KR101627073B1 - Manufacturing method for hybrids of abalone - Google Patents

Abstract

The present invention relates to a method for producing hybrid abalones comprising the following steps: selecting parent shellfishes of abalones; stimulating the selected parent shellfishes by exposing to air in shade and exposing to air at high temperatures, and soaking the resultant products in sea water to induce spawning and ejaculation; collecting spawned eggs and released semen; intrauterinally inseminating the collected eggs and semen having an optimum concentration of spermatozoa; and hatching the fertilized eggs in the sea water having salinity in a range of 29-33 psu. Accordingly, the method for producing hybrid abalones: increases intrauterine insemination success rates and normal development rates of artificial fertilized eggs, thereby increasing productivity in aquaculture industry; and increases growth speed of abalones, and thus shortens the abalone supply cycle, thereby increasing economic capacity through the aquaculture industry.

Description

{Manufacturing method for hybrids of abalone}

The present invention relates to a method for producing hybrid plants by crossing four species of abalone. More specifically, the present invention relates to a method of producing hybrid seeds from four species of abalone, such as northern abalone, round abalone, horse abalone, The present invention relates to a method for producing abalone hybrids capable of producing aquacultural product having a large size and rapid growth rate by performing artificial insemination among them.

Abalone species belong to molluscs (Phylim Mollusca), Gastropoda, Archaeogastroppoda, Haliotidae and Haliotis, and about 100 species are known in 60 ~ 70 species (Lindberg 1992) have.

About 100 kinds of abalone are distributed throughout the world, and its main destinations are East Asia, Australia coast, New Zealand, and the Pacific coast of North America. In the abalone, which is distributed in Korea, there are recorded 6 species of horse abalone, abalone, round abalone, northern abalone, 5th instar and 5th instar macroscopic era in round abalone, and most of abalone and shellfish in the south coast and Jeju island But the northern abalone appears in the waters of the central and northern regions including Ulleungdo.

The industrialization of abalone farming in Korea started in 1972 with the study on the production of abalone and shellfish seedlings in the cultivation field of Bukjeju National Fisheries Research and Development Agency. After that, in the 1980s, the seedlings were produced at 11 cultivation sites under the National Fisheries Research and Development Agency (KFDA), and they were laid out for the aquaculture fishermen.

In 2000, caged aquaculture technology was developed in Heuksan Island, and aquaculture using marine cage facilities was activated mainly in the western coast of the south coast. Since 2003, the production volume has increased explosively, which accounts for about 80% of domestic abalone production. In addition to the development of marine cage facilities, improvement of breeding techniques and development of aquaculture materials have made dramatic development in abalone farming in the last 9 years, with a 60-fold increase in aquaculture production, reaching the current abalone farming industry level.

However, various problems have arisen due to rapid growth. The first problem was that the farm environment was deteriorated due to the expansion of the cage facilities within the limited watershed, resulting in massive mortality caused by the inhabitants. Secondly, the productivity was greatly reduced due to the severe incapacitation due to the inbreeding of the domestic Anshan abalone ≪ / RTI > In addition, there is a problem in the development of the large abalone, and the large abalone-abalone fish is significantly reduced, resulting in a great difficulty due to the shortage of the production. Therefore, it is necessary to supply excellent breeding abalone through breed development .

Korean Patent No. 10-1231126 discloses a double-diploid aquatic animal having a different genome AB from a species, and has sex chromosomes of the XXXY type having fertility. A number of hybrids housed in a closed system (closed system) (Non-reduced number of eggs) of female (non-reduced number sperm) and female (female) are selected and the non-redundant egg is corrected to the non-suppression column, It discloses a double-bladder aquatic animal and its breeding method in which an embryonic zoo animal is produced and inbreeding is repeated but the influence of the subordinate weakness does not appear. Korean Patent No. 10-0676214 discloses an artificial embryo having an orange shell, which is selected as a seed, dried, exposed to UV-irradiation water under a thermal shock to artificially induce scattering, Breeding methods of various embryo cultivars having orange shells produced by abalone, larval or offspring breeding, modified by crossing or collective crossing, and produced by abalone cultivation, larvae, ≪ / RTI > Korean Patent No. 10-0724805 discloses a new hybrids produced by crossing a flounder and a strength leg, which is a kind of a sweatfly, and a method for producing such hybrids, wherein a female of a flounder (olive flounder, Paralichthys olivaceus) (starry flounder, Platichthys stellatus), and the hybridization between the flounder and the intact legs including the step of embryo hatching and breeding, Lt; / RTI > However, the above-mentioned prior art does not disclose a method for producing a hybrid of a new variety by artificial fertilization of egg and semen from egg abalone, round abalone, horse abalone, and four kinds of abalone of the present invention It shows a difference.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a method for collecting and collecting eggs and semen from selected females and males selected from four kinds of abalone such as northern abalone, round abalone, horse abalone, We propose a method for the production of abalone hybrids that can fertilize eggs with artificial fertilization in the range of 100,000-3,000,000 cells / cc and then fertilize artificially fertilized embryos in seawater in the range of 29 psu to 33 psu.

As a means for solving the above-mentioned problems, Inducing the selected mother liquor to stimulate a sound stimulation and a stimulation to stimulate the stimulation at a high temperature and immersing it in seawater to induce scattering and solving; Collecting and dispensing the eggs and semen that have been scattered and shaped; Artificially modifying the cumulated egg and the semen of a suitable sperm concentration; And hatching said embryos in seawater having a salinity ranging from 29 psu to 33 psu.

The step of screening the mother line includes male northern abalone and female king abalone, male round abalone and female northern abalone, male round abalone and female king abalone, male horse abalone and female round abalone, male king abalone and female northern abalone, And female round overturns. In the step of inducing scattering and flattening, the stimulation of the sound stimulation is performed for 30 minutes, the high temperature stimulation is performed at 30 ° C, the sperm concentration of semen is 100,000 to 3,000,000 cell / cc. < / RTI >

Hybrids produced by artificially fertilization of females and males selected selectively from four types of abalone, such as northern abalone, round abalone, horse abalone, and royal abalone, have a high incidence of artificial fertilization and artificial fertilization, have.

Fig. 1 shows the process of producing the abalone hybrid of the present invention.
Fig. 2 shows the fertilization rate of the spermatozoa in hybrid crossing between the northern abalone male and the seagrass.
FIG. 3 shows the fertilization rates of spermatozoa in a hybrid cross between a round abalone male and a female.
FIG. 4 shows the fertilization rate of the spermatozoa in hybrid crossing between male and female endometrium.
FIG. 5 shows the fertilization rates of spermatozoa in hybrid crosses between male and female.
Fig. 6 shows the salinization rates of round abalone ♀ x round abalone ♂ (obedient household) and round abalone ♀ x king abalone (mixed household).
FIG. 7 is a photograph showing the untreated male × × rounded abalone ♂ (obedient household) and the round abalone ♀ × king of abalone (hybrid household) at a salt concentration of 25 psu.
FIG. 8 is a graph showing the normal incidence rate of round abalone ♀ x round abalone male (purebred ancestor) and round abalone female x 王 king abalone male (hybrid household).
Fig. 9 shows malformed individuals of round abalone ♀ x round abalone (obedient household) and round abalone ♀ x Wang abalone (hybrid household) according to salinity concentration.
Fig. 10 is a graph showing the result of the first measurement of the first sowing household.
Fig. 11 is a graph showing the results of the second-order measurement of the primary picking household.
Fig. 12 is a graph showing the results of the second-order measurement of the primary picking household.
Fig. 13 is a graph showing the growth change over 90 days for each household in the primary seedling household.
FIG. 14 is a photograph showing the shape of a spatula growing and growing for 90 days in each household of the primary pickling household.
15 is a graph showing the results of the first measurement of the secondary seedling household.
16 is a graph showing the result of the second measurement of the second picking household.
FIG. 17 is a graph showing the growth change over 90 days for each household in the secondary seedling household.
Fig. 18 is a photograph showing the appearance of a spatula growing and growing for 90 days in each household of the secondary pickling household.
Fig. 19 is a photograph showing the results of the second measurement of the third order picking household and the external shape of the spatula. Fig.

The present invention relates to a method for producing a hybrid by embedding semen and eggs in four types of abalone, such as northern abalone, round abalone, horse abalone, and king abalone. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to specific embodiments and accompanying drawings.

Ⅰ. How to Produce Abalone Hybrid

The method of producing an abalone hybrid of the present invention comprises the steps of stimulating the mother to stimulate the desiccation and inducing the scattering and infiltration by immersing the mother in a seawater; Collecting and filling egg and semen from the egg-laying mother who has been induced to scatter and shape; Artificially correcting the cumulated egg with a semen of a suitable sperm concentration; And hatching the embryo in seawater having a salinity ranging from 29 psu to 33 psu.

Hereinafter, a more specific experimental example will be described as follows. In the step of selecting the mother of the present invention, the collection of the mother group was collected for four types of Northern abalone, round abalone, horse abalone, and king abalone. Table 1 shows the size of the leg according to the age of the four abalones collected as a parent group.

The Northern abalone was collected mainly from the seed island of the Samchonpo coastal area of Sangcheon - si, Gyeongsangnam - do, and the natural mountain mainly collected from the coast of. Samcheonpo coast where northern abalone is sampled is a sea area where abundant artificial abalone discharge is not done. Collecting is carried out by the sea dawdlers at the depth of 8.0 ~ 14m in Junggu Island and 5.0 ~ 13.0m in the coast of Seed Island. After the purchase, the offspring of the naturally abalone abalone was sampled for the exclusion of the pesa from the reared breeding tank during the adaptation period (about 15 days of adaptation), and morphometric measurements and age were evaluated for the dead animals.

Size of legs according to age of four kinds of abalone kind age 2 3 4 5 6 7 8 9 Sum Abalone
H. discus hannai Population
(Marie) 23 30 12 2 2 69 Chief
(mm) 79.4
(70 to 84) 85.9
(80 to 92) 97.5
(93 to 105) 112.0
(111 to 113) 125.5
(125 to 126) Width
(mm) 53.9
(49-65) 58.6
(55 to 69) 65.4
(62 to 69) 74.5
(74 to 75) 85.0
(82-88) Each
(mm) 15.7
(14-19) 17.1
(15-24) 65.4
(62 to 69) 25.5
(24-27) 25.5
(24-27) Round abalone
H. discus Population
(Marie) 4 18 41 44 34 20 16 3 180 Chief
(mm) 72.8
(64 to 77) 83.6
(79-88) 91.6
(77-98) 99.5
(90 to 107) 112.7
(108-120) 127.2
(120 to 138) 137.7
(130 to 144) 149.7
(145 to 156) Width
(mm) 51.5
(44 to 56) 58.9
(54 to 67) 64.6
(55 to 70) 70.0
(63 to 76) 80.0
(74 ~ 94) 90.7
(84 to 102) 98.1
(90 to 102) 104.3
(101 to 107) Each
(mm) 13.3
(11 to 16) 15.2
(13-18) 17.3
(11-22) 19.2
(10 to 23) 23.8
(18 ~ 31) 30.2
(25 ~ 34) 31.6
(27 to 36) 35.0
(32-39) Horse abalone
H. gigantea Population
(Marie) One 20 25 11 8 7 72 Chief
(mm) 87.0 101.6 (90 ~ 115) 109.9
(99-117) 120.4
(117 to 128) 132.9
(125 to 142) 142.6
(132-155) Width
(mm) 65.0 76.9
(62 to 88) 84.2
(75 to 92) 91.3
(87-95) 100.8
(96 to 107) 110.6
(103 to 120) Each
(mm) 13.0 18.0
(15-21) 19.3
(15-24) 21.5
(20-24) 25.5
(20 to 30) 26.7
(24-29) King Abalone
H. madaka Population
(Marie) 2 2 16 14 8 5 3 4 54 Chief
(mm) 79.0
(77 to 81) 87.5
(84 to 91) 103.3
(94 to 109) 113.6
(102 to 120) 122.3
(110 to 124) 128.0
(125-132) 134.7
(134-135) 159.5
(151 to 165) Width
(mm) 55.0
(55 to 55) 64.5
(59-70) 77.6
(67 to 86) 84.7
(71 to 99) 91.4
(85-95) 91.0
(85 to 103) 98.0
(91 to 104) 119.5
(110 to 124) Each
(mm) 13.5
(13-14) 16.0
(14-18) 21.5
(17-25) 23.3
(18-28) 26.1
(22 to 32) 26.2
(23-29) 28.0
(27 to 30) 36.8
(35-39)

In order to secure the mother of abalone, horse abalone, and king abalone, we purchased abalone, which is supposed to be natural, from the marine products committee collecting the abalone caught by the sea lions in the village of Jeju. Jeju Island is located in Jeju City, Udo Island, Seogwipo City, Onpyeong, Shinheung, Hasee, Junghwan, Gangjeong, Haeye, Segye, Hamo and Gapa fishing villages. Sampling depths were mainly from 3.0 ~ 15.0m depths and deep water depths for larger objects. The distribution of round abalone was distributed evenly throughout the Jeju waters, and the sampling depth was collected mainly at 3.0 ~ 8.0m. King Abalone was mainly collected in Gangjeong, Jukhwan, and Great Pyongchon, and the depth of the water was 5.0 ~ 10.0m. Horse abalone was collected mainly in Gapa, Mossoo, Gangjeong and Gimjeon, and the depth of water was 7.0 ~ 15.0m.

Fig. 1 shows the process of producing the abalone hybrid of the present invention. Four species of abalone, such as northern abalone, round abalone, horse abalone, and king abalone, designated as the mother group, were raised at the Fish Seed Research Center and matured among the seeds of each breed were selected for a total of three times.

The scattering and emulsifying step of the present invention can be performed by the extraction of the shade, the extraction of the high temperature, and the seawater immersion process. Females were separated from males and females and housed in containers. After clearing the shade, it moved to the room where the temperature could be controlled, and the high temperature intermittent stimulation of about 30 ° C was performed for one hour, and then immersed in the UV treated seawater. The shaded area and high temperature discharge were done in the air, and the luminous intensity of the shaded area was substantially 0 lx, and the luminous intensity value was substantially 0 lx even at the high temperature.

Mother spawning was performed at intervals of one hour, and UV sprays were replaced when spawning and cocking were not performed. In the case of spawning, the eggs were collected and placed in a separate container. The spermatozoa were diluted to the planned concentration and used for artificial fertilization. Table 2 shows mating households and their contents in interspecific mating of the abalone mother selected as the parent group. The interspecific crosses were divided into three parts, and the contents were as follows.

Interspecific crosses of abalone mother selected as parent group and their content Turn Time Modification time Mating household First cross breed
(2014. 10. 23-24) First period
10/23 22:00 Northern abalone × round abalone Round abalone × round abalone King Abalone × round abalone Second period 10/24 00:00 Northern abalone × Northern abalone Round abalone × Northern abalone King Abalone × Northern Abalone Third period 10/24 01:00 Northern abalone × round abalone Round abalone × round abalone King Abalone × round abalone The fourth period 10/24 02:00 Horse abalone × Northern abalone Northern Front × Abalone Abalone King Abalone × horse abalone Second interspecific crossing
(Apr. 11, 2014) First period 11/10 18:00 King Abalone × King Abalone Horse abalone × king abalone King Abalone × horse abalone Horse abalone × Northern abalone Second period 11/10 19:00 Horse abalone × Northern abalone Horse abalone × round abalone Horse abalone × horse abalone Northern abalone × king abalone Third period 11/10 21:00 Round abalone × horse abalone Horse abalone × round abalone King Abalone × round abalone Third interspecific crossing
(2014. 11. 21 ~ 22) First period 11/21 19:00 Horse abalone × king abalone Second period 11/21 21:00 Round abalone × king abalone Third period 11/21 22:00 Round x horse abalone Horse abalone × king abalone The fourth period 11/22 00:00 Round abalone × king abalone Northern abalone × horse abalone Horse abalone × horse abalone Round abalone × horse abalone

The first interspecific crosses were conducted between October 23rd and October 24th, 2014. Parents were selected from five to ten species of Northern abalone, round abalone, horse abalone, and king abalone arm, respectively, and artificial insemination was carried out four times in total. The first phase was performed at 22 o'clock on the 23rd day. Artificial fertilization of northern abalone ♂ x round abalone ♀, round abalone ♂ x round abalone ♀, and king abalone ♂ x round abalone were performed. The second period was at 00:00 on the 24th, and artificial fertilization of northern abalone ♂ × northern abalone ♀, round abalone ♂ × northern abalone ♀, northern abalone ♂ × northern abalone was performed. The third phase was performed at 01:00 on the 24th day. Artificial fertilization of Northern abalone ♂ × round abalone ♀, round abalone ♂ × round abalone ♀, and royal abalone ♂ × round abalone were performed. The fourth period was performed at 02 o'clock on the 24th day, and artificial fertilization of horse abalone ♂ × northern abalone ♀, northern abalone ♂ × horse abalone ♀, and king abalone ♂ × horse abalone were performed.

Second interspecific crossing took place on November 10, 2014. Mother - of - pearl, rounded abalone, horse abalone, and king abalone arm were selected from 5 ~ 10, respectively. The first phase was performed at 18 o'clock, and artificial insemination of 전 × × × ×, 전 전. ×, × × × ×, 전.. ×, The second phase was performed at 19 o'clock, and artificial insemination of horse abalone ♂ x northern abalone ♀, horse abalone ♂ x round abalone ♀, horse abalone ♂ x horse abalone ♀, northern abalone ♂ x king abalone were performed. The third phase was performed at 21 o'clock, and the round abalone ♂ × horse abalone ♀, horse abalone ♂ × round abalone ♀, and royal abalone ♂ × round abalone ♀ were artificially fertilized.

Third cross breeding was conducted between November 21 and 22, 2014. Mothers were divided into 5 ~ 10 male and 5 male, respectively, and 4 artificial inseminations were carried out. The first stage was performed at 19:00 on the 21st day, and artificial insemination of horse abalone ♂ × king abalone was performed. The second phase was performed at 21:00 on the 21st day, and artificial fertilization of round abalone ♂ × king abalone was performed. The third phase was performed at 22:00 on the 21st day. Artificial insemination of round abalone ♂ × horse abalone ♀, horse abalone ♂ × king abalone was performed. The fourth period was at 00:00 on the 22nd, and the round abalone ♂ × king abalone ♀, northern abalone ♂ × horse abalone ♀, horse abalone ♂ × horse abalone ♀, round abalone ♂ × horse abalone fertilization were performed.

In order to determine the fertilization rate according to the optimum sperm concentration in the artificial fertilization step of the present invention, hybridization of each breed was carried out by examining the fertility of each hybrid species according to the sperm concentration. Table 3 shows the fertilization rate in the range of 100,000-4,000,000 cells / cc for the spermatozoa of the male when each strain was hybridized.

The rate of fertilization (100,000 ~ 4 million cells / cc) female
cock Northern abalone Round abalone Horse abalone King Abalone Northern abalone 86.2% 38.6 to 44.1% 3.4% 63.9% Round abalone 79.4% 18.7 to 41.6% 0% 52.7% Horse abalone 3.4% 76% 61.6% 0% King Abalone 85.7% 17.5% to 71.2% 21.7% 77.5%

Fig. 2 shows the fertilization rate of the spermatozoa in hybrid crossing between the northern abalone male and the seagrass. The fertilization rate of round abalone eggs was 44.1% at 100,000 cell / cc of spermatozoa when the male abalones were crossed with eggs and hybrids. In the case of 1 million cell / cc spermatozoa, the rate of round abortion was 38.6%, that of northern abortion was 86.2%, and that of horse abductor was 3.4%. The fertilization rate was 63.9% at 3 million cells / cc and the fertilization rate was 0% at 4 million cell / cc. The concentration was found to be 100,000 to 3 million cells / cc.

At the concentration of 100,000-3,000,000 cells / cc, northern abalone sperm showed 38.6% ~ 63.9% of the fertilization rate except for the abalone egg. Horse abalone ♀ "Hybrid production of household is considered to be difficult.

As a result, it is appropriate that female abalone and artificial insemination should be performed properly when the Northern abalone is selected as males, and the concentration of the blood should be 3 million cells / cc.

FIG. 3 shows the fertilization rates of spermatozoa in a hybrid cross between a round abalone male and a female. In the case of crossbreeding of round - breeding male spermatozoa with the eggs of each breed, the fertilization rate with rounded abalone egg was 41.6% at 100,000 cell / cc. In the case of 1 million cell / cc spermatozoa, 18.7%, 52.7% and 79.4% of round abalone and ovary showed the highest fertilization rate, respectively. In the case of 3 million cell / cc of spermatozoa, the rate of reproductive failure was 46.4% and the rate of fertilization was 0% at 4 million cells / cc. Even at 6 million cells / cc, the fertilization rate of horse overturned egg was 0%, and the optimum sperm concentration was 100,000-300,000 cells / cc when artificially fertilized with round abalone.

In the case of round abalone, the fertilization rate of northern abalone and king abalone was 50% or more at 1 million cell / cc, and successful hybridization was possible. Horse abalone ♀ "Hybrid production of household is considered to be difficult.

As a result, when the round abalone was selected as males, it was appropriate to overturn the female northern North Jeongbuk or the female king and artificial fertilization. When the female North Jeonbuk was selected in the artificial fertilization stage, the concentration was 1 million cells / cc, When the abalone was selected, it would be appropriate to have 100 ~ 3 million cells / cc.

FIG. 4 shows the fertilization rate of the spermatozoa in hybrid crossing between male and female endometrium. In the crossbreeding between male spermatozoa of each species and the eggs of each varieties, fertilization rate of 1.0% cell / cc was 3.4% for northern abalone and 0% for king abalone. In the case of 3 million cell / cc spermatozoa, the rate of round abalone was 76%, that of horse abalone was 61.6%, and that of king abalone was 0%. In case of sperm concentration 4 million cell / cc, All of the king rollovers showed 0% fertilization.

It was concluded that the production of hybridization of "horse abalone ♂ × 王 전" "family is difficult due to the lack of fertilization with the king abalone egg.

As a result, it is appropriate that round overturning and artificial insemination should be carried out when the male abalone is selected as males, and that the concentration of the cells should be 3 million cells / cc.

FIG. 5 shows the fertilization rates of spermatozoa in hybrid crosses between male and female. In the case of crossbreeding of the male abalone spermatozoa with the eggs of each breed, 85.7% of the northern abalone, 77.5% of the abalone, 17.5% of the abalone and 17.5% And 17.1%, respectively. At 3 million cell / cc concentration, it showed 71.2% rounded overturning and 21.7% at 4,000,000 cell / cc. It showed relatively good fertilization rate at high concentration of spermatozoa. The male spermatozoa showed higher fertilization rate than the other spermatozoa in hybrid crossing, and showed a northern abalone egg and especially high fertilization rate.

As a result, when selecting the male abalone as a male, it is appropriate that the female northern North or female round abalone and artificial insemination should be performed, and when the female northern North Korea is selected in the artificial insemination stage, the concentration of the fish is 1 million cells / When round abalone was selected, it would be appropriate to have 3 million cells / cc.

In order to investigate the fertilization rate of fertilized eggs according to the salinity concentration in the artificial fertilization step of the present invention, hybrid fertilization between the round abalone female and the round abalone male and the hybrid cross between the round abalone female and the royal abalone male were carried out, Normal incidence was examined.

1. Investigation of the rate of fertilization according to salinity

The salinity conditions used in the experiments were 33 psu, 31 psu, 29 psu, 27 psu and 25 psu. In case of 33 psu, the filtered natural seawater was used as it was and the other salt conditions were prepared by mixing filtered seawater and distilled water .

After receiving the eggs of round abalone collected in 100 ml beakers filled with each seawater of different salinity, the round abalone and the king abalone sperm were added at a concentration of 500,000 cells / ml, respectively, to obtain a round abalone ♀ × round abalone ♂ , And ♀ × round abalone (♂ × 王) were artificially fertilized (water temperature 20 ℃).

After 90 seconds of fertilization, sera were performed and three sera were performed every 3 minutes. Three replicates for each salinity were set up. After fertilization for 2 hours after fertilization, 67 ~ 156 eggs were randomly sampled from each experiment and the fertilization rate was measured.

Fig. 6 shows the fertilization rates of round abalone ♀ x round abalone (obedient household) and round abalone ♀ x king abalone (mixed household), Fig. 7 shows round abalone ♀ x round abalone ♂ ) And round abalone ♀ × Wang abalone ♂ (hybrid household). A-1 and 2 shown in Fig. 7 are enlarged photographs of the untreated male × x rounded abalone with x 40 times and x 100 times, respectively. B-1 and 2 are photographs showing that the male abalone ♀ x 王 복 Respectively, at a magnification of × 40 and × 100, respectively. Scale bar = A, B-1: 200 m; A and B-2 = 100 mu m.

Round abalone ♀ x round abalone ♂ The fertilization rate was as high as 98.4% and 92.5% for 33 psu and 31 psu, respectively, and slightly lower than 80.7% and 83.6% for 29 psu and 27 psu, respectively. And 52.8% for 25 psu, respectively. The round abalone ♀ × king abalone ♂ in the cases of 33 psu and 31 psu in the fertilization rate was as high as 96.2% and 90.8%, respectively, and in the case of 29 psu and 27 psu, 86.7% and 80.4% respectively. 25 psu was the lowest at 64.3%.

2. Normal incidence by salinity

The salinity conditions were 33 psu, 31 psu, 29 psu, 27 psu and 25 psu, respectively. In the case of 33 psu, the filtered natural seawater was used as it was. The other salinity conditions were filtration seawater And distilled water.

In the natural seawater of 33 psu, the round abalone ♀ × round abalone ♂ and the round abalone ♀ × walleye abalone were artificially fertilized at 90,000 sec / Conferences were held.

After 2 hours of fertilization, it was observed under a microscope. Normal fertilized eggs were selected and stored in a 100 ml beaker filled with 20 ° C seawater for each salinity. When the larval stage occurred, normal developmental and teratogenic developmental groups were distinguished Respectively. Each experimental group was equipped with three repetitions.

Fig. 8 is a graph showing the normal incidence rate of round abalone ♀ x round abalone ♂ (obedience household) and round abalone ♀ x king abalone ♂ (hybrid household), and Fig. 9 is a graph showing the incidence of round abalone ♀ x round abalone Family household) and round abalone ♀ × Wang abortion ♂ (hybrid household).

A-1 shown in Fig. 9 is a round abalone ♀ x round abalone ♂ normal (35 psu), A-2 is a round abalone ♀ x round abalone ♂ anomaly (27 psu), A-3 is a round abalone ♀ x round abalone (25 psu), B-1 represents round abalone ♀ x Wang abortus ♂ normal (35 psu), B-2 round abalone ♀ x Wang abortion ♂ deformed (27 psu), B- ♀ × Worl-overturned body (25 psu). Also, the scale bar shown in the drawing is 100 탆.

In the graph, the normal incidence of round abalone ♀ × round abalone ♂ was 99.5% ~ 91.2% at 33 psu, 31 psu and 29 psu, but it was very low at 29 psu and 0.6% In the case of psu, no normal occurrence was observed.

The normal incidence of round abalone × male abalone males was higher than that of 33 psu, 31 psu, and 29 psu, which was 99.3% ~ 93.6%, and there was no significant difference, and the normal incidence was as low as 19.4% at 27 psu. At 25 psu, no normal individuals were observed.

As a result of examining the fertilization rate and normal incidence according to the salt concentration, it is appropriate to correct fertilized eggs that have hybridized with the male abalone and the female abalone at 29 ~ 33 psu salinity.

In the incubation step of the present invention, the fertilized eggs that have undergone the artificial fertilization step were housed in the individual containers before being housed in the larvae, and only the hatched individuals were housed in the aquaculture tank at the completion of hatching. The water quality control was carried out by small amount of runoff before the seedlings, and the dead larvae which had settled on the bottom were removed by siphoning. The larvae developed by the pickling tiller were collected using a 100 ㎛ mesh muller bag. According to the amount of larvae, 200,000 or more were collected in a concrete tank of 1m × 7m × 0.6m and 0.8m × 6m × It was housed in a 0.3 m FRP tank.

Ⅱ. Abalone production results

The interspecific hybridization was carried out from 1 to 3 times, and the larvae hatched were sown according to each tea. Table 4 shows the amount of larvae hatched by interspecific crosses from 1 to 3, respectively.

Hybrid household and sowing time female
cock Northern abalone Round abalone Horse abalone King Abalone Northern abalone ■ Primary seedling: 280,000 ■ Primary seedling: 280,000 ■ Primary seedling: 5,000 ■ Second cultivation: 190,000 Round abalone ■ Primary seedling: 280,000 ■ Primary seedling: 290,000 - ■ Third seedling: 200,000 Horse abalone ■ Primary cultivation: 40,000 grains
■ Second grazing: 100,000 grains ■ Second cultivation: 240,000 ■ Second cultivation: 110,000 grains - King Abalone ■ Primary seedling: 280,000 ■ Primary cultivation: 250,000 grains
■ Second seedling: 290,000 ■ Primary seedling: 1,500 grains
■ Second cultivation: 260,000 ■ Second batch: 270,000

The first interspecific crosses were conducted between October 23rd and October 24th, 2014. Five to ten female abalone, round abalone, horse abalone, and king abalone arm and water were prepared, respectively, and spawning and irregularity were induced, followed by artificial insemination. In females, induction was possible in northern abalone, round abalone, and female abalone except for king abalone. In males, all four abalone were inducible.

First interspecific mating result Northern abalone ♂ × Northern abalone ♀, Northern abalone ♂ × Abal abdomen ♀, Northern abalone ♂ × Horse abalone ♀, Round abalone ♂ × Round abalone ♀, Round abalone ♂ × Northern abalone ♀, Abalone ♀, king abalone ♂ × round abalone ♀, king abalone ♂ × horse abalone ♀, horse abalone ♂ × northern abalone ♀.

Second cross breeding took place on November 10, 2014. 5 ~ 10 male and female abalone, round abalone, horse abalone, and king abalone arm and water were prepared, respectively. After spawning stimulation process, induction of blast and occlusion was induced and artificial insemination was carried out. In all four kinds of males and females, Hybrid cultivation was carried out mainly on hybrid households where the amount of seedlings was insufficient in the first seedling.

× × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × × Northern abalone ♀, northern abalone ♂ × king abalone ♀.

The seedlings were carried out on November 13th, and the seedlings were king 전 × 전 전 전 ♀::::::::: ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♀ ♂ × horse abalone ♀: 110,000, abalone ♂ × round abalone ♀: 240,000, horse abalone ♂ × northern abalone ♀: 100,000, northern abalone ♂ × king abalone ♀: 190,000.

The third interspecific cross breeding was conducted between November 21 and 22, Females were divided into two groups: horse abalone, king abalone, and male abalone, round abalone, and horse abalone, respectively.

Two kinds of females and three males were breeding and breeding, and hybrid breeding was carried out mainly on hybrid households where the amount of seedlings was insufficient during the first and second sowing.

As a result of hybridization between the three species, hybrid cross breeding attempts were made on horse abalone ♂ × horse abalone ♀, horse abalone ♂ × king abalone ♀, round abalone ♂ × king abalone ♀, round abalone ♂ × horse abalone ♀, northern abalone ♂ × horse abalone ♀ Total 5 households were carried out, but no correction was made in the rest of the household except for the round abalone ♂ × Wang Abalone ♀ hybrid household.

The harvesting was carried out on November 24th, and the seedlings were round abalone ♂ × king abalone ♀: 200,000. Hybrid crosses of "horse abalone ♂ × king abalone ♀", "round abalone ♂ × horse abalone ♀" and "northern abalone ♂ × horse abalone ♀" showed very low fertilization rates even in the first and second hybrid crosses, It is necessary to apply various conditions for

In order to investigate the growth of the spore produced by the first to third interspecific crosses of the present invention, it was carried out once a month at intervals of one month. The first day of the month was 26 days, the second month was 13 days, the third month was 24 days, and 10 ~ 15 eggs were randomly collected from each household. Nikon, V-12B).

Fig. 10 is a graph showing the result of the first measurement of the first sowing household. The results of the first one-time growth survey after 30 days after the first harvesting of the household were taken as follows: Northern abalone ♂ × round abalone ♀: 1340.3 ± 200.5㎛, Wang abalone ♂ × Northern abalone ♀: 1362.7 ± 267.3㎛, Northern abalone ♂ × Northern abalone ♀: 1339.8 ± 449.0 ㎛, King Abalone ♂ × round abalone ♀: 1172.5 ± 149.7 ㎛, round abalone ♂ × northern abalone ♀: 1040.4 ± 189.3 ㎛, round abalone ♂ × round abalone ♀: 1122.1 ± 180.9 ㎛.

The size of the spatula is "King Abalone ♂ × Northern abalone ♀> Northern abalone ♂ × round abalone ♀ Northern abalone ♂ × Northern abalone ♀> King abalone ♂ × round abalone ♀ Round abalone ♀ × round abalone ♀> Round abalone ♂ × Northern abalone ♀ "There was a difference in growth in the order. Horse abalone ♂ × Northern abalone ♀, Northern abalone ♂ × Horse abalone ♀, King abalone ♂ × Horse abalone ♀ In the case of the abalone,

Fig. 11 is a graph showing the results of the second-order measurement of the primary picking household. After 60 days, 60 days after the seedling growth, the Northern abalone ♂ × round abalone ♀: 3663.3 ± 461.5㎛, King Abalone ♂ × Northern abalone ♀: 3245.0 ± 410.9㎛, Northern abalone ♂ × Northern abalone ♀: 2696.3 ± 229.9㎛, King Abalone ♂ × Round Abalone ♀: 2433.7 ± 589.8㎛, Round abalone ♀ × Northern abalone ♀: 2064.8 ± 301.1㎛, Horse abalone ♂ × Northern abalone ♀: 1699.3 ± 265.1㎛, Northern abalone ♂ × Horse abalone ♀: 1687.5 ± 341.6 Respectively.

The size of the spatula is "Northern abalone ♂ × round abalone ♀> Northern abalone ♂ × Northern abalone ♀ Northern abalone ♂ × Northern abalone ♀> Northern abalone ♂ × round abalone ♀> Northern abalone ♀ × Northern abalone ♀> Northern abalone ♂ × Northern abalone ♀> northern abalone ♂ × horse abalone ♀ "There was a difference in growth in order.

The round abalone, which was able to measure once, could not be measured because it was difficult to find the sparrow in the case of round abalone. ♀ Abal abortion ♀ × Northern abalone ♀ × Northern abalone ♂ × Horse abalone ♀ Measurements were made possible by the discovery of spear. In the case of 전 전 × × × 말 ♀ ♀ ♀ ♀ ♀................

Fig. 12 is a graph showing the results of the second-order measurement of the primary picking household. After 90 days, 90 days, after 3 days of growth, the northern abalone ♂ × round abalone ♀: 9235.0 ± 1456.9㎛, King Abalone ♂ × Northern abalone ♀: 7107.9 ± 941.3㎛, Northern abalone ♂ × Northern abalone ♀: 5480.3 ± 635.0㎛, King abalone ♂ × round abalone ♀: 3953.2 ± 874.7㎛, horse abalone ♂ × northern abalone ♀: 5073.1 ± 874.7㎛, round abalone ♂ × northern abalone ♀: measured as 5924.0 ± 1138.9㎛.

The size of the spatula is "Northern abalone ♂ x round abalone ♀> Northern abalone ♂ × Northern abalone ♀> Northern abalone ♀ × Northern abalone ♀> Northern abalone ♂ × Northern abalone ♀> Northern abalone ♂ × Northern abalone ♀> ♀ "There was a difference in growth in the order. Round abalone ♂ × round abalone ♀, 王 전 × × × × × 여전히 여전히 여전히 여전히 여전히 여전히 여전히 여전히 여전히...........................

FIG. 13 is a graph showing a growth change over 90 days for each household in the primary picking household, and FIG. 14 is a photograph showing a state of a spatula. 14, A-1, 2 and 3 represent the 30th, 60th, and 90th days after the seedling of the Northern abalone ♂ × round abalone ♀, and B-1, 2 and 3 represent the abalone ♂ × Northern abalone ♀, 60 days and 90 days. In addition, C-1, 2 and 3 are round abalone ♂ × Northern abalone ♀ 30 days after sowing. 60 days. D-1, 2, and 3 of 90 days represent the northern abalone ♂ × northern abalone ♀ represent 30 days, 60 days, and 90 days after the embryo, E-1, 2 and 3 represent the 王 전 × Days, 60 days, and 90 days, and F-1 and 2 represent 60 days and 90 days after horse embryo ♂ × Northern abalone ♀ embryo. The scale bar shown in Fig. 14 indicates 1,000 mu m.

15 is a graph showing the results of the first measurement of the secondary seedling household. After 30 days, after 30 days of growth, the results of the 1st growth test showed that the horse abalone ♀ × Northern abalone ♀: 1203.8 ± 140.5 ㎛, King abalone ♂ × Horse abalone ♀: 1123.7 ± 147.4 ㎛, King abalone ♂ × Round abalone ♀: 1018.5 ± 111.2 ㎛, Northern abalone ♂ × Royal abalone ♀: 1017.3 ± 134.8㎛, Royal abalone ♂ × Royal abalone ♀: 1013.4 ± 94.4㎛, Horse abalone ♂ × Round abalone ♀: 969.3 ± 60.8㎛.

The size of the spatula is "Abalone ♂ × Northern abalone ♀> Abalone ♂ × Horse Abalone ♀> Abalone Abalone ♂ × Abalone Abalone ♀ Abalone Abalone ♂ × Abalone Abalone ♀ × Abalone Abalone ♂ × Abalone Abalone Female ♀ Abalone ♂ × Round Abalone ♀ "There was a difference in growth in the order. In the case of horse abalone ♂ × horse abalone ♀, it was impossible to measure because it was difficult to find spider in the water tank.

16 is a graph showing the result of the second measurement of the second picking household. After 60 days, 60 days after the seedling growth, the results of the growth survey showed that horse abalone ♂ × northern abalone ♀: 2987.3 ± 436.3 ㎛, king abalone ♂ × horse abalone ♀: 2803.4 ± 320.5 ㎛, king abalone ♂ × round abalone ♀: 2836.7 ± 384.9 ㎛, Northern abalone ♂ × Royal abalone ♀: 2496.6 ± 512.2㎛, King abalone ♂ × Royal abalone ♀: 2166.3 ± 265.0㎛, Horse abalone ♂ × Round abalone ♀: 2482.8 ± 435.4, ㎛, Horse abalone ♂ × Horse abalone ♀: 2380.4 ± Lt; / RTI >

The size of the spatula is "Abalone ♂ × Northern Abalone ♀> Abalone Abalone ♂ × Abalone Abalone ♀ × Abalone Abalone ♂ × Abalone Abalone ♀ Abalone Abalone ♂ × Abalone Female Abalone Female Abalone ♂ × Abalone Abalone Female ♀ Abalone Horse Abalone Female Abalone × ♀> King Overture ♂ × King Overture ♀ "There was a difference in growth in order.

FIG. 17 is a graph showing the growth change over 90 days for each household in the secondary picking household, and FIG. 18 is a photograph showing the state of the spatula. 18, A represents abalone ♂ × 60 days after the overthrow of the northern abalone, B represents 60 days after 王 × abalone ♂ × round abalone 채 embroidery, C represents 60 days after 王 abalone ♂ x horse abalone ♀ embroidery, Northern abalone ♂ × king abalone ♀ 60 days after embroidery, E is abalone ♂ × round abalone ♀ 60 days after embroidery, F is horse abalone ♂ × horse abalone ♀ 60 days after embryo G is king abalone ♂ × king abalone ♀ embroidery Respectively. Also, the scale bar indicates 1,000 mu m.

Fig. 19 is a photograph showing the results of the second measurement of the third order picking household and the external shape of the spatula. Fig. In the case of the 3rd seedling family, it was a family of round abalone ♂ × king abalone. It was difficult to detect the seedlings in the tank when 30 days after harvesting. As a result of the growth survey, round abalone ♂ × king abalone ♀: 1069.1 ± 183.5 ㎛. The scale bar shown in Fig. 19 (B) indicates 1,000 mu m.

By providing a hybrid production method that can hybridize four kinds of abalone, such as northern abalone, round abalone, horse abalone, and king abalone, the artificial fertilization rate is high and the normal incidence of artificial embryo is high, It is possible to increase productivity of abalone aquaculture industry as well as to contribute to the increase of fishermen's income by improving the international competitiveness of aquaculture technology as well as development of fisheries field and expansion of aquaculture industry. There is a possibility of award.

Claims (4)

Selection of abalone to choose one of male northern abalone and female king abalone, male round abalone and female northern abalone, male horse abalone and female round abalone, male king abalone and female northern abalone hybridization;
Placing the selected abalone capsules in a container, stimulating the sound discharge for 30 minutes, inducing high-temperature intermittent stimulation for 1 hour at 30 ° C, and then immersing in seawater to induce scattering and uniformity;
Collecting the scattered and fixed eggs, receiving the eggs in a separate container, and diluting the spermatozoa to a predetermined concentration;
Artificial fertilization of the collected egg and the semen collected at a spermicide concentration of 1 million to 3 million cells / cc;
And hatching the embryo in seawater having a salinity ranging from 29 psu to 33 psu. delete delete delete

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