Method for inducing triploid of Malus salicina by hydrostatic pressure method

Abstract

The invention discloses a method for inducing triploids of Malus salicina by using a hydrostatic pressure method. Relates to the technical field of aquatic genetic breeding. Comprising the following steps: preparing parent fish; collecting eggs for fertilization, starting timing after the eggs and the eggs are mixed, and controlling the water temperature to be 6-8 DEG C; pressurizing treatment: pressurizing is started after 15-20 min after fertilization, the pressure is selected to be 60-65 kg / cm < 2 >, the continuous pressurizing time is 5-6 min, and the fertilized eggs subjected to pressurizing treatment are obtained; hatching fertilized eggs; juvenile fish breeding. The growth speed of the obtained triploid is obviously improved compared with that of a diploid, the fish quality is improved, the defects of domestic salmon and trout breeding varieties are overcome, and the method has important significance in promoting industrial development of domestic salmon and trout and has wide market application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic genetic breeding, and more particularly to a method for inducing triploidy of Masson salmon by utilizing a hydrostatic pressure method. Background Art

[0002] Masu salmon (Oncorhynchus masou), belonging to the Salmonidae family, is anadromous fish distributed only in the waters of the Asian side of the North Pacific Ocean. It lives in the ocean for about 4 years and then migrates to the rivers where it was born to lay eggs and reproduce.

[0003] Salmonids have a high mortality rate after sexual maturity and reproduction, especially migratory salmon species, where the broodstock dies immediately after reproduction, which is called "maturity death". They only reproduce once in their lifetime, which is an unchangeable genetic characteristic. Although the landlocked population of Salmon does not migrate to the sea, it still maintains the characteristic of "maturity death". Based on the theory and practice of fish genetic breeding, artificial induction technology is used to change the chromosome set in somatic cells from the ordinary two sets to three sets, and meiosis cannot form normal gametes, resulting in the inability of gonads to mature, thus solving the problem of "maturity death", extending the life cycle, and avoiding the problems of growth stagnation and fish quality decline during the gonad development stage and spawning season of ordinary diploid fish.

[0004] There are mainly biological, physical and chemical methods for triploid breeding of fish. Physical methods are mostly used in production. The principle is to inhibit the discharge of the second polar body of the fertilized egg, so that two sets of female nuclear chromosomes and one set of male nuclear chromosomes are retained in the fertilized egg, thereby producing triploids. It mainly includes temperature shock method and hydrostatic pressure method.

[0005] The temperature shock method does not have unified dedicated equipment, is greatly affected by human operation, and has limitations such as unstable effects.

[0006] The hydrostatic method is a method of induction that is completed by special equipment, the procedure is easy to standardize, the damage to the embryo is less than that of the temperature shock method, and it is highly operable and conducive to large-scale production. The hydrostatic method is currently the most advanced method of triploid breeding for salmon and trout that is generally recognized internationally and within the industry.

[0007] There are many studies on triploid breeding technology for salmon and trout at home and abroad. The heat shock and hydrostatic pressure methods for rainbow trout and Atlantic salmon are very successful. For the study of triploid salmon in China, the only method used is heat shock induction, but the effect is not very ideal. There is no report on hydrostatic pressure induction of salmon.

[0008] The technical core of the hydrostatic pressure method is to set accurate induction parameters, namely the start time, duration and pressure of the treatment. The determination of the induction parameters requires a lot of experimental research in the early stage, and also has high requirements for the performance of the press. The start time of the treatment is particularly important. Accurately positioning the start time of the induction treatment before the discharge of the second polar body is the key to obtaining a good induction effect. The discharge time of the second polar body is very short, and the development of the fertilized egg is not completely synchronized, which makes it difficult to determine the start time of the treatment. Studies generally believe that 15 to 40 minutes after fertilization of salmonids is the effective time for the discharge of the second polar body and artificial intervention. This time range spans a large range and covers the requirements of most species. Not only do the requirements vary greatly between different species, even for the same species, a time difference of a few tenths of a minute will bring different results. There are two parameters for inducing triploid fish by the hydrostatic pressure method: duration and pressure. Insufficient duration and insufficient pressure will lead to a low triploid rate. Too long duration and excessive pressure will not only fail to increase the triploid induction rate, but also cause chromosome breakage or loss, form aneuploidy, and damage the embryo to affect the hatching rate. Only the best combination of the three parameters can achieve the ideal result, so other types of induction schemes cannot be simply applied. Usually, the best scheme is the one with a triploid rate of 40%, a hatching rate of 60%, and the highest comprehensive score.

[0009] Therefore, whether it is possible to provide a method for inducing triploidy of Masson salmon using the hydrostatic pressure method is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0010] In view of this, the present invention provides a method for inducing triploidy of Masson salmon by using a hydrostatic pressure method.

[0011] In order to achieve the above object, the present invention adopts the following technical solution:

[0012] A method for inducing triploidy of a salmon using a hydrostatic pressure method comprises the following steps:

[0013] (1) Preparation of broodstock;

[0014] (2) Egg collection and fertilization: start timing after the sperm and eggs are mixed, and control the water temperature at 6-8°C;

[0015] (3) Pressurization: When the time reaches 15-20 minutes after fertilization, start pressurization at a pressure of 60-65 kg / cm 2 , the pressurization time is continued for 5 to 6 minutes, and the fertilized eggs after pressurization are obtained;

[0016] (4) Hatching of fertilized eggs;

[0017] (5) Breeding of juvenile fish.

[0018] Preferably: step (1) broodstock preparation comprises:

[0019] 11) Control the water temperature of the broodstock to 14-17°C, pH 7.6-8.5, dissolved oxygen 6-10 mg / L, and then carry out artificial breeding;

[0020] 12) During the parent fish breeding stage, the broodstock are selected every 1 to 2 months, and the fish are bred separately according to size and fed with a special feed for salmon and trout: crude protein ≥ 45.0%, crude fat ≥ 10.0%, crude fiber ≤ 5.0%, crude ash ≤ 14.0%, moisture ≤ 10.0%, feeding rate 1.0%, and feeding twice a day;

[0021] 13) Reduce the feeding amount by half in the first two weeks of the spawning period, and stop feeding 5 days before spawning;

[0022] 14) After reaching one year old, they must go through a 2-month hibernation period at a water temperature of 0-4℃. Before the gonads mature and secondary sexual characteristics appear, broodstock are selected and paired with males in a 1:1 ratio, and males weighing less than 250g are eliminated. When they are 2 years old, females from the same batch weighing more than 500g and with round abdomens are selected as broodstock, with a sexual maturity coefficient of more than 20%.

[0023] Preferably: step (2) egg collection and fertilization comprises:

[0024] 21) Control the water temperature to 8-10℃;

[0025] 22) The eggs of female fish are collected by squeezing or laparotomy, and the eggs are placed in physiological saline; the semen of male fish is collected by squeezing to avoid water droplets;

[0026] 23) The sperm:egg ratio is 1:20-25. Mix the sperm and eggs and stir gently for 10-20 seconds. Add clean water to cover the eggs. Stir for another 10-20 seconds. Add clean water 3-5 times to wash the eggs. Add clean water and let it stand. Start timing after the sperm and eggs are mixed, and control the water temperature at 6-8℃.

[0027] Preferably: step (4) fertilized egg hatching comprises:

[0028] 41) Filtered water for hatching, dissolved oxygen ≥ 6 mg / L, water temperature 5-8°C;

[0029] 42) No vibration behavior is allowed from the beginning of incubation to the beginning of emergence;

[0030] 43) Control water flow and disinfection;

[0031] 44) After the eyes have opened, the eye opening rate is calculated and when the accumulated temperature reaches 350-390℃, the seedlings will hatch and emerge.

[0032] Preferably: step (5) fry culture comprises:

[0033] The fry are cultivated in flowing water. When more than 80% of the fry float to the surface, they are fed with starter feed at a daily feeding rate of 8-10%, and they are fed once every 3 hours.

[0034] The present invention also provides application of any of the above methods in aquaculture.

[0035] It can be seen from the above technical scheme that compared with the prior art, the present invention discloses a method for inducing triploid salmon by hydrostatic pressure method, and the technical effect achieved is that the growth rate of triploid is significantly improved compared with diploid, and the quality of fish meat is improved, which makes up for the shortcomings of domestic salmon and trout breeding varieties, is of great significance for promoting the industrialization development of domestic salmon and trout, and has broad market application prospects. DETAILED DESCRIPTION

[0036] The following will be described clearly and completely in conjunction with the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] The embodiment of the present invention discloses a method for inducing triploidy of Masu salmon by hydrostatic pressure method. Raw materials, reagents and tools not mentioned in the embodiment are all commercially available and will not be described in detail here.

[0038] Example 1

[0039] A method for inducing triploidy of a salmon using a hydrostatic pressure method comprises the following steps:

[0040] (1) Broodstock preparation:

[0041] The quality of broodstock is the primary link in fish breeding, and the degree of egg development directly affects the breeding results.

[0042] Masu salmon is a cold-water fish. The water quality required for broodstock cultivation must meet the basic requirements for cold-water fish farming:

[0043] The suitable water temperature for growth is 14-17℃, pH7.6-8.5, dissolved oxygen 6-10mg / L, and mountain spring water is best. The sizes of Masu salmon vary greatly among individuals, and broodstock need to be artificially selected. In the broodstock breeding stage, selection is generally carried out once every 1-2 months, and large and small are bred separately. Feed salmon and trout with special compound feed, crude protein ≥45.0%, crude fat ≥10.0%, crude fiber ≤5.0%, crude ash ≤14.0%, moisture ≤10.0%, feeding rate of about 1.0%, and feed twice a day. The feeding amount is halved in the first two weeks of spawning, and feeding is stopped 5 days before spawning.

[0044] Masu salmon mature sexually at 2 years old, and after reaching 1 year old, they have to go through a hibernation period of about 2 months at a water temperature of 0-4℃. The sex ratio is unbalanced, with females accounting for only 1 / 3. Before the gonads mature and secondary sexual characteristics appear, the sexes are matched and the excess males are eliminated. The abdomen of the female fish is round and light in color. The abdomen of the male fish is narrow, the color gradually turns black, the snout gradually protrudes forward and teeth grow. Due to different breeding environments, mainly the different suitable growth periods, the individual sizes of broodstock vary greatly (in the place of origin of Jilin, natural river water breeding, the 2-year-old size generally does not exceed 250g. In the mountain spring water breeding environment in Liaoning, Hebei, Shandong and other places, the weight of 2-year-olds is mostly between 500 and 1000g). Select female fish with a weight of more than 500g and a round abdomen from the same batch as broodstock, and the general sexual maturity index (GSI) is above 20%.

[0045] (2) Egg collection and fertilization:

[0046] The water temperature during the breeding period should not exceed 10℃ (preferably 8-10℃). Female fish are squeezed or laparotomy to collect eggs. Male fish semen is collected by squeezing. The amount of male semen is large, and one fish can be collected three times. When collecting eggs for fertilization, the ratio of male to female is n:3, that is, one basin (containing saline) of eggs collected by several female fish should be matched with the semen of 3 male fish, that is, the ratio of sperm: eggs is 1:20-25. Before collecting semen, wipe the water off the male fish with a dry towel, and be careful to prevent semen from getting wet. The action of squeezing eggs should be gentle, and the damage to eggs caused by artificial squeezing should be minimized, and the influence of egg fluid on the fertilization process should be reduced. After filtering out the body cavity, pour the eggs into a basin filled with saline and squeeze in semen.

[0047] Mix the sperm and eggs and stir gently for 10-20 seconds, add clean water to cover the eggs, stir for another 10-20 seconds, add clean water 3-5 times to wash the eggs, filter out excess semen, white eggs, residual blood, etc., add clean water and let it stand. Start timing with a timer after the sperm and eggs are mixed. The best water temperature for egg collection is 6-8℃. Avoid direct sunlight during egg collection and standing.

[0048] (3) Pressurization treatment

[0049] The press machine selected is the Canadian TRC fish egg breeding pressurized chamber (TRC-APV-K).

[0050] Place the fertilized eggs in the egg filter bucket equipped with the pressure chamber. The amount of eggs placed cannot exceed the upper limit specified. The egg volume of a 2.7L press generally does not exceed 800g. Close the pressure chamber and adjust the drain valve to the closed state.

[0051] 15-20 minutes after fertilization, start pressurization (and timing), the pressure should be 60-65kg / cm 2 , the pressurization time is 5 to 6 minutes.

[0052] After the time is reached, the pressure is manually released, the pressure cabin cover is opened, the fertilized eggs are taken out, the dead eggs are picked out, the pressure damage rate is counted, and the number of fertilized eggs is determined.

[0053] (4) Hatching of fertilized eggs

[0054] After pressure treatment, the fertilized eggs are placed in an incubator for hatching as soon as possible.

[0055] For incubators, choose traditional salmon and trout hatching buckets or "shelf-type" three-dimensional incubators, preferably CATAQUA "shelf-type" incubators.

[0056] The water for hatching needs to be filtered to reduce the adhesion of sediment. The dissolved oxygen should be ≥6mg / L, and the best water temperature for hatching is 5-8℃, and the highest temperature should not exceed 10℃.

[0057] The embryonic development period is relatively sensitive to external environmental stimulation, and there are obvious differences in different development stages. From the beginning of incubation to before the eggs emerge, all vibration behaviors such as picking eggs and stirring are prohibited. Control the flow of incubation water and do not flush the eggs. During the incubation period, prevent the growth of water molds and disinfect: soak in 500ppm formaldehyde once a week before the eggs emerge, each time for 5 minutes.

[0058] When the accumulated incubation temperature reaches 100℃.d, take 30 eggs and soak them in identification solution for 5 minutes. Those with white spots are fertilized eggs, and the fertilization rate is calculated. (Formula for fertilization rate identification solution: 7 grams of sodium chloride, 50 ml of glacial acetic acid, 1 liter of distilled water). For incubation in a hatching bucket, after the fry have eyes, pour the fertilized eggs into a small parallel tank (45cm×30cm) and continue to incubate with flowing water, and pick out dead eggs and unfertilized eggs. For incubation in a "shelf-type" incubator, continue to incubate in the original incubator until the fry float up and then move them into the cultivation container. After the fry have eyes, calculate the eye rate. When the accumulated temperature reaches 350-390℃.d, hatch the fry, count the unhatched eggs, and calculate the hatching rate.

[0059] (5) Juvenile fish cultivation

[0060] Juveniles are cultured in indoor glass fiber reinforced plastic tanks (45cm×200cm) or in a 200cm diameter nursery basin with flowing water. Feeding is an important part of daily management. Juveniles eat a small amount of food at a time, but their digestion and metabolism are fast. The principle of small amounts and multiple times should be adopted. After juveniles float more than 80%, they are directly fed with salmon and trout starter feed (micro-particle feed, crude protein content ≥60%) (Aller, Denmark), with a daily feeding rate of 8-10%, and once every 3 hours. Before each feeding, use a straw to suck out the remaining bait, feces and dead fish.

[0061] Technical effect verification

[0062] Ploidy detection

[0063] Pacfec CyFIow Cube 8 flow cytometer was used to detect DNA content, determine ploidy, and calculate triploid rate. Ploidy detection can be performed in the juvenile stage, with 30 randomly sampled and 3 common diploids as controls. It is also possible to anesthetize the juveniles alive after they grow to 8-10 cm, and collect blood from the tail vein for testing. Flow cytometric analysis showed that the relative DNA content of Masu salmon was 33 for diploid and 51 for triploid.

[0064] Determining the best solution

[0065] The comprehensive score was calculated based on the triploid rate of 40% and the hatching rate of 60% to determine the best induction parameters. Under the condition of water temperature of 6-8℃, 15-20 minutes after fertilization, 60-65kg / cm 2 By continuously applying pressure for 5 to 6 minutes, 90 to 100% triploid fish can be obtained, with a hatching rate of up to 85% and a comprehensive score of 90, which is the best effect.

[0066] Comparative experiment 1

[0067] The method of Example 1 was used to culture triploid Masu salmon in a fishery in Liaoning. The water source was underground spring water with a stable water temperature of 8-10°C in winter and 14-15°C in summer. After 15 months of culture, the diploid fish weighed 0.2-0.5kg and the triploid fish weighed 0.8-1.5kg. The diploid fish weighed 0.3-1.0kg at the age of 2, and the triploid fish weighed 1.0-2.2kg. Under the same conditions, the triploid grew more than twice as fast as the diploid.

[0068] Comparative experiment 2

[0069] 13-14 minutes after fertilization, use 60-65 kg / cm 2 The pressure (optimal pressure) was continuously increased for 5 to 6 minutes (optimal continuous pressurization time), and the highest rate of triploid fish was 50 to 55%, and the hatching rate was up to 80%. This indicates that the pressurization time was too early, and the second polar body of some fertilized eggs had not yet formed, and the triploid rate was low, but the effect on hatching was small.

[0070] 15 to 20 minutes after fertilization (optimal start time), use 70 to 75 kg / cm 2 When the pressure is continuously applied for 5 to 6 minutes (the optimal continuous pressurization time), no more than 60% of the triploid fish are obtained, 20% of the embryos with incomplete chromosomes such as chimeras are obtained, the hatching rate is no more than 40%, and the death rate of fertilized eggs increases after pressurization, indicating that excessive pressure causes great harm to the embryos.

[0071] 15 to 20 minutes after fertilization (optimal start time), use 60 to 65 kg / cm 2When the pressure (optimal pressure) is continuously applied for 7 to 8 minutes, the obtained triploid fish does not exceed 70%, the embryos with incomplete chromosomes such as mosaics reach 15%, and the hatching rate does not exceed 45%, indicating that too long pressurization time has a great impact on the embryo.

[0072] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0073] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for inducing triploidy of Masu salmon using hydrostatic pressure method, characterized in that: The following steps are involved: (1) Preparation of broodstock; (2) Egg collection and fertilization: start timing after the sperm and eggs are mixed, and control the water temperature at 6-8°C; (3) Pressurization: When the time reaches 15-20 minutes after fertilization, start pressurization at a pressure of 60-65 kg / cm 2 , the pressurization time is continued for 5 to 6 minutes, and the fertilized eggs after pressurization are obtained; (4) Hatching of fertilized eggs; (5) Breeding of juvenile fish.

2. The method according to claim 1, characterized in that The broodstock preparation in step (1) comprises: 11) Control the water temperature of the broodstock to 14-17°C, pH 7.6-8.5, dissolved oxygen 6-10 mg / L, and then carry out artificial breeding; 12) During the parent fish breeding stage, the broodstock are selected every 1 to 2 months, and the fish are bred separately according to size and fed with a special feed for salmon and trout: crude protein ≥ 45.0%, crude fat ≥ 10.0%, crude fiber ≤ 5.0%, crude ash ≤ 14.0%, moisture ≤ 10.0%, feeding rate 1.0%, and feeding twice a day; 13) Reduce the feeding amount by half in the first two weeks of the spawning period, and stop feeding 5 days before spawning; 14) After reaching one year old, they must go through a 2-month hibernation period at a water temperature of 0-4℃. Before the gonads mature and secondary sexual characteristics appear, broodstock are selected and paired with males in a 1:1 ratio, and males weighing less than 250g are eliminated. When they are 2 years old, females from the same batch weighing more than 500g and with round abdomens are selected as broodstock, with a sexual maturity coefficient of more than 20%.

3. The method according to claim 2, characterized in that The egg collection and fertilization in step (2) comprises: 21) Control the water temperature to 8-10℃; 22) The eggs of female fish are collected by squeezing or laparotomy, and the eggs are placed in physiological saline; the semen of male fish is collected by squeezing to avoid water droplets; 23) The sperm:egg ratio is 1:20-25. Mix the sperm and eggs and stir gently for 10-20 seconds. Add clean water to cover the eggs. Stir for another 10-20 seconds. Add clean water 3-5 times to wash the eggs. Add clean water and let it stand. Start timing after the sperm and eggs are mixed, and control the water temperature at 6-8℃.

4. The method according to claim 3, characterized in that The fertilized egg hatching in step (4) comprises: 41) Filtered water for hatching, dissolved oxygen ≥ 6 mg / L, water temperature 5-8°C; 42) No vibration behavior is allowed from the beginning of incubation to the beginning of emergence; 43) Control water flow and disinfection; 44) After the eyes have opened, the eye opening rate is calculated and when the accumulated temperature reaches 350-390℃, the seedlings will hatch and emerge.

5. The method according to claim 4, characterized in that The fry culture in step (5) comprises: The fry are cultivated in flowing water. When more than 80% of the fry float to the surface, they are fed with starter feed at a daily feeding rate of 8-10%, and they are fed once every 3 hours.

6. Use of the method according to any one of claims 1 to 5 in aquaculture.