The first step in artificial breeding of sea cucumber is to obtain a large quantity of high-quality, mature gametes (fines, eggs), which is the key to the success of artificial breeding. There are the following ways for the pro-egg picking:
1. The time for natural spawning and pro-species acquisition is appropriate, and the gonadal development is fully mature, and natural spawning methods can be adopted.
The advantage of this method is that it is simple and can basically meet the needs of nursery. Stichopus japonicus often spawns and ejaculates in the evening or at night. It is not possible to artificially control spawning time and fecundity, and it is sometimes the case that excessive production of eggs and excessive semen cause water spoilage resulting in the disintegration of fertilized eggs. Therefore, night watch personnel should be arranged to observe at any time, collect in time, or reach the amount of eggs, remove the pro-ginseng to other pools, or remove excess male ejaculation and sea cucumbers from the ovulation pool.
Second, artificial stimulation of spawning The commonly used methods for the induction of spawning of sea cucumbers include temperature difference method, ultraviolet seawater immersion method, and dripping water stimulation method.
1. Differential temperature method
The operating temperature difference is preferably increased or decreased by 5°C to 8°C up or down in the seawater temperature at which the sea cucumber is stored. When inducing, the parent can be directly placed in high temperature water (direct temperature rise); or the parent can be treated with low water temperature first, then treated with high water temperature (heating after warming first); or it can be repeated with high and low water temperature. (High and low temperature interactive operation). The treatment time for a single rise (fall) water temperature is usually from 1 hour to overnight, depending on the status of the parent. If signs of spawning are found, individuals should be removed and placed in a normal water temperature environment.
2. UV seawater immersion method
This method uses seawater irradiated by an ultraviolet lamp to induce the reproduction of the ginseng (the mechanism is still unknown). Normally 10 liters of seawater is irradiated with an 8-watt ultraviolet lamp. At a flow rate of 10 liters/minute, it is considered to have reached saturation after one hour of circulation. Full aeration during the cycle, so that the dissolved oxygen in the water is sufficient. After the preparation of "ultraviolet seawater" is completed, the parent compound is placed in it, and the maximum limit is one for each liter of ultraviolet water. Normally, male individuals will ejaculate 0.5 hours after induction and female individuals will take longer (45 minutes to several hours). During the induction process, it is necessary to carefully observe the status of the parental reference. In the case of self-cutting or water-absorbing individuals, they need to be immediately removed to the normal seawater environment.
3. Dry trunk water stimulation method
It is usually done after the evening. First dry the seawater in the storage tank, allow the pro-reference to dry for 45 minutes to 1 hour, and then use water to impact for 10 minutes to 15 minutes, or run water for 40 minutes to 50 minutes. Then add the pro-ginseng into the pool with fresh filtered seawater. . In general, about 2 hours, the pro-ginseng began crawling toward the surface pool wall, moving frequently, often lifting the head and swinging around the surface of the sea water, the male began to row fine, and the female ginseng began to lay eggs 0.5 hours later. In the actual production, dry, flowing water and heating and cooling methods are often used in combination.
4. Potassium chloride injection method
This method is suitable for individuals with small body size and high maturity. The mechanism of action is to increase the concentration of potassium ions in the body fluids of the paraffin body, to contract the body wall muscles, and to extrude the gametes by squeezing the genital nests. The potassium chloride injection concentration is 0.5 mol/l to 1 mol/l, the parental wet weight is 0.5 kg, the total injection volume is 5 ml, and 1 ml to 2 ml dose should be injected every 10 minutes to Avoid overstimulation. In order to prevent the ginseng from being excreted from the anus, it is advisable to fix the anus on the water surface with a needle handle. If the gonads have a high degree of maturation, they can be excreted in a short time. On the other hand, if the pro-growth is not mature enough, it will cause self-cut.
5. Anatomy This method
It is suitable for individuals who are large individuals and who experience difficulty in reproduction (usually female, the egg does not reach the final stage of maturity). Using a blade from the rear of the parent's tentacles on the back of the 1/4 body, cut out about 3 cm long openings, and gently push the middle part of the ginseng body to let the genitalia flow out.
The gonads harvested were immediately cleaned several times with clean seawater to prevent the pro-gazed body fluids from affecting the quality of the gonads. The eggs obtained at this time are usually not fertilized (not reaching the final maturity) and must be matured before they can be fertilized. The ripening methods include drug soaking method and ovary chopping method, which are described as follows:
(1) Drug soaking The ovaries are probed with probes, and the released eggs are prepared with DTT, BAL or L-Cysteine ​​and other drugs in a concentration of 0.1 mol/l of seawater solution. After soaking for 15 minutes, the eggs are repeatedly washed with clean seawater. 1 hour, then insemination. When the drug soaking method is used, the dosage and the soaking time must be strictly observed so as not to poison the egg. Place the collected eggs in a container and add a small amount (about the same volume of egg) of clean seawater. Immediately add the sperm (the amount of eggs in a female, add 1 drop of undiluted semen or a few drops of diluted sperm) and light Stir gently (shake) and then add diluted seawater in an equally proportional amount every 5 minutes.
(2) Precipitation and egg-cleaning method The eggs were soaked in clean seawater, and the clean semen was diluted and activated by adding 10 volumes of seawater. Sperm dilution (the amount of eggs in one female, add a few drops of sperm diluent) is added to the egg fluid and the mixture is fully fertilized by gentle agitation. After that, the mixture of eggs and eggs is left to stand until the fertilized eggs precipitate, and the upper layer contains the spermatozoa seawater, and the cleaned fertilized eggs are added by adding clean seawater.
1. The time for natural spawning and pro-species acquisition is appropriate, and the gonadal development is fully mature, and natural spawning methods can be adopted.
The advantage of this method is that it is simple and can basically meet the needs of nursery. Stichopus japonicus often spawns and ejaculates in the evening or at night. It is not possible to artificially control spawning time and fecundity, and it is sometimes the case that excessive production of eggs and excessive semen cause water spoilage resulting in the disintegration of fertilized eggs. Therefore, night watch personnel should be arranged to observe at any time, collect in time, or reach the amount of eggs, remove the pro-ginseng to other pools, or remove excess male ejaculation and sea cucumbers from the ovulation pool.
Second, artificial stimulation of spawning The commonly used methods for the induction of spawning of sea cucumbers include temperature difference method, ultraviolet seawater immersion method, and dripping water stimulation method.
1. Differential temperature method
The operating temperature difference is preferably increased or decreased by 5°C to 8°C up or down in the seawater temperature at which the sea cucumber is stored. When inducing, the parent can be directly placed in high temperature water (direct temperature rise); or the parent can be treated with low water temperature first, then treated with high water temperature (heating after warming first); or it can be repeated with high and low water temperature. (High and low temperature interactive operation). The treatment time for a single rise (fall) water temperature is usually from 1 hour to overnight, depending on the status of the parent. If signs of spawning are found, individuals should be removed and placed in a normal water temperature environment.
2. UV seawater immersion method
This method uses seawater irradiated by an ultraviolet lamp to induce the reproduction of the ginseng (the mechanism is still unknown). Normally 10 liters of seawater is irradiated with an 8-watt ultraviolet lamp. At a flow rate of 10 liters/minute, it is considered to have reached saturation after one hour of circulation. Full aeration during the cycle, so that the dissolved oxygen in the water is sufficient. After the preparation of "ultraviolet seawater" is completed, the parent compound is placed in it, and the maximum limit is one for each liter of ultraviolet water. Normally, male individuals will ejaculate 0.5 hours after induction and female individuals will take longer (45 minutes to several hours). During the induction process, it is necessary to carefully observe the status of the parental reference. In the case of self-cutting or water-absorbing individuals, they need to be immediately removed to the normal seawater environment.
3. Dry trunk water stimulation method
It is usually done after the evening. First dry the seawater in the storage tank, allow the pro-reference to dry for 45 minutes to 1 hour, and then use water to impact for 10 minutes to 15 minutes, or run water for 40 minutes to 50 minutes. Then add the pro-ginseng into the pool with fresh filtered seawater. . In general, about 2 hours, the pro-ginseng began crawling toward the surface pool wall, moving frequently, often lifting the head and swinging around the surface of the sea water, the male began to row fine, and the female ginseng began to lay eggs 0.5 hours later. In the actual production, dry, flowing water and heating and cooling methods are often used in combination.
4. Potassium chloride injection method
This method is suitable for individuals with small body size and high maturity. The mechanism of action is to increase the concentration of potassium ions in the body fluids of the paraffin body, to contract the body wall muscles, and to extrude the gametes by squeezing the genital nests. The potassium chloride injection concentration is 0.5 mol/l to 1 mol/l, the parental wet weight is 0.5 kg, the total injection volume is 5 ml, and 1 ml to 2 ml dose should be injected every 10 minutes to Avoid overstimulation. In order to prevent the ginseng from being excreted from the anus, it is advisable to fix the anus on the water surface with a needle handle. If the gonads have a high degree of maturation, they can be excreted in a short time. On the other hand, if the pro-growth is not mature enough, it will cause self-cut.
5. Anatomy This method
It is suitable for individuals who are large individuals and who experience difficulty in reproduction (usually female, the egg does not reach the final stage of maturity). Using a blade from the rear of the parent's tentacles on the back of the 1/4 body, cut out about 3 cm long openings, and gently push the middle part of the ginseng body to let the genitalia flow out.
The gonads harvested were immediately cleaned several times with clean seawater to prevent the pro-gazed body fluids from affecting the quality of the gonads. The eggs obtained at this time are usually not fertilized (not reaching the final maturity) and must be matured before they can be fertilized. The ripening methods include drug soaking method and ovary chopping method, which are described as follows:
(1) Drug soaking The ovaries are probed with probes, and the released eggs are prepared with DTT, BAL or L-Cysteine ​​and other drugs in a concentration of 0.1 mol/l of seawater solution. After soaking for 15 minutes, the eggs are repeatedly washed with clean seawater. 1 hour, then insemination. When the drug soaking method is used, the dosage and the soaking time must be strictly observed so as not to poison the egg. Place the collected eggs in a container and add a small amount (about the same volume of egg) of clean seawater. Immediately add the sperm (the amount of eggs in a female, add 1 drop of undiluted semen or a few drops of diluted sperm) and light Stir gently (shake) and then add diluted seawater in an equally proportional amount every 5 minutes.
(2) Precipitation and egg-cleaning method The eggs were soaked in clean seawater, and the clean semen was diluted and activated by adding 10 volumes of seawater. Sperm dilution (the amount of eggs in one female, add a few drops of sperm diluent) is added to the egg fluid and the mixture is fully fertilized by gentle agitation. After that, the mixture of eggs and eggs is left to stand until the fertilized eggs precipitate, and the upper layer contains the spermatozoa seawater, and the cleaned fertilized eggs are added by adding clean seawater.
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