KR101432387B1 - The method for repetitively over-ovulating and the method for repetitively obtaining fertilized eggs using Bombina orientalis - Google Patents

Abstract

The present invention relates to a method for repeatedly inducing ovulation and producing embryos in a mushroom frog, comprising the steps of: inducing superovulation by administering hCG to a female bovine frog ( Bombina orientalis ); Obtaining an oocyte from the superfamily induced guarless frog; Resting the bovine frog induced by the superovulation; And inducing superovulation again by administering hCG to the resting mothless frog. The present invention also provides a method for inducing repeated ovarian hyperstimulation and repetitive embryo production. According to the present invention, it is possible to obtain a large amount of healthy oocytes and healthy embryos in a stable manner despite the repetitive scattering by minimizing the stress applied to the mushroom frog, thereby remarkably enhancing the adult survival rate after embryo development .

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for inducing repetitive ovarian hyperstimulation,

The present invention relates to a method for inducing ovulation of a mushroom frog and a method for producing an embryo, and more particularly, to a method for repeatedly inducing ovulation and producing an embryo from a mushroom frog.

The amphibian laboratory animals that have been mainly used in the field of conventional embryology and toxicology studies were exotic species of Xenopus. Amphibians in Genopus have been widely used because they produce many embryos, are easy to breed, and are easy to induce ovulation using hormones. Studies on induction of ovulation and artificial insemination of amphibians of genus Xenopus have been actively conducted, but the research on native amphibians of Korea is still poor. Therefore, it will be necessary to establish the method of securing and using the species which can be easily used as experimental animals, such as amphibians of the Genovese genus, among domestic native amphibian species.

On the other hand, Bombina orientalis , one of the native amphibians of Korea, is a small frog whose body length is less than 5 ㎝ belongs to the frog of the Yangshu River frog and the frog frog. It lives in Northeast Asia. In Korea, Jeju is known as the southern limit of the forest, which is often found in the forest forest area near the agricultural area and the forest valley area where the first grade flows. Black patterns are irregularly distributed on the background of cyan, and the abdomen is characterized by an irregular black pattern on a red or yellow background, and many objects are traded as pets worldwide.

The mushroom frog is easy to handle in the laboratory because it is slow in movement and small in size, and its early occurrence and transformation time is fast compared with other species of frog neck. Therefore, it has many merits as a study model of amphibians. Despite the above advantages, it is easy to collect and collect in spring season (April to June) in Korea, but after the breeding season, it moves to the forest valley, which is the main habitat, Therefore, it is easy to sample breeding hormones such as the breeding hormone and gonadal changes of this species, but since it hibernates in the deep rocks around the valley from the autumn, there are many difficulties in securing individuals for research on reproduction physiology and hibernation ecology in hibernation . Furthermore, because of the ecological habit of spawning at low water level while performing semi-land life, the frog frog is mainly used for underwater life and spawning activity is also induced by ovarian induction and artificial There is a problem that the technique of modification can not be applied directly to the frog frog.

Although there is a growing need to establish methods for securing and using species for domestic amphibian species as experimental animals, the most suitable methods for inducing ovulation and artificial insemination for mushroom frogs, which have many merits as experimental animals, It is necessary to develop optimized ovulation induction and artificial fertilization techniques for using the insect frog as an experimental animal.

It is an object of the present invention to provide a repetitive method of inducing a superovulation and a method of producing a reproductive embryo optimized for a domestic native amphibian,

In order to accomplish the above object, one aspect of the present invention provides a method for preventing ovarian hyperstimulation, comprising: inducing superovulation by administering hCG to a female bulbous frog ( Bombina orientalis ); Obtaining an oocyte from the superfamily induced guarless frog; Resting the bovine frog induced by the superovulation; And administering hCG to the resting mushroom frog to induce superovulation again.

According to another aspect of the present invention, there is provided a method for inducing superovulation, comprising administering hCG to a female bovine orientalis ; Inducing egg scattering and fertilization from the superovulation-induced female mushroom frog to form an embryo; Obtaining said embryo; Resting the scattered-induced female mamma frog; And inducing superovulation again by administering hCG to the resting female shaman frog, and scattering the egg to obtain an embryo again.

According to the present invention, it is possible to obtain a large amount of healthy oocytes and healthy embryos in a stable manner despite the repetitive scattering by minimizing the stress applied to the mushroom frog, thereby remarkably enhancing the adult survival rate after embryo development . Since the size of the embryo obtained according to the present invention is larger than that of existing experimental species, it is necessary to perform initial occurrence observation, extraction of various explants during embryo development, study of germination using the same, and analysis of vertebrate animal development toxicity It is useful for the purpose of ① embryo toxicity experiment, ② ensuring laboratory tadpole, ③ analysis of transformation process by observation of physiological / histological changes due to metamorphosis, and ④ securing laboratory objects in laboratories Can be used.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

FIG. 1 is a flow chart showing the reproductive coagulation induction and reproductive embryo production process of the present invention.
FIG. 2 is a graph showing the number (A) of scattered eggs and the hatching rate (B) of embryos in the case of a single scan and a double scan.
FIG. 3 and FIG. 4 are graphs showing the results of measurement of the number of eggs scattered during repetitive coarctation induction according to rest periods.
FIG. 5 is a diagram showing the composition for disturbance used in the method for producing embryos according to the natural crossing of the present invention.

Hereinafter, the present invention will be described in detail.

One. Repeated superovulation induction method of mushroom frog

The present invention relates to a method for inducing superovulation by administering hCG to a female bulbous frog ( Bombina orientalis ); Obtaining an oocyte from the superfamily induced guarless frog; Resting the bovine frog induced by the superovulation; And administering hCG to the resting mushroom frog to induce superovulation again.

The method of inducing repetitive ovarian hyperstimulation of the mushroom frog of the present invention comprises the steps of: 1) inducing superovulation in a female mushroom frog; 2) obtaining an oocyte from the superfamily induced guarless frog; 3) resting the bovine frog induced by the superovulation; And 4) inducing superovulation again in the resting mussel frog.

The above steps 1) and 4) induce superovulation in the female mushroom frog, and the induction method is the same except that the induction time point of the superovulation is before and after the rest of the step 3) Step 4) will be described together.

The superovulation of steps 1) and 4) is induced by administration of hCG. The hCG refers to a human chorionic gonadotrpin. When administered to the female mushroom frog, the hCG stimulates the ovary of the female mushroom frog and promotes oocyte maturation and ovulation.

The administration of hCG in steps 1) and 4) above can be carried out by a single dose or a double dose method. The single administration means a single administration of the hCG to a female mushroom frog, and the double administration means administration of the hCG twice to a female mushroom frog. In the case of the single administration, oocyte maturation and ovulation are accelerated by one administration, and in the case of the double administration, oocyte maturation is induced by the first administration and ovulation is promoted by the second administration. In the case of the single administration, since the oocyte maturation and the promotion of ovulation must be simultaneously induced by only one administration, a larger amount of hCG is administered than in the case of the double administration. Both of these methods can induce superovulation of female mushroom frogs. However, since the amount of hCG suitable for the induction of oocyte maturation and ovulation is different, Positive oocytes can be obtained. In addition, in the case of the above-mentioned double administration, it is possible to further increase the survival rate of the post-administration animal because the stress caused by excessive hormone administration is not given to female frog frog.

When the hCG of the step 1) and the step 4) is singly administered to female female frog frogs, the dose of the hCG is preferably 400 IU to 600 IU, and the dosage of the hCG is 450 IU to 550 IU It is most preferable that the dosage of hCG is 500 IU, but is not limited thereto. When the hCG is administered to female female shaman frogs, it is preferable that the hCG is first administered in an amount of 1 IU to 20 IU and secondarily administered in an amount of 200 IU to 400 IU, 15 IU, and more preferably 250 IU to 350 IU, and the hCG is first administered in an amount of 10 IU and second administered in an amount of 300 IU Most preferred, but not limited thereto. As described above, in the case of the single administration, since the oocyte maturation and promotion of ovulation must be simultaneously induced by only one administration, the amount of hCG administered is higher than that of the double administration. In a specific example of the present invention, a comparison between the egg production rate and the hatching rate of the embryo according to the administration method of the hCG showed that a double administration of the single administration resulted in a greater number of oocytes in a healthy state Reference).

The administration of hCG in step 1) and step 4) can be administered to any one or more sites selected from the group consisting of dorsal lymphocytes, abdominal cavity, dorsal muscle and dorsal lymphocyte of a female mushroom frog, The administration is more preferably to the peritoneal or dorsal lymphocyte of the female mushroom frog, and the administration of the hCG is most preferably, but not necessarily, administered to the dorsal lymphocyte of the female mushroom frog. Since the administration of the abdominal cavity may have a great influence on the organ of the female mushroom frog, it is preferable to administer the hCG to the dorsal lymph node to obtain repetitive embryos. In the specific example of the present invention, it was confirmed that the number of oocytes to be ovulated and the number of embryos in which the animal pole was normally raised after fertilization were smaller than that in the case of injecting the dorsal lymphocytes in the dorsal lumen (see Table 1).

The step 2) can be performed by physically stimulating and scattering the abdomen of the female bovine frog, from which the superovulation has been induced in step 1). In the female frog frog induced by the superovulation in the above step 1), about 15 to 20 hours after the hCG administration, the total excreting females of the female are protruded outward. Therefore, when egg is scattered through physical stimulation of the subject's abdomen, it is preferably performed at about 15 to 20 hours after administration of hCG.

The rest of the above step 3) includes all the steps in which the female ovarian frog induced superovulation is relieved of the stress caused by ovarian hyperstimulation and restored to a state in which the ovary develops again. That is, the rest of the step 3) is a process in which the induction of the superovulation in the step 4) is restored to a state in which the induction of the superovulation is possible again. Preferably, the rest period is 4 to 5 months, and the rest period is 5 months. In a specific example of the present invention, it was confirmed that the resting period was different from that of the resting period, and as a result, the best recovery from the stress was obtained when the resting period was about 5 months (see Table 2).

The method for inducing repetitive ovarian hyperstimulation of the insect frog comprises the steps of: 5) resting the ovarian frog induced by the superovulation in step 4) for 4 to 5 months; and 6) resting the hCG And then inducing superovulation again. That is, a large amount of oocytes can be obtained by inducing superovulation in a female frog frog, restoring it for a sufficient rest period, and then repeating the process of inducing superovulation again. This repeated induction of ovarian hyperstimulation is only possible if the female shaman frog is fully recovered from the stress it receives during the ovulation phase, and the rest phase of the ovulation performed during the ovulation phase is very important. In other words, a healthy oocyte can be obtained in large quantities only when the individual is well recovered through sufficient rest. In a specific example of the present invention, the results of verifying the ovulation induction effect by varying the period of rest included between the induction of superovulation, it was confirmed that the restoration is best recovered from the stress when the rest period is about 4 to 5 months 3 and 4).

2. How to Produce Repeated Embryos of Mushroom Frog

In another aspect of the present invention, there is provided a method for preventing ovarian hyperstimulation, comprising the steps of: administering hCG to a female bulbous frog ( Bombina orientalis ) to induce superovulation; Inducing egg scattering and fertilization from the superovulation-induced female mushroom frog to form an embryo; Obtaining said embryo; Resting the scattered-induced female mamma frog; And inducing superovulation again by administering hCG to the resting female shaman frog, and scattering the egg to obtain an embryo again.

The method for producing repetitive embryos of a mushroom frog of the present invention comprises the steps of inducing superovulation in a female mushroom frog, 2 ') inducing egg scattering and fertilization in the superfamily induced mushroom frog to obtain embryos, ) Obtaining the embryo, 4 ') resting the scattered female mamma frog and 5') inducing superovulation and scattering again in the resting mamma frog to obtain embryos.

In the recurring Embryo Production method of the ladybird frog as to re-induction of ovulation and spawning jaesanran and the step 5 ') of the egg, ovulation induction, wherein the step 2) "), the first step is repeated for the" 1 ladybird frog Quot; method of inducing superovulation ". Therefore, the description of the item " 1. Method for inducing repetitive superovulation of frog frogs " is omitted, and a detailed description thereof will be omitted. In the following, only the constitution specific to the reproductive embryo production method of the insect frog will be described below.

In addition, the above step 5 ') is a step of inducing re-superovulation and a property column in a female mushroom frog to obtain an embryo, wherein the induction time of the superovulation is adjusted before and after the rest of the step 4' This same step, hereinafter referred to as step 1 ') and step 5', will be described together.

The embryos of step 2 ') and step 5') can be generated by natural mating of male mushroom frog and female mushroom frog induced by superovulation in step 1 '). The generation of embryos through natural crossing can utilize male mushroom frogs in which spermatogenesis is induced. In other words, it is possible to generate embryos by inducing the embryo formation of the male mushroom frog induced by spermatogenesis and the female mushroom frog induced by superovulation in step 1 '). The spermatogenesis of the male mushroom frog can be induced by single administration of 300 IU of hCG to the abdominal cavity or dorsal lymph node of the male mushroom frog.

The inclusion induction of the male shaman frog and female shaman frog can be performed in a water bath.

The water tank has a structure for a frying egg. The confusion structure is for attaching the egg laid out in the female shaman frog, and in the case of the frog frog, it is not scattered in the water like the Genovese genus amphibians, but has the habit of egg scattering by attaching the egg to the structure, The pull-in structure can be induced only if a drag structure is provided. The confusion structure may be a sponge, and the average pore size of the sponge is preferably 0.5 mm to 1.5 mm. In a specific embodiment of the present invention, a sponge having an average pore size of 1.18 mm was used as the confusion structure.

The water tank may have an area of 20 cm x 20 cm to 40 cm x 70 cm. The area of the water tank is determined according to the size of the frog-free frog, and can be suitably selected according to the judgment of the person skilled in the art upon induction of inclusion. The water bath also has water of a height of 3 cm to 10 cm, preferably 4 cm to 6 cm. In the case of a frog frog, it is not an aquatic organism living mainly underwater like an amphibians in Xenophus, but an ecological habit that sprays in low water level while doing semi-land life. Therefore, .

Embryos of step 2 ') and the step 5') can be produced by the in vitro modification (in vitro fertilization). The in vitro fertilization is induced by treating the sperm suspension obtained in the male mugwort frog with eggs obtained in the female mugwort frog induced with the superovulation in step 1 '). The generation of embryos through in vitro fertilization can utilize male mushroom frogs in which spermatogenesis is induced. That is, a fertilized egg can be produced by treating a sperm suspension obtained from a male spermatogonial frog induced spermatogenesis with an egg obtained from a female spermatogonial frog induced by a superovulation in the above step 1 '). The spermatogenesis of the male mushroom frog can be induced by single administration of 300 IU of hCG to the abdominal cavity or dorsal lymph node of the male mushroom frog.

The rest of the step 4 ') includes all the steps in which the female ovarian frog induced superovulation is relieved of the stress caused by the ovarian hyperstimulation and restored to a state in which the ovary develops again. That is, the rest of the step 4 ') means a process in which the induction of the superovulation in the step 5') is restored to a state where it can be re-enabled. Preferably, the rest period is 4 to 5 months, and the rest period is 5 months.

Hereinafter, the present invention will be described in detail with reference to examples.

It is to be understood, however, that the following examples are intended to assist the understanding of the present invention and are not intended to limit the scope of the present invention.

Screening of mushroom frog individuals

The larvae collected from Gapyeong area in Gyeonggi - do were kept in the breeding room for more than one year. Record the back marks of the female and male frog frogs over 2.5 years of age as digital photographs, individually number them, and record the date of hCG administration. At the time of selection of the experimental donors, breeding log records of each individual were checked, and individuals whose hCG injected at least 3 months or more were selected from the above individuals.

Induction of repeated ovulation

<2-1> hCG  Administration and Superovulation  Judo

10000 IU of hCG (Daesung Microbiology Research Institute) was diluted with a sterilizing diluent to prepare hCG having a final concentration of 2 IU / 占 퐇 and 0.1 IU / 占,, wiping off the water of the individual to fix it so as not to slip, The female shaman frogs were injected.

<2-1-1> Abdomen Lymphatic sac , Abdominal cavity, Back  Muscle and Back Lymphatic sac  Single shot

Ventral lymphsac (n = 6), abdominal cavity (n = 6), dorsal muscle (n = 6) of 24 female mushroom frogs selected in Example 1 We administered 500 IU of hCG to the dorsal muscle (n = 6) or dorsal lymphsac (n = 6). To minimize the loss of hCG into the breeding water, I put it.

<2-1-2> Abdomen Lymphatic sac , Abdominal cavity, Back  Muscle and Back Lymphatic sac   Double injection

Ventral lymphsac (n = 6), abdominal cavity (n = 6), dorsal muscle (n = 6) of 24 female mushroom frogs selected in Example 1 10 IU and 300 IU of hCG were administered to the dorsal muscle (n = 6) or dorsal lymphsac (n = 6) at intervals of 24 hours. Immediately after the injection, they were placed in a waterless bath for 10 minutes.

<2-2> Obtaining Eggs

After 18 hours from the injection of hCG as in <2-1> above, the abdomen of the female shaman frog was rubbed in one direction from the side of the anus, artificially scattered on a slide glass washed with sterilized distilled water, To minimize oocytes, the contralateral armpit area was rubbed in the same manner and scattered on a slide glass to obtain eggs.

<2-3> Analysis of effects of injection site and injection method

The quality of egg of frog frog obtained as in <2-2> was analyzed. From the total of 48 females of <2-2>, the quality of oocytes and embryos according to different hCG injection sites were analyzed. A total of 48 females and 1 males were used. After 18 hours of hCG injection, the abdomen of the females was pressed to induce spawning artificially, and the spawning eggs were artificially fertilized with the same individual spermatozoa The quality of the eggs was analyzed indirectly by analyzing the state of embryos. Sperm used for artificial fertilization for analyzing the quality of the oocyte were modified to each oocyte derived from 48 females by using sperm obtained from testes of a single individual to ensure homogeneity of the sperm. The effect of injection site and injection method on fertilized embryos were analyzed.

First, the egg quality, animal pole diffraction, and first egg scattering efficiency according to the injection site were analyzed with the embryo. We randomly selected 100 artificially scattered oocytes from four groups of female mushroom frogs according to the injection site, and identified the number of abnormal oocytes through the pigment distribution and morphology. Then, ② the number of oocytes that did not rise above the animal pole at 1 hour after artificial insemination. ③ The number of embryos that did not undergo the first cleavage after artificial fertilization was determined. As a result, it was confirmed that the most and healthy eggs were obtained when injected into the dorsal lymphocyte (Table 1).

Injection site Oocyte Animal pole 2 cells (2-cell) Abdominal lymph node 92/100 ^a 87/92 ^b 58/87 ^c Abdominal cavity 85/100 ^a 81/85 ^b 77/81 ^c Dorsal muscle 83/100 ^a 66/83 ^b 37/66 ^c Dorsal lymphocyte 93/100 ^a 91/93 ^b 90/91 ^{c a} , percentage of abnormal eggs among 100 randomly selected oocytes
^b , the percentage of oocytes in which the animal pole does not face upward after normal fertilization
^c , percentage of embryos in which the first pole of the embryo has not advanced,

Next, we compared the average number of oocytes obtained from the dorsal lymphocyte with single injection and double injection, and the hatching rate of the embryos after fertilization. The hatchability was confirmed by measuring the individual survival rate at each time point hatching through the two-cell aeration and blastocyst, compared to the number of embryos. As a result, from the aspect of the yield of eggs, about 190 eggs or more were obtained on an average per subject in the case of double injection, whereas only about 140 eggs per subject were obtained in a single injection (Fig. 2 (A)). In terms of the hatching rate of the embryo, about 90% or more of the hatching eventually occurred in the case of double injection, whereas in the case of single injection, only about 75% of the hatching eventually occurred (Fig. 2B) ).

From the above results, it can be seen that the most and healthy egg can be obtained when the dorsal lymph node is double-injected.

<2-3> Iterative Superovulation  Analysis of influence of induction and rest period

As shown in <2-2> above, female frog frog scattered eggs were divided into five groups according to the rest period, and they were rested for 65 days, 95 days, 120 days, 140 days and 170 days respectively. During this period, crickets and wheat worms were fed two times / week for female animals, and vitamin and calcium powder were supplied for food supply. The breeding temperature was maintained at 22 ℃. Then, in order to induce secondary ovarian hyperstimulation in the same manner as described in <2-1> and <2-2>, and to confirm the change in the number of spawning according to the resting period, the ovarian weight and length were measured And the ratio was compared.

As a result, at the induction of ovarian hyperstimulation, mushroom frog individuals in the resting period of 120 days and 140 days showed the highest ratio of ovarian weight to body weight (Table 2). From the above results, it can be seen that the ovaries develop well in the individuals who have rested for 4 to 5 months.

Recovery period
(Work) Length (mm) Weight (g) Ovarian weight (g) Weight / length
* 100 Ovary Weight / Weight
* 100 Ovarian weight / length
* 100 65 41.95 10.03 0.64 23.91 6.34 1.51 95 44.05 8.16 0.83 18.52 10.12 1.87 120 45.05 9.61 1.06 19.00 10.21 2.35 140 48.1 10.54 1.22 21.91 11.59 2.54 170 42.85 7.21 0.51 16.83 7.07 1.19

 In addition, the female frog frog having spawned eggs as described above in <2-2> was divided into a group having a rest period of 90 to 120 days, a group having a rest period of 121 to 150 days, a group having a rest period of 151 to 180 days Group with rest period, and group with rest period exceeding 180 days. Each of the four groups divided in detail as described above was grouped into two groups and rested for 90 to 150 days (Group 1) and 150 days (Group 2) And <2-2>, respectively. The second group of non - frog frogs which were induced by the second overhaul were again rested for the second time and the third overgrowth was induced. Then, the number of eggs laid out in the four groups was compared and analyzed.

As a result, in the first group, fewer oocytes were induced at first than in the second group, but the increase in the number of oocytes induced by repeated induction of superovulation was larger, 1 group (Fig. 3). In addition, among the individuals of the first group, the experiment group (groups C and D) having the rest period of 121 to 150 days scattered more eggs than the experiment group (groups A and B) having the rest period of 90 to 120 days (Fig. 4).

Repeated embryo production

<3-1> Natural Breeding Natural Mating )

<3-1-1> Inducing spermatogenesis

10000 IU of hCG (Daesung Microbiology Research Institute) was diluted with a sterilizing diluent to prepare hCG having a final concentration of 2 IU / 占 퐇 and 0.1 IU / 占,, wiping off the water of the individual to fix it so as not to slip, 300IU of hCG was injected into the dorsal lymphocyte to the male frog frog and placed in a waterless tank for 10 minutes immediately after the injection to minimize the dissolution of hCG into the breeding water.

<3-1-2> Enclosure  Derivation of induction and embryo

Male individuals inducing spermatogenesis as in the above <3-1-1> and female animals induced with superovulation such as <2-1-2> and <2-1-4> in separate water tanks , A confluent structure, and then formed a scattering environment with a light-dark cycle of 12 hours in an incubator set at a temperature of 22 ° C. The size of the water tank was varied according to the size of the object as shown in Table 3 below.

Object Size (g) Tank size (width × length × water level) <5 20 cm x 20 cm x 4-5 cm 5 to 15 30 cm x 40 cm x 4-5 cm > 15 40 cm x 70 cm x 4-5 cm

As a result, from the time when 18 hours had elapsed due to the inclusion of male and female, female mushroom frog started to scatter 20 to 30 oocytes, and after confirming that fertilization by natural crossing occurred, the embryo of the mushroom frog produced was obtained.

<3-1-3> Effect according to kind of confusion structure

In order to compare the scattering efficiency according to the type of confusion structure, induction of male and female shelling was performed as in <3-1-2>, and the types of confusion structures contained in the water bath were classified as 1.18 mm and 0.2 Mm sponge, and rice straw.

As a result, it was confirmed that it is effective to use a sponge having an average pore size of 1.18 ㎜ in terms of ease of recovery of embryo and ease of washing in reuse.

<3-1-4> Culture of embryos

The embryo obtained in the above <3-1-2> was cultured in a darkness period of 12 hours in an incubator adjusted to a temperature of 22 ° C to 23 ° C. For culture, 0.1X MMR was used until the blast phase was reached, and 0.3X MMR was used after blastocyst stage. The culture medium was changed once a day, and when replacing the culture medium, the dead jelly layer turned white due to mold and so forth was removed and the contamination of other embryos was prevented. In addition, when the jelly layer turned white, the jelly layer of the embryo was peeled off when the inside of the animal was alive and moving.

<3-2> In Vitro  Modified ( In Vitro fertilization , IVF )

<3-2-1> Trial removal

The endothelium and endothelium of a male mushroom frog which had been elongated 18 hours after inducing spermatogenesis were dissected as described in <3-1-1>, and the intestines of the two membranes and abdomen were removed to extract testis between the kidneys .

<3-2-2> Preparation of Sperm Suspension

Among the extracted testicular tissues extracted from the above <3-2-1>, a small testis that has not properly received hCG stimulation and was not developed properly was removed, and 2 ml of 0.1X MMR or 10% steinberg solution solution) and finely squeezed with a dissector to make a sperm suspension. The prepared sperm suspension was allowed to stand at room temperature until it was processed into oocytes.

<3-2-3> Obtain egg and embryo

After 18 hours from the injection of hCG as in <2-1> above, the abdomen of the female shaman frog was rubbed in one direction from the side of the anus, artificially scattered on a slide glass washed with sterilized distilled water, To minimize oocytes, the contralateral armpit area was rubbed in the same manner and scattered on a slide glass to obtain eggs. The sperm suspension prepared in the above <3-2-2> was uniformly sprayed on the thus obtained oocytes with a pipette, and at the same time, the oocytes were arranged as a single layer to induce the fertilization of sperm and oocytes.

<3-2-4> Culture of embryos

The embryo obtained in the above <3-2-3> was cultured in a darkness period of 12 hours in an incubator adjusted to a temperature of 22 ° C to 23 ° C. For culture, 0.1X MMR was used until the blast phase was reached, and 0.3X MMR was used after blastocyst stage. The culture medium was changed once a day, and when replacing the culture medium, the dead jelly layer turned into white due to fungi, etc., was removed and the contamination of other embryos was prevented. In addition, when the jelly layer turned white, the jelly layer of the embryo was peeled off when the inside of the animal was alive and moving.

Natural mating and IVF Comparison of

The hatching success rate of the embryos obtained in the same manner as in the above <3-1> and the embryos obtained in <3-2> were compared.

As a result, the hatching success rate of the embryos obtained by IVF was only about 63.4%, while the hatching success rate of the embryos obtained by natural crossing reached 90.2% (Table 4). From the above results, it was confirmed that the embryo obtained by natural hybridization had a higher hatching success rate than the embryo obtained by IVF.

Object number
IVF Natural breeding Scattered water Successful hatching Hatching success rate Scattered water Successful hatching Hatching success rate One 201 120 59.7 291 260 89.3 2 392 250 63.8 131 121 92.4 3 220 150 68.2 133 113 85.0 4 108 67 62.0 154 145 94.2 Average 230 147 63.4 177 160 90.2

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It will be possible to change it appropriately.

Claims (9)

delete delete delete Inducing superovulation by administering hCG to a female bulbous frog ( Bombina orientalis );
Inducing egg scattering and fertilization from the superovulation-induced female mushroom frog to form an embryo;
Obtaining said embryo;
Resting the scattered-induced female mamma frog; And
Administering hCG to the resting female shamanic frog to induce superovulation again, and scattering the egg to obtain an embryo again,
The scattering and correction of the egg is carried out by inducing folding of the male mushroom frog and the female mushroom frog guided by the superovulation in a water tank provided with a structure for concealing the egg, and the structure for confusion is a sponge A method of producing repetitive embryos of a mushroom frog. delete delete delete delete 5. The method of claim 4, wherein the sponge has an average pore size of 0.5 mm to 1.5 mm.

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