A method for producing tetraploid largemouth bass by inducing double hydrostatic shock

Abstract

The present invention discloses a method for producing tetraploid largemouth black bass by inducing a double hydrostatic shock method. The method first obtains high-quality fertilized eggs through natural fertilization: the steps of injecting hormones and squeezing the abdomen to obtain sperm and eggs in the traditional method are avoided, thereby significantly reducing the damage to the parent fish. Combined with the innovative double hydrostatic shock method, by optimizing the pressure intensity and processing time, the damage to the fertilized eggs is further reduced, the second cleavage is successfully inhibited, and the tetraploid induction rate can be as high as 24.44%. The method can be applied to the tetraploid production of largemouth black bass: the operating method of the present invention is simple and easy, has strong repeatability, and has a high induction rate. These characteristics make the method of the present invention have a high application value in largemouth black bass breeding, and provide strong support for the breeding of tetraploid largemouth black bass.

Description

Technical Field

[0001] The invention relates to the technical field of fish breeding, and in particular to a method for producing tetraploid largemouth seabass by inducing a double hydrostatic shock method. Background Art

[0002] Largemouth Bass Micropterus salmoides , commonly known as California bass, is one of the important economic aquaculture species in my country. It grows rapidly, has strong adaptability, and has delicious meat without intramuscular spines, which is deeply loved by consumers. According to statistics, the national largemouth bass production in 2022 has reached 800,000 tons. However, with the expansion of largemouth bass farming scale and the increase in farming density, problems such as germplasm degeneration and frequent diseases have gradually become prominent, especially in the production season. The breeding activities of largemouth bass consume a lot of energy and cause damage to the fish body, which seriously affects the breeding benefits. Polyploidization technology, as one of the important means of modern fish genetic improvement, effectively changes fish traits by inducing chromosome doubling, providing a new way for fish breeding. The induction of tetraploid largemouth bass through polyploidization technology and hybridization with normal diploid largemouth bass to produce infertile triploid largemouth bass is expected to provide new ideas for solving the above problems.

[0003] Temperature shock and hydrostatic shock are the most commonly used methods to induce tetraploid fish. The principle of hydrostatic shock is to use high-intensity pressure to destroy the microtubule configuration in the fertilized egg cell and destroy the formation of spindle fibers, thereby temporarily blocking the first cleavage process and achieving the purpose of chromosome doubling to form tetraploid. Hydrostatic shock has the advantages of uniform pressure, little damage to fertilized eggs, high induction rate and low deformity rate. It has been successful in inducing tetraploidy in many different fish, such as turbot. Scophthalmus maximus Applying 75 MPa pressure to the fertilized egg at 80 mpf (minutes post-fertilization) for 6 minutes to inhibit the first cleavage can produce tetraploids with an induction rate of more than 80%; Monopterus albus Fertilized egg at 85mpf, 750kg / cm 3 After 5 minutes of treatment, the first cleavage was successfully inhibited to form tetraploids, with an induction rate of 23.33% and a hatching rate of 4.7%; Oncorhynchus mykissAfter the fertilized eggs were treated with hydrostatic pressure of 65Mpa for 6 minutes, tetraploids were obtained with an induction rate of 25%. The parameter conditions in the hydrostatic pressure method have a significant impact on the induction results. If the pressure intensity is too low, it will not be able to effectively inhibit egg cleavage; conversely, if the pressure intensity is too high, the hatching rate of fertilized eggs will drop significantly, and it will be difficult to obtain a sufficient number of fry. In addition, variables such as the start time of treatment, treatment duration, and water temperature will also have an important impact on the induction effect. Given that the optimal induction parameters of fertilized eggs of different species of fish are different, a large number of experimental explorations are needed to accurately determine the optimal parameters of hydrostatic shock for each type of fish fertilized eggs, so as to achieve efficient induction of tetraploids.

[0004] In the process of tetraploid induction in fish, the quality of gametes directly affects the success rate of induction. In order to obtain fertilized eggs, the commonly used method is artificial insemination technology, which injects hormones into the parent fish and squeezes out sperm and eggs. This method can ensure the high synchronization of the development of fertilized eggs and the precise fertilization time. However, for largemouth bass, its ovaries have the characteristics of asynchronous maturation, containing oocytes at different developmental stages. Largemouth bass will not discharge all eggs at once during the spawning season, but will discharge mature eggs in small quantities and multiple times. In actual operation, the artificial insemination effect of largemouth bass is not ideal. Often only a small number of eggs can be squeezed out or even no eggs can be squeezed out. Among the squeezed eggs, only a small part reaches the mature state for fertilization, and the rest are mostly immature eggs. In addition, the egg squeezing process will cause irreversible damage to female fish. In contrast, it is more difficult for male fish to squeeze out sperm. Even after hormone injection, male fish still have difficulty squeezing out sperm. Although viable sperm can be obtained by removing the testis and grinding it, this operation will lead to the death of male fish. In addition, the sperm obtained in this way is very easy to be activated prematurely due to contamination by body fluids or tissue fluids, and the activated sperm of largemouth bass will be inactivated within 2 to 3 minutes. Even if artificial insemination is successfully carried out, the quality of fertilized eggs is uneven, and the fertilization rate and hatching rate are low. A female largemouth bass can squeeze out 200 to 2000 eggs after induced spawning, but about 40% to 60% of the eggs cannot survive, and each female fish can only produce about 12 fry in the end, while the fry yield of natural fertilization is as high as about 1000. Natural fertilization not only produces a small number of eggs and is unstable, but also has a low synchronization of egg development, and it is difficult to accurately determine the starting time of fertilization. Therefore, to date, there has been no report on the use of natural fertilization for polyploid induction experiments.

[0005] Compared with triploidy and gynogenetic development, the mortality rate of fertilized eggs after tetraploidy induction is higher and the induction success rate is relatively low. This means that higher quality and quantity of fertilized eggs are needed for induction. However, the difficulty of artificial insemination of largemouth bass makes it particularly difficult to obtain high-quality fertilized eggs. Researchers in related fields have always lacked in-depth understanding of the cleavage interval of largemouth bass at different water temperatures, the impact of fertilized egg quality on the induction success rate, and the exploration of tetraploidy induction conditions. Although some researchers have tried to use the cold shock method to inhibit the first cleavage of largemouth bass fertilized eggs to produce tetraploids, these attempts have all failed due to the difficulty in obtaining gametes for artificial insemination, the high mortality rate of fertilized eggs, and the failure to detect tetraploids in offspring (Induction of Tetraploidy and Gynogenesis toProduce Sterile All-Female Largemouth Bass - OHIO STATE UNIVERSITY, https: / / portal.nifa.usda.gov / web / crisprojectpages / 1020978-induction-of-tetraploidy-and-gynogenesis-to-produce-sterile-all-female-largemouth-bass.html, Dabrowski, et al. 2019). At present, there are no reports of successful induction of tetraploidy in largemouth bass at home and abroad. Summary of the invention

[0006] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide a method for producing tetraploid largemouth seabass by inducing double hydrostatic shock method.

[0007] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0008] The present invention first provides a method for producing tetraploid largemouth bass by inducing a double hydrostatic shock method, comprising the following steps:

[0009] S1. Obtaining fertilized eggs obtained by natural fertilization of largemouth bass;

[0010] S2. Within 50 to 65 minutes after fertilization, the fertilized egg is subjected to double hydrostatic shock treatment; the double hydrostatic shock treatment is 6000 to 8000 psi for 0.5 to 1 minute, slowly depressurizing to normal pressure, and then 6000 to 8000 psi for 0.5 to 1 minute after an interval of 5 to 10 minutes;

[0011] S3. The fertilized eggs treated with double hydrostatic pressure were hatched to obtain tetraploid largemouth bass.

[0012] The present invention first obtains fertilized eggs through natural fertilization of largemouth black bass. The present invention's research shows that the fertilized eggs obtained by natural fertilization are higher than those obtained by artificial insemination in terms of fertilization rate, survival rate and hatching rate, and under hydrostatic pressure treatment, the hatching rate of naturally fertilized fertilized eggs is better than that of artificial insemination, indicating that it has higher tolerance to hydrostatic pressure treatment. On the premise of obtaining high-quality fertilized eggs, the present invention studies the first cleavage interval (FCI) of largemouth black bass fertilized eggs under different water temperatures, and creates a double hydrostatic shock method, exploring parameters such as the starting time of pressure treatment, pressure intensity, pressure interval time, and pressure application time. By increasing the number of pressures, slowing down the speed of pressure increase and decrease, and reducing the pressure application time, the damage to the fertilized eggs during the pressure application process is reduced, and the hydrostatic pressure inhibition effect of cleavage is improved, and finally a method for producing tetraploid largemouth black bass is obtained.

[0013] Furthermore, in step S1, the largemouth bass broodstock need to be nutrient-enriched to ensure that they are strong and sexually mature. The selected high-quality broodstock are placed in a constant temperature (20°C) flowing water pool to maintain their optimal reproductive state.

[0014] Furthermore, the step S1 is to collect naturally fertilized eggs of largemouth bass using an artificial fish nest connected to an underwater camera; the underwater camera is used to observe the spawning process and determine the fertilization time of the fertilized eggs.

[0015] In the breeding environment, artificial fish nests are placed and connected to underwater cameras, which can monitor the spawning behavior of parent fish in real time and capture the spawning moment in time without disturbing the parent fish. Specifically, the underwater camera can accurately capture the exact moment of parent fish spawning and record the fertilization start time. Fertilized egg quality detection: detect the hatching rate of naturally fertilized and artificially fertilized eggs under different pressure intensity treatments, and explore the first cleavage interval of largemouth bass fertilized eggs and the first cleavage cycle under different water temperatures.

[0016] Preferably, the artificial fish nest is a bowl-shaped structure, covered with an easy-to-adhere gauze membrane. This special artificial fish nest is different from the traditional sheet-shaped artificial fish nest. It greatly improves the collection rate of fish eggs and is connected to an underwater camera. After the camera observes the ovulation of the parent fish, the gauze membrane with fertilized eggs adhered to it is immediately taken out to collect the fertilized eggs. After replacing the gauze membrane, the parent fish will continue to lay eggs in the fish nest. The naturally fertilized eggs collected in this way minimize the human interference in the spawning process of the parent fish. The use of the special artificial fish nest to collect naturally fertilized eggs avoids the steps of injecting hormones and squeezing the abdomen to obtain sperm and eggs in the traditional method, thereby significantly reducing the damage to the parent fish. The amount of naturally fertilized eggs obtained by a single pair of parent fish is more than 10,000, and the fertilization rate and hatching rate of naturally fertilized eggs without applying hydrostatic pressure are more than 90%. After hydrostatic pressure treatment, the hatching rate of naturally fertilized eggs obtained by this method can reach up to 30%, and the maximum tolerable pressure can reach 12,000 psi and fry can still be hatched. Its pressure tolerance is much greater than that of artificially fertilized eggs. In addition, the fertilization time of naturally fertilized eggs obtained by this method can be accurately determined, and more than 80% of fertilized eggs synchronously complete the first cleavage within 6 minutes, which can replace the traditional artificial insemination method.

[0017] Furthermore, the step S2 is to apply double hydrostatic shock treatment to the fertilized egg within 50 to 60 minutes after fertilization.

[0018] Preferably, step S2 is to apply double hydrostatic shock treatment to the fertilized egg within 50 to 55 minutes after fertilization. More preferably, the fertilized egg is applied double hydrostatic shock treatment within 55 minutes after fertilization.

[0019] Preferably, the dual hydrostatic pressure treatment parameters in step S2 are 6000 psi for 1 min, slowly releasing the pressure to normal pressure, and then 6000 psi for 1 min after an interval of 10 min.

[0020] Furthermore, the pressure increase rate during the double hydrostatic pressure treatment in step S2 is 200-400 psi / s.

[0021] Preferably, the pressure increase rate during the dual hydrostatic pressure treatment is 300 psi / s.

[0022] Furthermore, the temperature of the double hydrostatic shock treatment in step S2 is 20-22°C.

[0023] Preferably, the water temperature is kept constant during the double hydrostatic shock treatment, with the fluctuation not exceeding 0.5°C.

[0024] Furthermore, the pressure change rate of the pressure relief in step S2 is 200-400 psi / s.

[0025] Preferably, the pressure change rate of the pressure relief is 300psi / s.

[0026] That is, the hydrostatic pressure increase and decrease process in the double hydrostatic shock treatment needs to be carried out slowly, and the water temperature in the pressure chamber is kept constant throughout the whole process, with a fluctuation of no more than 0.5°C.

[0027] Furthermore, after the treatment in step S3, the first cleavage of the fertilized egg proceeds normally, but the second cleavage is delayed.

[0028] Furthermore, after the fertilized eggs are hatched in step S3, ploidy identification and ploidy composition determination are performed to identify whether they are tetraploid fish. Among them, the largemouth bass successfully hatched by doubling includes tetraploid largemouth bass, and the number of chromosomes is 92 according to karyotype analysis, and the DNA content is twice that of diploid largemouth bass detected by flow cytometry.

[0029] Preferably, the ploidy identification and determination of ploidy composition are performed by flow cytometry and chromosome karyotype analysis.

[0030] The present invention also provides application of any of the above methods in polyploid breeding of largemouth bass.

[0031] Furthermore, the polyploid breed is a tetraploid and / or a triploid breed.

[0032] Furthermore, the triploid breeding is to obtain a triploid largemouth seabass by performing interploid hybridization between a tetraploid largemouth seabass obtained by any of the above methods and a diploid largemouth seabass.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention adopts naturally fertilized largemouth bass fertilized eggs, whose maximum tolerance pressure can reach 12000psi, and then innovatively adopts a double hydrostatic shock method, by optimizing the pressure intensity and processing time, further reducing the damage to the fertilized eggs, successfully inhibiting the second cleavage, and successfully inducing largemouth bass tetraploids, and the tetraploid induction rate can reach up to 24.44%. The operating method of the present invention is simple and easy, has strong repeatability, and has a high induction rate. These characteristics make the method of the present invention have a high application value in actual production, and provide strong support for the large-scale production of tetraploid largemouth bass. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the morphology of the first cleavage of the fertilized egg of largemouth bass; A to D are the morphology of fertilized eggs of 60, 75, 80, and 85 mpf, respectively, and the scale is 3 mm.

[0036] Figure 2 It is the interval between the first cleavage of fertilized eggs of largemouth bass under different water temperatures.

[0037] Figure 3 To investigate the tolerance of artificially inseminated and naturally fertilized eggs to hydrostatic pressure treatments of different intensities.

[0038] Figure 4 To synchronize the development of artificial insemination and naturally fertilized eggs.

[0039] Figure 5 The figure is for the ploidy identification of largemouth bass; A is the metaphase of embryonic cell division of diploid largemouth bass (2n=46), B is the metaphase of embryonic cell division of tetraploid largemouth bass (4n=92), C is the relative DNA content of diploid largemouth bass fry detected by flow cytometry, and D is the relative DNA content of tetraploid largemouth bass fry. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0042] Example 1

[0043] 1. Broodstock breeding and screening

[0044] Largemouth black bass broodstock are raised in earthen ponds before the birth season. They are fed with compound feed twice a day and nutritionally enhanced twice a week, including feeding fresh fish and egg yolk powder. At the same time, the aquaculture water quality is strictly monitored, with a water temperature of 26±2℃, dissolved oxygen ≥6.0 mg / L, pH 7.2-7.6, ammonia nitrogen ≤0.6 mg / L, and nitrite 0.03-0.20 mg / L. Sexually mature and healthy largemouth black bass broodstock are selected from the earthen pond and transferred to a constant temperature (20℃) flowing water pool for breeding to ensure that they are in the best reproductive state. Brown sheets are laid on the bottom of the pond to facilitate spawning of broodstock, and underwater cameras are used to monitor the spawning behavior of broodstock in real time.

[0045] 2. Mating of largemouth bass broodstock and collection of fertilized eggs

[0046] Twenty pairs of sexually mature largemouth bass broodstock were selected and kept in a 20-cubic-meter pool with a constant water temperature of 20°C. The broodstock were stimulated to estrus by avoiding light and keeping running water. An underwater camera was connected to the artificial fish nest and placed at the bottom of the pool. The behavior of the broodstock was continuously observed through the connected screen; the artificial fish nest was a bowl-shaped structure covered with an easy-to-adhere gauze membrane. When the female fish laid eggs, the male fish simultaneously discharged sperm, and the fertilized eggs adhered to the gauze membrane of the artificial fish nest. Within 5 minutes of observing the female fish laying eggs, the artificial fish nest was quickly picked up, and the midpoint of the time period between the moment the female fish began to lay eggs and the moment the fish nest was picked up was regarded as the fertilization time. The gauze membrane with fertilized eggs was carefully collected, and the gauze membrane was replaced and the artificial fish nest was returned to its original position so that the broodstock could continue to lay eggs.

[0047] 3. Observation of the first cleavage interval at different water temperatures

[0048] The first cleavage interval (FCI) refers to the time required for the embryo to complete the first cleavage and enter the two-cell stage after fertilization, which is an important basis for designing the starting time of hydrostatic induction. Largemouth bass that have successfully paired in the pool were selected, and a mixed oxytocin of luteinizing hormone-releasing hormone analog (LRH A2), human chorionic gonadotropin (HCG) and diometron (DOM) was injected into the base of their pectoral fins using a single needle method to induce fertilization. The injection dose for female fish was LRH A2 20-25μg / kg, HCG 1500-2000IU / kg, and DOM 5mg / kg; the dose for male fish was halved. After 24 hours of the effect period, the parent fish was gently caught with a scoop net, and the abdomen was gently pressed to squeeze out sperm and eggs into a stainless steel basin. After the sperm and eggs were mixed, clean water was added to activate them, and they were gently stirred with a feather. The activation time was recorded as the fertilization time. The fertilized eggs were washed three times with clean water for observation, and their development was observed under a microscope at different water temperatures of 17, 18, 19, 20, 21, and 22°C until they completed the first cleavage, with observations every 5 minutes. Since the cleavage of the fertilized egg is a process of gradual morphological changes, a morphological judgment standard for the completion of the first cleavage of a single fertilized egg was established: a single fertilized egg at 20°C water temperature was used as an observation example. When the fertilized egg cleaved 60mpf, the protoplasm layer of the fertilized egg first protruded ( Figure 1 Middle A), the fertilized egg showed egg cracks at 75 mpf ( Figure 1 Middle B), 80mpf egg cleavage gradually deepens, and cells begin to separate ( Figure 1 Middle C), 85mpf two cells are completely separated ( Figure 1 D) Figure 1 The morphology of the fertilized egg in D was used as the standard to judge the completion of the first cleavage of a single fertilized egg. 50 fertilized eggs were observed each time, and the time required for more than 40 (80%) fertilized eggs to complete cleavage was regarded as the first cleavage interval. Figure 2As the water temperature rises, the speed of the first cleavage gradually increases, and the time required is reduced from 120 minutes to 65 minutes.

[0049] 4. Quality testing of fertilized eggs from natural fertilization and artificial insemination

[0050] The method for collecting fertilized eggs from natural fertilization is as described in step 2, and the method for collecting fertilized eggs from artificial insemination is as described in step 3

[0051] (1) Hatching rate and maximum stress tolerance test:

[0052] Fertilized eggs from three pairs of broodstock were collected from each group. After the collected fertilized eggs were placed in 20℃ clean water for incubation for 55 minutes, half of them were taken out and placed in the pressure chamber of the hydrostatic pressure gauge, and were treated with 6000psi, 8000psi, 10000psi, and 12000psi pressures for 1 minute. The fertilized eggs after treatment were taken out and placed back in clean water for continued incubation; the other half were not treated and incubated normally. The egg laying amount and body weight of each group, as well as the fertilization rate, survival rate and hatching rate of fertilized eggs were recorded. The results showed that under the condition of no hydrostatic pressure, the fertilization rate, survival rate and hatching rate of the fertilized eggs in the artificial insemination group were lower than those in the natural fertilization group (as shown in Table 1). In addition, the number of fertilized eggs obtained by natural fertilization was significantly more than that by artificial insemination. In actual operation, the eggs artificially squeezed out by female fish contain a large number of immature eggs, which cannot be fertilized and hatched, and are difficult to separate from normal eggs. Unfertilized eggs quickly die and turn white, which damages the water quality and affects the normal hatching of surviving eggs. Female fish also die after squeezing eggs. Figure 3 As shown in the figure, after applying hydrostatic pressure of different intensities, the hatching rate of the natural fertilization group was higher than that of the artificial insemination group in each pressure intensity group. The hatching rate could still reach about 30% under the treatment of 6000psi for 1min, and the fry could still be hatched under the highest pressure treatment of 12000psi, while the artificial insemination group could not hatch fry after the treatment of 8000psi for 1min. The morphology of the fertilized eggs after pressurization was observed and it was found that the artificial insemination group had ruptured fertilized eggs. It is speculated that this is because the fertilized eggs are immature or of poor quality and cannot withstand high water pressure, resulting in rupture of the egg membrane and overflow of the contents. This shows that the hatching rate of naturally fertilized eggs is better than that of artificial insemination, and they have a higher tolerance to hydrostatic pressure treatment.

[0053] Table 1. Spawning of artificially inseminated and naturally inseminated largemouth bass broodstock

[0054]

[0055] (2) Observation on the synchronization of development between artificial insemination and natural fertilization:

[0056] Developmental synchrony of fertilized eggs For artificially inseminated fertilized eggs, all fertilized eggs can be regarded as being fertilized at the same time, in which the first cleavage intervals of different embryos are relatively close, and the cleavage is completed almost at the same time, and the developmental synchrony is relatively high. However, in naturally fertilized fertilized eggs, different fertilized eggs are discharged from the female fish body one after another, and their developmental synchrony may be poor (affected by the ovulation speed and time of the female fish). In order to detect the developmental synchrony of naturally fertilized eggs and artificially fertilized eggs obtained by this method, artificially inseminated and naturally fertilized fertilized eggs were taken, the fertilization time was recorded, and their morphological changes were continuously observed under a microscope. The first cleavage interval completion rate (Rate of FCI) was used to judge the developmental synchrony of fertilized eggs. 200 fertilized eggs were taken from each of the natural fertilization group and the artificial insemination group in a plate, and the water temperature in the plate was controlled at 20°C. The cleavage of the fertilized eggs was continuously observed until all the fertilized eggs completed cleavage. After it was observed that the fertilized eggs completed cleavage, the percentage of fertilized eggs that completed cleavage was recorded every 2 minutes.

[0057] The results are as follows Figure 4 As shown, the first fertilized egg completed the first cleavage at 81 minutes in artificial insemination, and all the fertilized eggs completed cleavage at 87 minutes, which took 6 minutes. The natural fertilization group took 8 minutes, but more than 80% of the fertilized eggs in the natural fertilization group completed cleavage at 6 minutes. This shows that the developmental synchronization of naturally fertilized fertilized eggs adopted by this method is similar to that of artificial insemination, which meets the requirements of tetraploid induction.

[0058] In summary, the fertilization rate and hatching rate of the naturally fertilized eggs collected by this method were all above 90%, and the highest hatching rate was still 30% after hydrostatic pressure treatment. The developmental synchronization of the fertilized eggs was similar to that of artificial insemination, reaching the quality standard of fertilized eggs required for tetraploid induction.

[0059] Example 2

[0060] Exploration of optimal parameters for hydrostatic shock treatment:

[0061] The water temperature in the pressure chamber of the hydrostatic pressure gauge was controlled at 20°C. One minute before the set induction start time, the brown sheet with fertilized eggs attached was placed in the pressure chamber of the hydrostatic pressure gauge in advance, and the pressure was slowly increased within 30 seconds (the speed was controlled at 300psi / s), so that when the induction start time point was reached, the pressure value in the chamber just reached the pressure required for induction. After the pressure was increased, the pressure was slowly released within 30 seconds, the gauze membrane was removed, the fertilized eggs were evenly spread in the net box, and placed in 20°C water for incubation.

[0062] Incubation of fertilized eggs: Fertilized eggs are placed in 20℃ water for aeration and incubation, and anti-mold drugs are sprayed. During this period, whitish dead eggs are sucked out every 12 hours to prevent the dead eggs from rotting and mold, which will have an adverse effect on the live eggs, and the water quality is measured. After 60 hours, the fry are hatched. After 6 days, the fry that float to the surface are transferred to the open pond to be fed with Artemia.

[0063] Calculation of fertilization rate, survival rate and hatching rate of fertilized eggs: scrape 100 to 200 eggs from the brown sheet and place them in a culture dish for incubation to count the number of eggs. The incubation water temperature is controlled at 20°C. White eggs during the incubation process are unfertilized eggs and dead eggs. 12 hours after fertilization, the percentage of surviving eggs to the total number of eggs is the fertilization rate. 24 hours after fertilization, the percentage of total white eggs to the total number of eggs is the survival rate. After 48 hours, the percentage of hatched fry to the total number of eggs is the hatching rate.

[0064] (1) Determination of the optimal induction hydrostatic pressure and treatment time: 4 combinations of pressure and time parameters were set, double hydrostatic shock group: 6000psi, lasting 1min, twice, 10min interval (hereinafter referred to as 6000psi 1min×2), 8000psi lasting 30s, twice with 10min interval (hereinafter referred to as 8000psi 30s×2); single hydrostatic shock group: 6000psi 2.5 min, 8000psi 1min. 55min after fertilization, the fertilized eggs were placed in the hydrostatic pressure instrument cavity and subjected to different pressure treatments. After the induction, the fertilized eggs were grouped and placed in different cages for incubation, and the fertilization rate, survival rate, and hatching rate were recorded. After 48 hours, the fertilized eggs hatched into fry. The experimental results of each group were statistically analyzed. The results showed that a certain proportion of tetraploid fry could be obtained after the fertilized eggs were treated with three parameters: 8000psi 30s×2, 6000psi 1min×2, and 8000psi 1min. Among them, the proportion of tetraploid fry was the highest when the 6000psi 1min×2 was treated. Therefore, the optimal parameter for tetraploid induction of largemouth bass was determined to be 6000psi 1min×2 (see Table 2), and this parameter was used to determine the optimal induction starting time point.

[0065] (2) Determination of the optimal induction start time: At different times of 50, 55, 60, and 65 minutes after fertilization, the largemouth bass eggs were placed in a hydrostatic pressure instrument for pressurization treatment. The pressure parameter was: 6000psi 1min×2. After the induction, the fertilized eggs were divided into groups and placed in different cages for incubation. The fertilization rate, survival rate, and hatching rate were recorded. After 48 hours, the fertilized eggs hatched into fry, and the experimental results of each group were statistically analyzed.

[0066] (3) Ploidy identification:

[0067] 1) Embryo karyotype analysis

[0068] The diploid largemouth bass has 46 chromosomes (e.g. Figure 5(A), the number of chromosomes of tetraploid largemouth bass is 92, so the ploidy can be identified by making chromosome karyotype slides and observing and calculating the number of staining of largemouth bass. Fertilized egg embryos that have developed to the somites stage are selected for chromosome preparation. The fertilized eggs are squeezed to remove the embryos, and the embryos are soaked in 0.01% colchicine for 1 hour, then transferred to 0.0375MKCL solution and placed at room temperature for 30 min. After hypotonic treatment, the embryos are removed from the hypotonic solution and fixed with freshly prepared Carnoy's fixative (methanol: acetic acid, 3:1) at -20°C for 3 times, each time for 20 minutes. After fixation, the embryos are removed with pointed forceps, placed in 50% glacial acetic acid for 5 minutes of dissociation, and the embryos are crushed with the tip of the forceps to make a cell suspension. The cold drop method is used, over-dried, Gissam stained, and examined under a microscope. This method is used to identify the ploidy of fertilized eggs. When there are 92 chromosomes, it is determined to be tetraploid (such as Figure 5 Middle B).

[0069] 2) Flow cytometry to identify ploidy

[0070] This method uses fry one day after hatching for DNA content detection. After the fertilized eggs are hatched, the fry are taken 1 to 3 days after hatching, rinsed with distilled water, frozen and stored in 5% DMSO solution, and washed with distilled water after thawing. The fry are injected into a 1.5 mL centrifuge tube containing 0.5 mL PI (Biosharp) dye solution (75μmol / L) through a 1 mL syringe, stained for 30 minutes in the dark, and the relative DNA content is detected and analyzed using a flow cytometer (Beckman). Normal diploid fry are used as controls. If the relative DNA content of the induced fry is twice that of the diploid fry, it can be determined to be tetraploid (such as Figure 5 C, 5D) and the tetraploid proportion was calculated.

[0071] The fertilization rate, survival rate, hatching rate and tetraploidy rate (the proportion of tetraploid fry detected to the total number of fry detected) of the above experiment were statistically analyzed. The results are shown in Table 3. The experimental results show that a certain proportion of tetraploids can be produced by induction at 50, 55, 60, and 65 minutes after fertilization, among which the tetraploidy rate of the 55-minute group can reach as high as 24%. Therefore, the optimal starting time for induction of tetraploidy in largemouth bass is determined to be 55 minutes after fertilization.

[0072] Table 2. Tetraploidy induced by double hydrostatic shock and single hydrostatic shock with different parameters in largemouth bass

[0073]

[0074] Note: Different lowercase letters indicate significant differences ( p <0.05).

[0075] Table 3. Induction of tetraploidy in largemouth bass by different treatment start times

[0076]

[0077] Note: Different lowercase letters indicate significant differences ( p <0.05).

[0078] (4) Observation of hydrostatic shock inhibition of cleavage: To verify whether hydrostatic shock treatment successfully inhibits cleavage of fertilized eggs, an experiment was conducted. The fertilized eggs of the same pair of broodstock were treated as follows: the fertilized eggs were divided into 4 portions, each with 100 fertilized eggs. Three of them were subjected to different shock treatments 42.5 minutes after fertilization: double hydrostatic shock (6000psi 1min×2 intervals of 5min), single hydrostatic shock (6000psi 2.5min), and cold shock (4℃ 20min). The remaining portion was not treated as a control group. After treatment, the cleavage of fertilized eggs in each group was continuously observed, and the proportion of fertilized eggs reaching the one-, two-, three-, and four-cell stages in the first three cleavage cycles was counted.

[0079] The results of the control group showed that the first cleavage cycle was 65 minutes, and the second and third were 20 minutes. The second cleavage of 43% of the fertilized eggs in the double hydrostatic shock was inhibited and stayed in the second cell (as shown in Table 4), while the single hydrostatic shock was only 10%. The cold shock treatment inhibited the development of 20 minutes of fertilized eggs, simulating the inhibition of the cleavage cycle by hydrostatic shock. Subsequent observations showed that the cold shock group gradually recovered the normal cycle, and the development speed caught up with the normal fertilized eggs in the control group, while some fertilized eggs in the double hydrostatic shock group always lagged behind by one cycle, which shows that the double hydrostatic shock inhibited the second cleavage, and its inhibition rate was higher than that of the single hydrostatic shock, which may also be the reason for its high tetraploid induction rate.

[0080] Table 4. The proportion of embryos at different stages in fertilized eggs at different times

[0081]

[0082] Example 3

[0083] (1) Mating of largemouth bass broodstock and collection of fertilized eggs

[0084] Twenty pairs of selectively mature largemouth bass broodstock were raised in a 30-cubic-meter canvas pool with a sunshade net, the water temperature was maintained at 22°C, light was avoided, and running water was maintained to stimulate estrus. An underwater camera was tied to the artificial fish nest and placed at the bottom of the pool, and the behavior of the broodstock was continuously observed through the screen connected to the camera. After the female fish released eggs, the male fish released sperm synchronously, and the fertilized eggs adhered to the gauze membrane of the artificial fish nest. At this time, the artificial fish nest was quickly picked up and the timing was started from the start time of fertilization, and the gauze membrane with fertilized eggs was carefully collected.

[0085] (2) Double hydrostatic shock treatment

[0086] The water temperature of the site was measured to be 22°C. The first cleavage time T0 of the fertilized egg at 22°C was 65 minutes after fertilization, which was faster than the first cleavage time of the fertilized egg at 20°C, which was 85 minutes. The best time to treat it would also be advanced. It is known that the best time to treat at 20°C is 55 mpf. According to the formula: 55×65 / 85≈42 mpf, the best time to treat at 22°C is 42 minutes after fertilization. The interval time of double hydrostatic pressure is also shortened to 7.5 minutes according to the formula: 10×65 / 85≈7.5 minutes. The fertilized egg was placed in the hydrostatic pressure instrument. At 42 minutes after fertilization, the pressure was increased to 6000 psi, and the pressure was released after 1 minute of treatment. After an interval of 7.5 minutes, the pressure was increased to 6000 psi again, and the pressure was reduced after 1 minute of treatment. The pressure increase and decrease speed was maintained at 300 psi / s.

[0087] (3) Hatching of fertilized eggs and rearing of fry

[0088] The fertilized eggs were placed in 22℃ water for aeration and incubation, and anti-mildew drugs were sprayed. During this period, the whitish dead eggs were sucked out every 12 hours to prevent the dead eggs from rotting and mildewing, which would have an adverse effect on the live eggs. The water quality was also measured. After 48 hours, the fry broke the membrane and hatched. After 5 days, the fry completely absorbed the yolk and floated to the surface. The fry were transferred to the open pond to be fed with Artemia.

[0089] (4) Calculation of fertilized eggs, hatching rate, and tetraploidy rate

[0090] The fertilization rate is the ratio of the number of dead fertilized eggs 6 hours after fertilization to the total number of fertilized eggs, the hatching rate is the ratio of the number of fry hatched from fertilized eggs to the total number of fertilized eggs, and the tetraploidy rate is the ratio of the number of tetraploid fry detected to the total number of fry detected. Ploidy identification was performed using a flow cytometer. The fry were taken 2 days after hatching, rinsed with distilled water, and frozen in a 5% DMSO solution. After thawing, the fry were injected into a 1.5mL centrifuge tube containing 0.5mL PI (Biosharp) dye solution (75μmol / L) through a 1mL syringe. After staining in the dark for 30min, the tube was tested on the machine. Diploid fry were used for comparison to determine whether they were tetraploid, and the tetraploid ratio was calculated.

[0091] In this embodiment, 5 batches of fertilized egg induction were carried out, and a total of about 25,000 fry were obtained, with a hatching rate of 10.5% and a tetraploid rate of 22.3%.

[0092] Example 4

[0093] This embodiment is basically the same as the embodiment 3, except that the parameters of the double hydrostatic shock treatment in the method for producing tetraploid largemouth seabass by double hydrostatic shock are:

[0094] The fertilized eggs were placed in a hydrostatic pressure instrument and the pressure was increased to 6000 psi. After 1 minute of treatment, the pressure was released. After an interval of 5 minutes, the pressure was increased to 6000 psi again and treated for 1 minute before the pressure was lowered. The rate of pressure increase and decrease was maintained at 200 psi / s.

[0095] The method described in this embodiment can inhibit the cleavage of fertilized eggs and induce the formation of tetraploid largemouth bass.

[0096] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for producing tetraploid largemouth bass by inducing double hydrostatic shock, characterized in that: The following steps are involved: S1. Obtaining fertilized eggs obtained by natural fertilization of largemouth bass: using an artificial fish nest connected to an underwater camera to collect fertilized eggs obtained by natural fertilization of largemouth bass; the underwater camera is used to observe the spawning process and determine the fertilization time of the fertilized eggs, and the artificial fish nest is a bowl-shaped structure covered with a gauze membrane that is easy to adhere to; S2. Within 50 to 65 minutes after fertilization, the fertilized egg is subjected to double hydrostatic shock treatment; the double hydrostatic treatment parameters are 6000 psi treatment for 1 minute, slowly decompressing to normal pressure, and then 6000 psi treatment for 1 minute after an interval of 10 minutes; S3. The fertilized eggs treated with double hydrostatic pressure were hatched to obtain tetraploid largemouth bass.

2. The seed production method according to claim 1, characterized in that: The step S2 is to apply double hydrostatic shock treatment to the fertilized egg within 55 to 60 minutes after fertilization.

3. The seed production method according to claim 1, characterized in that: The pressure increase rate during the double hydrostatic pressure treatment in step S2 is 200-400 psi / s.

4. The seed production method according to claim 1, characterized in that: The temperature of the double hydrostatic shock treatment in step S2 is 20-22°C.

5. The seed production method according to claim 4, characterized in that: The water temperature is kept constant during the double hydrostatic shock treatment, with fluctuations not exceeding 0.5°C.

6. The seed production method according to claim 1, characterized in that: The pressure change rate of the pressure relief in step S2 is 200-400 psi / s.

7. Application of the method according to any one of claims 1 to 6 in triploid breeding of largemouth bass, characterized in that: The tetraploid largemouth bass obtained by the method described in any one of claims 1 to 6 is subjected to inter-polyploid hybridization with the diploid largemouth bass to obtain the triploid largemouth bass.

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