Application of sperms of river perches in inducing gynogenesis of micropterus salmoides

Abstract

The invention belongs to the field of fish germplasm improvement and innovation, and discloses an application of river perch sperms in inducing gynogenesis of largemouth bass, which comprises the following steps: obtaining mature largemouth bass ova and river perch sperms through artificial spawning induction; the method comprises the following steps: collecting river perch semen, diluting, oscillating, irradiating and inactivating with an ultraviolet lamp, mixing with mature largemouth bass ova, inseminating, and hatching fertilized ova to obtain gynogenesis largemouth bass. According to the method, the sperms of the river perches are used for stimulating gynogenesis of the micropterus salmoides for the first time, the gynogenesis micropterus salmoides is successfully obtained, and the heterologous sperms in the far geographic space are used as the stimulation source of the artificial gynogenesis ovum for the first time. The method not only provides an innovative germplasm resource basis in the aspect of improving germplasm of the micropterus salmoides, but also has important significance in the research aspects of fish genetic breeding and'micro-hybridization among fish species in far geographic space '.

Description

Technical Field

[0001] The present invention belongs to the field of fish germplasm improvement and innovation, and particularly relates to the application of perch sperm in inducing gynogenesis of largemouth bass. Background Art

[0002] The largemouth bass ( Micropterus salmoides ), belonging to Perciformes ( Perciformes ), Percoidei ( Percoidei ), Centrarchidae ( Centrachidae ), Micropterus ( Micropterus ), also known as California bass, black bass or large-mouth bass, is native to the Mississippi River system in North America and has a chromosome number of 2n = 46. It was introduced into Guangdong Province, China in 1983. Due to its rapid growth, short breeding cycle, delicious meat and no intermuscular spines, it is highly favored by consumers and has now formed large-scale farming in areas such as Guangdong, Jiangsu, Zhejiang, Sichuan and Fujian in China. Currently, the largemouth bass ranks 16th in the world in terms of the output value of freshwater aquaculture fish (FAO), and the demand for largemouth bass in China is very large, ranking among the top in the latest fishery aquaculture fish. However, in recent years, due to factors such as increasing aquaculture density, deteriorating water environment, and germplasm degradation, and the largemouth bass is an introduced foreign species, restricted by the introduced varieties and quantities, the genetic diversity of germplasm resources is seriously insufficient, and there is an urgent need to expand the germplasm in the process of largemouth bass farming to solve the industrialization bottleneck problem.

[0003] The perch ( Perca fluviatilis ), belonging to Perciformes, Percoidei, Percidae ( Percide ), Perca ( Perca ), also known as red perch or five-striped black, is widely distributed in various parts of Europe and the northern Asian basins. In China, it is mainly distributed in the Wulungu River and Irtysh River basins in Xinjiang in the northwest region. Through artificial introduction or spread, it is currently also distributed in some rivers in the northeast region of China, with a chromosome number of 2n = 48. The perch has a fast growth rate, its taste is smooth, with few spines, the taste is delicious, the meat quality is excellent, it contains only about 1% fat, and is rich in essential amino acids for the human body and high-content meat protein, with high nutritional value, and is deeply loved by consumers and farmers, and is one of the important international economic famous and excellent fish.

[0004] Artificial gynogenesis technology is of great significance in fish genetic breeding and is an important way for fish genetic improvement. Generally speaking, artificial induction of gynogenesis refers to the use of experimental means to enable sperm to activate the development of eggs, but the sperm nucleus does not fuse with the egg nucleus, and the eggs develop into embryos or individuals relying only on the genetic material of the female parent. In the laboratory of the present invention, multiple new fish species have been created through artificial gynogenesis technology for a long time. Through in-depth research on the offspring and breeding results, it has been found that the genetic composition of gynogenetic offspring not only has all the genetic information from the female parent, but also has chromosomes or DNA fragments of heterologous sperm. These infiltrated heterosperm genetic fragments can affect the phenotypes of gynogenetic individuals, especially in traits such as growth rate, meat quality, and disease resistance, showing a "micro-hybridization" effect. However, during the implementation of artificial induction of gynogenesis, the selection of heterologous sperm is the key to preparing a gynogenetic population. The selection of appropriate heterologous sperm requires a certain degree of affinity between the two parents and a large geographical distance. At the same time, during the experimental process, there are too many subjective human operations, especially in steps such as the period of inducing the parents to spawn, the selection and inactivation treatment degree of heterologous sperm, the cold treatment temperature and time of fertilized eggs, etc. These steps are also difficult problems in creating a gynogenetic population.

[0005] Therefore, aiming at the innovation of the germplasm of Micropterus salmoides and the technical operation problems of the gynogenesis technology of Micropterus salmoides, cultivating new gynogenetic germplasm of Micropterus salmoides with good meat quality and strong stress resistance has great significance in production applications. Summary of the Invention The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide an application of perch sperm in inducing gynogenesis of Micropterus salmoides.

[0006] To solve the above technical problem, the technical solution proposed by the present invention is: An application of perch sperm in inducing gynogenesis of Micropterus salmoides, comprising the following steps: (1) Obtain mature eggs of Micropterus salmoides and perch semen through artificial spawning induction; (2) Dilute the collected perch semen with pre-cooled Hank's solution, spread the dilution to form a liquid thin layer, perform oscillation treatment, and at the same time irradiate with an ultraviolet lamp for inactivation treatment; (3) Mix the perch semen after inactivation treatment in step (2) with the mature eggs of Micropterus salmoides in step (1) for fertilization, and then incubate the fertilized eggs with normal temperature water. The hatched normal fry are gynogenetic Micropterus salmoides.

[0007] The present invention utilizes the embryonic development characteristics of the hybrid embryos obtained by crossing heterologous sperm from different geographical spaces with the eggs of Micropterus salmoides to determine the affinity between the hybrid parents, so as to select suitable artificial gynogenetic heterologous sperm sources. Finally, using the sperm of Perca fluviatilis as the stimulator has obvious advantages. First, Perca fluviatilis and Micropterus salmoides belong to the same order Perciformes and suborder Percoidei, but different families. The sperm of Perca fluviatilis can activate and initiate the development of the eggs of Micropterus salmoides, but viable hybrid fry cannot be obtained, which is convenient for the identification of the obtained gynogenetic fry. Second, there is a strong reproductive isolation between Perca fluviatilis and Micropterus salmoides in geographical space. Using fish species from distant geographical spaces as the heterologous sperm sources for artificial gynogenesis is more likely to achieve the effect of germplasm improvement. Perca fluviatilis has delicious meat, low fat content, strong disease resistance and cold tolerance. The gynogenetic Micropterus salmoides fry obtained using the sperm of Perca fluviatilis as the inducer has strong cold tolerance, and the adult fish has a lower fat content than the commercial feed bass, and a high content of essential amino acids for the human body. This is the first time to stimulate the gynogenesis of Micropterus salmoides with the sperm of Perca fluviatilis and successfully obtain gynogenetic Micropterus salmoides, and it is also the first time to use heterologous sperm from a distant geographical space as the stimulator for artificial gynogenetic eggs. This not only provides an innovative germplasm resource basis for improving the germplasm of Micropterus salmoides, but also has important significance in the research of fish genetic breeding and "interspecific micro-hybridization of fish in distant geographical spaces".

[0008] For the above application, preferably, in step (1), the specific operation steps for obtaining mature eggs of Micropterus salmoides and sperm of Perca fluviatilis are as follows: Select the same-aged Micropterus salmoides of age I or II and male Perca fluviatilis as parents for intensive cultivation, and induce spawning of female Micropterus salmoides and male Perca fluviatilis during the peak production period of Micropterus salmoides to obtain mature eggs and semen.

[0009] Preferably, in step (1), the specific steps for artificial spawning induction are as follows: During the peak self-spawning period of Micropterus salmoides, inject a mixed spawning inducer of LRH-A2 and DOM into the broodstock for spawning induction. The injection amount of LRH-A2 for female Micropterus salmoides is 10 - 12 μg / kg, the injection amount of DOM is 2.5 mg / kg, and the effective time is 24 - 26 hours; at the end of the self-spawning period of Micropterus salmoides, a combination of HCG, LRH-A2 and DOM is used as the spawning inducer, and the injection amounts are 1500 - 2000 - IU / kg, 10 - 12 μg / kg, and 2.5 mg / kg respectively, and the effective time is 36 - 48 hours; the injection amount of LRH-A2 for male Perca fluviatilis is 5 - 6 μg / kg.

[0010] Preferably, in step (2), the precooling temperature of Hank's is 4 - 6°C. Each liter of Hank's solution contains 1.36 M NaCl, 12.6 mM CaCl2, 53.6 mM KCl, 4.93 mM MgCl2, 4.07 mM MgSO4, 4.41 mM KH2PO4, 41.66 mM NaHCO3, 3.36 mM Na2HPO4, and 55.5 mM D - Glucose. It is made up to 1 L and stored in a 4°C refrigerator. The Hank's solution and semen are diluted at a volume ratio of 15 - 20∶1.

[0011] Preferably, in step (2), the thickness of the liquid thin layer is 2 - 3 mm.

[0012] Preferably, in step (2), the shaking speed of the shaker for the shaking treatment is 100 - 130 r / min.

[0013] Preferably, in step (2), the specific steps of the inactivation treatment are as follows: Inactivation treatment is carried out by irradiating with a 40W ultraviolet lamp. The distance between the ultraviolet lamp and the shaker is 18 - 20 cm. The inactivation treatment time is based on the sperm motility observed under the microscope being 10% - 15% of the motility before inactivation, and the approximate treatment time is 5 - 7 min.

[0014] The degree of sperm inactivation, cold shock temperature, and treatment time are the key to cultivating gynogenetic largemouth bass. The present invention compared the sperm motility at different gradients and found that the sperm motility of 10% - 15% of the motility before inactivation had the best effect. The sperm in this state was slightly jittery when activated with water under microscopic examination. By directly fertilizing largemouth bass eggs without any chromosome doubling treatment, it was statistically found that the fertilization rate reached about 90%, and the formed fry were all haploid (showing haploid syndrome phenotypes such as bent tails and short tails). At the same time, the present invention also conducted experiments by controlling the cold shock treatment as a single variable and found that it was most suitable to cold - treat the fertilized eggs for 10 - 12 min at a water temperature of 5 - 7°C. If the cold shock treatment time is too long or the water temperature is too low, the cold stress damage to the fertilized eggs is too strong and it is not easy to cross the gastrula stage; if the cold shock treatment time is too short or the water temperature is too high, it is easy to form haploid fry.

[0015] Preferably, in step (3), the specific steps of the fertilization are as follows: Put it into normal - temperature water and stir for fertilization for 2 - 4 min, and then put the fertilized eggs into cold water at 5 - 7°C for cold shock for 10 - 12 min.

[0016] Preferably, in step (3), when the fertilized eggs are hatched, the embryos that stop developing are removed regularly to maintain the microenvironment of the hatching water body. The normal fry hatched in the culture dish are transferred to a fry storage basin (the size of the fry storage basin is 50 cm × 30 cm × 30 cm, and each basin can hold 500 fry). When the hatched fry can swim horizontally, the fry are transferred to a fry acclimation pond for cultivation and acclimation. After the acclimation is completed, they are transferred to a earthen pond for feeding.

[0017] Preferably, the acclimation specifically includes the following steps: the volume of the fry acclimation pond is 1 m × 1 m × 1.5 m. Create a dark environment in the fry acclimation pond, install a light above the middle of the pond (the illumination range is 1 / 3 of the water surface area), and install an oxygen pump device and a flowing water device; when feeding, turn on the light and the flowing water, sprinkle live small plankton collected from the pond onto the illuminated area, and knock on an object to make a sound. After the fry have aggregated and fed in the illuminated area, turn off the flowing water and the light, and one feeding is completed; feed 3 - 4 times a day. In the first 2 - 3 days, feed on live small plankton. After the fry form a conditioned reflex (when the light is turned on, the sound is knocked, or the flowing water is turned on, the female nucleus development bass fry aggregate in the illuminated area waiting for feeding), gradually add floating feed, and the acclimation of the female nucleus development bass fry can be completed in 6 - 8 days.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The method of the present invention uses perch as heterologous sperm to successfully induce the female nucleus development of largemouth bass to obtain offspring of female nucleus development largemouth bass, and optimizes the artificial operation steps in the female nucleus development experiment. About 1000 offspring of female nucleus development can be obtained in each batch (2 female parents), providing feasibility and convenience for subsequent acclimation (aggregation effect).

[0019] 2. The present invention compares the induced spawning effects before and after the self - spawning stage of largemouth bass, and summarizes the advantages and disadvantages of egg quality in different stages, laying a good foundation for obtaining high - quality eggs by artificial induced spawning.

[0020] 3. Perch is a cold - water fish, which has a fast growth rate, smooth taste, few thorns, delicious taste, excellent meat quality, only contains about 1% fat, and is rich in essential amino acids for the human body and high - content meat protein, with high nutritional value. By using perch as heterologous sperm to carry out the female nucleus development of largemouth bass in a "micro - hybridization" female nucleus development method, the cold tolerance of the hatched female nucleus development fry is significantly enhanced, and the growth rate of the acclimated largemouth bass is fast, and its fat content is significantly lower than that of largemouth bass fed with ordinary feed. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 are the real-time characteristics of the embryonic development of the hybridization of different geospatial allosperm and Micropterus salmoides; Figure 2 is the external view of gynogenetic Micropterus salmoides; Figure 3 is the external view of Perca fluviatilis; Figure 4 is the external view of common Micropterus salmoides; Figure 5 is the electrophoresis map of 5S rDNA PCR amplification of common perch, gynogenetic perch and Perca fluviatilis; Figure 6 is the flow cytometry graph of Perca fluviatilis; Figure 7 is the chromosome number of Perca fluviatilis; Figure 8 is the flow cytometry graph of common Micropterus salmoides; Figure 9 is the chromosome number of common Micropterus salmoides; Figure 10 is the flow cytometry graph of gynogenetic Micropterus salmoides; Figure 11 is the chromosome number of gynogenetic Micropterus salmoides; Figure 12 is the embryonic development map of hybridization and gynogenesis. Detailed implementation manners

[0023] To facilitate the understanding of the present invention, the following will describe the present invention more comprehensively and meticulously in combination with the accompanying drawings of the specification and the preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0024] Unless otherwise defined, all the technical terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0025] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments and equipment used in the present invention can be obtained through the market or can be prepared by existing methods.

[0026] Appropriate heterologous semen and high-quality mature eggs are prerequisites for the gynogenesis of Micropterus salmoides. In the present invention, heterologous sperm from different spatial and geographical locations were collected and hybridized with Micropterus salmoides eggs, and the affinity between the hybrid parents was judged by observing the characteristics of embryonic development in real time, so as to select a suitable heterologous sperm source for artificial gynogenesis of Micropterus salmoides - Perca fluviatilis. This is the first time to realize the use of heterologous sperm from a far geographical space as a stimulant for artificial gynogenetic eggs.

[0027] Perca fluviatilis and Micropterus salmoides both belong to Perciformes, Percoidei, and they are in different families. Through preliminary experiments, with Micropterus salmoides as the female parent and Perca fluviatilis as the male parent for hybridization, it was found that the sperm of Perca fluviatilis could activate the eggs of Micropterus salmoides, but the fertilized eggs could not cross the gastrula and died; at the same time, the present invention also carried out hybridization preliminary experiments between different orders and suborders such as Cyprinidae fish (Megalobrama amblycephala, Erythroculter ilishaeformis, Cyprinus carpio, etc.), Tilapia (Perciformes, Labroidei), and found that these heterologous sperm could not activate the eggs of Micropterus salmoides, so the gynogenetic breeding of Micropterus salmoides could not be carried out. Therefore, the sperm of Perca fluviatilis can activate the eggs of Micropterus salmoides and cannot form viable offspring, which is a suitable heterologous sperm for the gynogenetic breeding of Micropterus salmoides. At the same time, the present invention compared the induced spawning effects in the pre-spawning period, peak spawning period, and late spawning period of self-produced Micropterus salmoides, and found that the egg quality was the best, the normal spawning rate was the highest, and the spawning amount was the largest in the peak spawning period of self-produced Micropterus salmoides.

[0028] Example: From April to June 2024, in the present invention, sperm of Perca fluviatilis (2n = 48) was selected as a stimulant from many heterologous sperm source fish species, and an artificial gynogenesis experiment was carried out on Micropterus salmoides (2n = 46), and a utilization method of heterologous sperm applied to the germplasm improvement of Micropterus salmoides was explored. The semen of Perca fluviatilis of the present invention and the eggs of Micropterus salmoides were obtained through artificial induced spawning.

[0029] An application of sperm of Perca fluviatilis in inducing gynogenesis of Micropterus salmoides of the present invention includes the following steps: (1) Collection of parents: Before winter of the current year, sexually mature heterologous fish species from all over the country were collected, including traditional Cyprinidae fish in Hunan area such as Carassius auratus, Cyprinus carpio, Erythroculter ilishaeformis, Megalobrama amblycephala, etc., Tilapia in Guangxi area, Perca fluviatilis in Xinjiang area, Siniperca chuatsi in Hunan area, and Lepomis cyanellus in Guangdong area and other Perciformes fish, Ictalurus punctatus in Guangdong area, Pelteobagrus fulvidraco in Hubei area and other Siluriformes fish.

[0030] (2)Artificial induced spawning: During the peak spawning period of Micropterus salmoides in the breeding base of the current year (from mid-April to the end of April), a mixed inducing agent of luteinizing hormone releasing hormone analogue (LRH-A2) and domperidone (DOM) was injected into the broodstock for induced spawning. The injection amount of LRH-A2 for female Micropterus salmoides was 10 - 12 μg / kg, the injection amount of DOM was 2.5 mg / kg, and the effective time was 24 - 26 hours; at the end of the natural spawning period of Micropterus salmoides, a combination of HCG, LRH-A2 and DOM was used as the inducing agent, and the injection amounts were 1500 - 2000 - IU / kg, 10 - 12 μg / kg, and 2.5 mg / kg respectively, and the effective time was 36 - 48 hours; the injection amount of LRH-A2 for male heterologous fish species was 5 - 6 μg / kg.

[0031] (3)Selection of the source of heterologous sperm for artificial gynogenesis: The collected mature male heterologous semen was mixed with mature high-quality eggs of Micropterus salmoides respectively for artificial insemination, and then the embryonic development of each combination was observed in real time ( Figure 1 ), and the appropriate source of heterologous sperm for artificial gynogenesis of Micropterus salmoides was selected based on the embryonic development characteristics and the degree of geographical isolation between the two parents. Finally, Perca fluviatilis was selected as the heterologous sperm source fish species for the artificial gynogenesis of Micropterus salmoides.

[0032] (4)Collection and inactivation of Perca fluviatilis semen: When the water temperature was 18℃ - 20℃, the effective time of the inducing drug for Micropterus salmoides was about 24 hours. When the effective time of the drug was reached, the abdomen of the female Micropterus salmoides was gently squeezed to extrude a small amount of egg grains for preliminary inspection of the egg quality. The best egg grains were transparent, round and semi-viscous, and then the gynogenesis experiment could be carried out: First, collect Perca fluviatilis semen, dilute it with Hank’s at a volume ratio of 1:15, spread the diluted semen evenly in a pre-cooled culture dish to a thin layer (2 - 3 mm), place it on a shaker with an ice plate, the rotation speed of the shaker was 100 - 130 r / min, and at the same time, inactivate it with a 40W ultraviolet lamp. The distance between the ultraviolet lamp and the shaker was 18 - 20 cm, and the inactivation time was approximately 5 - 7 min. The sperm motility was observed under an optical microscope to judge the degree of sperm inactivation, and it was observed every 30 s - 60 s. When the sperm motility was 10% - 15% of the motility before inactivation (under microscopic examination, the activated sperm trembled slightly in place), it was okay. The inactivated semen was stored in a light-proof tube at 4℃; Formulation of Hank’s solution: Each liter of Hank’s solution contains 1.36M NaCl, 12.6mM CaCl2, 53.6mM KCl, 4.93mM MgCl2, 4.07mM MgSO4, 4.41mM KH2PO4, 41.66mM NaHCO3, 3.36mM Na2HPO4, 55.5mM D-Glucose, and the volume was made up to 1L and stored in a 4℃ refrigerator.

[0033] (5) Egg activation and cold treatment: Squeeze the eggs of Micropterus salmoides into a clean, dry and smooth iron basin, add the inactivated semen of Perca fluviatilis, gently stir with a feather, then add normal temperature water to fertilize them. Then lay them flat in a petri dish containing normal temperature water for fertilization for 2 - 4 minutes. Then replace the normal temperature water in the petri dish with cold water at 5 - 7 °C for cold shock treatment for 10 - 12 minutes. Then replace the cold water in the petri dish with normal temperature water for static incubation. The normal fry hatched are the gynogenetic Micropterus salmoides ( Figure 2 ).

[0034] (6) Rearing: When the fry hatched can swim horizontally, transfer the fry to a fry rearing pond for cultivation and domestication. After domestication is completed, transfer them to a soil pond for rearing.

[0035] The process of domesticating gynogenetic Micropterus salmoides fry specifically includes the following steps: The volume of the fry rearing pond is 1m × 1m × 1.5m. Create a dark environment in the fry rearing pond, install a light above the middle of the pond (the illumination range is 1 / 3 of the water surface area), and install an oxygen pump device and a flowing water device. When feeding, turn on the light and the flowing water, sprinkle live small plankton such as water fleas collected from the pond onto the illuminated area, and make a sound by knocking on an object. After the fry have clustered and fed in the illuminated area, turn off the flowing water and the light, and one feeding is completed. Feed 3 - 4 times a day. In the first 2 - 3 days, feed on live small plankton. After the fry form a conditioned reflex (when the light is on, the sound is knocked or the flowing water is turned on, the Micropterus salmoides fry cluster in the illuminated area waiting to be fed), gradually add floating feed, and the domestication of gynogenetic Micropterus salmoides fry can be completed in 6 - 8 days.

[0036] Before the gynogenetic breeding experiment of Micropterus salmoides, the present invention evaluated the egg quality of Micropterus salmoides at different stages, as shown in Table 1.

[0037] Table 1: Induced spawning output and egg quality of Micropterus salmoides at different stages (peak and end of natural spawning period)

[0038] Note: Egg quality is comprehensively evaluated by the fertilization rate and seedling emergence rate of artificially self-fertilized eggs in practice As shown in Table 1, during the peak period of natural spawning of Micropterus salmoides, its egg quality is the best and the quantity is the largest.

[0039] Meanwhile, in order to find a suitable sperm stimulation source for gynogenesis of Micropterus salmoides, the present invention conducted hybridization experiments on combinations of Oreochromis niloticus, Siniperca chuatsi, Perca fluviatilis, Pelteobagrus fulvidraco, Ictalurus punctatus and Cyprinidae (Cyprinus carpio, Megalobrama amblycephala and Erythroculter ilishaeformis, etc.) × Micropterus salmoides. The observation criteria for preliminary judgment of hybridization affinity by whether different heterologous sperms can initiate the embryonic development of Micropterus salmoides eggs are shown in Table 2: Table 2: Fertilization rate, gastrulation rate and seedling emergence rate of different heterologous sperms and Micropterus salmoides eggs

[0040] Perca fluviatilis ( Figure 3 ) and Micropterus salmoides ( Figure 4 ) both belong to the order Perciformes, suborder Percoidei. They are from different families. Through preliminary experiments, hybridization was carried out with Micropterus salmoides as the female parent and Perca fluviatilis as the male parent. It was found that the sperm of Perca fluviatilis could activate the eggs of Micropterus salmoides, but the fertilized eggs could not pass through gastrulation and died. At the same time, the present invention also conducted preliminary hybridization experiments between different orders and suborders, such as Cyprinidae fish (Megalobrama amblycephala, Erythroculter ilishaeformis, Cyprinus carpio, etc.), Tilapia (order Perciformes, suborder Labroidei), and Siluriformes fish (Pelteobagrus fulvidraco and Ictalurus punctatus). It was found that these heterologous sperms could not activate the eggs of Micropterus salmoides, so gynogenesis breeding of Micropterus salmoides could not be carried out. Therefore, the sperm of Perca fluviatilis can activate the eggs of Micropterus salmoides, and no offspring can be formed by hybridization. It is a suitable heterologous sperm for gynogenesis breeding of Micropterus salmoides.

[0041] During the actual operation of artificial gynogenesis, the present invention optimized the experimental conditions of artificial gynogenesis. This includes the fertilization rate and hatching rate of the eggs of Micropterus salmoides by sperm with different gradient viabilities, as shown in Table 3: Table 3: Fertilization rate and hatching rate of the eggs of Micropterus salmoides by sperm with different gradient viabilities during the actual operation of artificial gynogenesis

[0042] As shown in Table 3, during the actual operation of gynogenesis of Micropterus salmoides, the optimal sperm viability is 10% - 15% of the viability before inactivation.

[0043] At the same time, the present invention also compared the hatching rate and survival rate under different gradient cold treatment temperatures, as shown in Table 4: Table 4: Hatching rate and survival rate under different gradient cold treatment temperatures during the actual operation of artificial gynogenesis

[0044] As shown in Table 4, the optimal cold shock temperature is 5°C - 7°C. If the temperature is too low, the damage to the eggs is too large and the gastrulation rate is low; if the temperature is too high, the efficiency of inhibiting the extrusion of the second polar body is low and haploid lethality is likely to occur.

[0045] The present invention statistically analyzed the survival rates of the gynogenesis seedling population and the self-crossed seedling population before and after "spring cold snap", as shown in Table 5.

[0046] Table 5: Survival rates of the gynogenesis seedling population and the self-crossed seedling population before and after "spring cold snap"

[0047] As shown in Table 5, the cold stress tolerance of the self-crossed population seedlings is lower than that of the gynogenesis population seedlings. The survival rate of the self-crossed population seedlings is 45.55%, and the survival rate of the gynogenesis population seedlings is 71.54%.

[0048] Four months after fry rearing, the present invention detected the measurable traits of gynogenetic largemouth bass and compared them with the parents, as shown in Table 6.

[0049] Table 6: Meristic traits of common largemouth bass, perch and gynogenetic largemouth bass

[0050] The present invention used 5s rDNA molecular specific markers to identify the germplasm of gynogenetic perch. It was found that the 5s rDNA electrophoresis bands of gynogenetic largemouth bass were consistent with those of the parental largemouth bass, and were significantly different from the 5s rDNA bands of the parental perch.

[0051] 5s rDNA molecular specific marker method: Cut the caudal fins of common largemouth bass, gynogenetic largemouth bass and perch respectively, and extract genomic DNA using a kit. Use 5s rDNA specific primers (5s-F: 5’-CAGGTTGGTATGGCCGTAAGC-3’ and 5S-R: 5’-GCTATGCCCGATCTCGTCTGA-3’) for PCR amplification reaction. The reaction conditions were pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 59.2 °C for 30 s, extension at 72 °C for 40 s, final extension at 72 °C for 10 min, and 35 cycles were experienced. The PCR products were separated by 1.5% agarose gel electrophoresis, linked to the Pmd18-T vector after purification, and single clone colonies were picked for Sanger sequencing. The electrophoresis results are compared as Figure 5 shown. Three types of 5S rDNA bands were obtained for common largemouth bass, two types of 5S rDNA were cloned for perch, and the electrophoresis results of gynogenetic largemouth bass were all consistent with those of common largemouth bass.

[0052] Meanwhile, the present invention used common largemouth bass as a reference and detected the DNA contents of gynogenetic largemouth bass and perch by flow cytometry. Figure 6 、 Figure 8 and Figure 10 are the flow cytometry diagrams of perch, common largemouth bass and gynogenetic largemouth bass respectively. The ratio of the average DNA content of gynogenetic largemouth bass to that of common largemouth bass is 1, and the ratio to the average DNA content of perch is not 1, preliminarily verifying that gynogenetic largemouth bass is diploid like common largemouth bass and the genetic material comes from common largemouth bass.

[0053] The steps of flow cytometry DNA content determination method are as follows: Collect about 0.01 ml of blood from the caudal vein of the fish with a disposable syringe containing 0.1 ml of anticoagulant ACD. Take 1 - 2.5 μL of blood sample and dissolve it into an Eppendorf tube containing 300 - 500 μL of DAPI solution. Leave it in the dark to stain the sample for about 5 - 10 min, and then perform the detection on the machine.

[0054] In order to further determine the ploidy of gynogenetic largemouth bass, in the present invention, chromosome preparations were respectively made for common largemouth bass, gynogenetic largemouth bass and perch. Figure 7 、 Figure 9 and Figure 11 They are respectively the chromosome maps of perch, common largemouth bass and gynogenetic largemouth bass. The number of chromosomes of perch is 48, and the number of chromosomes of gynogenetic largemouth bass is the same as that of common largemouth bass, which is 46.

[0055] The steps of the kidney cell chromosome preparation method are as follows: Inject phytohemagglutinin (PHA) into the experimental fish twice at a dose of 1 mg / mL. The injection interval is 12 - 24 h. 2 - 6 h before dissection, inject colchicine into each sample at a dose of 4 - 6 μg / g. Grind the kidney tissue with 0.8% NaCl, then perform hypotonic treatment with 0.075 M KCl at 37°C for 40 - 60 min, and then fix it 3 times in methanol - acetic acid with a ratio of 3:1. Drop two to three drops of cell solution onto a pre - cooled glass slide and dry it over a flame, and then stain it with Giemsa stain for 1 hour. Observe and photograph the metaphase chromosomes with Pixera Pro 600ES (US).

[0056] Artificial gynogenesis technology has great significance in fish breeding and is an important way for fish genetic improvement. Artificial gynogenesis refers to the technology that uses experimental means to enable sperm to activate the development of eggs, but the sperm nucleus does not fuse with the egg nucleus, and the eggs develop into embryos or individuals only relying on the genetic material of the female parent. Generally speaking, the genetic information of gynogenetic individuals comes from the female parent, and the sperm storing the genetic information of the male parent only participates in the activation of eggs during the embryonic development of eggs, and the genetic information in the sperm does not participate in the composition of the genetic material of the offspring. However, more and more evidence proves that sperm also makes a certain contribution to the genetic material composition of gynogenetic offspring. They are not only all - female populations, but also some traits of allogynogenetic offspring are significantly different from those of the female parent, such as growth rate, body shape, body color, isoenzymes, etc., and this phenomenon is called "heterosperm biological effect", which is a scientific means with strong practicability and has potential application value in aspects such as producing pure lines, chromosome manipulation, genetic analysis and sex control.

[0057] Micropterus salmoides was introduced into China in the 1980s and has been highly favored. However, in recent years, due to factors such as increasing aquaculture density, deteriorating water environment, and germplasm degradation, and since Micropterus salmoides is an introduced foreign species, restricted by the varieties and quantities introduced, the genetic diversity of its germplasm resources is seriously insufficient. There is an urgent need to expand the germplasm during the aquaculture process of Micropterus salmoides to solve its industrialization bottleneck problem. Using different heterologous sperm to stimulate the gynogenesis of Micropterus salmoides to expand the germplasm and solve its industrialization bottleneck problem is an extremely effective way. The present invention collects heterologous fish species from different geographical spaces, explores a set of methods to judge the affinity between heterologous sperm and eggs based on embryonic development characteristics, and combines the degree of geographical reproductive isolation between the parents. It selects Perca fluviatilis as the stimulating source for the gynogenesis of Micropterus salmoides, which has multiple advantages. First, Perca fluviatilis itself has a fast growth rate, its taste is smooth, with few spines, delicious, the meat quality is excellent, it contains only about 1% fat, and is rich in essential amino acids for the human body and high-content meat protein, with high nutritional value. Through the "micro-hybridization" method of heterologous sperm gynogenesis, the germplasm of Micropterus salmoides can be improved in terms of meat quality and growth rate. Second, Perca fluviatilis belongs to cold-water fish, while the fry of Micropterus salmoides is not cold-tolerant. Especially in spring, the frequent occurrence of cold snaps in spring is extremely likely to cause a large number of stress deaths of fry. The gynogenetic Micropterus salmoides fry developed with Perca fluviatilis semen as the stimulating source has significantly improved resistance and cold tolerance. Third, the convenience of obtaining Perca fluviatilis semen. The semen concentration of Perca fluviatilis is high and the quantity is large, which is conducive to carrying out artificial gynogenesis experiments. Moreover, it is easy to judge the degree of sperm inactivation during the semen inactivation treatment, and the inactivation treatment time is short. Fourth, when directly hybridizing Micropterus salmoides with Perca fluviatilis as the male parent, there are no surviving offspring ( Figure 12 ).

[0058] In the operation of artificial gynogenesis of Micropterus salmoides, the present invention controls a single variable in terms of the degree of sperm inactivation and the cold treatment temperature, and successfully explores the optimal degree of sperm inactivation and cold treatment temperature. And in terms of grasping the best period for artificial induced spawning of Micropterus salmoides, this set of procedures can be applied to the optimization of artificial gynogenesis operations of other fish, which has great reference value.

Claims

1. The use of perch sperm in inducing gynogenesis of largemouth bass, characterized in that: The steps include: (1) Obtaining mature largemouth bass eggs and river bass semen through artificial induced spawning; (2) Diluting the collected perch semen with pre-cooled Hank's solution, spreading the diluted solution to form a thin liquid layer, shaking it, and irradiating it with ultraviolet light to inactivate it; (3) The inactivated perch semen of step (2) is mixed with the mature largemouth bass eggs of step (1) for fertilization, and then the fertilized eggs are hatched with water at room temperature to hatch normal fry, i.e., gynogenetic largemouth bass.

2. The use according to claim 1, characterized in that: In step (1), the specific operation steps for obtaining mature largemouth bass eggs and river bass sperm are as follows: select largemouth bass and male river bass of the same age of age I or II as parents for intensive breeding, and induce spawning of female largemouth bass and male river bass during the peak period of largemouth bass production to obtain mature eggs and sperm.

3. The use according to claim 1, characterized in that: In step (1), the specific steps of artificial induction of spawning are as follows: at the peak of spontaneous spawning of largemouth bass, a mixed oxytocin of LRH-A2 and DOM is injected into the broodstock for induced spawning, the injection amount of LRH-A2 for female largemouth bass is 10-12 ug / kg, the injection amount of DOM is 2.5 mg / kg, and the effect time is 24-26 hours; at the end of spontaneous spawning of largemouth bass, HCG, LRH-A and DOM are combined as oxytocin, the injection amounts are 1500-2000-IU / kg, 10-12 ug / kg, 2.5 mg / kg, respectively, and the effect time is 36-48 hours; the injection amount of LRH-A2 for male river bass is 5-6 ug / kg.

4. The use according to claim 1, characterized in that: In step (2), the precooling temperature of Hank's solution is 4-6°C, and each liter of Hank's solution contains NaCl 1.36M, CaCl2 12.6mM, KCl 53.6mM, MgCl2 4.93mM, MgSO44.07mM, KH2PO4 4.41mM, NaHCO3 41.66mM, Na2HPO4 3.36mM, and D-Glucose 55.5mM, and the volume is fixed to 1L in a 4°C refrigerator; the Hank's solution and semen are diluted at a volume ratio of 10-15:

1.

5. The use according to claim 1, characterized in that: In step (2), the thickness of the liquid thin layer is 2-3 mm.

6. The use according to claim 1, characterized in that: In step (2), the shaking table speed of the shaking treatment is 100-130r / min.

7. The use according to claim 1, characterized in that: In step (2), the specific steps of the inactivation treatment are as follows: irradiation with a 40W ultraviolet lamp for inactivation treatment, the distance between the ultraviolet lamp and the shaker is 18-20cm, and the inactivation treatment time is based on the sperm motility observed under a microscope being 10%-15% of the motility before inactivation, and the approximate treatment time is 5-7min.

8. The use according to claim 1, characterized in that: In step (3), the specific steps of fertilization are as follows: placing the fertilized egg in room temperature water and stirring for fertilization for 2-4 minutes, and then placing the fertilized egg in 5-7°C cold water for cold shock for 10-12 minutes.

9. The use according to claim 1, characterized in that: In step (3), when the fertilized eggs are hatched, the embryos that have stopped developing are removed regularly and the microenvironment of the hatching water is maintained. When the hatched fry can swim horizontally, the fry are transferred to the fry training pond for cultivation and domestication. After the domestication is completed, they are transferred to the earthen pond for feeding.

10. The use according to claim 9, characterized in that: The domestication specifically comprises the following steps: creating a dark environment in the training pond, installing a light above the middle of the pond, and installing an oxygen pump device and a water flow device; turning on the light and the water flow during feeding, sprinkling small living plankton collected from the pond into the illuminated area, knocking on objects to make sounds, and turning off the water flow and the light after the fry have finished feeding in clusters in the illuminated area, thus completing one feeding; training feeding 3-4 times a day, feeding on small living plankton for the first 2-3 days, and gradually adding suspended feed after the fry form conditioned reflexes, and the domestication of gynogenetic seabass fry can be completed in 6-8 days.

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