuously maintain the sleep state to the dorsal T ₁ AM of the ^first administration to the subcutaneous region of the thorax.

During the awakening period, the light / dark cycle 12h: 12h was maintained without administering T ₁ AM. In addition, the internal temperature of the kennel (30 ± 2 ℃) was increased by circulation of hot water through a copper pipe (inner diameter of 7 mm) to maintain the body temperature of 36 ℃ or more. During this time only sterile water was supplied but no feed. In addition, antibiotic Baytril (Bayer HealthCare, Monheim, Germany) 0.2 ml kg to handle, such as colitis that may result in the ^fasted-1 was subcutaneously injected.

The experimental conditions of the dormant phase 2 was performed in the same manner as the dormant phase 1, and T ₁ AM was repeatedly administered starting with the first intraperitoneal injection. During recovery after dormancy, T ₁ AM was discontinued, the temperature in the kennel was raised to the hot water circulation, and the light / dark cycle was maintained. In addition, sterilized water and high-fat feed were re-supplied and antibiotics were injected subcutaneously at a 24-hour cycle on days 6 and 7.

Body temperature change in the MT group of Example 1 is shown in FIG. Experimental parameters (feed, water, light / dark cycle, number of injections, etc.) were performed as shown in FIG. Hot water was supplied using a copper tube to immediately recover the body temperature around the mice in the awakening period. The results showed for the first time that multiple T ₁ AM uptake effectively increased the animal's dormant state in the long term without any adverse effects after or during 5 days of administration (BD of FIG. 2). After the first T ₁ AM administration 50 mg / kg in the first and second dormant phase, the subcutaneous administration of T ₁ AM was performed every 3-6 hours and body temperature was maintained at 28-33 ° C.

The synergistic effects of T ₁ AM, fasting and dark conditions tended to slowly recover body temperature after dormancy, and decreased the need for drug administration.

Endogenous arousal during the day is deliberately included to follow the actual hibernating animal. The mean temperature of the MT group increased to 35 ° C. by exposing heat to the surroundings of the mouse kennel. Importantly, MT animals restored to normal body temperature exhibit spontaneous behavior, faeces excretion, and food seeking. During the awakening period, antibiotics are administered during the enteritis phase. As mentioned above, during hibernation, the actual hibernating animal can restore homeostasis by several physiological actions, including the immune response, so that it can survive even after hibernation. Periodic arousal helps to reactivate the pre-dormant immune system to avoid infectious diseases. In the present invention, the administration of antibiotics during the awakening period is to enhance the immunity against enteritis.

In the recovery period for 5 days after administration, the MT group maintained the body temperature 32-37 ° C. under the following conditions [Fig. 2B-D]: Feeding and water supply, light / dark cycle recovery, and circulation of hot water on the first day of recovery, the first day And second day antibiotics.

Repeated administration of T ₁ AM thus induces dormancy continuously, and by including an awakening step in the middle, it was possible to induce at least long-term operation surface.

Comparative example  2: Awakening  Including but not administered a hibernate inducer

For the control group, DMSO / physiological saline (6: 4) solution was intraperitoneally or subcutaneously injected in the same manner as when T ₁ AM was administered. The same procedure as in Example 1 was conducted except for T ₁ AM administration.

Body temperature change of the control is shown in A of FIG. The body temperature of the control group decreased by an average of 3 ° C. during the day. These mice were 2 days of dormancy (average body temperature 32.7 ° C.), 1 day of awakening (35 ° C.), 2 days of rest 2 (average temperature of 32.7 ° C.), and The average body temperature of 32 ° C. was maintained for a total of 10 days during the recovery period.

Comparative example  3: Awakening  Omitted and multi-dose hibernation-inducing substance

The same procedure as in Example 1 was conducted except that the arousal step was omitted.

During the preliminary experiment, which did not include the endogenous arousal period, some MT mice died for 3-4 days in the sustained dormant period. Two of the eight died due to excessive expansion of the bladder and six died of obvious enteritis (mainly duodenum).

Test Example  1: weight check

During the first 5 days, the MT group (Example 1) had less weight loss than the control group (Comparative Example 2) [0.79-0.68-fold reduction (P <0.05, Table 1)].

[Table 1]

This means that if the experiment is extended for a longer period of time, the MT group (Example 1) has a higher survival rate than the control group (Comparative Example 2). Because both groups were given water, the weight loss was partially alleviated during the awakening period. During the initial recovery period, body weight returned to normal levels with active food intake and urine excretion.

Test Example  2: check for nerve reactions

Three molecular markers were examined to investigate what cytoprotective activities or functional changes brain tissues undergo during prolonged (5 days) dormancy.

NSE (Neuron-specific enolase) was used to investigate the glycolysis function of brain tissues using glucose as an energy metabolism agent, and S100B (S100 calcium binding protein B) was used for the proliferation of glial cells. NSP was used as an indicator of neuronal survival activity, and HSP (heat shock protein) was used to investigate the oxidative stress response of brain tissue. In addition, since S100B is also used as an indicator molecule of brain tissue damage caused by hypoxia or local ischemia, the amount of S100B in the blood was also measured in this experiment.

Brain tissues and serum were obtained at 0, 2, 3, 5, and 7 days from the experimental group administered with T ₁ AM and quenched with liquid nitrogen. Frozen brain tissue was homogenized in a 10-fold volume of lysis buffer and subjected to immunoblotting analysis.

The lysis buffer composition was as follows: 20 mM HEPES (pH = 7.4), 75 mM NaCl, 2.5 mM MgCl ₂ , 0.1 mM EDTA, 0.05% (v / v) Triton X-100, 20 mM β-glycerophosphate, 1 mM Na ₃ VO ₄ , 10 mM NaF, 0.5 mM DTT, 0.4 mM PMSF (phenylmethylsulfonyl fluoride), and protease inhibitor Complete Mini (Roche, Penzberg, Germany). Two major serum proteins, albumin and immunoglobulin, were removed with a ProteoPrep Immunoaffinity Albumin & IgG Depletion Kit (PROT-IA, Sigma, Saint Louis, MO) for serum sample preparation.

Proteins in brain tissue lysates and serum samples were electrophoretically separated on 8-13% SDS polyacrylamide gels, followed by electrophoresis to nitrocellulose membrane GE Healthcare, Fairfield, CT, USA. (blotting) the membrane by electrotransfer. Then, blot was confirmed by staining with Ponceau S, and the dye was sufficiently removed using 1 × TBST, and then blocked with 5% (v / v) skim milk solution. Then, the primary antibody (primary antibody) was added to diluted skim milk and reacted at 4 ℃ for 12 hours. After the reaction, the cells were washed 5 times at 10 minute intervals using 1X TBST, and a horseradish peroxidase-labeled secondary antibody corresponding to the host source of the primary antibody was reacted at room temperature for 1 hour. After the reaction with the antibody was repeated washing with 1X TBST, the ECL detection kit (Amersham) was induced by the antigen-antibody reaction to induce color development, and exposed to the X-ray film to obtain a result. To rerobing the loading control, put the stripping buffer (Stripping buffer, Thermo, NMR, IL) into the membrane to confirm the result and react in a shaker for 15 minutes at room temperature, and then 5 minutes with 1X TBST for 5 minutes. After washing at intervals and blocking again, the primary antibody was reacted. For loading control, β-tubulin was used, and the intensity of the band appeared at this time was used as a standardization index. Band intensities of each protein were quantified with a Bio-Rad densitometer (Hercules, CA, USA). S100B band intensities were normalized to total protein stained with Ponceau S. The primary antibodies used in the experiment were mouse anti-NSE and rabbit anti-S100 beta monoclonal antibodies (ABcam, Cambridge, UK), and mouse anti-HSP72 and anti-HSP90 monoclonal antibodies from Stressgen (Victoria, BC, Canada). , And mouse anti-β-tubulin monoclonal antibody was purchased from SIGMA (Saint Louis, US). As secondary antibodies, HRP-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from Cell Signaling Technology (Beverly, CA, US).

The expression levels of NSE and S100B in brain tissue lysates were not significantly changed during the experimental period of 7 days or more.

Although the activation of NSE tended to decrease on day 5, the results for NSE confirmed that the whole glycolytic activity in the brain was not affected by the dormant state induced by T ₁ AM. The results for S100B suggest that neuronal protection by glial cells (glia cells) and the vascular-brain barrier remain intact during extended dormancy. In addition, serum samples were not significantly different at S100B levels during the test period. It can be seen that brain damage caused by repeated T ₁ AM treatments is negligible.

Experiments with HSPs provide an understanding of the cytoprotective activity of the brain due to heat and oxidative stress.

The magnitude of the temperature change (above 8 ° C.) between normal and hypothermia (B-D in FIG. 2) is sufficient to give the MT group tremendous thermal stress. Our data show that HSP72 increased 2.0-2.1 times compared to day 0 at day 3 (awakening) and at day 7 (recovery). In contrast, HSP90 did not show a statistically significant change (D-F in FIG. 3). This means that while HSP72 enhances survival protection during mobile phase, HSP90 has a limited role in brain tissue.

The results of neuronal response experiments, along with monitoring of body temperature, body weight and food intake-excretion activity for 10 days, support this experiment in that the brain maintains functional and cytoprotective activity by repeated T ₁ AM administration.

Therefore, the method of inducing moving surface using T ₁ AM multi-administration and dormant-wake cycle according to the present invention is a reliable method because it has no effect on the cranial nerve response in inducing continuous dormancy of animals that are not hibernating.

Claims (18)

A dormant step of sleeping by administering a hibernate inducer; And
Awakening step without hibernation-inducing substance
Moving surface induction method of non-hypnotic animals, except for human. A dormant first step of dormant by administering a hibernate inducer;
An awakening step without a hibernation inducing substance;
A dormant second step of dormant by administering a hibernate inducer;
Moving surface induction method of non-hypnotic animals, except for human. The method according to claim 1 or 2,
Animal is a moving surface induction method of a non-hypnotic animal is a mammal. The method according to claim 1 or 2,
Hibernate substances are non-hypnotic, such as T ₁ AM (3-iodothyronamine), DADLE ([D-Ala2, D-Leu5] enkephalin), 5'-AMP (5'-adenosine monophosphate) or H ₂ S (hydrogen sulfide) Method of inducing moving surface of animal. The method according to claim 1 or 2,
Movable surface induction method of non-hypnotic animals administered at least one hibernation inducing substance. The method according to claim 1 or 2,
A method of inducing a moving surface of a non-hypnotic animal that remains dark in the dormant phase. The method according to claim 1 or 2,
The dormant step is a method of inducing a moving surface of a non- hibernant animal to adjust the ambient temperature to 20 to 25 ℃. The method of claim 2,
Method of inducing movable surfaces of non-hypnotic animals, which stops supplying water and food one day before the first stage of sleep. The method according to claim 1 or 2,
The awakening step is a method of inducing moving surfaces of non-hypnotic animals to be carried out for 24 hours or less. The method according to claim 1 or 2,
Method of deriving the moving surface of a non-hypnotic animal which supplies water to the arousal stage. The method according to claim 1 or 2,
Movable surface induction method of non-hypnotic animals administered antibiotics in the arousal stage. The method according to claim 1 or 2,
Movable surface guidance method of a non-hypnotic animal which controls light / dark cycle at arousal stage. The method according to claim 1 or 2,
Moving surface induction method of a non- hibernant animal to adjust the ambient temperature to 28 to 32 ℃ during the awakening step. The method of claim 2,
A dormant second step, further comprising a recovery step of not administering a hibernate inducer. 15. The method of claim 14,
Method of guiding the moving surface of a non-hypnotic animal that supplies food and water during the recovery phase. 15. The method of claim 14,
Movable surface induction of non-hypnotic animals administered antibiotics in the recovery phase. 15. The method of claim 14,
Movable surface induction method of non-hypnotic animals controlling light / dark cycle during recovery phase. 15. The method of claim 14,
Movable surface guidance method of non-hypnotic animals which adjusts ambient temperature to 28-32 degreeC in a recovery stage.

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