Detailed Explanation of Key Technical Points for Artificial Ovulation Induction in Mullet

(1) Basic Principles Artificial ovulation induction is also called induced spawning. Commonly known as spawning induction, etc. It refers to the use of artificial methods to inject drugs into broodstock fish whose gonads have developed to maturity, stimulating the gonads to further mature and release, thereby obtaining mature eggs (or male fish sperm). Accurately grasping the spawning induction season, selecting broodstock suitable for induced spawning, injecting effective doses within the appropriate temperature range - these are the necessary conditions for artificial induced spawning. Whether the spawning induction technique is appropriate or not affects the fertilization rate of fish eggs, the hatching rate, as well as the spawning success rate and post-spawning survival rate of broodstock. Therefore, induced spawning is an important link in artificial propagation.

In the natural environment, when broodstock whose gonads have developed to stage IV are stimulated by certain external ecological conditions (such as water temperature, light, tidal currents or water flow, rainfall, salinity, etc.) acting on the exteroceptors of the broodstock (such as vision, hearing, lateral line, skin, etc.), excitement is produced, and through afferent nerves the received information (stimuli) is transmitted to the central nervous system (brain). Under the control of the central nervous system, the neurosecretory cells of the nutritive nucleus in the hypothalamus are activated, secreting the gonadotropin-releasing hormone (GnRH) stored within them, which travels via the hypophyseal portal vein to the pituitary gland, prompting the pars intermedia of the pituitary to secrete large amounts of gonadotropin (GTH). These hormones reach the gonads (ovary or testis) through body fluid circulation.

After the gonads receive the action of hormones, they rapidly develop and mature and begin to ovulate. At the same time, the gonads secrete sex steroid hormones (steroids), which cooperate with the action of pituitary gonadotropin to make the broodstock exhibit spawning behavior and enter the activity of egg-laying and sperm release. If the gonadal hormone secretion is excessive, then through a feedback mechanism, a signal is sent to the hypothalamus or pituitary, temporarily inhibiting the secretion of GnRH and gonadotropin GTH, thus forming a mutually coordinated and mutually restraining system. The first half of the process, from exteroceptors (sensory organs) to the hypothalamus, is transmitted by nerves; the second half of the process, from the pituitary gland to the sex cells, is entirely transmitted by chemical substances. The transition from nerve transmission to hormone transmission occurs at the contact surface between the hypothalamus and the pituitary.

The above explains that the process of gonadal development in fish is, to a large extent, constrained by the pituitary gland. And the secretory activity of the pituitary is, to a large extent, controlled through the central nervous system by relevant external factors. For mullet broodstock that have reached sexual maturity age, under pond culture conditions, although the female fish ovary has developed to stage IV, without the comprehensive ecological conditions that exist in nature, they cannot spawn naturally in the pond (the ovary cannot transition from stage IV to the spawning state of stage V) to reproduce offspring.

The basic principle of artificial induced spawning is based on the above-mentioned biological principles of natural reproduction in mullet and grey mullet, taking into account the deficiencies of pond culture ecological conditions, and adopting artificial methods to inject a certain dose of spawning agent into the broodstock body, so that as it flows with lymph and blood, this hormone is distributed throughout the fish's body, partially replacing the pituitary secretory activity of the live fish body, and prompting the fish's own pituitary to carry out secretory activity, thereby replacing those comprehensive ecological conditions required for natural reproduction, while retaining only those ecological factors necessary for affecting metabolism (such as water temperature, oxygen, etc.), prompting the broodstock gonads to mature and spawn and release sperm.

For artificial propagation of mullet and grey mullet, the following basic conditions should be met during induced spawning: the broodstock gonads must have developed to the end of stage IV, that is, when the oocytes are fully filled with yolk and the nucleus has migrated to the animal pole, induced spawning will be effective. Gonads that are less developed have poor sensitivity to hormones, or do not respond at all. Therefore, the gonadal development status of broodstock is the primary condition. Under this premise, injecting effective and appropriate amounts of hormones into broodstock to promote sex cell maturation, and acting on the relevant organs that control spawning activity, to strengthen the body's neuro-humoral regulation, causing spawning behavior and completing the spawning process. In addition, certain environmental conditions such as water temperature, oxygen, salinity, etc. must also be coordinated.

(2) Spawning Agents (Exogenous Hormones) There are three types of effective spawning agents widely used in production. One type is fish pituitary (mainly mullet, carp, and crucian carp pituitary); another type is mammalian gonadotropin preparations; the third type is synthetic. 1. Fish Pituitary The fish pituitary is the most important endocrine gland in the fish body. It is located on the ventral surface of the diencephalon and is connected to the hypothalamus. The pituitary can secrete various hormones with different uses. Among them, the hormones related to fish reproduction are collectively called "gonadotropins." It is generally believed that gonadotropins contain two effective substances, namely follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Follicle-stimulating hormone functions to promote the growth, development, and maturation of sperm and egg cells. Luteinizing hormone in females promotes corpus luteum formation and ovulation, while in males it stimulates sperm release and the secretory function of the interstitial cells of the testis. Therefore, injecting pituitary into broodstock can promote the development of sperm and egg cells and promote sperm release and ovulation in the fish.

Pituitary extraction and preservation: The gonadotropin content in fish pituitary varies somewhat with season. Generally, the best time to collect pituitary from mullet and carp is in winter or just before the reproductive season, when the gonadotropin content in the pituitary is relatively high and the dried product granules are plump. The fish from which the pituitary is extracted should preferably be sexually mature individuals. The fish should be fresh, but pituitary from fish that have not been dead long and still maintain considerable freshness can still be used. When extracting the pituitary, first use a knife to split open the skull, remove the fat in the cranial cavity to expose the entire fish brain, then use tweezers to lift the spinal cord connected behind the brain, gently turn the brain over, and a heart-shaped white small granule will be visible, which is the pituitary. Carefully use tweezers to gently separate the connective tissue beside the pituitary, then remove the pituitary. The removed pituitary should remain intact and unbroken. Place it on the back of the hand, roll it with tweezers several times to remove the connective tissue and fat attached to the pituitary, then place it in a small bottle containing acetone or anhydrous alcohol at 20 times the volume of the pituitary for degreasing and dehydration. Replace with new liquid after 8-12 hours, repeat two or three times. Finally, take out the soaked pituitary, place it on absorbent paper to dry, then put it into a small bottle, cover it, seal it, and record the fish name, size, number of pituitaries, and date of extraction, etc., and store in a desiccator for future use. The bottle mouth can also be sealed with paraffin and stored in a cool, dry place. Pituitary preserved in this way will not lose effectiveness for at least two years.

2. Mammalian Gonadotropin Preparations The main ones used in fish are HCG and Synahorin.

(1) Human Chorionic Gonadotropin (HCG): This hormone is a gonadotropin secreted by the trophoblast of the chorion of pregnant women, generally extracted from the urine of pregnant women. In the urine of pregnant women at 3-5 months, the hormone content is relatively high, and the main component is luteinizing hormone (LH). When injected into broodstock, it has the effect of promoting ovulation and sperm release in fish. It is the main spawning agent in artificial propagation. Currently, commercially available veterinary (or fish) gonadotropin products are white, grayish-white, or pale yellow powder, or foam-like solid, easily soluble in water. The hormone easily loses effectiveness when exposed to heat or moisture, so the product should be sealed and stored in a cool, dry place.

(2) Synahorin: This product is made by mixing 1/10 mammalian anterior pituitary gonadotropin and 9/10 HCG. Generally, rabbit units (RU) are used as the potency standard. This product alone is ineffective in inducing reproduction in grey mullet and must be used in combination with fish pituitary. If supplemented with vitamin E, the effect is even better.

3. Synthetic Hormones Gonadotropin-releasing hormone and its analogs. Gonadotropin-releasing hormone (LH-RH) is a natural neurohormone product extracted from the hypothalamus of animals. It can control and regulate the pituitary, stimulate the pituitary to secrete luteinizing hormone and follicle-stimulating hormone, and then stimulate ovulation and sperm formation. Inspired by this, China artificially synthesized luteinizing hormone-releasing hormone (LRH) and its analogs (LRH-A). They have the same biological activity as natural LH-RH. Through experiments on induced spawning in fish, it has been proven that LRH and its analogs have good maturation-inducing effects on both male and female broodstock. In particular, LRH-A has activity hundreds of times higher than LRH. As a fish spawning agent, its effect is very significant and it has more practical value. It is currently widely used in production. The quality of the hormone is related to the spawning induction effect. Before use, the hormone must be carefully subjected to potency determination. Generally, the method of toad ovary in vitro ovulation is used, that is, the minimum dose at which female toads weighing over 50g at 20°C temperature release over 50% of eggs in vitro within 24 hours is called the potency of that batch of hormone, called one toad unit (approximately equivalent to 500 international units).

(3) Selection of Spawning Induction Season Selecting the most suitable season for induced spawning is one of the keys to the success of artificial propagation. If the induced spawning time is too early, most female fish ovaries have not reached the sensitive period, making the induced spawning effect poor; if induced spawning is too late, the ovaries become overripe and similarly cannot achieve successful induced spawning. The period during which good induced spawning results can be maintained from gonadal maturity to the beginning of gonadal degeneration is generally only about 20 days for mullet, so it should be carried out promptly within the spawning induction season. The spawning induction season for mullet varies with water temperature in different regions. In Jiangsu, induced spawning is generally carried out from late April to early May, while in Shandong it is from early May to late May.

The induced spawning effect is closely related to water temperature. Mullet induced spawning is generally carried out at water temperatures of 15-22°C, with 16-20°C being the most suitable. Water temperatures below 14°C or above 24°C will not produce good results. The water temperature for grey mullet induced spawning is preferably 15-20°C.