KR101805034B1 - A method for triploid fertilized egg of Onchorhynchus mykiss - Google Patents

Abstract

The present invention relates to a method for producing triplicate rainbow trout embryos. In the case of using the method of preparing the triplet rainbow trout embryo of the present invention, it is possible to obtain a triploid rainbow trout embryo in an efficient manner in Korea, thereby solving the low hatching rate due to the transfer of the triplet rainbow trout embryo introduced in foreign countries, The introduction of intrinsic disease related to rainbow trout, which can be introduced together according to the present invention, can be fundamentally solved. In addition, it can solve the problems of ineffective diapause (winter egg) in the domestic aquaculture industry and can be usefully used in related business.

Description

Technical Field [0001] The present invention relates to a method for preparing triploid fertilized eggs of Onchorhynchus mykiss,

The present invention relates to a method for producing triplicate rainbow trout embryos.

Rainbow trout is an exotic species imported from the United States in the 1960s, but it is a well-adapted freshwater fish species domestically and internationally. The rainbow trout has a spawning season mainly around December, and a normal embryo is obtained using mature male gametophytes (eggs and sperm) obtained from the rainbow trout mother in the spawning season. The chromosome image of the embryo obtained at this time is called the diploid with 2n. The diploid embryo grows up to around 500 g in the first year and spawning in the second year at more than 1300-1500 g. However, since the edible portion of rainbow trout is only about 40% less than 1 kg, it grows up to 1.5 kg, and about 60% of the edible portion appears, so it can grow beyond the spawning season until the following year. However, during the spawning season, the mortality rate of males is high, and in females, the efficiency of breeding is very low due to weight loss due to spawning, so breeding for 2-3 years using normal diploids is recognized as a very inefficient method.

There are techniques to make a triple body using various methods at home and abroad, and apply it to various fish species such as loach and induce triple body. However, in the production of rainbow trout triploid embryos, very low triploid production rates of less than 40% or 70% are observed, and it has been reported that triploid eggs grow and have normal eggs and sperm. Therefore, it is necessary to produce an efficient method of producing the triploid embryo.

Therefore, the inventors of the present invention were studying a method for producing triploid rainbow trout eggs efficiently, giving a fixed temperature at the time of fertilization of diploid embryos of rainbow trout, setting a certain period of time after fertilization, and then giving warming temperature and time conditions. Thereafter, the above conditions were repeated to induce the female to induce femaleization, and as a result of crossing with the male of the diploid body, it was confirmed that triploid rainbow trout embryos could be produced effectively, thus completing the present invention.

It is also an object of the present invention to provide a method for producing triplicate rainbow trout embryos.

In order to solve the above-mentioned problems, the present invention provides a method for producing a diploid embryo, comprising the steps of: (1) inducing a diploid embryo using a male Onsolehynchus mykiss diploid; (2) inducing a diploid embryo of step (1) to a tetraploid state after being corrected for 6.5 to 7.5 hours; And (3) cross-crossing the male and female diploid crossbreds to produce a triploid rainbow trout embryo.

In the case of using the method of preparing the triplet rainbow trout embryo of the present invention, it is possible to obtain a triploid rainbow trout embryo in an efficient manner in Korea, thereby solving the low hatching rate due to the transfer of the triplet rainbow trout embryo introduced in foreign countries, The introduction of intrinsic disease related to rainbow trout, which can be introduced together according to the present invention, can be fundamentally solved. In addition, it can solve the problems of ineffective diapause (winter egg) in the domestic aquaculture industry and can be usefully used in related business.

FIG. 1 is a graph showing the results of confirming the hatching rate and the doublet induction ratio according to each elapsed time after the modification of the diploid embryo.
FIG. 2 is a graph showing the results of confirming mortality, hatching rate, and inbreeding rate after two days from the diploid embryo after each heating temperature condition and heating time condition.
Fig. 3 is a diagram showing the induction of a doublet in a thermostat equipped with an automatic stirrer.
FIG. 4 is a graph showing the comparison of the sizes of red blood cells in the blood of diploid, triplet and chondroblast.
FIG. 5 is a diagram showing a result of repeated verification 1 to 5 times in order to verify the established doublet induction condition according to the present invention. FIG.
FIG. 6 is a graph showing the results of confirming sex ratios of the complexes according to the respective integration temperature conditions.
FIG. 7 is a graph showing the results of confirming sex ratios of the complexes according to the respective accumulated water temperature conditions and the concentrations of the magnetic hormones (estrogen).
8 is a diagram showing the male and female gonads of the rainbow trout (left, female, right, male).
FIG. 9 is a graph showing the result of repeated verification 1 to 5 times according to the established induction conditions of the present invention. FIG.

(1) inducing diploid embryos using Onchorhynchus mykiss diploid female and male; (2) inducing a diploid embryo of step (1) to a tetraploid state after being corrected for 6.5 to 7.5 hours; And (3) cross-crossing the male and female diploid crossbreds to produce a triploid rainbow trout embryo.

In the present invention, the "rainbow trout ( Onchorhynchus mykiss "is one of the representative freshwater fishes that grow well in clear and clean fresh water of salmon and fish and cold regions. The rainbow trout has a purplish color and is composed of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA ), Etc., And its food composition is very similar to salmon in Korea.

In the present invention, the term "diploid" is a cell or an individual of a period having two pairs of homologous chromosomes in the life cycle, and is a concept of number-of-parentheses. The cells of diploid cells undergo meiosis during the formation of the actor itself, and four consecutive cells with half of the chromosomes are formed by two successive cell divisions. When two male and female sex actors meet and are fertilized, a diploid body develops again.

In the present invention, the term "triploid" refers to an individual having a chromosome number three times that of the basic number, which is usually caused by hybridization of diploid and tetraploid.

In the present invention, the term "tetraploid" means a chromosome four times as large as the basic number. It exists in nature, but it can be artificially created by colchicine.

The modification of the diploid embryo of step (1) is carried out at a temperature of 10 to 20 캜, preferably at 10 to 18 캜, but is not limited thereto.

The quiescence is allowed to stand for 6.5 to 7.5 hours after the modification, preferably for 7 hours. When left for less than 6.5 hours or for more than 7.5 hours, the degree of chelation is significantly lowered.

The step (2) may be performed at a temperature of 20 to 40 ° C for 10 to 40 minutes, preferably at a temperature of 25 to 35 ° C. When the temperature is less than 25 ° C, ℃, the hatching rate is reduced. Further, it is preferable to carry out the warming treatment for 15 to 35 minutes. When the heating time is less than 15 minutes, the induction ratio of the doublet is lowered, and when the warming treatment is performed for more than 35 minutes, the 2-day mortality and hatching rate are inhibited.

The method according to any one of claims 1 to 3, further comprising the step of femaleizing the doublet after step (2), wherein the embryotization is carried out under the conditions of an integrated water temperature of 800 to 1000 캜, preferably an integrated water temperature of 850 to 900 캜, The rate of females is lowered when the accumulated water temperature exceeds 1000 ° C.

The femaleization can be carried out by feeding the treated food with 0.1 to 20 ppm of a hormone (estrogen) under the above-mentioned cumulative water temperature condition, preferably 1 to 15 ppm, and when the concentration is less than 1 ppm, Is significantly reduced.

In the present invention, the above-mentioned "cross-breeding between the male and female diploids" means cross-breeding of diploid female or male, using the female and the remaining males out of the doublet derived from the diploid embryo of the present invention Can be crossed into a diploid (female) × trimeshed (male), a trimeshed (female) × trimeshed (male), and a trimeshed (female) × trimeshed (male).

In one embodiment of the present invention, in order to inhibit the progression of the first disturbance of the rainbow trout-diploid embryo and induce it to effectively become a quadruple, the modification is induced at a temperature of 10-16 캜, and 6.5-7.5 hours And it was confirmed that it is most effective to perform the heating treatment at 24-32 ° C for 20-35 minutes. Also, as a result of repeating the verification test of the above-mentioned doublet induction condition, it was confirmed that the doublet inducing condition of the present invention stably and effectively induces a doublet. In order to induce female multiplication, it was confirmed that female feeding was active when a feed containing 10 ppm of estrogen was fed and the female cumulation was induced at a suitable cumulative water temperature of about 880 ° C. Also, as a result of repeating the verification test for the female induction condition, it was confirmed that the female induction condition of the present invention stably and effectively became female.

Further, as a result of crossing the female quadriceps and the male diploid, it was confirmed that triploid embryos were effectively produced. Therefore, when using the method for producing triplet rainbow trout embryo of the present invention, it is possible to obtain a triploid rainbow trout embryo in an efficient manner in Korea, thereby solving the low hatching rate due to the transfer of the triplet rainbow trout embryo introduced from abroad, It is possible to fundamentally solve the introduction of intestinal diseases related to rainbow trout which can be introduced together with introduction of foreign countries. In addition, it can solve the problems of ineffective diapause (winter egg) in the domestic aquaculture industry and can be usefully used in related business.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention as defined by the appended claims. It will be obvious to you.

Example  1. Rabbit induction of diploid embryos of rainbow trout

1-1. Production of diploid embryos

We used diploid females and males of unshaped fish farms in Yuncheon - ri, Jecheon - city, Chungbuk province. Individuals weighing 830 g and 720 g, respectively, were used in the production of diploid embryos. Water temperature was 12-14 ℃ during fertilization and embryo embryos were produced by embryo pressing and embryo harvesting.

1-2. Confirmation of the induction of a doublet by elapsed time after correction

Using the diploid embryo of 1-1 described above, induction to a doublet by elapsed time after the modification was confirmed.

Specifically, the diploid embryos of 1-1 described above were allowed to stand for 6, 6.5, 7, 7.5 and 8 hours after the fertilized eggs were fertilized, and the induction rate to the doublet was confirmed. The results are shown in Fig.

As shown in FIG. 1, the hatching rate did not show any difference according to all treatment times, but the haplotype induction ratio was continuously increased from 6 hours after the modification, and was found to be 98.1 ± 2.71% when 7 hours had elapsed. In addition, it was confirmed that the induction ratio of the doublet was 43.3% when 7.5 hours passed and 0% at 8 hours. As a result, it was confirmed that the elapsed time after modification was a very important factor in the production of the chides, and the optimum elapsed time was 7 hours.

1-3. Judgment of induction of a doublet according to heating temperature condition and heating treatment time

As shown in 1-2 above, 100 diploid embryos after 7 hours of lapse of time after conditioning at a temperature of 12 ° C were subjected to heating (24, 28 and 32 ° C) and heating treatment time conditions (15, 20, 25, 30 and 35 hours) were given to induce the induction of the embryos to the quadrants by the suppression of the first embryos by the above warming conditions.

Specifically, 100 diploid embryos at 7 hours after the modification at 12 DEG C were subjected to warming temperature conditions of 24, 28, and 32 DEG C, followed by heating treatment times of 15, 20, 25, 30, and 35 hours, Were determined. The results are shown in Fig.

As shown in Fig. 2, the mortality rate within 2 days was the lowest in the group treated at 24 캜. In addition, the hatching rate remained constant over time in the 24 ° C-treated group, and the hatching rate remained high in the treated group at 28 ° C and 32 ° C before 30 minutes, Respectively. In addition, the induction ratios were steadily increased until 30 minutes heat treatment at 24 ℃ treatment group, and the induction rate after 25 minutes treatment treatment at 28 ℃ treatment group showed the maximum value. In addition, it was confirmed that more than 90% Therefore, it was confirmed that the induction of the doublet was most effective in the condition of heating at 28 ° C for 25 minutes.

1-4. Repeated verification of the doublet induction condition

As in the above example, the doublet induction conditions were established and verified repeatedly once to five times to verify the doublet induction rate.

Specifically, the embryo was shaken at a speed of 50 rpm in a thermostat equipped with an automatic agitator as shown in FIG. 3, and a thermostat was installed beside the breeding tank to minimize the distance of transport of the fertilized eggs. The distance of final embryo transfer was within 2 m. To verify the induction conditions of diploids, diploids were repeatedly tested 1, 2, 3, 4, and 5 times, and all of the embryos at each condition were subjected to the induction treatment of triploids. Embryos were placed in plastic baskets, It was allowed to shake until hatching. The hatching took place around 14-16 days at 14-16 ℃, and it took 5-7 days before the injury. After injury, they were grafted using Aller Future Larvae Ex. Immediately after the addition of the embryos, the embryos were placed in the aquariums divided by the number of induction of each doublet. The size of the diploid, triploid and triplet was measured as an average of the longest diameter using the ImageJ program. The size of the triplet and the size of the triplet were measured using an ImageJ program at 200x magnification under a light microscope Respectively. Thereafter, each of the squares was randomly sampled by 20 rats each. The results are shown in Fig. 4 and Fig.

As shown in FIG. 4, the size of red blood cells in the blood of the diploid of the rainbow trout of the present invention was confirmed, and it was confirmed that the average long diameter was 16.2 μm, the triplet was 21.3 μm, and the doublet was 22.4 μm.

Further, as shown in Fig. 5, it was confirmed that 100% of the doublet inducing effect was observed under the conditions of 1, 3, and 4 times of verification of each of the doublets. Also, it was confirmed that 95% and 90% of the doublet induction ratios were observed under the repeating verification conditions of 2 and 5 times of the doublet induction condition, and as a result, it was confirmed that a total of 97% Therefore, it was confirmed that the ligand inducing conditions established in the present invention effectively and stably induce the ligand.

Thus, in order to inhibit the first round progression of the diploid transplanted diploid embryo and effectively induce it to become a doublet, It was confirmed that the most efficient method is to conduct the heat treatment at 24-32 ℃ for 20-35 minutes under 6.5-7.5 hours condition.

Example 2: Femaleization of a doublet-derived rainbow trout

2-1. Fertilization of the quadrilateral according to the cumulative temperature condition

In order to confirm the femaleization of the rats according to the cumulative temperature conditions, feeds after the inoculation of the rats induced by the rats in Example 1 were performed, feeds treated with 10 ppm of magnetic hormone (estrogen) were fed, The percentages of males and females were determined at 200, 400, 600, 800 and 1000 ° C, respectively. The results are shown in Fig.

As shown in FIG. 6, no difference was observed in the ratio of 50:50 or 45:55 (male: female) at the accumulated water temperature of 200 ° C, 400 ° C and 600 ° C, but it was confirmed that 15% of males appeared at 800 ° C . Also, it was confirmed that 90% of females and 10% of males were at 1000 ° C, and the highest proportion of females was confirmed. Therefore, based on the above results, it is confirmed that the most suitable integrated water temperature for female cutting is 880 ℃ when the accurate integrated water temperature is calculated using Broken line model.

2-2. Maternalization of the quadrants according to the magnetic hormone concentration

In order to confirm the femaleization of the chondroitin according to the concentration of the magnetic hormone, treatment was carried out at a concentration of 1 and 10 ppm (mg / kg of feed) of the hormone (estrogen) in an integrated water temperature environment of 600 ° C or more according to the result of the above 2-1 The fed diets were fed to the rats. Fig. 7 shows the result of the femaleization of the resulting ridges.

 As shown in Fig. 7, it was confirmed that female transplants were carried out from male to female at 10 ppm at 600 ° C and 1 ppm at 800 ° C and 1000 ° C. In addition, 10% of the males that were not genetically converted at 10 ppm in the cumulative water temperature of 800 ° C or more were all transgenic to the female. Therefore, it was confirmed that the optimum induction condition was 880 ℃ for the integration temperature and 10% for the concentration of estrogen.

2-3. Repeated verification of the number of female induction times

In order to confirm the femaleization of the rats according to the number of female induction times, the cumulative temperature was 880 DEG C, 10 ppm (mg / kg of feed) of the hormone (estrogen) according to the female induction conditions established in 2-2 above, Were fed to the rats. Thereafter, repeated induction of 1, 2, 3, 4, and 5 cycles of induction of female induction was performed to confirm induction of female induction. Twenty animals were arbitrarily sampled for each female induction frequency group to confirm the sex ratio. Specifically, the sex ratio was grown up to about 10 g for 3 months, and the gonads were placed on a slide glass with covering each individual, covered with a cover glass, magnified 100 times under an optical microscope, As shown in Fig. The results are shown in Fig.

As shown in FIG. 9, it was confirmed by repeated test according to the induction conditions of the present invention that more than 80% of females were induced in the experimental group. Therefore, it was confirmed that the induction condition of the present invention stably and effectively induces femaleization of the ridges.

Example 3. Confirmation of fertility, hatching rate and survival rate by alternate crossing between female or male rats and diploid of the present invention

The fertilization rate, hatching rate and survival rate of the female and male of each of the twigs of Example 2 and the female and male of the diploid were respectively confirmed.

The female quadriceps of Example 2 was positive until 46 months after fertilization. Then, as shown in the following Table 1, the females and males of the diploid breeding trout of Example 2 and the females and males of the diploid females were cross-crossed, respectively, and the fertility, hatching rate and survival rate . The results are shown in Table 1.

Mating form Fertility (%) Hatching rate (%) Lifetime survival rate (%) Diploid (female) x diploid (male) 95 93 92 Diploid (female) x square (male) 72 91 91 Rectal (female) × diploid (male) 93 92 92 Zodiac (female) × zodiac (male) 53 91 89

As shown in Table 1, it was confirmed that the mating of the cross female and the male heterozygote showed 93% fertilization rate, 92% hatching rate and 91% survival rate, and compared with the result of the conventional diploid female and male crossing, , The hatchability and the survival rate were similar to each other, it was confirmed that the trichome female females induced by the method of the present invention can effectively induce triploid rainbow trout embryos when mated with male diploids.

As can be seen from the above series of results, after the first incomplete progress of the rainbow trout-embryo embryo was suppressed by the temperature, the treatment frequency and the processing time condition of the present invention to produce a trout trout, the trout had an estrogen concentration And breeding of the trichome rainbow trout embryo can be effectively carried out when the female is crossed with the diploid male and the diploid male.

Claims (7)

(1) inducing diploid embryos using Onchorhynchus mykiss diploid female and male;
(2) leaving the diploid embryo of step (1) under conditions of 7 hours after the modification;
(3) heating the diploid embryo of step (2) to a tetraploid state by heating at 24 to 32 ° C for 20 to 35 minutes; And
(4) cross-crossing the male and female diploid crossbreds to produce triploid rainbow trout embryos. The method according to claim 1,
Wherein the modification of the diploid embryo of step (1) is carried out at a temperature of 10 to 20 캜. delete delete The method according to claim 1,
The method of claim 1, further comprising the step of: after the step (3), femaleizing the doublet. delete delete

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