Artificial ovulation induction technique of barracuda

(1) the basic principle of artificially induced ovulation is also called induced labor. Commonly known as aphrodisiac, green, and so on. The artificial method is to inject drugs into the mature gonadal parent fish to stimulate the gonads to further mature and discharge, so as to obtain mature eggs (or male fish sperm). Accurate grasp of the spawning season, selection of parent fish suitable for spawning, and the use of effective dose injection in the appropriate temperature range is a necessary condition for artificial spawning. Whether the spawning technique is appropriate or not affects the fertilization rate and hatching rate of fish eggs, as well as the acquisition rate and postpartum survival rate of parent fish. Therefore, induced labor is an important part of artificial reproduction.

In the natural environment, the parent fish whose gonads develop to stage *, when certain external ecological conditions (such as water temperature, light, tidal current or current, rainfall, salinity, etc.) stimulate the external sensory organs (such as vision, hearing, lateral line, skin, etc.) of the parent fish, it produces excitement and transmits the received information (stimulation) to the central nervous system (brain) through human nerves. Under the control of the central nervous system, the neurosecretory cells in the nutritional nucleus of the hypothalamus are stimulated to secrete the gonadotropin-releasing hormone (GTH-RH) stored in the hypothalamus and through the portal vein of the pituitary to the pituitary, prompting the interpituitary to secrete a large amount of gonadotropin (GTH). These hormones reach the gonad (ovary or testis) through the circulation of body fluids.

After the gonads are affected by hormones, they develop rapidly and begin to ovulate. At the same time, gonadal secretion of sex retention hormones (steroids), in conjunction with the gonadotropin of the pituitary gland, make parent fish in estrus and enter the activities of spawning and ejaculation. If the secretion of gonadal hormone is excessive, it will send a signal to the hypothalamus or pituitary through feedback, temporarily inhibiting the secretion of GTH-RH and gonadotropin GTH, thus forming a system that coordinates and restricts each other. The first half of the process from the external receptors (senses) to the hypothalamus is conducted by nerves; the second half of the process from the pituitary gland to sex cells is transmitted entirely by chemicals. The transition from nerve conduction to hormone transfer occurs on the interface between hypothalamus and pituitary gland.

The above results show that the process of gonadal development in fish is largely restricted by the pituitary gland. To a large extent, the secretory activity of pituitary gland is controlled by external motivation through the central nervous system. For the parent fish that reach the age of sexual maturity, under the condition of pond culture, although the ovaries of the female fish develop to the Ⅳ stage, they do not have the same comprehensive ecological conditions as in nature, and cannot lay eggs on their own in the pond (the ovaries cannot transition from the Ⅳ stage to the Ⅴ stage of the spawning state) and reproduce.

The basic principle of artificial induction of labor is that according to the above biological principles of natural reproduction of mullet and angled fish and considering the deficiency of ecological conditions of pond culture, an artificial method is adopted to inject a certain dose of oxytocin into the parent fish. With the flow of lymph and blood, the hormone is distributed to the whole body of the fish, partly replacing the secretory activity of the pituitary gland of the live fish, and promoting the secretory activity of the pituitary gland of the fish body itself. So as to replace the comprehensive ecological conditions needed for natural reproduction, and retain only the necessary ecological factors that affect metabolism (such as water temperature, oxygen, etc.) to promote gonadal maturation, spawning and ovulation of their parent fish.

The artificial reproduction of mullet and angled fish should have the following basic conditions, that is, the gonads of parent fish should be effective only when the gonads of parent fish develop to the end of stage Ⅳ, that is, when the yolk in the oocyte is full and the nucleus is biased to the animal. Poorly developed gonads are less sensitive to hormones or do not accept them at all. Therefore, the gonadal development of parent fish is the first condition. Under this premise, an effective and appropriate amount of hormone is injected into parent fish to promote sexual cell maturation and act on the relevant organs that control spawning activity, so as to strengthen systemic neurohumoral regulation, cause estrus behavior and complete the process of spawning. In addition, it is necessary to cooperate with certain environmental conditions such as water temperature, oxygen, salinity and so on.

(2) there are three kinds of effective oxytocin widely used in the production of oxytocin (exogenous hormone). One is the pituitary of fish (mainly the pituitary of barracuda, carp and crucian carp); the other is mammalian gonadotropin products: the third category is 1. Fish pituitary is the most important endocrine gland in fish, which is located on the ventral side of diencephalon and connected with hypothalamus. The pituitary gland can secrete a variety of hormones with different uses, among which the hormones related to fish reproduction are always called "gonadotropins". It is generally believed that gonadotropin contains two effective substances, namely follicular growth hormone (FSH) and luteinizing hormone (LH). Follicle-stimulating hormone, whose function is to promote the growth, development and maturation of sperm and egg cells. Luteinizing hormone promotes luteal formation and ovulation in females and stimulates the secretion of spermatozoa and testicular interstitial cells in males. Therefore, injecting pituitary into the parent fish can promote the development of sperm and eggs and promote the ovulation and ovulation of the fish.

Extraction and preservation of pituitary gland: the content of gonadotropin in pituitary gland of fish varies with different seasons. In general, the time to collect the pituitary gland of shuttle and carp is best in winter or near the reproductive season, when the content of gonadotropin in pituitary is higher and the particles of dry products are full. The fish with pituitary gland removed had better be sexually mature individuals, and the fish should be fresh, but the pituitary gland, which maintains considerable freshness, can still be used shortly after the fish's death. When removing the pituitary gland, first split off the skull with a knife, peel off the fat in the brain cavity to expose the whole fish brain, then use tweezers to pick up the spinal cord connected to the back of the brain, gently open the brain, it can be seen that there is a heart-shaped small white particle, that is, the pituitary gland. Carefully remove the connective tissue next to the pituitary gland with tweezers, and then remove the pituitary gland. The removed pituitary gland is required to remain intact, put it on the back, roll it several times with tweezers, remove the connective tissue and fat attached to the pituitary gland, and then put it into a small bottle of acetone or anhydrous alcohol with a volume of 20 times the pituitary gland for degreasing and dehydration. The new fluid is replaced once after 8 min for 12 hours, two or three times in a row. Finally, the impregnated pituitary gland is removed, put on absorbent paper to absorb dry, and then put into the vial to cover. And record the name, size, quantity of pituitary gland and date of digging, etc., and store them in the dryer. The mouth of the bottle can also be sealed with paraffin wax and stored in a cool and dry place so that the preserved pituitary gland will not fail for at least two years.

2. Mammalian gonadotropin products HCG and Sinahelin are mainly used in fish.

(1) Chorionic gonadotropin (HCG): this hormone is a kind of gonadotropin secreted by the trophoblast of placental chorion of pregnant women and is generally extracted from the urine of pregnant women. (3) the content of hormone in the urine of pregnant women who were 5 months old was higher, and the main component was luteinizing hormone (LH), which was injected into the parent fish and could promote ovulation and ovulation. It is the main oxytocin in artificial reproduction. At present, the veterinary (or fish) gonadotropin sold in the market is white, grayish white, light yellow powder, or foamy solid, soluble in water, and the hormone is easy to fail in case of heat or moisture, so the product should be sealed and stored in a shady place.

(2) Sinahorin: this product is a mixture of mammal anterior lobe gonadotropin of 1x10 and HCG of 9amp10. Generally, the rabbit unit (RU) is used as the titer standard. The product used alone to induce reproduction of grey mullet is ineffective and must be mixed with the pituitary gland of fish. If supplemented with vitamin E, the effect is better.

3. Synthetic hormone gonadotropin-releasing hormone and its analogue gonadotropin-releasing hormone (LH-RH) is a natural product of neurohormones extracted from animal hypothalamus. It can control and regulate the pituitary gland, stimulate the secretion of luteinizing hormone and follicle-stimulating hormone, and then stimulate ovulation and spermatogenesis. Inspired by this, luteinizing hormone-releasing hormone (LRH) and its analogue (LRH-A) have been synthesized in China. It has the same biological activity as natural LH-RH. Through the experiment of stimulating fish to spawn, it is proved that LRH and its analogues have good ripening effect on both male and female parent fish. In particular, the activity of LRH-An is hundreds of times higher than that of LRH, and it has been widely used in production because of its remarkable effect and more practical value as an accelerant for fish. The quality of hormone is related to the effect of induced labor, and the titer of hormone must be carefully identified before use. The method of dropping eggs from toad ovaries in vitro is generally used. even if the female toad weighs more than 50g at a temperature of 20C, the lowest dose of more than 50% of the eggs in vitro within 24 hours is called the titer of this batch of hormones, it is called a toad unit (equivalent to about 500 international units).

(3) the selection of the most suitable season for induced labor is one of the keys to the success of artificial reproduction. If the time of induction of labor is too early, most of the ovary development of female fish does not reach the sensitive stage, so that the effect of induction of labor is not good; if the induction of labor is too late, the ovary is overmature and the success of induction of labor can not be achieved either. From the gonadal development and maturity of the parent fish to the gonadal degeneration, the time to maintain a good spawning effect is generally only about 20 days, so it should be carried out in the spawning season. The spawning season of angled fish varies due to different water temperatures. Jiangsu is generally from the end of April to early May, while Shandong is from early May to late May.

The effect of induced labor is closely related to the water temperature. The induction of spawning is generally carried out when the water temperature is 15 ℃ 22 ℃, and the most suitable temperature is 16 ℃ 20 ℃. If the water temperature is lower than 14 ℃ or higher than 24 ℃, it will not have good effect. The best water temperature for spawning of mullet is 15 ℃ and 20 min.