Method for inducing gynogenesis of acrossocheilus fasciatus by using opsariichthys bidens sperms

Abstract

The invention provides a method for inducing gynogenesis of acrossocheilus fasciatus by using opsariichthys bidens sperms, and relates to the technical field of fish genetic breeding, and the method comprises the following steps: taking acrossocheilus fasciatus ova and inactivated opsariichthys bidens sperms for mixed fertilization, adding water to activate the ova, placing the activated ova in ice water, carrying out cold shock treatment for 10-50 minutes, and then placing the ova in normal-temperature water for incubation and seedling emergence. Heterologous sperms and opsariichthys bidens sperms are used as induced sperms, so that the phenomenon that homologous sperms which are not thoroughly inactivated are mixed to form normal diploids is avoided, and complicated verification is eliminated. By inducing gynogenesis, all-female breeding can be achieved, and the breeding yield is remarkably increased. By utilizing a gynogenesis method to perform acrossocheilus fasciatus improved variety breeding, homozygosis can be quickly realized, the breeding time is shortened, and the method has incomparable superiority. The success of inducing the gynogenesis of the acrossocheilus fasciatus is significant for improving the variety of the acrossocheilus fasciatus and increasing the breeding yield, and the problem that a method for inducing the gynogenesis of the acrossocheilus fasciatus by using the heterologous sperms is lacked in the prior art is solved.

Description

Technical Field

[0001] The invention relates to the technical field of fish genetic breeding, in particular to a method for inducing gynogenetic development of glaucus glabery with sperm of glaucus glabery. Background Art

[0002] Acrossocheilus fasciatus belongs to the Cyprinidae, Barbinae, and Acrossocheilus genus. It is commonly known as stream grouper, rose fish, etc. It is a stream fish distributed in the southeast region of my country. Acrossocheilus fasciatus is delicious and tender. It has traditional consumption habits in Zhejiang, Anhui, Jiangxi and other places and is very popular among the general public. In recent years, with the expansion of consumption forms such as farmhouses, the demand for Acrossocheilus fasciatus has grown rapidly. Relying on catching wild fish is far from meeting market needs and has seriously damaged fishery resources. Therefore, the artificial breeding industry has rapidly emerged. Now broodstock breeding, artificial insemination, artificial seedling breeding, pond and factory-scale water breeding, etc. have formed a one-stop production. Because female Acrossocheilus is large and male is small, the weight of mature females is more than 30% larger than that of males, and the sexual growth dimorphism is very obvious. Therefore, cultivating new germplasm of all-female Acrossocheilus fasciatus and giving full play to the characteristics of large females and fast growth are of great significance to improving breeding output and economic benefits.

[0003] Gynogenetic development is a special sexual reproduction mode of fish. Because sperm is exposed to ultraviolet light, the genetic material loses its activity. Although the sperm enters the egg, the sperm nucleus does not fuse with the egg nucleus, and only plays a stimulating role in activating the egg. When the egg releases the second polar body under fertilization, it is affected by external forces such as cold shock or hydrostatic pressure, which prevents the release of the second polar body and forms a doubling of chromosomes, becoming a gynogenetic diploid. In this process, if sperm of the same species is used for activation, gynogenetic development can also be induced. However, when inducing with inactivated sperm of the same species, there is a risk that ultraviolet inactivation is not complete to form normal fertilization and develop into a normal diploid. If heterologous sperm that cannot hybridize is used for activation, the induction is successful, resulting in gynogenetic diploid. If the inactivation is not complete, it will become a hybrid and no offspring will be produced. Therefore, the selection of heterologous sperm that cannot hybridize but can induce egg development has become an important choice for inducing gynogenetic development. So far, there has been no report on the induction of gynogenetic development in light-lipped fish using heterologous sperm.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for inducing gynogenetic development of glaucus glabripennis with sperm of mackerel, so as to solve the problem that there is a lack of methods for inducing gynogenetic development of glaucus glabripennis with heterologous sperm in the prior art.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] The invention provides a method for inducing gynogenetic development of light lip fish by using sperm of black carp, comprising the steps of taking eggs of black carp and inactivated sperm of black carp for mixing and fertilizing, adding water to activate the eggs, placing the activated eggs in ice water, subjecting them to cold shock treatment for 10 to 50 minutes, and then placing them in water at normal temperature for hatching and germination.

[0008] Furthermore, the volume ratio of the inactivated horsemouth fish semen to the smooth lip fish eggs is 1:5-10;

[0009] Preferably, the eggs are taken from female fish over 2 winters old;

[0010] Preferably, the eggs are plump, have uniform diameters, and are fully mature.

[0011] Furthermore, the method for preparing the inactivated croaker semen comprises placing the diluted croaker semen in a culture dish, shaking it horizontally, and irradiating it with an ultraviolet lamp to inactivate it;

[0012] Preferably, the thickness of the diluted Carcharodon carpio semen in the culture dish is 0.07-0.1 mm, preferably 0.08 mm;

[0013] Preferably, the horizontal oscillation speed is 60 to 120 r / min;

[0014] Preferably, the power of the ultraviolet lamp is 30W;

[0015] Preferably, the irradiation time of the ultraviolet lamp is 45 to 180 seconds, preferably 90 seconds;

[0016] Preferably, the irradiation dose of the ultraviolet lamp is 40 to 95 mJ / cm 2 ;

[0017] Preferably, the ambient temperature of the ultraviolet lamp irradiation is 18-26°C.

[0018] Furthermore, the diluent used to dilute the semen of the black carp includes the following components at the following concentrations: NaCl 8.0-8.2 g / L, CaCl 2 0.14~0.15g / L, KCl 0.40~0.45g / L, MgCl 2 6H 2 O0.10~0.15g / L, MgSO 4 7H 2 O0.20~0.25g / L, KH 2 PO 4 0.06~0.065g / L, NaHCO 3 0.35~0.36g / L, Na 2 HPO 4 ·H2 O 0.06-0.065 g / L and D Glucose 1.0 g / L;

[0019] Preferably, the dilution multiple of the semen of the black carp is 20 to 50 times.

[0020] Furthermore, the mixed fertilization also includes stirring with feathers to ensure sufficient contact between sperm and eggs.

[0021] Furthermore, the time for adding water to activate the eggs is 2 to 10 minutes.

[0022] Furthermore, the water temperature for activating eggs is 22-25°C.

[0023] Furthermore, the temperature of the ice water for cold shock is 0-6°C.

[0024] Furthermore, the incubation method is micro-flow water or still water;

[0025] Preferably, the incubation time is 48 to 72 hours;

[0026] Preferably, the incubation period also includes oxygenation.

[0027] Furthermore, the water temperature for hatching seedlings is 22-26°C.

[0028] The present invention provides a method for inducing gynogenetic development of light-lipped fish with sperm of croaker, using heterologous sperm, croaker sperm as inducing sperm, croaker and light-lipped fish both belong to the Cyprinidae, but light-lipped fish belongs to the subfamily Cyprininae, croaker belongs to the subfamily □, light-lipped fish and croaker are distantly related, have reproductive isolation, and cannot hybridize. Therefore, the surviving offspring obtained by inducing gynogenetic development with croaker are gynogenetic individuals. It can avoid the mixing of inactivated homologous sperm into normal diploids, thereby eliminating complicated verification. The successful induction of gynogenetic development in light-lipped fish provides a key technology for creating a new germplasm of all-female light-lipped fish. By inducing gynogenetic development, fully feminized breeding can be achieved, significantly improving breeding yield. Using the gynogenetic method to select and breed light-lipped fish can quickly achieve homozygosity, shorten the breeding time, and has incomparable advantages. The successful induction of gynogenetic development in smooth lip fish is of great significance for the variety improvement and increasing of aquaculture yield of smooth lip fish, and solves the problem of the lack of methods for inducing gynogenetic development in smooth lip fish using heterologous sperm in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the following briefly introduces the drawings required for use in the specific implementation or the prior art description. Obviously, the drawings described below are some implementations of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 A comparison diagram of the appearance of the gynogenetic fry of the smooth lip fish provided in Example 3 of the present invention, the mother fish, and the male horsemouth fish providing inactivated sperm;

[0031] Figure 2 The relative content detection result of DNA of the gynogenetic fry of the glossophrys glabrescens of the present invention;

[0032] Figure 3 A comparison diagram of the gynogenetic normal fry of the glossophrys of the present invention and the deformed fry that has not been subjected to cold shock treatment;

[0033] Figure 4 The present invention provides a comparison diagram of gonad sections of gynogenetic smoothlip fish and common smoothlip fish. DETAILED DESCRIPTION

[0034] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0035] The methods and techniques of the present invention are generally performed unless otherwise indicated according to conventional methods well known in the art and as described in various general and more specific references.

[0036] The invention provides a method for inducing gynogenetic development of light lip fish by using sperm of black carp, comprising the steps of taking eggs of black carp and inactivated sperm of black carp for mixing and fertilizing, adding water to activate the eggs, placing the activated eggs in ice water, subjecting them to cold shock treatment for 10 to 50 minutes, and then placing them in water at normal temperature for hatching and germination.

[0037] This method uses heterologous sperm, sperm of the croaker as induced sperm. Both the croaker and the light-lipped fish belong to the Cyprinidae, but the light-lipped fish belongs to the subfamily of the Hypocrida, and the croaker belongs to the subfamily of the □. The light-lipped fish and the croaker are distantly related, have reproductive isolation, and cannot hybridize. Therefore, the surviving offspring obtained by inducing gynogenesis with the croaker are gynogenetic offspring. It can avoid the mixing of inactivated homologous sperm into normal diploids, thereby eliminating complicated verification. The successful induction of gynogenesis in the light-lipped fish provides a key technology for the creation of a new germplasm of all-female light-lipped fish. By inducing gynogenesis, fully feminized breeding can be achieved, significantly increasing the breeding yield. Using the gynogenetic method for the breeding of light-lipped fish can quickly achieve homozygosity, shorten the breeding time, and has incomparable superiority. The successful induction of gynogenetic development in the light-lipped fish is of great significance for the variety improvement and the increase of breeding yield of the light-lipped fish, and solves the problem of the lack of methods for inducing gynogenetic development of light-lipped fish with heterologous sperm in the prior art.

[0038] The duration of the cold shock treatment may be 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min or 50 min, or any time between 10 and 50 min, preferably 30 min.

[0039] In some specific embodiments, the volume ratio of the inactivated horse mackerel semen to the smooth lipped fish eggs is 1:5-10.

[0040] The volume ratio of the inactivated black carp semen to the smooth lipped fish eggs may be, but is not limited to, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5 or 1:10, or any ratio between 1:5 and 10.

[0041] In some specific embodiments, the eggs are obtained from female fish that are more than 2 winters old.

[0042] Among them, female fish over 2 winters old are cultivated in flowing water, and nutrition is strengthened. Two months before spawning, multivitamins containing vitamins E and A are added to promote gonadal development. There is no need to inject oxytocin, and the eggs can be squeezed out directly.

[0043] In some specific embodiments, normal mature eggs with yellow color, plump ova, uniform egg diameter, and crystal clearness are selected for inducing gynogenetic development.

[0044] Experiments have shown that abnormally mature eggs with uneven and incomplete ovum cannot be used to induce female nuclear development.

[0045] In some specific embodiments, the method for preparing the inactivated black carp semen comprises placing the diluted black carp semen in a culture dish, shaking it horizontally, and inactivating it by irradiating it with ultraviolet light.

[0046] The horizontal shaking includes placing the culture dish on a horizontal shaker and shaking it to inactivate the sperm uniformly. The inactivated horsemouth fish semen is stored at an ambient temperature of 10°C.

[0047] In some specific embodiments, the thickness of the diluted carp semen in the culture dish is 0.07-0.1 mm.

[0048] The thickness of the diluted croaker semen in the culture dish may be, but is not limited to, 0.07 mm, 0.075 mm, 0.08 mm, 0.085 mm, 0.09 mm, 0.095 mm or 0.1 mm, and may be any value between 0.07 and 0.1 mm, preferably 0.08 mm.

[0049] In some specific embodiments, the horizontal oscillation has a rotation speed of 60 to 120 r / min.

[0050] The number of revolutions of the horizontal oscillation may be, but is not limited to, 60 r / min, 70 r / min, 80 r / min, 90 r / min, 100 r / min, 110 r / min or 120 r / min, and may also be any value between 60 and 120 r / min.

[0051] In some specific embodiments, the power of the ultraviolet lamp is 30W; in some specific embodiments, the irradiation time of the ultraviolet lamp is 45 to 180s; in some specific embodiments, the irradiation dose of the ultraviolet lamp is 40 to 95mJ / cm 2 ; In some specific embodiments, the ambient temperature of ultraviolet light irradiation is 18 to 26°C.

[0052] The irradiation time and irradiation dose of the ultraviolet lamp can inactivate sperms, and the sperms have normal activity. Most sperms in the irradiated semen have good activity and are in a linear motion state under a microscope.

[0053] The irradiation time of the ultraviolet lamp may be, but is not limited to, 45 s, 50 s, 70 s, 90 s, 100 s, 120 s, 140 s, 160 s or 180 s, and may be any time between 45 and 180 s, preferably 90 s.

[0054] The irradiation dose of the ultraviolet lamp can be but is not limited to 40mJ / cm 2 、45mJ / cm 2 、50mJ / cm 2 、60mJ / cm 2 、70mJ / cm 2 、80mJ / cm 2 、90mJ / cm2 or 95mJ / cm 2 , or 40~95mJ / cm 2 Any value in between.

[0055] The ambient temperature of the ultraviolet lamp irradiation can be but not limited to 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C or 26°C, and can also be any temperature between 18 and 26°C.

[0056] In some specific embodiments, the diluent used to dilute the semen of the black carp includes the following components at the following concentrations: NaCl 8.0-8.2 g / L, CaCl 2 0.14~0.15g / L, KCl 0.40~0.45g / L, MgCl 2 6H 2 O 0.10~0.15g / L, MgSO 4 7H 2 O 0.20~0.25g / L, KH 2 PO 4 0.06~0.065g / L, NaHCO 3 0.35~0.36g / L, Na 2 HPO 4 ·H 2 O 0.06~0.065g / L and DGlucose 1.0g / L.

[0057] In some specific embodiments, the dilution factor of the black carp semen is 20 to 50 times.

[0058] The dilution multiple can be, but is not limited to, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times or 50 times, or any multiple between 20 and 50 times. The specific multiple can be adjusted within a range according to the concentration of sperm of different male fish to ensure a stable inactivation effect.

[0059] In some specific embodiments, the mixed fertilization further comprises stirring with feathers to allow the sperm and egg to fully contact each other.

[0060] In some specific embodiments, the time for adding water to activate the eggs is 2 to 10 minutes.

[0061] The time for adding water to activate the eggs may be, but is not limited to, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min, or may be any time between 2 and 10 min.

[0062] In some specific embodiments, the temperature of the water used to activate the eggs is 22-25°C.

[0063] The water temperature for activating the eggs may be, but is not limited to, 22°C, 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C or 25°C, or may be 22-25°C.

[0064] In order to ensure the formation of diploid, in some specific embodiments, the temperature of the ice water for cold shock is 0-6°C.

[0065] The temperature of the ice water for cold shock may be, but is not limited to, 0°C, 0.5°C, 1°C, 1.5°C, 2°C, 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, 5°C, 5.5°C or 6°C, or any temperature between 0 and 6°C.

[0066] In some specific embodiments, the incubation method is micro-flowing water or still water.

[0067] Experiments have shown that a membrane can be hatched out after incubation for 48 hours. In some specific embodiments, the incubation time is 48 to 72 hours.

[0068] Among them, they float up after 72 hours and start to eat Artemia or mealworm pulp. Artemia larvae are obtained by hatching Artemia eggs with salt water equivalent to the salinity of seawater. Mealworm pulp is made by adding water to mealworms and crushing them with a beater. After filtering out the coarse particles, the remaining pulp can be directly fed into the nursery pond. After 7 to 10 days of cultivation with Artemia larvae or mealworm pulp, mix it with juvenile beetles and feed powder to make dough and feed it in the pond. The fry of the smooth lip fish will gather around and nibble on it.

[0069] In some specific embodiments, the incubation period also includes oxygenation to ensure that there is sufficient oxygen in the water.

[0070] In some specific embodiments, the water temperature for hatching seedlings is 22-26°C.

[0071] Among them, the water temperature for hatching seedlings can be but not limited to 22°C, 22.5°C, 23°C, 23.5°C, 24°C, 24.5°C, 25°C, 25.5°C or 26°C, and can also be any temperature between 22 and 26°C, preferably 24 to 26°C.

[0072] The technical solution of the present invention will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than 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.

[0073] Cultivation and selection of broodstock: Two months before breeding, carry out nutritional fortification, mix vitamin E, A, etc. into the bait, and increase the water flow to stimulate the water flow and promote the development of gonads. Select female fish with a strong body, no deformity, no injury, and a soft abdomen as broodstock for egg collection. In the breeding season, eggs can be squeezed out by gently pressing the abdomen, and there is no need to induce labor. Cultivate the mackerel alone, increase the water flow one month before spawning, and stimulate it to fully develop its gonads. Select male individuals with bright body colors as broodstock, and squeeze out semen by gently pressing the abdomen.

[0074] Semen diluent (Hank's solution) includes: NaCl 8.0g, CaCl 2 0.14g, KCl 0.40g, MgCl 2 6H 2 O0.10g, MgSO 4 7H 2 O 0.20g, KH 2 PO 4 0.06 g, NaHCO 3 0.35g, Na 2 HPO 4 ·H 2 O 0.06g and D Glucose 1.0g, make up to 1L, store in refrigerator for later use.

[0075] Example 1 Optimization of sperm inactivation time

[0076] Collecting the semen of the black carp: Gently press the abdomen to squeeze out the semen of the black carp, collect the semen with a syringe, put it in a 100ml beaker, dilute it 50 times with Hank's solution, stir and mix it thoroughly. The dilution multiple is determined by the concentration of the semen. The concentration of black carp semen is not as thick as other fish, but the sperm is very active. Store it at about 10℃ for later use.

[0077] The diluted sperm was placed in a culture dish, and the semen thickness was about 0.08mm. The culture dish was placed on a horizontal shaker, and the shaker speed was 60r / min. At room temperature below 26℃, the shaker was shaken while irradiation was performed. The irradiation time was 0s, 30s, 45s, 60s, 90s, 120s, 150s and 180s, and the irradiation dose was 80mJ / cm 2 The irradiated semen was used for fertilization. At 25°C, the embryos developed for 40 hours. The irradiation time and the normal embryo rate are shown in Table 1.

[0078] Table 1 Normal embryo rate under different irradiation time

[0079] Group Irradiation time Number of observed samples Normal embryo rate% 1 0(control group) 135 92 2 30s 138 14 3 45s 114 35 4 60s 126 52 5 90s 143 67 6 120 128 42 7 150 134 30 8 180 148 25

[0080] The normal embryo rate of the control group without ultraviolet irradiation was 92%, while that of the control group after irradiation for 30 seconds was 14%, and that of the control group after irradiation for more than 45 seconds was more than 35%. The normal embryo rate was the highest when the control group was irradiated for 90 seconds, reaching 67%. Based on the above results, it can be seen that the irradiation time for sperm inactivation of horse mackerel is 45 seconds to 180 seconds, and the optimal irradiation time is 90 seconds.

[0081] Example 2 Optimization of cold shock conditions

[0082] Chromosome doubling is mainly composed of three factors, namely the start time, duration and cold shock temperature of the cold shock treatment. In this embodiment, the water temperature parameter for the light lip fish is selected as 2±1°C. The evaluation standard is the normal embryo rate before hatching, because normal embryos and deformed embryos can be clearly distinguished before hatching.

[0083] The experimental results showed that under the conditions of 2±1℃ and 3min starting time (treatment time for adding water to activate the eggs), the normal embryo rate of the cold shock duration 30min group was 46.5%, and that of the 45min group was 12.8%. The normal embryo rate of the 30min group was significantly higher than that of the other groups. The results showed that the optimal duration of induction was 30min. Under the condition of a duration of 10min, the groups with a starting time of 3min, 5min, 7min and 9min could induce gynogenetic development and had a certain number of normal embryos, but the normal embryo rate of the 5min group was the highest, which was the optimal cold shock starting time, that is, the optimal treatment time for adding water to activate the eggs was 5min.

[0084] Example 3

[0085] A method for inducing gynogenesis of glaucus glabripennis by sperm of glaucus glabripennis comprises the following steps:

[0086] 1. Sperm collection and inactivation: Collect the semen of the black carp, gently press the abdomen to squeeze out the semen, use a syringe to collect the semen, put it in a 100ml beaker, dilute it 30 times with Hank's solution, stir and mix it thoroughly. The concentration of black carp semen is not as thick as other fish, but the sperm is very active. Store it at about 10℃ for later use.

[0087] Place the diluted sperm in a culture dish, the semen thickness is about 0.08mm. Place the culture dish on a horizontal shaker, the shaker speed is 60r / min. At room temperature below 26℃, shake the shaker while irradiating. The irradiation time is 90s, and the irradiation dose is 80mJ / cm 2. Before irradiation, the vitality of sperm is examined under a microscope, and almost all of them are in a state of active movement. If there is no abnormality, irradiation is carried out. After irradiation, the proportion of sperm moving in a straight line is examined under a microscope. If most of them are in a state of linear movement, they can be used for activation. If most of the sperm cannot move in a straight line, they cannot be used to induce female nuclear development. It is necessary to adjust the dilution concentration or change the irradiation dose. The irradiated semen is collected in a centrifuge tube and stored at about 10°C for the next fertilization step.

[0088] 2. Egg collection and activation

[0089] During the fertilization operation, use a towel to wipe the surface moisture of the mature female fish to prevent it from entering the eggs. Squeeze the eggs into a clean beaker or bowl and add inactivated semen. The ratio of semen to eggs is about 1:10, which is determined according to the concentration of semen. Mix the inactivated horsemouth fish semen with the eggs of the light lip fish and stir. The fertilization time is 3 minutes. Then add clean water not higher than 25℃ and stir to activate the eggs.

[0090] It should be noted that the squeezed eggs must be activated within 20 minutes, otherwise the hatching rate will drop significantly. The eggs of the light lip fish are sticky and need to be stirred with feathers to make them fully contact with clean water to activate the eggs. The activated eggs are placed in a prepared net bag so that they can be placed in ice water for cold shock treatment.

[0091] 3. Egg chromosomes double

[0092] After activating the eggs for 3 minutes, place the net bag containing the activated eggs directly into 2°C ice water for 30 minutes of cold shock treatment to double the chromosomes. The ice water should have a mass of more than 50 times that of the cold-shocked eggs so that the heat of the eggs can be quickly absorbed to achieve the purpose of cold shock.

[0093] 4. Cultivation of Gynogenetic Fry

[0094] After the female nucleus develops, the fry lie on the bottom of the pond, occasionally swimming, and begin to float after 2-3 days. After that, add artemia or mealworm pulp every day, and the fry begin to eat. The mealworm is crushed with a beater, and then the coarse particles are filtered out. The pulp can be used by the fry. After about 10 days of cultivation, they can eat fine powder or powder dough.

[0095] Comparative Example 1

[0096] The difference from Example 3 is that the egg chromosome doubling process in step 3 is not performed.

[0097] Experiment 1: Morphological comparison

[0098] The morphology of the gynogenetic fry obtained in Example 3 was compared with that of the female smooth-lipped fish providing eggs and the male horsemouth fish providing inactivated sperm. Figure 1As shown, A is the female smooth lip fish, B is the male croaker providing inactivated sperm, and C is the gynogenetic diploid smooth lip fish of Example 3. The morphology is the same as the female smooth lip fish, but completely different from the croaker providing sperm. It can be seen that the morphology of the gynogenetic fry is not affected by the induction of heterologous sperm, and the appearance and morphology are normal.

[0099] Experimental 2-ploidy detection

[0100] With the normal fertilized and developed smooth lipped fish as the control group, the 2-3 day embryos of Example 1 that were not treated with cold shock and the normal-sized fry of Example 3 that were treated with cold shock 2-3 days after hatching were taken, and the relative DNA content was detected by flow cytometry. The steps of the flow cytometer DNA content determination method are as follows: wash the fry with pure water several times and put them in a culture dish. Add an appropriate amount of CyStain UV Ploidy solution (Sysmex, Japan) to the culture dish, and chop the tissue with a double-sided blade. Then filter with a dense mesh and collect the filtrate. Add an appropriate amount of CyStain UV Ploidy solution to the filtrate before the measurement. According to the instructions of the flow cytometer (Sysmex, Japan) operating procedures, the DNA content of the smooth lipped fish cells in the filtrate was determined under a wavelength of 365nm UV-LED light. The results are as follows Figure 2 and Figure 3 As shown, Figure 2 A is comparative example 1, B is control group, and C is example 3; Figure 3 A is comparative example 1, and B is example 3.

[0101] Depend on Figure 2 It can be seen that the comparative example 1 that was not subjected to cold shock treatment was a haploid, while the example 3 that was subjected to cold shock treatment was a gynogenetic diploid, which was the same as the common diploid, and was a diploid.

[0102] Depend on Figure 3 It can be seen that some haploid embryos that have not been treated with cold shock can hatch out of the membrane, while others cannot. The embryos rotate in the egg membrane. Regardless of whether they hatch out of the membrane or not, the embryos are deformed, showing haploid syndrome, swollen yolk sac, bent tail and trunk, and die after 3-5 days.

[0103] Experiment 3 Sex identification and gonadal sectioning

[0104] In order to check the sex of the gynogenetic diploids, after the fry were cultured for 4 months, the gonads of the fry cultured in Example 3 were taken out for tissue sectioning and observed after HE staining. The results are as follows Figure 4 According to the results of gonadal tissue identification, all the gynogenetic diploids are ovarian tissue, that is, female.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for inducing gynogenesis of glaucus glabripennis by sperm of croaker, characterized in that: The method comprises the steps of mixing roe deer eggs and inactivated semen of horse mackerel for fertilization, adding water to activate the roe deer eggs, placing the activated roe deer eggs in ice water, subjecting them to cold shock treatment for 10 to 50 minutes, and then placing them in water at room temperature for hatching.

2. The method according to claim 1, characterized in that: The volume ratio of the inactivated croaker semen to the smooth lipped fish eggs is 1:5-10; Preferably, the eggs are taken from female fish over 2 winters old; Preferably, the eggs are plump, have uniform diameters, and are fully mature.

3. The method according to claim 1, characterized in that The method for preparing the inactivated carp semen comprises placing the diluted carp semen in a culture dish, shaking horizontally, and irradiating with ultraviolet light to inactivate the semen; Preferably, the thickness of the diluted Carcharodon carpio semen in the culture dish is 0.07-0.1 mm, preferably 0.08 mm; Preferably, the horizontal oscillation speed is 60 to 120 r / min; Preferably, the power of the ultraviolet lamp is 30W; Preferably, the irradiation time of the ultraviolet lamp is 45 to 180 seconds, preferably 90 seconds; Preferably, the irradiation dose of the ultraviolet lamp is 40 to 95 mJ / cm 2 ; Preferably, the ambient temperature of the ultraviolet lamp irradiation is 18-26°C.

4. The method according to claim 3, characterized in that: The diluent used for diluting the semen of the horsemouth fish includes the following components in concentration: NaCl 8.0-8.2 g / L, CaCl2 0.14-0.15 g / L, KCl0.40-0.45 g / L, MgCl2·6H2O0.10-0.15 g / L, MgSO4·7H2O 0.20-0.25 g / L, KH2PO4 0.06-0.065 g / L, NaHCO3 0.35-0.36 g / L, Na2HPO4·H2O 0.06-0.065 g / L and D Glucose 1.0 g / L; Preferably, the dilution multiple of the semen of the black carp is 20 to 50 times.

5. The method according to claim 1, characterized in that: The mixed fertilization involves stirring with feathers to allow the sperm and eggs to come into full contact.

6. The method according to claim 1, characterized in that The time for adding water to activate the eggs is 2 to 10 minutes.

7. The method according to claim 6, characterized in that The water temperature for activating eggs is 22-25℃.

8. The method according to claim 1, characterized in that: The temperature of the ice water for the cold shock is 0-6°C.

9. The method according to claim 1, characterized in that: The incubation method is micro-flow water or still water; Preferably, the incubation time is 48 to 72 hours; Preferably, the incubation period also includes oxygenation.

10. The method according to claim 1, characterized in that The water temperature for hatching and germination is 22-26℃.

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