KR102319352B1 - Nonhormonal Method for Masculinization of Chum Salmon - Google Patents

Abstract

The present invention relates to a method for inducing masculinization of a chum salmon capable of inducing masculinization through water temperature control and differentiation of food sources after hatching without using sex hormones. The method according to the present invention increases a male proportion to 85% or more by managing fish culture at high temperature of 15℃ or higher at a time when having an egg yolk after hatching the chum salmon; increases the male proportion to 95% or more by supplying seonji and lecithin at high water temperature after eating raw food in an early stage of hatching; artificially increases the male proportion by suppressing conversion of male sex hormones of the chum salmon to magnetic hormones through control of water temperature and feed supply instead of using the sex hormones; massively produces PDRN, which is a genetic fragment for industrialization, at a low cost by securing many semen and testes of males; and is used in a field of food and medicine without restrictions.

Description

Nonhormonal Method for Masculinization of Chum Salmon

The present invention relates to a method for inducing maleization of juvenile salmon, without using sex hormones, and to a method of inducing maleization through regulation of water temperature after hatching and differentiation of food sources.

Gender control in fish has been studied for several decades, and the method is mainly applied by controlling the amount of estradiol and testosterone. It is undesirable in terms of food safety to artificially control sex using

Most of the sex control of rainbow trout uses a method by controlling chromosomes, and UV treatment of male semen to abolish X or Y chromosome activity in sperm to prevent the division of X chromosome in eggs by high pressure or high pressure. High-temperature treatment to make females with XX chromosomes, or inactivate the X chromosomes of eggs and control the division of X or Y chromosomes of sperm to create individuals with XX or YY chromosomes, and super males with YY chromosomes (However, the method of making 100% males is used as a method of fertilization to males that produce males with XY chromosomes no matter how they are bred.

Salmon ( Oncorhynchus keta ) is rarely farmed for food, but in the case of rainbow trout ( Oncorhynchus mykiss ), males have a high mortality rate depending on the maturity of the spawning season. They are farmed in populations intended for human consumption.

The use of sex hormones in the manufacture of food or medicine is permitted only for patients, and the use of non-prescribed sex hormone preparations is strictly regulated. Accordingly, in the aquaculture industry, human chorionic gonadotropin ( hCG) was developed and used to promote spawning, but other hormone preparations such as follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone and estrogen are used in the food sector. not allowed in

However, if you naturally induce quantitative changes in sex hormones in the body, such as by changing the breeding environment of living organisms to control sex, you can escape from the limiting factors of artificial sex hormone use, for example, genetic sex (at the time of fertilization) there trying to control (environmental resistance) due to environmental factors such as temperature regulation before development, and the goldfish or tilapia although progress has specific studies, salmonids, in particular, presents a limit temperature in a fraction if the salmon (Oncorhynchus keta) However, it does not reach the level of technology that can be commercialized.

Korean Patent Publication No. 1805034 (Manufacturing method of fertilized egg of triploid rainbow trout) Korean Patent Publication No. 1892109 (Injectable composition containing letrozole or anastrozole)

Ohnam Kwon, Shinji Adachi, 2009. Effect of oral administration of 17α-methyltestosterone and estradiol-17β for sex reassignment of bester sturgeon fry. Korean Society of Fisheries Science 42, 585-590. Baker IJ, Solar II and Donaldson EM, 1988. Masculinization of chinook salmon (Oncorhynchus tshawytscha) by immersion treatments using 17α-methyltestosterone around the time of hatching. Aquaculture 72, 359-367. Dongsoo Kim, Changhwa Jeong, Inbae Kim, Induction of female triploid rainbow trout. Korean Genetic Society 15, 213-218. Baroiller JF, D’Cotta H and Wessels S, 2009. Tilapia sex determination: Where tmeperature and genetics meet. CBP A 15, 30-38. Long BJ, Jelovac D, Handratta V, Thiantanawat A, MacPherson, Ragaz J, Golouubeva OG and Brodie AM, 2004. Therapeutic strategies using the aromatase inhibitor letrozole and tamoxifen in an breat cancer model. J Natl Cance Inst 96, 456-465.

The present invention is to solve the above problem, and to provide a method for naturally inducing maleization of the salmon by changing the breeding environment without using sex hormones.

In order to solve the above problem, the present invention is to fertilize and hatch at a water temperature of 12 ℃ or less using mature eggs and semen of cumulus salmon, and then culture the hatched larvae at a water temperature of 15 ℃ or more from 8 to 12 days after hatching, After 18~22 days of hatching, culture at 10~15 ℃ of water temperature, supply live feed for 25~35 days after 13~17 days of hatching, stop supplying live feed after supplying live feed, and seonji, lecithin for 3~4 months Alternatively, it provides a non-hormonal method for increasing the maleing ratio of sardines, comprising the process of supplying a mixture thereof together with feed in an amount of 1 to 5% by weight of the fish body weight every day.

In this case, the water temperature for fertilization and incubation is preferably 10 to 12 ℃, and the water temperature after 8 to 12 days of incubation is preferably 15 to 18 ℃.

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The method according to the present invention raises the proportion of males to 85% or more by managing the aquaculture at high temperature of 15 ℃ or higher during the period when the yolk is added after hatching of the juvenile salmon. The proportion of males can be increased to more than 95%.

In addition, instead of using sex hormones, it is possible to artificially increase the ratio of males by inhibiting the conversion of male hormones into female hormones through control of water temperature and feed supply, so as to secure as much of the male semen and testes as possible. It is possible to mass-produce PDRN, a gene fragment for industrialization, at low cost, and can be used without restrictions in the food and pharmaceutical fields.

1 and 2 are graphs showing the fertilization rate and average fertilization time for each water temperature of fertilized salmonid eggs.
3 to 5 are graphs showing the hatching rate, first hatching date, and hatching period by water temperature of fertilized salmonid eggs.
6 is a photograph showing a microscopic observation of the male and female gonads confirmed by the gonad compression method.
7 is a graph showing the maleization ratio for each water temperature of juvenile salmon.
8 and 9 are graphs showing the maleing ratio by period and period in which fertilized/hatched larvae were exposed to high temperature (15.6 °C) at 12 °C.
10 to 12 are graphs showing the maleing ratio (12 ℃ / 15.6 ℃) and the survival rate according to the difference in the additional food source supplied to the juvenile salmon after yolk absorption.

Males of salmonid fish are recognized as biomaterials for producing polydeoxyribonucleotide (PDRN) and polynucleotide (PN), which are recently spotlighted biomaterials.

PDRN exists in tissue regeneration (anagenesis) active substances in living organisms, and as a cell growth activator, it is effective in the regeneration and inflammation relief of human tissues such as ligaments, tendons, and skin. Due to difficulties in productivity, etc., fish PDRN was suggested as an alternative.

Among fish, semen and testis of salmon and trout have a high nucleic acid content and their DNA combination is 96% or more identical to that of the human body, so their utilization is very high. The global market is very large.

However, since the value as a culture other than those produced in nature decreases, artificial culture is not carried out, and the male of rainbow trout matures to sexual maturity 2 years after hatching and begins to produce sperm. Males in the spawning season have fewer feeding activities and use a lot of energy for sexual maturity, so they die a lot as their body gets thinner and thinner.

Most of the salmon produced in Korea are triploid or sterilized to prevent disruption of domestic business and resource management and genetic resources in other countries (importing countries) in the exporting country. forming the price level.

The present invention proposes a method for efficiently producing males of juvenile salmon to secure high-quality medical material, PDRN raw material, at low cost, and induces maleization through water temperature treatment after hatching and differentiation of food sources.

To this end, by reducing the amount of female hormones in fish by inhibiting aromatase, which converts male hormones to female hormones after hatching, by maintaining the culture water temperature above 15 °C after hatching. Naturally increase the proportion of males.

In the case of salmon ( Oncorhynchus keta ), aquaculture management at 10 ° C after fertilization until hatching and hatching was a common method to secure survival, but 15 ° C to inhibit the aromatase activity of hatched larvae with yolk for maleing. Management at a higher water temperature can effectively increase the male induction rate.

In addition, in order to increase the proportion of males in the male-dominated group secured by water temperature control, kettle blood plasma and lecithin are additionally supplied as food for 3 to 4 months after the supply of live feed to produce aromatase. By supplying letrozole, which acts as an aromatase inhibitor, the amount of sex hormones is reduced and the proportion of males is naturally increased.

Glands and lecithin contain letrzole, which acts as an aromatase inhibitor, and yolk sac lecithin can be used as lecithin. By inhibiting the activity of aromatase, which is an aromatase involved in

Specifically, in order to secure the survival rate in the hatching management process of the freshwater salmon, it is preferable to manage it at a water temperature of 12 ° C or less, preferably 10 to 12 ° C, and 15 ° C or more after hatching, preferably to increase the maleing ratio Aquaculture management at a water temperature of 15 to 18 ℃ can increase the male ratio to more than 85%, and in addition, additionally supply glandular fat and lecithin at a ratio of 1 to 5% by weight of fish body weight for 3-4 months separately from food 95 More than % male populations can be obtained.

More specifically, after collecting the mature eggs and semen of the salmon, fertilization and incubation at a water temperature of 12 ° C or less, preferably 10 to 12 ° C. From then on, when exposed to a high temperature of 15 ° C. or more, preferably 15 to 18 ° C., the male ratio can be effectively increased.

In addition, even when exposed to high temperature only from the 8th to 12th days after hatching to the 18th to 22nd days after hatching, the proportion of males increases due to environmental influences. As the ratio can be increased, if the water temperature is high, keep the water temperature above 15℃ from the 8th to 12th day after hatching until the freshwater salmon becomes an adult fish. It is desirable to lower the water temperature from the 18th to 22nd days after maintaining 15 °C or higher, and since the water temperature above 20 °C adversely affects the survival rate, breed at around 13 °C according to the characteristics of the fish farm water, preferably at 10-15 °C. By maintaining the water temperature, it is possible to reduce the energy cost required to maintain the water temperature.

In addition, from the 13th to the 17th day after hatching, that is, after the larvae have absorbed more than 80% of the egg yolk, supply raw food suitable for the size of the mouth, and stop the supply of live food from the 25th to 35th days after the hatchling 3 If feed for fry for ~4 months and supplementary supply of glandular fat and lecithin in an amount of 1~5% by weight of fish body weight every day, the maleing ratio increased by high temperature treatment can be further increased.

In addition, it is effective to increase the maleization ratio by supplying a mixture of the gland and lecithin rather than separately supplying the gland and lecithin.

In addition, even when the water temperature is maintained at 15 ℃ or higher and the sardines and lecithin are additionally supplied, the survival rate of juvenile salmon is 85% or more. The method did not adversely affect the spawning mortality rate of juvenile males.

Hereinafter, the present invention will be described in more detail based on the following examples.

However, the following examples are only for illustrating the present invention, and the present invention is not limited by the following examples, and can be replaced with other examples that are substituted and equivalent within the scope without departing from the technical spirit of the present invention. It will be apparent to those of ordinary skill in the art to which the present invention pertains.

<Example 1> Incubation in the eyes of each water temperature

Eggs and semen were collected from females and males of wild-caught freshwater salmon, and fertilized eggs were generated by accommodating them at water temperatures of 10, 12, 15 and 18 °C. was significantly lower at 10% (FIG. 1), and the period to induction was 25 days at 10 °C and the shortest 15 days at 18 °C (FIG. 2).

The hatching rate by water temperature continuously exposed from the fertilized egg of juvenile salmon was significantly highest at 65% at 10 °C, and the lowest at 18 °C (3%) (Fig. 3), and hatched larvae at 10 °C. was observed at 52 days after fertilization, and at 18 °C, the shortest significantly observed at 29 days (Fig. 4).

Incubation period was incubated for 26 days from the 52nd day to the 78th day at 10 °C, and incubated for a total of 8 days from the 29th day to the 37th day at 18 °C (FIG. 5).

<Example 2> Maleization ratio of carnivores exposed to high temperature

For the food of hatched brood fish, the initial frozen copepod and feed for fry (trout fry feed No. 1 and 2, dominance feed, Korea) were supplied. Males and females were distinguished by checking the eggs and sperm of

The male ratios of brood fish cultured at 10 °C and 12 °C were significantly low at 48% and 49%, respectively, and significantly higher maleization of 83% and 85%, respectively, in broodstock cultured at 15 °C and 18 °C. ratio (Fig. 7).

The change in the maleing ratio was observed by exposing the broccoli fertilized and hatched at a water temperature of 12 ℃ to a high temperature of 15.6 ℃. When looking at the time of exposure to high temperature, maleization was observed immediately after hatching and when exposed to high temperature from the 5th day. The ratio did not show a significant difference at 49~51%, but when exposed from the 10th day, it showed a high maleization rate of 88%, and when exposed from the 15th day, it was 72%, which is lower than the result from the 10th day, but 0~ It showed a higher maleization rate than the case of exposure after 5 days and 20 days (FIG. 8).

In addition, the maleing ratio for each period of exposure to high temperature was confirmed in the case of fertilization and hatching management at 12 ℃ water temperature, exposed to high temperature of 15.6 ℃ for 5 days, and then cultured while feeding at 13 ℃ water temperature.

When exposed to high temperature during 0-5 days after hatching and when exposed to high temperature on 5-10 days after hatching, 49-51%, showing a significantly lower maleing rate together with exposure on 20-25 days On the other hand, exposure to high temperature on the 10th to 15th days showed the highest maleization rate of 89%, and exposure to high temperature on the 15th to 20th days showed a high maleization rate of 75% (FIG. 9).

<Example 3> Maleization ratio according to the supply of additional food sources of larvae

The whole process of spawning, hatching, and growth is divided into the case of cultured at 12 ℃ water temperature and the case of spawning and hatching at 12 ℃ water temperature and then grown at 15.6 ℃ water temperature, supply additional food sources and observe the maleing ratio. did.

Frozen copepot (live food) is supplied to the freshwater salmon right before injury, and live feed is stopped from the 25th to 35th day of the live feed. , Korea) or testosterone smear-treated fry (T 10 ppm) was supplied, and fresh wire and egg yolk lecithin were additionally supplied when feeding fry.

All experimental fry were housed in a 1×1 m net by 100, and frozen copepods were divided into 6% of the weight of the fish at 09:00 and 16:00, and 3% each was supplied twice daily, and feed (fry for fry) Or testosterone 10 ppm smear feed) was also supplied twice at 9:00 and 16:00, each 1.5% of the body weight.

It was eaten in an amount of 2% of the weight of the fish body once a day along with the feed for the fry, and the fresh scallops were put into the feeding box, and the egg yolk lecithin was sprinkled on water once a day at the same weight as the fresh spears for consumption. 2% of the compound feed port in which egg yolk lecithin was mixed with 1% of each fish body weight was supplied once a day.

At 4 months after yolk absorption, the gonads of the fry were removed and males and females were screened, and when cultured at 12 °C, the male ratio was 48% in the control group (frozen copepods and feed without additional food sources), and testosterone. When 10 ppm feed was supplied, a high maleization rate of 92% was shown, and when pigeon fat and lecithin were supplied together with fry feed, respectively, the maleization ratio was 78~82%, and fresh fish fat and lecithin were supplied together. In one case, it showed a high maleization rate of 95% (FIG. 10).

And when cultured at 15.6 ℃ immediately after hatching, it was 82.5% in the control group, and high maleing ratio of 95.5% in the testosterone 10 ppm feed group, whereas when the glandular fat and lecithin were additionally supplied, it increased to 88.3%. was present, but there was no significant difference, and in the supply group mixed with glandular gland and lecithin, it showed a significantly high maleization ratio (96.3%) (FIG. 11).

In addition, when looking at the survival rate when spawned and hatched at a water temperature of 12 ℃ and grown at a water temperature of 15.6 ℃, the control group, fresh wire, lecithin and mixed feed showed a high survival rate of 86~88%, but testosterone 10 ppm feed It is lowered to 79% in the case of supplying , it can be seen that the supply of testosterone has a negative effect on the survival rate of the fry (Fig. 12).

Claims (7)

After fertilization and incubation at a water temperature of 12 ℃ or less using matured eggs and semen, hatched larvae are cultured at a water temperature of 15 ℃ or higher from 8 to 12 days after hatching, and from 18 to 22 days after hatching, a water temperature of 10 to 15 cultured at ℃,
From 13 to 17 days after hatching, feed live feed for 25 to 35 days, stop feeding live feed after supplying live feed, and for 3 to 4 months, take saliva, lecithin, or a mixture thereof in an amount of 1 to 5% by weight of the fish body weight every day. A non-hormonal method of increasing the maleing ratio of sardines, comprising the process of feeding them together with feed. The method according to claim 1,
The fertilization and hatching water temperature is a non-hormonal method of increasing the maleization rate of salmon, characterized in that 10 ~ 12 ℃. The method according to claim 1,
The non-hormonal method of increasing the maleization rate of chum salmon, characterized in that the water temperature after 8 to 12 days of hatching is 15 to 18 ℃. delete delete delete delete

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