In natural waters, mature gonads of gonads secrete gonadotropins under the stimulation of certain environmental conditions such as water velocity, water temperature, and water level, which can cause ovaries or testes to complete maturation, ovulation or The ejaculation process. However, it is difficult to reproduce the broodstock naturally using artificially simulated ecological conditions. Therefore, artificial injection of hormones is used in production to promote the maturation, ovulation and spawning of female brood fish eggs, and to promote the maturation of spermatozoa and platoon sperm. The level of oxytocin technology directly affects the rate of inducing, fertilizing, and hatching. Therefore, artificial oxytocin must be strictly followed in accordance with the operating specifications and technical requirements.
As early as 1977, Godinho et al. used chorionic gonadotropin (HCG) to elicit the success of the fine-scaly dagger, and observed the development of early embryos. Later, in 1981, Castagnolli and Donaldson used carp pituitary extract (SG). -G100). In 1985 Romagosa et al. used squid crude pituitary products (SPE) and Bernardina et al. in 1986 to elicit production of L. striata with luteinizing hormone-releasing hormone analogues (LRH-A2). success.
The Zhejiang Provincial Institute of Freshwater Fisheries, in July 1999 in China, artificially propagated the fine-scale oystercatcher and achieved success for the first time.
(A) The choice of broodstock
In order to ensure the success of oxytocin production, the maturity of female and male broodstock must be selected. Only mature broodstock can properly ovulate or ejaculate and artificially inseminate in suitable conditions such as water temperature, water level, oxytocin and oxytocin production techniques. Generally, broodstock maturation can be identified based on the appearance of the broodstock or a combination of digging observations.
1. The appearance of the fine scales before the sexual maturity; it is difficult to distinguish between male and female. When sexual maturity is reached, the individual females are larger, the abdomen is enlarged, the posterior abdomen is soft and elastic, and the outline of the ovary on both sides of the abdomen is obvious, and the genital mastoid is large and purple-red. Such females meet the conditions for oxytocin production. If the front abdomen is full and the female with a small back and belly is not soft, it is still immature. Females whose abdomen is too soft and relaxed are broodstock that have been absorbed and degenerated by the ovaries; sexually mature males have relatively small individuals, narrow abdomens, and genitalia are concave in triangles. If there is more milky semen flowing from the abdomen, It shows that the maturity of the fish is good and can be used for oxytocin production. Such as light pressure male abdomen after the abdomen, can not squeeze out semen or squeeze a small amount of semen and semen into the water into a thin line does not spread out, this shows that the male fish is not yet fully mature, can not be used.
2. Egg-digging observation In order to more accurately identify the maturity of female broodstock, oocyte retrieval may also be performed using an oocyte. The digger can be made of plastic pipe, copper rod or duck goose feather tube, and in the front part is a hole with a length of 1.5 cm and a width of 0.3 cm. Use a digger to gently insert the female fish's gonads. When the digger is successfully inserted into the genital hole of the fish, it will be rotated for 1 week. After gently pulling out the digger, the eggs will be removed. If the genital pores are too tight, it indicates that the female broodstock is poorly matured and should not be inserted with force. Eggs were examined by microscopy. The eggs were emerald green. The eggs were of uniform size and flow freely. Eggs with an egg diameter of 939.0 microns had high fertilization rates. For this kind of egg, the egg diameter will increase to 1001.6 microns before ovulation.
The fertility of the brooded broodstock is proportional to the age. The 3rd instar female brood is the absolute amount of 297308 eggs/tail, and the 4th instar female brood is the absolute amount of 377643 eggs/tail.
(two) male and female matching group
Before the oxytocin production, the number of male and female broodstock is systematically matched with the matching group. In order to facilitate the spawning and fertilization of brooded broodstock, the number of male and female broodstock is generally more than that of female broodstock. The ratio of male to female broodstock is 1:2. It is important to note that the size of individuals participating in the same batch of spawning male and female broodstock should be as close as possible.
(III) Oxygen production and injection volume
The commonly used oxytocins include: pituitary (PG), chorionic gonadotropin (HCG), luteinizing hormone-releasing hormone analogue (LRH-A2), domoxone (DOM) and mixed hormones.
The type of oxytocic acid and the effective dose of oxytocic acid should be selected according to the number of broodstock, maturity, sex and individual size, as well as the timing of urine production and water temperature. In the case of early induction or low water temperature or poor broodstock maturity, the oxytocin dose should be increased appropriately; otherwise, it should be reduced appropriately. It has been proved that the two-needle injection method uses two needles at intervals of 6 hours and 12 hours, and has the same ripening effect. This indicates that the ripening rate is determined by the first needle, and the second is very effective in inducing ovulation.
(D) Oxygen Production Method
The commonly used oxytocin method is: First, a small amount of oxytocin is used for ripening, and then after a certain period of time, oxytocic acid is injected for oxytocin production. That is: commonly referred to as two injections.
1. Preparation of oxytocin The pituitary gland (PG) is ground into a powder with a clean mortar, and the finer it is, the better it is; then it is formulated with a physiological saline solution to form a suspension. Human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LRH-A2) were dissolved in physiological saline. In general, the total amount of oxytocin is calculated according to the weight of the bred broodstock, supplemented by 5% supplemented by 10% and consumed as an injection. The amount of normal saline injected is generally 2 ml per broodstock, and should not be too thick or too thin, so as not to affect the oxytocin effect.
2. Injectable methods Body cavity injections and muscle annotations are all available, and body cavity injections are often used in production. Before injection, syringes and needles should be sterilized with water. When the body cavity is sterilized, the needle is usually inserted at the base of the pectoral fin or pelvic fin without a phosphorus depression, and the depth is 11.5 cm. Note that the heart cannot be stabbed and the injection is slowly injected. The intramuscular injection site is between the lateral line and the dorsal fin. When the injection, the needle is used to provoke the scales and penetrate the scales forward into the muscles, or from the dorsal fin base behind the scales and the fins parallel to the needle depth of 1.52 cm . The two stitches are 814 hours apart.
(E) Effect time and heat
The time interval from the last injection of the oxytocide to oviposition is called the effect time. The length of the effect time is closely related to the water temperature, the type of oxytocin, the number of injections, the dose, and the gonad maturation of the broodstock, especially the relationship between the water temperature, the type of oxytocin and the dose. In general, the water temperature is high and the effect time is short; otherwise, it is long. Injection of the pituitary gland is faster than other oxytocide injections. After the broodstock was injected into the microfluidic water production tank with a water temperature of 2330°C, the effect time was 714 hours. At the optimal breeding water temperature of 2528°C, the effect time was 810 hours.
After the broodstock is injected into the pool, they first roam, and after the effect time, the male and female fish begin to chase the female broodstock, that is, heat. When the estrus reaches its climax, the brooding fish swims in the water with excitement, stimulating water into waves, or pulling out water. Flushing has a certain stimulating effect on the estrus of broodstock. Generally 2 hours before estrus, the flow rate of water in the spawning pool is increased, and the broodstock chasing, spawning or platooning and other reproductive activities are promoted. After estrus and spawning, give micro-flowing water.
(6) Natural spawning and artificial insemination
1. Naturally spawning estrus escort broodstock to achieve high levels of excitement, the male and female broodstock swims quickly, the body clings or twists together, and finally the female and male fish close to the abdomen, pectoral fins open close together, tail tail tightly hooked, At this time, the female fish spawn, the male fish ejaculate, and the fins stir the water. The egg and sperm are fully in contact and fertilized.
2. Artificial insemination Artificially collected eggs, sperm collection, insemination, to obtain fertilized eggs is called artificial insemination. In order to improve the artificial insemination rate, we must first prepare the time for judging the spawning of the broodstock, timely fishing, picking eggs, and sperm extraction. Second, when performing artificial insemination, we must master more skilled operating techniques. General artificial insemination methods are dry, semi-dry and wet methods, of which dry insemination is the most commonly used method. The specific approach is:
(1) Oviposition When the broodstock is elicited, when the female and male broodstock chase intensely, or when the hen and broodstock have no chase when the effect time is reached, it is necessary to promptly check whether the female broodstock lays eggs. The examination can be carried out in water, with the abdomen facing up and the abdomen gently pressed by hand. If more eggs are produced from the reproductive hole, the eggs can be picked. If spawning is rare, the broodstock can be raised for about half an hour before picking eggs.
Collect the color of fish eggs, such as greenish gray, free, uniform size, full, rounded round, fast water absorption, uniform expansion and swell, ovarian fluid was viscous, moderate amount, this is a high quality egg, fertilization rate; Such as the egg was dull, with "white eggs", egg size is uneven, poor elasticity or inelastic, the egg membrane is easy to rupture, water absorption is slow and incomplete, ovarian fluid is less or more like water, no stickiness, this is Poorly matured eggs or over-ripened eggs have low fertilization rates or are not fertilized.
(2) Semen of mature male and female fish is generally collected at any time. If the semen is milky or thick, it is a high-quality semen; if the semen is thin and slightly yellow, it is usually over-ripe and the quality is poor. Semen collection, generally using 510 ml syringe (without needle) inhaled artificially squeezed semen for storage. Semen can also be collected in advance and placed in the preservation solution. The preservative solution was prepared by 2.9 g of glucose, 1.0 g of sodium citrate, 0.2 g of sodium bicarbonate, 0.03 g of potassium chloride, and 0.3 g of sulfanilamide, plus 100 ml of distilled water. When saving, add fresh semen, add 3 times the preservation solution to dilute, then gradually cool to 3 °C, and put it in the refrigerator or thermos for future use. Generally, sperm can live for 1015 days and can be used for artificial insemination when the temperature rises after use.
(3) Dry insemination The mature ovulated female broodstock and mature male broodstock are caught, and the water of the broodstock fish body and the drain hole is wiped off with a dry towel, and the eggs of the female broodstock are squeezed into the dried white enamel basin. At the same time, the male and female fish are squeezed into the container into the semen or injected into the semen collected in advance, immediately with feather or hand evenly stirred for l2 minutes, add a small amount of physiological saline and then gently stirred, the fertilization process will be carried out at this time. In the process of artificial insemination, avoid direct sunlight, so as to avoid ultraviolet rays killing sperm and affecting the fertilization rate.
Third, hatching
The fertilized egg undergoes a series of changes under the appropriate environmental conditions and finally breaks out of the membrane and becomes a fry, which is the embryonic development process. The hatching in production actually includes the entire period before the fish in the lower pond. The fertilized eggs of the ichthyosis are hatched and run in running water.
(a) embryonic development
When the fertilized eggs hatched, when the water temperature was 2528°C, the membrane rupture time was 1620 hours; when the water temperature was 29°C, the membrane rupture time was 13 hours. The fish that had just been filmed had a length of 56 mm.
1. The fertilized egg is a gray-gray, non-viscous egg, and the egg membrane is thin and transparent. It is a floating fish egg. The just-grown ovum has a diameter of 1.11.2 mm. Each gram of egg contains 9001000 eggs. The fertilized eggs swelled to 56 mm after 30 minutes of absorption.
2. Embryonic development Scientists from the Brazilian New Variety Research Group of the Freshwater Fisheries Research Institute in Zhejiang Province conducted preliminary observations and measurements on the embryonic development of L. striata in 1999.
After hatching the fish fry, after 45 days of incubation, the waist point of the fish fry is obvious. The yolk sac is about to be absorbed and the digestive tract is connected. At this time, the fry can prepare the lower pond and enter the fry cultivation stage.
(b) Incubation equipment
The commonly used incubating equipment for artificial propagation of fine-spotted oysters is: hatching tanks, hatching barrels, and hatching rings.
1. Incubation barrel (cylinder) The incubator barrel is generally welded by galvanized iron sheet. It is funnel-shaped and consists of a barrel body, a filter hood, a bracket, and an inlet and outlet pipe. The water flows through the bottom of the cylinder and flushes the eggs, causing the eggs to tumbling up and down, in a state of suspension. The hatching water is filtered out by the filter cover and flows through the outlet. Incubation tanks are generally made from water tanks. Influent water is passed through the conduit in the middle of the tank. The water from the upper part of the conduit rises up to the bottom. The eggs are kept rolling and the water flows out through the filter. Per cubic meter of water, 800,200,000 fertilized eggs can be placed.
2. Large-scale hatchery equipment suitable for large-scale production of hatchery loops is mostly a hatchery ring of a brick-cement mortar-maturing structure. The small ones are also made of plastic or tin. Generally, the loop is 23 rings, and there are also single rings or 3 rings. Above the ring. The water inlet pipe is located at the bottom of the ring road, and the water circulates in the ring road. Then it overflows or overflows through the drainage pipe. The water inlet and outlet water are each a system, all controlled by a gate valve outside the ring road. In order to prevent the occurrence of dead angle in the water flow, the density of oviposition should not be too large. Generally, it is appropriate to lay eggs of 1 million and 1.2 million grains per cubic meter of water.
(III) Incubation Management
1. Choosing water for hatching When selecting hatchery water, it requires fresh water, non-toxic, non-polluting and invincible pests. The oxygen content is above 5 mg/l, the pH is moderate, and the pH is 6.57.5.
2.Prevention of predatory organisms Since the fry of the first film is very delicate, the organs are not fully developed, the resistance to the bad environment is poor, and it is easy to be killed by biological predators. Therefore, the 120-mesh nylon sieve should be used to filter the water before entering to prevent water. The adults and larvae of sword leeches, leeches, aquatic insects cause damage to fish eggs and fry.
3. Water temperature requirements The fertilized eggs hatched at different water temperature conditions, the embryonic development speed is also different. Within a certain temperature range, the incubation time will be shortened as the water temperature rises. When the incubation water temperature is lower or higher than the required water temperature or sudden changes in water temperature, embryonic development may be arrested, or deformed embryos may increase, affecting hatching emergence rate. The optimum incubation temperature for the fertilized eggs was 2528°C.
4. Dissolved oxygen embryo development requires oxygen supply, and the oxygen requirement gradually increases with the development process. The production requires that the oxygen content of the incubation water should be no less than 5 mg/L. At the same time, it is also required to ensure that eggs or seedlings do not accumulate. Otherwise, oxygen-deprivation suffocation and death will occur even in high dissolved oxygen.
5. When flowing water is incubated, the flow rate determines the amount of oxygen in the water and the distribution density of fertilized eggs. If the flow rate is too slow, eggs will suffocate and die. Too fast will prematurely rupture the membrane and the embryo will die. Therefore, during the incubation process, water flow regulation is a very important task. The flow rate of water can be adjusted according to the exchange condition of water. Generally, it is exchanged once in 15 minutes, and once in 3040 minutes. When laying eggs, eggs are required to flow with the water to ensure the embryo's need for oxygen. With the development of the embryo, the oxygen requirement of the embryo will also increase, and the flow rate must be gradually increased. When the membrane is released, the flow rate should be appropriately slowed to increase the concentration of hatching enzymes. After filming, the fry has weak vigor. Most of the time it is in the lower layer of the water. In order to prevent the fry from sinking and dying, the flow rate should be increased appropriately to facilitate the floating and even distribution of fry.
(IV) Fertilization rate, hatching rate and emergence rate
1. Fertilization rate Generally, the fertilization rate is calculated from the embryonic development to the middle and late stages of the original intestine. 200,500 fish eggs were randomly selected and placed in petri dishes. The total number of eggs and the number of normal fertilized eggs were counted (repeated counts were repeated several times), and the percentage of oviparous eggs that were normally developed was calculated as the percentage of the total eggs. The fertilization rate.
Fertility rate = (number of eggs fertilized) 100%
2. Hatching rate To improve the level of hatchery management, the hatching rate must be calculated.
Hatching rate = (total number of hatched fry eggs) l00%
3. Emergence rate The actual hatching rate in production is difficult to calculate. The production rate is mainly calculated by the seedling rate. The emergence rate is the percentage of the total number of fertilized eggs that emerged.
Seedling rate = (number of seedlings fertilized) 100%
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