阅读说明：本技术 一种促进粘性卵孵化同步发育的方法 (Method for promoting synchronous development of adhesive eggs during incubation ) 是由 陈春娜 黄颖颖 龙治海 陈先均 李正一 徐飞 张俊林 苏旭涛 肖宇 于 2021-09-13 设计创作，主要内容包括：本发明公开了一种促进粘性卵孵化同步发育的方法,包括以下步骤：繁殖前准备、人工催产、人工授精、布卵、晾卵、出膜,其中晾卵为当受精卵中发育到囊胚末期的卵占50％以上时,将粘有受精卵的粘连物从孵化容器内拿出,置于通风、干燥、自然光照且无阳光直射的地方,晾干粘连物表面水分即可。本发明可使得受精卵分裂与发育间的时间间隙缩短,从而促使后期发育和出膜的同步；技术操作简单、安全、规范；适用范围广,可广泛应用于粘性卵的同步孵化。(The invention discloses a method for promoting synchronous development of adhesive egg incubation, which comprises the following steps: preparing before propagation, artificially hastening parturition, artificially inseminating, distributing eggs, airing eggs and taking out membranes, wherein the egg airing is realized by taking out the adhesion substance adhered with the fertilized eggs from an incubation container when more than 50 percent of eggs growing to the last stage of blastocyst in the fertilized eggs are put in a place which is ventilated, dried, naturally illuminated and has no direct sunlight, and airing the water on the surface of the adhesion substance. The invention can shorten the time gap between division and development of the fertilized eggs, thereby promoting the synchronization of later development and membrane emergence; the technical operation is simple, safe and standard; the application range is wide, and the method can be widely applied to synchronous hatching of the viscous eggs.)

1. A method of promoting the synchronous development of hatching of a viscous egg, comprising: the method comprises the following steps:

(1) preparation before propagation: selecting a proper age parent fish for laying sticky eggs and putting the fish into an oxytocic pool for culturing;

(2) artificial hastening parturition: respectively injecting spawning-inducing medicines into the male and female parent fishes, putting the male and female parent fishes into an spawning-inducing pool, and standing for medicine effect time;

(3) artificial insemination: and (3) mixing female fish and male fish according to the ratio of 1: 1-5: 1, polyculture in proportion, and performing artificial insemination to obtain fertilized eggs;

(4) laying eggs: uniformly sticking the oosperms which generate viscosity on the adhesion substances, standing in water for 3-5 minutes, and then putting into an incubation container for incubation;

(5) airing eggs: when the fertilized eggs grow to the eggs at the last stage of blastocyst stage and account for more than 50 percent, taking out the adhesion objects adhered with the fertilized eggs from the hatching container, placing the adhesion objects in a ventilated, dry and natural-illumination place without direct sunlight, and airing the moisture on the surfaces of the adhesion objects;

(6) film discharging: and (4) placing the eggs dried at the same time into the same net cage, continuously culturing until the fry is out of the film, and transferring the fry with water into a new net cage for fry culture.

2. The method of promoting synchronous development of hatching of sticky eggs according to claim 1, wherein: and (2) keeping a running water or circulating water state in the spawning inducing pool in the step (1).

3. The method of promoting synchronous development of hatching of sticky eggs according to claim 1, wherein: the oxytocic is at least one of PG, HCG, LRH-A and DOM.

4. The method for promoting synchronous development of hatching of adhesive eggs as claimed in claim 1, wherein the artificial insemination method in the step (3) is as follows: obtaining semen and ovum by artificial extrusion, placing semen and ovum into a container, adding clear water, and shaking the container to mix well.

5. The method of promoting synchronous development of hatching of sticky eggs according to claim 1, wherein: the density of the fertilized eggs on the adhesion substance in the step (4) is 5-10 granules/cm²。

6. The method for promoting synchronous development of hatching of the adhesive eggs, according to claim 1 or 5, wherein: the adhesion substance is a sticky egg plate or a palm sheet.

7. The method of promoting synchronous development of hatching of sticky eggs according to claim 1, wherein: and (5) when eggs are aired in the step (5), the adhesion is in a suspension state.

8. The method of promoting synchronous development of hatching of sticky eggs according to claim 1, wherein: and (5) airing the eggs for 20-30 minutes.

Technical Field

The invention belongs to the technical field of fish culture, and particularly relates to a method for promoting synchronous development of hatching of adhesive eggs.

Background

In the artificial breeding process, the situation that the ovulation time of fishes producing viscous eggs is inconsistent or the fishes produce eggs for multiple times often occurs, so that the interval time of different batches of viscous eggs is too long, the fishes are firstly hatched, opening and food transfer are asynchronous, and a series of problems such as large eating size, discrete growth, increased breeding cost and the like are brought to later production.

Therefore, in order to promote the synchronous development of eggs and shorten the interval time between the fish fry and the membrane as much as possible, in actual production, practitioners often control the ovulation time of fish by adjusting the dosage of an oxytocic drug, but the following disadvantages still exist in the actual production:

(1) practitioners determine the injection dosage by judging the development degree of the parent fish, so that the method has strong experience and unsatisfactory effect.

(2) The difficulty of the actual operation process is increased by the difference of the injection dosage of the medicine.

(3) Great uncertainty is brought to the manual induced spawning work.

Disclosure of Invention

Aiming at the prior art, the invention provides the method for promoting the synchronous development of the hatching of the viscous eggs, which can effectively shorten the interval time of the fry in the membrane, and has the advantages of simple method, unified, standard and reliable operation flow.

In order to achieve the purpose, the invention adopts the technical scheme that: there is provided a method of promoting synchronous development of hatching of a viscous egg, the method comprising the steps of:

(1) preparation before propagation: selecting a proper age parent fish for laying sticky eggs and putting the fish into an oxytocic pool for culturing;

(2) artificial hastening parturition: respectively injecting spawning-inducing medicines into the male and female parent fishes, putting the male and female parent fishes into an spawning-inducing pool, and standing for medicine effect time;

(3) artificial insemination: and (3) mixing female fish and male fish according to the ratio of 1: 1-5: 1, polyculture in proportion, and performing artificial insemination to obtain fertilized eggs;

(4) laying eggs: uniformly sticking the oosperms which generate viscosity on the adhesion substances, standing in water for 3-5 minutes, and then putting into an incubation container for incubation;

(5) airing eggs: when the fertilized eggs grow to the eggs at the last stage of blastocyst stage and account for more than 50 percent, taking out the adhesion objects adhered with the fertilized eggs from the hatching container, placing the adhesion objects in a ventilated, dry and natural-illumination place without direct sunlight, and airing the moisture on the surfaces of the adhesion objects;

(6) film discharging: and (4) placing the eggs dried at the same time into the same net cage, and transferring the fry with water into a new net cage for fry cultivation after the fry is subjected to film forming.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, in the step (1), the spawning pool is kept in a running water or circulating water state.

Further, the oxytocic is at least one of PG, HCG, LRH-A and DOM.

Further, the artificial insemination method in the step (3) comprises the following steps: obtaining semen and ovum by artificial extrusion, placing semen and ovum into a container, adding clear water, and shaking the container to mix well.

Further, the density of the fertilized eggs on the adhesion substances in the step (4) is 5-10 granules/cm²。

Further, the adhesion substance is a sticky egg plate or a palm sheet.

Further, in the step (5), the adhesive is in a hanging state when eggs are aired.

Further, the egg airing time in the step (5) is 20-30 minutes.

The invention has the beneficial effects that: because the hatching speeds of different eggs are different, in order to enable the eggs in different states to tend to be uniform, the invention adopts an egg airing mode, fertilized eggs are moved out of a water body in a short time, the hatching environment of the fertilized eggs is changed, the speed difference among the fertilized eggs is shortened at a certain stage, the hatching speed of the eggs in a development phase is delayed, and meanwhile, the eggs in a division phase are not influenced, so that the eggs in different phases tend to be uniform. The egg drying method fully utilizes the characteristics of the viscous eggs and the early development characteristics of embryos, the viscous eggs are viscous and can be well attached to adhesive substances, the mucus on the surface of an egg membrane can greatly reduce the loss of water, and meanwhile, the protective effect is also achieved on the egg membrane, so that the egg drying method can adapt to the egg drying in the absence of water in a short time; during the early development of the embryo, the embryo is in the cell division stage, and the egg at the cell division stage cannot be influenced by too large environment due to the spontaneous division so as to delay the development. Therefore, the time interval between the division and the development of different fertilized eggs can be effectively shortened by airing the eggs.

The invention has the characteristics of simple, safe and standard technical operation; the characteristics of the viscous eggs and the early development characteristics of the embryos are fully utilized, the development is delayed and the progress of the division is not influenced by the change of the hatching environment in the division and development transition period of the fertilized eggs, so that the time gap between the division and the development of the fertilized eggs is shortened, and the synchronization of the later development and the membrane emergence is promoted; the application range is wide, and the method can be widely applied to synchronous hatching of the viscous eggs.

Detailed Description

The method for promoting the synchronous development of the incubation of the viscous eggs comprises the following steps:

(1) preparation before propagation: selecting mature and non-damaged parent fish of suitable age for laying adhesive eggs in advance, and placing the fish into a prepared spawning induction pond, wherein the pond is kept in a running water or circulating water state.

(2) Artificial hastening parturition: according to the technical standard of induced spawning, after injecting corresponding doses of induced spawning drugs into male and female parent fishes according to the weight and the types of the fishes, putting the fishes into an induced spawning pond, and standing for drug effect time.

(3) Artificial insemination: according to the actual water temperature, after the effect time is reached, the female fish can be checked to see whether free eggs are discharged, if so, the female fish and the male fish are processed according to the ratio of 1: 1-5: 1 and preparing for artificial insemination. The artificial insemination method comprises the following steps: obtaining semen and ovum by artificial extrusion, placing semen and ovum into a container, adding clear water, and shaking the container to mix well.

(4) Laying eggs: when the fertilized eggs are sticky, the fertilized eggs are uniformly stuck on the adhesive substance, and the density of the fertilized eggs is controlled to be 5-10 granules/cm²And standing in water for 3-5And then putting the water in an incubation container for incubation, and simultaneously recording corresponding water quality information.

(5) Airing eggs: after the eggs absorb water and swell, randomly selecting 5-10 eggs from each hatching pool or hatching container every 1-2 hours for microscopic examination.

Secondly, when the fertilized eggs are observed by microscopic examination to be developed to more than 50 percent of eggs at the terminal stage of blastocyst, the egg airing operation can be carried out:

I. and respectively measuring the water temperature and the room temperature to ensure that the ambient temperature of the egg drying position is +/-3-5 ℃, and if the temperature difference is overlarge, adjusting by using equipment such as an air conditioner and the like.

And II, selecting a place which is ventilated and dried, is naturally illuminated and has no direct sunlight, arranging an iron wire above the place, taking out the whole adhesive such as an egg sticking plate or palm sheets stuck with the fertilized eggs from the incubation container, and hanging one end of the adhesive on the iron wire by a thin wire so as to avoid pressing the fertilized eggs.

And III, in the egg airing process, in order to avoid the adverse effect on the eggs caused by too long water leaving time of the fertilized eggs, the surface moisture of the adhesive such as the egg adhering plate or the palm sheet is just dried (generally for 20 to 30 minutes).

And IV, in large-scale production, the number of parent fishes bred every time is excessive or the parent fishes belong to fishes for multiple spawning, the spawning time interval is too long, the fertilized egg development time phase difference is large, and the eggs can be aired in batches according to the steps.

(6) Film discharging

And (3) putting the eggs dried at the same time into the same net cage in advance, and after the fry is released from the film, transferring the fry with water into a new net cage for fry cultivation.

The technical solution of the present invention will be specifically described below with reference to examples.

Example 1

Take procypris rabaudi as an example.

1. Preparation before reproduction

Selecting parent Cyprinus Carpio with high maturity of more than 4 years old and no injury in advance, placing into a prepared spawning induction pond, and keeping running water or circulating water state in the pond.

2. Artificial hasten parturition

After cold tide, the air temperature gradually rises, the water temperature is stabilized above 19 ℃ for a week, the induced spawning can be started, each kilogram of female fish is injected by adopting a 2-needle method according to 5mg of PG +1000IU of HCG +10ug of LRH-A, the injection dosage of male fish is halved, the total dosage of the first injection is 10%, and the residual dosage of the first injection is injected after 12 hours. After injection, the mixture is put into an oxytocic pool and is left for the drug effect time.

3. Artificial insemination

The drug effect time is about 15h when the water temperature is about 22 ℃, during which, 1 time of female fish is checked every 1h to see whether free eggs are discharged, if yes, the female fish and the male fish are processed according to the ratio of 3: a ratio of 1 in preparation for artificial insemination. After semen and ovum are obtained by artificial extrusion, dry insemination is adopted, the semen and the ovum are evenly mixed in a container, a small amount of clear water is added, and the container is quickly and lightly shaken to evenly mix the semen and the ovum.

4. Cloth ovum

Uniformly sticking the viscous fertilized eggs on the palm sheet, and controlling the density of the fertilized eggs to be 7 grains/cm²And standing in water for 4 minutes, putting the eggs into an incubation pool for incubation after the eggs are stuck, and simultaneously recording corresponding water quality information.

5. Egg airing device

After the eggs begin to grow after water absorption and expansion, randomly selecting 5 eggs from each hatching pool or hatching container every 1 hour for microscopic examination.

Secondly, when the fertilized eggs are observed by microscopic examination to be developed to more than 50 percent of eggs at the terminal stage of blastocyst, the egg airing operation can be carried out:

I. and respectively measuring the water temperature and the room temperature to ensure that the ambient temperature of the egg airing position is +/-4 ℃, and if the temperature difference is overlarge, adjusting by using equipment such as an air conditioner and the like.

II, selecting a place which is ventilated, dried and naturally illuminated and has no direct sunlight, arranging an iron wire above the place, taking out the whole palm piece stuck with the fertilized eggs from the hatching container, hanging one end of the palm piece with a thin wire on the iron wire, and suspending the palm piece in the air in half to avoid pressing the fertilized eggs.

And III, in the egg airing process, in order to avoid the adverse effect on the eggs caused by too long water leaving time of the fertilized eggs, the water on the surface of the palm pieces is just dried (generally about 25 minutes).

And IV, in large-scale production, the number of parent fishes bred every time is excessive or the parent fishes belong to fishes for multiple spawning, the spawning time interval is too long, the fertilized egg development time phase difference is large, and the eggs can be aired in batches according to the steps.

6. Film discharging

Before the fry is taken out of the film, the eggs dried at the same time are put into the same net cage in advance, and after the fry is taken out of the film, the fry with water is transferred into a new net cage for fry cultivation.

Example 2

Take procypris rabaudi as an example.

1. Preparation before reproduction

Selecting parent Cyprinus Carpio with high maturity of more than 4 years old and no injury in advance, placing into a prepared spawning induction pond, and keeping running water or circulating water state in the pond.

2. Artificial hasten parturition

After cold tide, the air temperature gradually rises, the water temperature is stabilized above 19 ℃ for a week, the induced spawning can be started, each kilogram of female fish is injected by adopting a 2-needle method according to 5mg of PG +1000IU of HCG +10ug of LRH-A, the injection dosage of male fish is halved, the total dosage of the first injection is 10%, and the residual dosage of the first injection is injected after 12 hours. After injection, the mixture is put into an oxytocic pool and is left for the drug effect time.

3. Artificial insemination

The drug effect time is about 15h when the water temperature is about 22 ℃, during which, 1 time of female fish is checked every 1h to see whether free eggs are discharged, if yes, the female fish and the male fish are processed according to the ratio of 1: a ratio of 1 in preparation for artificial insemination. After semen and ovum are obtained by artificial extrusion, dry insemination is adopted, the semen and the ovum are evenly mixed in a container, a small amount of clear water is added, and the container is quickly and lightly shaken to evenly mix the semen and the ovum.

4. Cloth ovum

Uniformly sticking the viscous fertilized eggs on the palm sheet, and controlling the density of the fertilized eggs to be 5 particles/cm²And standing in water for 3 minutes, putting the eggs into an incubation pool for incubation after the eggs are stuck, and simultaneously recording corresponding water quality information.

5. Egg airing device

After the eggs begin to grow after water absorption and expansion, randomly selecting 7 eggs from each hatching pool or hatching container every 2 hours for microscopic examination.

Secondly, when the fertilized eggs are observed by microscopic examination to be developed to more than 50 percent of eggs at the terminal stage of blastocyst, the egg airing operation can be carried out:

I. and respectively measuring the water temperature and the room temperature to ensure that the ambient temperature of the egg airing position is +/-4 ℃, and if the temperature difference is overlarge, adjusting by using equipment such as an air conditioner and the like.

II, selecting a place which is ventilated, dried and naturally illuminated and has no direct sunlight, arranging an iron wire above the place, taking out the whole palm piece stuck with the fertilized eggs from the hatching container, hanging one end of the palm piece with a thin wire on the iron wire, and suspending the palm piece in the air in half to avoid pressing the fertilized eggs.

And III, in the egg airing process, in order to avoid the adverse effect on the eggs caused by too long water leaving time of the fertilized eggs, the water content on the surface of the palm pieces is just dried (generally about 20 minutes).

And IV, in large-scale production, the number of parent fishes bred every time is excessive or the parent fishes belong to fishes for multiple spawning, the spawning time interval is too long, the fertilized egg development time phase difference is large, and the eggs can be aired in batches according to the steps.

6. Film discharging

Before the fry is taken out of the film, the eggs dried at the same time are put into the same net cage in advance, and after the fry is taken out of the film, the fry with water is transferred into a new net cage for fry cultivation.

Example 3

Take Procypris rabaudi as an example

1. Preparation before reproduction

Selecting parent Cyprinus Carpio with high maturity of more than 4 years old and no injury in advance, placing into a prepared spawning induction pond, and keeping running water or circulating water state in the pond.

2. Artificial hasten parturition

After cold tide, the air temperature gradually rises, the water temperature is stabilized above 19 ℃ for a week, the induced spawning can be started, each kilogram of female fish is injected by adopting a 2-needle method according to 5mg of PG +1000IU of HCG +10ug of LRH-A, the injection dosage of male fish is halved, the total dosage of the first injection is 10%, and the residual dosage of the first injection is injected after 12 hours. After injection, the mixture is put into an oxytocic pool and is left for the drug effect time.

3. Artificial insemination

The drug effect time is about 15h when the water temperature is about 22 ℃, during which, 1 time of female fish is checked every 1h to see whether free eggs are discharged, if yes, the female fish and the male fish are processed according to the ratio of 5: a ratio of 1 in preparation for artificial insemination. After semen and ovum are obtained by artificial extrusion, dry insemination is adopted, the semen and the ovum are evenly mixed in a container, a small amount of clear water is added, and the container is quickly and lightly shaken to evenly mix the semen and the ovum.

4. Cloth ovum

Uniformly sticking the viscous fertilized eggs on palm pieces, and controlling the density of the fertilized eggs to be 10 grains/cm²And standing in water for 5 minutes, putting the eggs into an incubation pool for incubation after the eggs are stuck, and simultaneously recording corresponding water quality information.

5. Egg airing device

After the eggs begin to grow after water absorption and expansion, randomly selecting 10 eggs from each hatching pool or hatching container every 1 hour for microscopic examination.

Secondly, when the fertilized eggs are observed by microscopic examination to be developed to more than 50 percent of eggs at the terminal stage of blastocyst, the egg airing operation can be carried out:

I. and respectively measuring the water temperature and the room temperature to ensure that the environment temperature of the egg airing position is +/-5 ℃, and if the temperature difference is overlarge, adjusting by using equipment such as an air conditioner and the like.

II, selecting a place which is ventilated, dried and naturally illuminated and has no direct sunlight, arranging an iron wire above the place, taking out the whole palm piece stuck with the fertilized eggs from the hatching container, hanging one end of the palm piece with a thin wire on the iron wire, and suspending the palm piece in the air in half to avoid pressing the fertilized eggs.

And III, in the egg airing process, in order to avoid the adverse effect on the eggs caused by too long water leaving time of the fertilized eggs, the water content on the surface of the palm pieces is just dried (generally about 30 minutes).

And IV, in large-scale production, the number of parent fishes bred every time is excessive or the parent fishes belong to fishes for multiple spawning, the spawning time interval is too long, the fertilized egg development time phase difference is large, and the eggs can be aired in batches according to the steps.

6. Film discharging

Before the fry is taken out of the film, the eggs dried at the same time are put into the same net cage in advance, and after the fry is taken out of the film, the fry with water is transferred into a new net cage for fry cultivation.

Comparative example 1

Take procypris rabaudi as an example.

1. Preparation before reproduction

Selecting parent Cyprinus Carpio with high maturity of more than 4 years old and no injury in advance, placing into a prepared spawning induction pond, and keeping running water or circulating water state in the pond.

2. Artificial hasten parturition

After cold tide, the air temperature gradually rises, the water temperature is stabilized above 19 ℃ for a week, the induced spawning can be started, each kilogram of female fish is injected by adopting a 2-needle method according to 5mg of PG +1000IU of HCG +10ug of LRH-A, the injection dosage of male fish is halved, the total dosage of the first injection is 10%, and the residual dosage of the first injection is injected after 12 hours. After injection, the mixture is put into an oxytocic pool and is left for the drug effect time.

3. Artificial insemination

The drug effect time is 15h when the water temperature is about 22 ℃, during which, 1 time of female fish is checked every 1h to see whether free eggs are discharged, if yes, the female fish and the male fish are processed according to the ratio of 3: a ratio of 1 in preparation for artificial insemination. After semen and ovum are obtained by artificial extrusion, dry insemination is adopted, the semen and the ovum are evenly mixed in a container, a small amount of clear water is added, and the container is quickly and lightly shaken to evenly mix the semen and the ovum.

4. Cloth ovum

Uniformly sticking the viscous fertilized eggs on the palm sheet, and controlling the density of the fertilized eggs to be 7 grains/cm²And standing in water for 4 minutes, putting the eggs into an incubation pool for incubation after the eggs are stuck, and simultaneously recording corresponding water quality information.

5. Film discharging

Before the fry is out of the film, eggs are put into the net cage in advance, and after the fry is out of the film, the fry with water is transferred into a new net cage for fry cultivation.

The following tests and analyses were carried out on the fishes cultivated in the examples and comparative examples:

statistics of fish fertilization rate, hatchability and 4-hour membrane output rate are shown in table 1. From the aspect of the membrane emergence rate, the membrane emergence rate in 4 hours is as high as 83% by adopting the method for hatching the adhesive eggs, and the table 1 shows that the method for hatching the adhesive eggs can effectively shorten the time gap between the division and the development of the fertilized eggs, promote the synchronous development of the fertilized eggs and lead the membrane emergence time of the fish fries to be similar.

TABLE 1 Fish weight gain, survival rate, mortality

Detailed description of the preferred embodiments	Fertilization Rate (%)	Hatching rate (%)	Film formation Rate (%) at 4 hours
				Example 1	73.7	63.5	83.2
Example 2	74.1	63.9	83.7
				Example 3	73.2	63.1	82.6
Comparative example 1	69.4	60.7	37.8

While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.