KR101632842B1 - Hybridized species of olive takifugu obscurus and takifugu rubripes, and process for preparing the same - Google Patents

Abstract

According to the present invention, provided is a production method of a crossbreed of Takifugu obscurus and Takifugu rubripes, which comprises the following steps: extracting ova and semen from female Takifugu obscurus and male Takifugu rubripes respectively; performing artificial fertilization to obtain fertilized ova; and incubating the fertilized ova and breeding the same. According to the present invention, a new crossbreed of Takifugu obscurus and Takifugu rubripes produced by the production method has an advantage of rapid growth rate, thereby allowing the promotion of existing puffer culture industry declining due to low production rate and low economical efficiency.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hybrids and a production method for producing hibiscus,

More particularly, the present invention relates to a method for hybridization of wild-type and frequently-heterozygous embryos, and more particularly, to a method of hybridizing wild-type embryos to embryos, Which is capable of producing a breed of aquaculture having a rapid growth rate, including a step of hatching and cultivating a habitat of a haploid and a haploid, and a production method thereof.

Fugu is a species that lives mainly in the warm fever of tropical and temperate regions. In Korea, And clothing About 6 species are caught and used for food. Currently, the amount of fur fish resources on the coast is rapidly decreasing every year due to overfishing. In order to meet the increasing market demand, it is required to expand the species of cultured fish catches and increase the production quantity.

In Korea, there are two types of puffer fish, two kinds of pufferfish, and two kinds of puffer fish. However, the aggressiveness and cannicalism phenomenon during the early development of pufferfish are very high compared with other fish species, It is difficult to industrialize because it takes a long period of time from slow to commercialization.

However, with the long-term effort of researchers, it has been successful in industrialization because of the complete breeding. Takifugu rubripes belongs to the family of the Hanwha family, and it is the most cultivated variety in the world among the swallows distributed in the coast of Korea, the western part of the Sea of Japan, the East China Sea and others. The morphological features of the blessing are blackish brown on the back of the body, and black dots of several eyeglasses size on the base of silver-gray color extend to one or two lines to the caudal fin. It takes 18 months to grow to a commercial size of 1 kg, and it belongs to a large species of bivalves.

On the other hand, Hwangbok is suffering from full style due to its biological characteristics and strong official behavior and aggression. Puffer (Takifugu obscurus) is dorsally of acronyms into a fish belonging to the pufferfish (Tetraodontidae) of Tetraodontiformes (Tetraodontiformes) the olive and the stomach is the radiance as a white, x has a wide serotti seonhwangsaek width. The main distribution area is the Yellow Sea such as South and South Seas in Korea and the East and South Seas in China, and it lives in the sea until it becomes a mother. From April to May as the spawning season, it reaches up to the midstream of the river, It is known to have ecology.

Unlike other fugu, Hwabok has high adaptability to salinity, so it can be cultured in sea water as well as in domestic water, and it is a high value added fish species with a selling price of about 50,000 won / kg. On the other hand, there is a disadvantage in that it requires long-term breeding for 30 months or longer in order to produce over 350 g commercially available yellowtail. Therefore, fisheries fishery for Hwangbok is a long-term rearing condition. Despite high productivity due to the occurrence of diseases, heating and manpower for wintering and high added value, Efforts are being made to popularize it.

 On the other hand, hybrids in fish have been studied in order to examine the taxonomic position between two geographically separated groups and to understand the genetic contamination of indigenous species by the introduction of alien species. Although there are many variables such as initial occurrence and maturity in artificial production of fish hybrids, researches are underway to develop new aquaculture species with the advantages of two species as aquaculture develops.

The ongoing work of this research can be explained by heterosis, in which some traits expressed in the first generation of hybrids are superior to the traits of the two species used as broodstock. However, because hybrid stress is not a phenomenon that occurs in all traits, hybrid studies of various species have continued to produce hybrids with superior traits. At present, there are a few studies on hybrid fishes in Korea, such as hybridomas and hybrid triploids of loach and mudfish, cytogenetic and molecular biology of flounder and marine flounder.

Therefore, if Hybrid and Hyungbok can produce hybrids with good quality traits through hybridization technique, it will be possible to solve the above problems and to have industrially high value as aquaculture species.

In Korean Patent No. 10-0724805, egg and semen are extracted from a male of a flounder (olive flounder, Paralichthys olivaceus) and a male of a starry flounder (Platichthys stellatus), and artificial insemination is performed between them to obtain an embryo , A hybrid of a flounder and a strong leg including hatching and cultivating an embryo, and a hybridization method thereof. Korean Patent Laid-Open Publication No. 10-2011-0005259 discloses a method of extracting egg and semen from male of Pagrus major (Temminck et Schlegel) and male of Acanthopagrus schlegeli (Bleeker), respectively, And a step of hatching and cultivating the embryo, and a method for producing the hybrid. Korean Patent Laid-Open Publication No. 10-2000-0063064 discloses a method of artificially modifying ovulation, fertilization, and fertilization by artificially inserting a perch, a perch, and a perch into an ovulation or fertilization, It is disclosed a cross-breeding method and a method of a pointy perch and a perch fish having an excellent character of a perch fish.

The present invention has been devised in view of the above-mentioned needs, and includes the steps of extracting eggs and semen from each of female and larvae of a wild boar and subjecting them to artificial fertilization to obtain fertilized eggs, and then hatching and growing fertilized eggs And to provide hybrids and production methods of Hwangbok and Hwangbok, which have disease tolerance due to hybridization with high growth rate.

In order to solve the above-mentioned problems, the present invention provides a method for producing a fertilized egg, comprising the steps of: collecting and filling eggs and semen from a female and a male; A step of hatching in fresh water, and a step of stocking the hatched fish and the fry in seawater.

The fresh water in the incubation step is at a temperature of 18 to 20 ° C and a salinity of 5 psu. The seawater supply stage of the fryer is 3 days after hatching. The temperature of the seawater is 22 to 24 ° C and the salinity is 31 psu. Is 3 days after hatching, and seawater has a temperature of 22 to 24 ° C and a salinity of 31 psu.

The present invention also relates to a method of extracting eggs and semen from the female and the male, respectively, obtained from the female and the male, Which is obtained by artificially fertilization and fertilization of fertilized eggs of heterozygous hybrids.

According to the present invention, the crossbreeding between the wild habitus and the common habitus is a new variety having an intermediate trait of Hwangbok and Mulberry. The growth is rapid and similar to the Hwangbok appearance with high industrial value.

FIG. 1 is a line graph showing weights of a hybrid of Hypoglycem and Haploid produced by the method of the present invention measured at intervals of 2 weeks from 2 weeks to 16 weeks.
FIG. 2 is a photograph showing the appearance and size of Hybok and Hapjong hybrids produced by the method of the present invention, and the appearance and size of the Hapjongbu and Hapjib tests.
Figure 3 shows flow cytometry diagrams of Hwangbok, Hwangbok, and Hybrid.
Fig. 4 shows the karyotype (A, B, C) and the dyed dolls (NORs) middle (D, E, F)
Fig. 5 shows the histological areas of the kidney, liver, and stomach, in the yellow fever, the bladder and the hybrid.
FIG. 6 shows triploids derived from external erythrocyte forms (A, B, C) and diploid gill filaments (NOR) (D, E, F) and yolk.
Fig. 7 shows the histological sections of the retina of the haploid, self-blasted, and hybrid.

The present invention relates to a hybrid and a production method of matured eggs and semen which have been artificially modified and extracted from Hwangbok and Mulberry. Hereinafter, the present invention will be described in detail with reference to specific examples.

Ⅰ. Hybrid production methods

One. Hwabok Often blessed  Hybrid production method

The present invention relates to a method for producing an egg yolk and a herbaceous plant, which comprises the steps of: Artificially correcting the obtained eggs and semen by a dry method; Hatching the embryo at a water temperature of 18 to 20 ° C and salinity of 5 psu in fresh water; The hatched hatchlings can be harvested in seawater having a salinity of 31 psu in a temperature range of 22 to 24 ° C after three days. Hereinafter, a more specific experimental example will be described as follows.

In the present invention, in the stage of egg and semen harvesting and fertilization, the female woolly female (337 ± 8.7 g) and the female male (1,876 ± 16.7 g) were 3 to 5 year old raised in Chungcheongnam-do Fisheries Research Institute Good individuals were selected and used in the experiments. Human chorionic gonadotropin (hCG; Sigma, USA) was injected into the abdominal cavity at 1,000 IU / kg for the induction of spawning of the female moth, and the abdomen was lightly pressed at the interval of 3 hours after 36 hours, Respectively. The sperm was sampled by abdominal compression method in males. The sperm were collected and diluted 10 times with 0.85% physiological saline.

The step of modifying the present invention can be carried out by artificially modifying the embryonic mature egg and the reproductive tract by a dry method in which the obtained egg and semen are brought into intimate contact with each other to induce fertilization.

In the hatching step of the present invention, the embryos were housed in freshwater and seawater in consideration of the hatching conditions of the interspecific embryos, respectively. As a control group, weapon fertilization and fertilization were artificially modified in the same manner, respectively. Hybridized embryos were also housed in seawater and freshwater, respectively.

The embryos of each experimental group were removed after 3 minutes of fertilization by stirring for 30 seconds in cray-earth-containing stocks. Immediately after the agitation, the cells were transferred to sterilized filtration broth and incubated in an incubator for incubation. The filtration of water and the removal of mucus by using a gauze remover can prevent the hatching rate from dropping due to clogging of the eggs. For the removal of gypsum, it is appropriate to remove the gypsum by mixing it with fertilized eggs using fuller's earth of 125-250 μm particle size and stirring for 3 minutes. Table 1 shows the environmental conditions during hatching in the embryos of each experimental group.

Embryo hatching condition of each experimental group Temp. (占 폚) Sal. (Psu) Water River puffer 19 5 supply seawater after 3 days from hatching Tiger puffer 19 31 - Hybrid (freshwater) 19 5 supply seawater after 3 days from hatching Hybrid (seawater) 19 31 -

The seawater supply step of the present invention is a step of supplying seawater having a salinity of 31 psu in the range of 22 to 24 ° C after 3 days after being hatched in the fresh water condition. After the hatching, a small amount of chlorella was added to the broodstock immediately after hatching, and the rotifer was supplied at the level of 3 to 10 individuals / mL from the third hatching stage. Artemia nauplius larvae Were fed at a level of 3 to 10 individuals / mL. The compound feeds were supplied from the 15th day of hatching. Feeding diets and compound feeds were equally supplied to all experimental sites.

Growth was measured at 2 - week intervals from 2 to 16 weeks during the rearing period. After the anesthesia with anesthetic, the length and weight of the fish were measured in mm and g. During the experimental period, the temperature of rearing water was 23.7 ± 0.84 and dissolved oxygen was 4.2 ~ 6.5 ppm.

2. Hwabok Often blessed  Hybrid production method result and morphology survey

The production results of the hybrids produced by the method of the present invention and the external shapes appearing from the siblings were examined. Table 2 shows the initial survival rates of the wild boar, the herbivore, the hybrid produced by the above method, the fertilization rate, the hatching rate, the hatching rate, and the 7 days after hatching.

Fertilization rate, hatching rate, hatching rate, chi and urticaria survival rate (7 days after hatching) Fertilization Eyed eggs Hatching Survival River puffer 86.4 77.7 67.2 94.9 Tiger puffer 88.8 73.1 58.5 90.8 Hybrid (freshwater) 85.7 71.6 61.7 83.4 Hybrid (seawater) 81.1 66.3 48.7 43.7

Compared with the control group, fertilized embryo, fertilization rate, hatching rate, hatching rate and survival rate, the freshwater embryos induced by hatching in fresh water were 85.7% fertilized, 71.1% fertilized, 71.7% hatching, and 83.4% There was no big difference.

The fertilized eggs of the experimental group showed 81.1% fertilization rate, 66.1% fertilization rate, 48.7% hatching rate and 43.7% survival rate. The fertilization rate and fertilization rate of control embryo were significantly higher than control There was no difference, but there was significant difference in hatching rate and survival rate. Based on the above results, it is considered appropriate to hybridize embryos in a fresh water environment controlled at a temperature of 19 ℃ and a salinity of 5 psu.

In order to determine the industrial value, the survival rate of the hybridized hatchlings in freshwater conditions was 83.4%, 94.9%, and the survival rate was about the same as that of the larvae which showed the survival rate of 90.8. .

Table 3 below shows the results of body weights measured at intervals of 2 weeks from 2 weeks to 16 weeks after hatching in each of wild boar, Bombyx mori, Hwangbok, and Hwangbok were measured at 2 week intervals for 2 to 16 weeks.

 Weighing results from 2 weeks to 16 weeks after hatching from yellow fever, Weeks after hatching Survival
(%) 2 4 6 8 10 12 14 16 River puffer (g) 0.07 1.27 2.04 3.3 5.48 8.2 12.3 16.8 62.5 Tiger puffer (g) 0.08 1.77 4.78 9.24 15.6 24.3 32.6 43.7 64.4 Hybrid (g) 0.07 1.57 3.86 6.87 12.7 19.8 27.8 37.4 61.8

The results of 16 weeks of growth experiment of Hwangbok, Hwangbok and Hwangburo according to the age of rearing showed no significant difference in body weight between Hwangbok, Hwangbong and Hyungbuk until 6 weeks after hatching. However, from 6 weeks after hatching, The weight of the hybrid and hybrid pufferfish was significantly higher than that of the puffer fish. On the other hand, no significant difference was observed during the breeding period when comparing the growth of the hybrids and hybrids.

 FIG. 2 is a photograph showing the appearance and size of Hybok and Hapjong hybrids produced by the method of the present invention, and the appearance and size of the Hapjongbu and Hapjib tests. At the top, there are typical river puffer and tiger puffer fish. The middle part and the lowermost part were compared with each of sixteen weeks old, three times, three times, and three larvae after hatching by the experimental method of the present invention.

The outline of the hybrids showed a distinct difference in the appearance of the frequent apparel with small olive spots on the back, but there was no significant difference between the apparel and the apparel. Therefore, the external shape of the hybrids derived from this experiment is expected to be industrially valuable as it is similar to that of female.

Ⅱ. Genetic analysis of crossbreeding between wild boar

The genetic characteristics of the hybrids produced by the method according to the present invention were investigated, and a genetic analysis was carried out for hybrid identification.

1. Genetic analysis experiment of crossbreeding of wild boar

 1) Chromosome number and karyotype analysis

For chromosome number and karyotype analysis of experimental Kim et al. (1982), a chromosome sample was prepared using renal tissue. The colchicine was intraperitoneally injected into 10 randomly sampled specimens at an appropriate concentration (1 to 10 μg / g) and left for 3 to 4 hours. After elongation, the kidneys were removed and treated for 30 minutes at room temperature (0.075 M KCl) Respectively. The kidney tissue cells treated with the stock solution were fixed with 3 times of methanol (glacial acetic acid = 3: 1).

Slide specimens were prepared by air drying and stained with 5% May-Grünbaldt Giemsa solution for 10-15 minutes. The developed specimens were counted under an optical microscope (× 1,000) and counted in about 50 readable chromosomes in each individual. The karyotype analysis was performed by Levan et al . (1964).

2) Red blood cell size measurement

The blood was collected from the microvessel vein of each individual, and then stained with 100% ethanol and stained with Giemsa solution. 120 red blood cells per each individual were measured, and the long diameter (a) and short diameter (b) of the red blood cells and nucleus were measured with a micrometer under an optical microscope (× 1,000). The surface area is calculated by the formula of abπ / 4 (Sezaki and Kobayashi, 1978) and the volume by the formula 4 (a / 2) × (b / 2) × π / 3 (Lemoine and Smith, 1980).

3) Nuclear organizer regions (NORs)

The gills are randomly extracted from the gills, and 10 of them are randomly extracted. The gill tissues are extracted and fixed in Carnoy solution (1: 3 acetic acid: ethanol) for more than 30 minutes. In the same way, the sample is stored and refrigerated until analysis.

Place each piece of tissue firmly immobilized on a slide glass, and after supplying enough of 45% acetic acid, disperse each tissue on slide glass according to the chopping method. After slicing, slide slides fixed with 95% ethanol were mixed with a 2: 1 mixture of 50% AgNO3 and gelatin solution (Howell et al., 1979) (Howell et al., 1979) After covering the cover glass, the plate was incubated in a slide warmer at 68 ° C for 100 seconds, washed with distilled water, and examined for NORs of red blood cells in an optical microscope (× 1,000).

4) Measurement of DNA content

Flow cytometry was performed to analyze the DNA content of Hwangbok, Hwangbok and Hybrid. To prevent blood clotting, 1 mL of disposable syringe was coated with ACD solution (0.48 g citric acid, 1.32 g trisodium citrate, 1.47 g glucose, 100 mL distilled water) and then 0.1-0.2 mL of blood Blood was collected at 1-2 × 10 ⁶ cells / mL using a hemocytometer and fixed in 75% ethanol.

For DNA staining, 100 μl of each sample was added to 2 ml of the Cystain DNA 2-step kit (Partec GmbH Munster, Germany), and the mixture was stained for 15-30 minutes in a water-cooled condition and used with a Partec PA-Ⅱ flow cytometer (Partec GmbH Munster, Germany) And the DNA content was measured.

5) Histological observation

Hwangbok, Hwangbok, and 10 interspecific hybrids were randomly extracted, and liver, ocular, intestine, and kidney were extracted and the cell nucleus size was measured by a histological method. Each organ was fixed in 10% neutral formalin. Twenty-four hours later, each sample was replaced with fresh 10% neutral formalin. Twenty-four hours after the change, the tissue was softened by adding Bouin's solution (750 ml of picric acid, 250 ml of formalin, 5 ml of acetic acid) for 24 hours.

After washing for 24 hours in water flowing through softened tissue, dehydration was carried out in 70, 80, 90 and 100% ethanol for 1 hour each. Dehydration was performed by adding industrial xylene and xylene for reagent for 1 hour each. Alkaline samples were placed in soft paraffin for 12 hours to infiltrate soft paraffin into the sample tissues. Hard paraffin was re-infiltrated into hard paraffin for 24 hours.

The sample that had been infiltrated was embedded in a square shape with a hard paraffin using a foraging machine (EG1150H, Leica, Germany), and the embedded sample was analyzed using a microtome (RM2235, Leica, Germany) And cut on a 6 μm scale, and then, the resultant was spread on a slide glass. The slide glass was stained with Hematoxylin-Eosin stain and examined. Retinal thickness, nucleus area of hepatocytes, nucleus length of intestinal epithelium, length and short axis of cells and nucleus of second proximal tubule of kidney, and area of the kidney were measured.

2. Hwabok Often blessed  Result of hybrid genetic analysis

1) Analysis of chromosome number and karyotype

Figure 3 shows flow cytometry diagrams of Hwangbok, Hwangbok, and Hybrid. The average of peak A was about 200, and the peak of B was hybrids close to 240, and the peak of C was 290.

Table 4 below shows different amounts of phosphorus among 100 cells of the gill tissue in the wild boar, the common breed, and the hybrid. The number of phosphorus in diploid is 1 or 2, and triploid phosphorus is 1, 2 or 3. Values represent the mean ± standard deviation of triplicate groups.

Among the 100 cells of the gill tissues in the hyacinth, Sample No. River puffer Hybrid Tiger puffer One        2 One 2 One 2 One 36 64 38 62 41 59 2 37 63 40 60 40 60 3 35 65 41 59 41 59 4 38 62 43 57 42 58 5 34 66 37 63 44 56 6 35 65 39 61 38 62 7 35 65 43 57 39 61 8 35 65 44 56 39 61 9 37 63 40 60 40 60 10 36 64 41 59 41 59 Mean ± SD ² 35.8 ± 1.22 64.2 ± 1.23 40.6 ± 2.27 59.4 ± 2.27 40.5 ± 1.72 59.5 ± 1.72

As shown in Table 4, in the amount of phosphorus in the gill tissue, 64.2 times on the second chromosome on the first chromosome of the gilbang, 59.5 times on the second chromosome on the first chromosome of 40.5 times, 40.6 times on the first chromosome of the hybrid, And 59.4 cycles on the second chromosome.

Fig. 4 shows the karyotype (A, B, C) and the dyed dolls (NORs) middle (D, E, F) A shows the middle stage (left) and karyotype (right) of yellow fever (2n = 44). B represents the middle stage (left) and karyotype (right) of the hybrid (2n = 48). C represents the middle stage (left) and karyotype (right) of the frequent duplication (2n = 48). D is the middle colored NORs of yellow birch, E is the middle colored NORs of the hybrid, and F represents the middle colored NORs of yellowish brown. Arrows indicate the chromosomes of the microscope. The bars represent 10 탆.

In most cases, hybrid chromosomes consist of a set of haploid chromosomes of the male and female horses used in the hybridization induction. Therefore, the hybridization is highly accurate by the chromosome number or karyotype analysis.

2) Red blood cell size measurement result

Table 5 shows the nucleus sizes of the proximal tubule of the renal tubule and the middle ear epithelium of the kidney of the present invention, Each value represents the mean ± standard deviation of the three groups. Superscripts in the same row show significant differences ( P <0.05). And surface area = 1/4 × abπ, vol = 4/3 × π (a / 2) × (b / 2) 2. was calculated by the following formula. a represents the maximum axis of the cell or nucleus, and b represents the minimum axis of the cell or nucleus.

Comparison of nuclear size of proximal tubule and middle ear epithelium of renal tubules River puffer Hybrid Tiger puffer Proximal tubule cell of kidney **   Major axis (탆) 3.7 ± 0.28 ^a 3.9 ± 0.47 ^ab 4.3 ± 0.81 ^b   Minor axis (㎛) 2.1 ± 0.11 ^a 2.3 ± 0.64 ^ab 2.8 ± 0.93 ^b Surface area (㎛ ² ) 6.1 ± 0.69 ^a 07.0 ± 0.57 ^ab 9.4 ± 0.51 ^b Volume (탆 ³ ) 9.8 ± 0.79 ^a 10.7 ± 1.68 ^ab 17.6 + 1.26 ^b Cell number in proximal tubule 12.5 ± 0.81 ^a 13.2 ± 1.02 ^ab 14. ± 51.10 ^b Hepatocytes of liver **   Major axis (탆) 4.6 ± 0.14 ^a 5.1 ± 0.77 ^ab 5.5 ± 0.95 ^b Minor axis (㎛) 3.4 ± 0.26 ^a 4.0 ± 0.45 ^ab 4.4 ± 0.50 ^b Surface area (㎛ ² ) 16.6 ± 1.90 ^a 16.0 ± 2.55 ^ab 18.9 ± 3.66 ^b Volume (탆 ³ ) 47.1 ± 8.29 ^a 42.7 ± 4.07 ^ab 55.7 ± 4.53 ^b Nuclear height of midgut epithelium 4.8 ± 0.25 ^a 5.4 ± 0.18 ^ab 6.0 ± 0.25 ^b

Fig. 5 shows the histological areas of the kidney, liver, and stomach, in the yellow fever, the bladder and the hybrid. (A) indicates a yellow fever, (B) indicates a hybrid, and (C) indicates a kidney tissue section of a bladder. (D), (E), and (F), respectively. (G) is yellowish, (H) is hybrids, and (I) is the intestinal tissue section. The abbreviations shown in FIG. 5 include BV, blood vessel, HC, nucleus of hepatocyte, ME, midgut epithelium, secondary proximal tubule (SPT) Quot;

As shown in Table 5 and FIG. 5, there was a significant difference in the sizes of renal tubular cells, hepatocytes, and intestinal epithelial cell nuclei, similar to the sizes of red blood cells and nuclei, And there was no significant difference between the three groups.

3) Results of nucleolar organizer regions (NORs) of red blood cells

Table 6 below shows a comparison of erythrocytes and nuclei in the wild boar, hybrids, and blastocysts. Each value represents the mean ± standard deviation of the three groups. Superscripts in the same row show significant differences ( P <0.05). And surface area = 1/4 × abπ, vol = 4/3 × π (a / 2) × (b / 2) 2. was calculated by the following formula. a represents the maximum axis of the cell or nucleus, and b represents the minimum axis of the cell or nucleus.

Comparison of Red Blood Cells and Nuclei in Hwangbok, Hybrid, and Self-mutilation River puffer Hybrid Tiger puffer Erythrocyte **   Major axis (탆) 7.5 ± 0.82 ^a 8.1 ± 0.99 ^ab 10.1 + 1.44 ^b   Minor axis (㎛) 5.5 ± 0.75 ^a 5.9 ± 0.85 ^ab 7.5 ± 0.51 ^b Surface area (㎛ ² ) 33.5 ± 6.33 ^a 37.5 ± 5.87 ^ab 59.5 ± 8.11 ^b Volume (탆 ³ ) 125.5 ± 35.97 ^a 147.6 ± 48.22 ^ab 297.3 ± 50.09 ^b Nucleus of erythrocyte **   Major axis (탆) 2.7 ± 0.37 ^a 3.4 ± 0.47 ^ab 4.5 ± 0.37 ^b   Minor axis (㎛) 1.7 ± 0.33 ^a 2.1 ± 0.34 ^ab 2.7 ± 0.34 ^b Surface area (㎛ ² ) 3.5 ± 0.81 ^a 5.6 ± 0.84 ^ab 7.8 ± 0.68 ^b Volume (탆 ³ ) 4.0 ± 1.48 ^a 09.5 ± 1.37 ^ab 17.2 ± 1.67 ^b

FIG. 6 shows triploids derived from external erythrocyte forms (A, B, C) and diploid gill filaments (NOR) (D, E, F) and yolk. A and D are hyacinth, B and E are hybrids, and C and F are frequent duplicates. In each air-dried group, stains appear and are stained with May-Grünwald Giemsa. Arrows indicate a stained nucleus doll, and the bars represent 10 μm.

As shown in Table 6 and FIG. 6, there were significant differences in the size of erythrocytes and nuclei between the ulnar nodules and the nodules in all evaluation items such as long axis, short axis, surface area and volume of erythrocytes and nucleus. However, the long axis, short axis, surface area, and volume of the red blood cells and nucleus of the hybrid were not intermediate between the three groups, and the easiest method for hybridization was Chevassus (1983 ), The hybrid produced by the method according to the present invention can be judged to be a hybrid of Hwangbok and Hwangbok.

4) Results of DNA content measurement

Table 7 below shows the DNA content of hybrids ( Takifugu obscures) , T. rubripes, and hybrids produced according to the method of the present invention.

The DNA content of the hybrid of the present invention, Sample No. DNA contents (pg / nucleus) River puffer Hybrid Tiger puffer One 1.81 2.15 2.62 2 1.85 2.15 2.62 3 1.84 2.16 2.65 4 1.82 2.14 2.63 5 1.85 2.14 2.64 6 1.83 2.16 2.63 7 1.84 2.13 2.66 8 1.84 2.15 2.65 9 1.84 2.14 2.65 10 1.86 2.16 2.66 Mean ± SD  1.84 + 0.019 2.15 + 0.020 2.64 ± 0.015

For the DNA content measurement in Table 7 above, the DNA content of loach ( Misudurnis mizolepis (2.81 pg / nucleus)) was used as a standard reference. The contents of DNA were 1.84 and 2.64 pg / nucleus, respectively, and 2.15 pg / nucleus, respectively, in hybrid. This result is considered to reflect the difference in chromosomal numbers (2n = 44), chromosomal numbers (2n = 48), and heterozygous chromosomal numbers (2n = 48) .

5) Histological findings

Table 8 below shows the thickness in the number of the constituent layers of the eyeball and the number of the outer nuclear layer in the hwabokgwa, haboki, and hybrids. Each value represents the mean ± standard deviation of the three groups. Superscripts in the same row show significant differences ( P <0.05).

In the number of each component layer and outer nuclear layer of the retina, River puffer Hybrid Tiger puffer Thickness of retina (占 퐉) 110.9 ± 2.15 ^a 128.7 ± 3.83 ^ab 130.7 ± 2.41 ^b Thickness of each layer of retina (%) Epithelial layer 22.5 ± 0.89 ^a 22.6 ± 1.76 ^a 23.0 ± 1.52 ^a Rod and cone layer 11.9 + 0.85 ^a 12.0 + 0.88 ^a 12.2 ± 1.04 ^a Outer limiting membrane 4.5 ± 0.40 ^a 04.9 ± 0.29 ^a 5.0 ± 0.33 ^a Outer nuclear layer 12.3 ± 0.43 ^a 12.6 ± 1.06 ^a 12.5 + 0.99 ^a Outer plexiform layer 4.1 ± 0.21 ^a 4.1 ± 0.37 ^a 4.3 ± 0.31 ^a Inner nuclear layer 13.1 ± 0.44 ^a 13.0 + 0.49 ^a 13.4 ± 0.81 ^a Inner plexiform layer 23.0 ± 0.42 ^a 23.2 ± 0.88 ^a 23.3 ± 0.71 ^a Ganglion cell layer 4.9 ± 0.14 ^a 4.8 ± 0.75 ^a 5.0 ± 0.69 ^a Optic nerve fiber layer 5.7 ± 0.56 ^a 5.9 ± 0.65 ^a 5.9 ± 0.51 ^a Number of outer layer cell nucleus 2 ^a 2 ^a 2 ^a * Each value is the means standard deviation of triplicated groups. Means in rows with the different superscript letters are significantly different ( P <0.05).

Fig. 7 shows the histological sections of the retina of the haploid, self-blasted, and hybrid. The letters A, B, and C denote hybrids. Each alphabetical abbreviation stands for the epithelial layer (EL), the ganglion cell layer (GCL), the innermost plexiform layer (INL) ), The outer plexiform layer (OPL), and the rod and cone layer (RCL). The rod refers to 10 占 퐉.

In Table 8 and FIG. 7, there was a significant difference in the retinal thickness between the yellowish and purple and hybrid groups, but there was no significant difference between the yellowish, There were no significant differences in the number of cells in the outer border membrane, outer granule layer, outer mesenteric layer, inner granule layer, inner mesenteric layer, ganglion cell layer, optic nerve fiber layer and outer layer.

As a result, the hybrids of ♀ and ♀ were similar to those of the parent, and the appearance was similar to that of the female, It is similar in terms of cost and growth. However, most of the hybrids were judged to be hybrids between Korean and Korean populations. Therefore, Hybridization and growth evaluation of ♀ and ♀ showed a possibility of industrialization of hybrid fugu .

By producing hybrids of Hwangbok and Suweonbok, it is possible to produce new varieties with high growth rate of short term and intermediate traits of Suwonbuk and Suweonbok by solving the problem of Hwabok style with low production efficiency and economical efficiency, , It is possible to contribute to the increase of income of fishermen by improving the international competitiveness of the aquaculture technology as well as the development of the fisheries field and expansion of the form base.

Claims (6)

And egg and semen from the female and the female, respectively.
Artificially correcting the learned egg and semen;
Hatching the artificially fertilized embryo in fresh water;
And a step of stocking the hatched fish and the fry in seawater.
The method according to claim 1, wherein the fresh water in the hatching step is a water temperature of 18 to 20 ° C and a salinity of 5 psu.
[3] The method according to claim 2, wherein the embryo and the fry are placed in seawater three days after hatching, and the seawater has a water temperature of 22 to 24 DEG C and a salinity of 31 psu.
This study was carried out to investigate the effects of fertilization and fertilization on the fertilization and fertilization.
It was obtained by hatching and fertilization of a hybrid egg and a heterozygous hybrid egg which were obtained by artificial insemination by extracting eggs and semen from each of female and larvae of Hwangbok.
The DNA content of the hybrid of the 5th and 5th lines of the present invention is 2.15 ± 0.02 pg / nucleus,

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