CN113317241B - Method for gonadotropic development and artificial induced spawning of parent fish of takifugu hexamaculatus - Google Patents
Method for gonadotropic development and artificial induced spawning of parent fish of takifugu hexamaculatus Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23K10/20—Animal feeding-stuffs from material of animal origin
- A23K10/22—Animal feeding-stuffs from material of animal origin from fish
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention provides a method for gonadotropic development and artificial spawning promotion of parent fish of Fugu hexamaculatus, which is suitable for the cultivation of the parent fish of Fugu hexamaculatus under the condition of artificial breeding environment, wherein a bait formula for nutrition enhancement in the cultivation process comprises the following components: adding a propagation enhancer, fish oil powder, vitamin C, vitamin E and taurine into the fresh shrimps or the fresh razor clams according to a certain proportion, and feeding the shrimps or the razor clams at regular time every day. Ripening is carried out on the parent fish after intensive culture for 35 days, and the ripening medicines are chorionic gonadotropin and luteinizing hormone releasing hormone A2. After gonad development and maturity, oxytocin No. 2 and chorionic gonadotropin I are used for induced spawning, parent fish self-spermiation, oviposition and fertilization are carried out after reaching induced spawning effect time, and then high-quality fertilized eggs are collected and selected for seed culture. The method can effectively improve the synchronization rate of egg particle development in the gonads of the takifugu rubripes, the group synchronization rate reaches more than 80%, and the egg diameter is obviously improved by 17% by egg laying.
Description
Technical Field
The invention belongs to the technical field of spawning promotion of fish parents, and particularly relates to a method for promoting gonadal development and artificial spawning promotion of parent fishes of takifugu hexamaculatus.
Background
Fugu hexamaculatus (Diodon holocata) is a kind of Fugu of Fuciformes of the class Phlophagoides, and Biodontidae, and is commonly named as Guaiqi Guaizi and Guaigu rubrum. The tropical marine bottom layer small-sized fish mainly inhabit shallow reef areas, soft bottom sea areas or open water areas. Generally, the fishes live alone and sometimes gather into groups, and the juvenile fishes live in oceanic floating sex life. The puffer fish has delicious meat quality, rich collagen in fish skin, high market popularity, 200 yuan/kg at most and great market potential. At present, all sources of the globefish are caught on the sea, and the resource amount is in a sharp reduction trend along with over catching by fishermen or environmental deterioration.
The takifugu rubripes is an economic variety with important development potential, and the total yield of the fish is not high and the supply and demand on the market are not high due to the unstable population quantity of the resources and obvious seasonal variation of the capture amount. The method is the only way to meet the supply quantity of the market and realize the large-scale seedling culture and the industrial culture. In order to achieve large-scale artificial seedling, sufficient supply of high-quality fertilized eggs needs to be realized, and the premise is to ensure that the gonads of the parents are well developed and supply the high-quality fertilized eggs continuously and stably. No research report on the aspect is found at present.
Disclosure of Invention
The invention aims to solve the technical problems and provides a method for promoting gonadal development and artificial spawning of parent fishes of fugu hexamaculatus, in particular to a method for enhancing nutrition and promoting artificial spawning of parent fishes for gonadal development. The nutrition strengthening method can promote the mature development of ovary or testis in stage III to ovary or testis in stage IV in the process of Fugu rubripes gonad development, and promote gonad development to reach stage V by induced spawning. Compared with the parent fish which is not subjected to nutrition enhancement, the parent fish which is subjected to nutrition enhancement by adopting the technology of the invention can reach the purpose of gonad maturation 7-10 days in advance, the group synchronization rate reaches more than 80%, the spawning rate, the fertilization rate and the hatching rate are obviously improved, and the artificial breeding success rate of the takifugu rubripes is effectively improved.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a method for gonadotropic development and artificial spawning of parent fish of Fugu hexamaculatus, which is suitable for the parent fish of Fugu hexamaculatus to be cultivated under the condition of artificial cultivation environment and comprises the following steps:
(1) parent fish selection
Selecting high-quality Fugu hexapetalus with body mass of over 1000g, body length of over 18cm, no injury on body surface, bright body color and good health activity as parent, culturing in specific culture environment with culture density of 5-7 tails/m 3 ；
(2) Parent fish fortification
Carrying out parent gonadotropic development nutrition enhancement within 1-2 months, and carrying out nutrition enhancement within an artificial breeding stage within 3-9 months;
the nutrition enhancing bait comprises two groups of A enhancing bait and B enhancing bait, wherein the A enhancing bait comprises fresh and live shrimps, live mixed crabs, a propagation enhancer, fish oil powder, vitamin C, vitamin E and taurine; the group B strengthening baits comprise fresh razor clams, squids, a propagation strengthening agent, fish oil powder, vitamin C, vitamin E and taurine;
before feeding each time, dissolving the propagation enhancer, fish oil powder, vitamin C, vitamin E and taurine in physiological saline water, and injecting into shrimp or razor clam to be fed;
the feeding mode is that the group A and the group B reinforced baits are fed alternately, the group A reinforced baits are fed at regular time every morning, and the group B reinforced baits are fed at regular time in the noon, and are all fed after being fed with enough food; meanwhile, culture management and disease prevention and control are carried out in the strengthening process;
(3) artificial ripening and spawning
After intensive culturing of parent fish for 35 days, uniformly ripening the parent fish in a parent fish pond once, selecting 65 units/kg chorionic gonadotropin I +1 microgram/kg luteinizing hormone releasing hormone A2 as ripening medicine, checking the parent fish once per week after ripening, selecting gonad mature, and hastening the parturition of the parent fish which reaches the parturition induction period,
the oxytocic drug is 5 mu g/kg of ovulation promoting hormone No. 2 and 350 units/kg of chorionic gonadotropin I, the oxytocic time is 10: 00-11: 00, and the effect time is 40-46 h; after the parent fish reaches the induced spawning effect time, the parent fish is self-spermiated, spawned and fertilized in the water body, fertilized eggs are collected after the parent fish spawns, the fertilization rate is checked, and high-quality fertilized eggs are selected for seedling cultivation.
Preferably, in the step (1), the conditions for parent fish cultivation are as follows: the diameter of the culture pond is 5m, and the water depth is 1.2 m; the water quality requirement of the culture pond is as follows: salinity is 28-32, the temperature of water is maintained at 24-26 ℃ for 1-2 months, the temperature of water is maintained at 26-28 ℃ for 3-9 months, the pH value is 7.8-8.5, the dissolved oxygen in the water body is maintained to be more than 5mg/L, and the illumination of the water surface layer is 500 lux.
Preferably, in step (2), the formula of the group A fortified bait is as follows: 50 percent of fresh and live shrimps, 40 percent of live mixed crabs, 4 percent of a propagation enhancer, 3 percent of fish oil powder, 1.5 percent of vitamin C, 1.2 percent of vitamin E and 0.3 percent of taurine,
the formula of the B group reinforced bait is as follows: 50% of fresh and alive razor clams, 40% of squids, 4% of propagation enhancer, 3% of fish oil powder, 1.5% of vitamin C, 1.2% of vitamin E and 0.3% of taurine;
before each feeding, uniformly mixing the propagation enhancer, the fish oil powder, the vitamin C, the vitamin E and the taurine in percentage by mass with 0.6 percent of normal saline according to the feeding amount, and averagely injecting the mixture into the shrimp bodies or the razor clam bodies to be fed;
when feeding, the feeding time is 7 points in the morning, and A group of baits are fed every day; feeding the group B baits at 12 noon, and feeding after full feeding.
Preferably, in the step (2), in the parent fish strengthening process, the cultivation management mode is as follows:
detecting water temperature, pH, dissolved oxygen and illumination every morning, and adjusting by a constant temperature machine, changing water, an inflator pump and shading cloth; draining water 30% at 16 pm every day, cleaning the garbage at the bottom of the pool, adding fresh water with the water temperature of 24-26 ℃ after cleaning, and after filling with the fresh water, flowing water in a trace amount, wherein the water temperature is maintained at 24-26 ℃;
the disease control method is as follows: soaking parent fish with 25ppm florfenicol for 12 hours every 6 days, changing water for 100 percent after 12 hours, and continuously using for 2 days; 0.45ppm copper sulfate is used for sprinkling once every 10 days; the pond is poured once every month.
Preferably, in step (3), the method for inspecting the parent fish comprises the following steps: selecting individuals with raised abdomen and reddish and prominent genital pores from parent fish groups; pushing with hand from fish belly to genital pore direction, calculating that white semen or transparent ovum has reached sexual maturity, and breeding in another pool for induced spawning.
After the parent fish is strengthened for 35 days according to the method, the strengthened and un-strengthened parent fish are dissected, the gonad development condition is compared, the gonad development of the parent fish after nutrient strengthening is obviously better than that of the parent fish of the un-strengthened group, the egg diameter of the parent fish of the strengthened group is uniform, and the parent fish starts to appear in a free state (figure 3), and the egg diameter of the parent fish of the control group is relatively non-uniform and does not appear in a free state (figure 2).
By comparing the effect of inducing spawning of parent fish with mature gonad, the egg diameter of the spawned parent fish is 1.8 +/-0.1 mm (figure 5), and the fertilized egg embryo develops well (figure 6); the egg diameter of parent fish eggs which are not induced to spawn is 1.5 +/-0.2 mm (figure 4).
Through statistical comparison, the nutrition strengthening method and the variety and dosage of the oxytocic are adopted, and the relative fecundity is improved by 18 percent (p is less than 0.05) compared with a control group; the individual spawning synchronous rate after nutrition enhancement and spawning induction is 80 percent and is obviously higher than 30 percent of that of a control group; the fertilization rate experimental group (51 +/-5.1%) is obviously higher than that of the control group (38 +/-2.9%); the spawning quality is obviously improved, and the spawning time is controllable.
The invention has the following beneficial effects:
(1) the invention belongs to the first nutrient enrichment culture of a parent of takifugu rubripes in China. The feed has the nutritional ingredients required by the development of the ovary of the takifugu rubripes by adopting scientific compatibility, the selected main raw materials of the fresh and live shrimps, the live crabs, the squids, the fresh and live razor clams and the like are aquatic organisms, and no anti-nutritional factors exist between the fresh and live shrimps, the live crabs, the squid, the fresh and live razor clams and the takifugu rubripes.
(2) The added propagation enhancer, fish oil powder, vitamin C, vitamin E, taurine and the like are all necessary nutritional components for fish gonad development, and can effectively promote the gonad development from stages II and III to stage IV; the fresh shrimps or the fresh razor clams are filled in the fish bait after being prepared according to the proportion, so that the fish bait is favorable for the swallowing behavior of the takifugu spinosus. The feeding method has the advantages of high nutrient content, good palatability and strong food calling property, avoids water pollution caused by loss of additives, and has good application prospect.
(3) The invention can effectively improve the gonad development of the takifugu hexamaculatus within 35 days by nutrition enhancement at the early stage of propagation, improve the relative brood amount by 20 percent, lead the gonad development to reach the IV stage about 7 days in advance, effectively improve the synchronization rate of the gonad development of parent groups and lay a good foundation for artificial seedling culture.
(4) The feed can be used for quickly recovering the gonads of parent fishes of the takifugu hexamaculatus during the breeding period, and achieves the purpose of multiple spawning in a moderate temperature period of 3-9 months of breeding.
(5) The species and the dosage of the selected ripening hormone can effectively improve the synchronization rate of gonad development of the parents, and the synchronization rate reaches over 80 percent; the selected induced spawning variety and the induced spawning dosage are suitable for spawning of the Fugu rubripes, and the high-quality fertilized eggs obtained through induced spawning have the fertility rate of over 60 percent and the hatching rate of over 85 percent.
In conclusion, the effective implementation of the method can provide technical support for improving the breeding success rate of the acanthopanax papulosus and vigorously developing the breeding of the acanthopanax papulosus, and the method has important economic and social meanings undoubtedly for promoting industrial development and has wide application prospect.
Drawings
Figure 1 shows the selected parent of takifugu hexamaculatus.
FIG. 2 shows the development of ovary of Fugu hexamaculatus fed by the nutrition enrichment method of the present invention.
FIG. 3 shows the ovarian development of Fugu hexamaculatus fed by the nutrition enrichment method of the present invention.
FIG. 4 is the measurement of the size of the ovum in the ovary of Fugu hexamaculatus in stage V, which is not fed by the strengthening method of the present invention, and the diameter of the ovum is 1.5 + -0.2 mm.
FIG. 5 shows the measurement of ovum size of Fugu hexamaculatus fed by the strengthening method of the present invention, and the diameter of the ovum is 1.80 +/-0.1 mm.
FIG. 6 is a fertilized egg from the blastoderm bulge resulting from induction of labor in accordance with the present invention.
Detailed Description
The following embodiments are implemented on the premise of the technical scheme of the present invention, and give detailed implementation modes and specific operation procedures, but the protection scope of the present invention is not limited to the following embodiments.
Example 1 comparison of Fugu hexamaculatus Carallus Carruth with respect to nutrient enrichment in gonadal development stage
(1) Selecting parent fish
Selecting high-quality Fugu hexamaculatus with body mass of more than 1000g, body length of more than 18cm, no injury on body surface, and good health and activity from domesticated and stabilized Fugu hexamaculatus (figure 1). Dividing the selected fish into an experimental group and a control group IIGroup, put into the pool with diameter of 5m and water depth of 1.2m, and put into the stocking density average pool with 5-7 tails/m 3 。
And (3) cultivating environmental conditions: salinity of 28-32, maintaining the water temperature through a constant temperature system, 24-26 ℃ for 1-2 months, 26-28 ℃ for 3-9 months, pH 7.2-8.5, dissolved oxygen content of more than 5mg/L, and illumination of the surface of the water layer of about 500 lux. Discharging water by about 30% at 16 pm every day, cleaning garbage at the bottom of the pool, adding fresh water with the temperature of 24-26 ℃ after cleaning, and pouring a small amount of running water after filling.
(2) Parent fish fortification
Baits during the cultivation of parent fish of the takifugu hexamaculatus, experimental group baits comprise fresh and live shrimps, live crabs, squids and fresh and live razor clams, and a propagation enhancer, fish oil powder, vitamin C, vitamin E and taurine are added as nutrition enhancers; the bait for the control group was fresh live shrimp, fresh alive razor clam, live crab, squid, and did not include nutrition enhancer.
In this example, the propagation enhancer was purchased from wakame industries co, and the main components were soybean lecithin, yeast for feed, calcium carbonate, guar gum, spirulina fine algae powder, rosemary extract, anhydrous silicic acid, garlic powder, various vitamins and various minerals, and the function was to promote gonad development.
The experimental group bait comprises A, B two groups, and the difference is that the group A adopts: 50% of fresh and live shrimps, 50% of fresh and live razor clams in the group B, and 40% of live mixed crabs, 40% of squids, 4% of propagation enhancer, 3% of fish oil powder, 1.5% of vitamin C, 1.2% of vitamin E and 0.3% of taurine in all the groups B. Before feeding, the propagation enhancer, fish oil powder, vitamin C, vitamin E and taurine are mixed with 0.6% normal saline and injected into shrimps or razor clam bodies.
Feeding is carried out at regular time every day, group A baits are fed 7 o 'clock earlier, group B baits are fed 12 o' clock at noon, and feeding is carried out after full feeding.
Soaking the mixture for 12 hours by using 25ppm of florfenicol every 6 days, changing water for 100 percent after 12 hours, and continuously using for 2 days; 0.45ppm copper sulfate is used for sprinkling once every 10 days; the pond is poured once every month.
Dissecting fish body after 35 days of culture, checking gonad development condition (figure 2, figure 3), and showing that the gonad development of experimental group is faster than that of control group, the egg diameter is uniform, and the fish starts to appear free; the control group had relatively uneven egg diameters and no free state was observed.
After intensive culturing of parent fish for 35 days, uniformly ripening the parent fish once, wherein the ripening medicine is chorionic gonadotropin I (65 units/kg) and luteinizing hormone releasing hormone A2(1 mu g/kg). After ripening, the parent fish is checked once a week to see whether the gonad development is mature or not and whether the period of spawning induction is reached or not.
Example 2 comparison of oxytocic patterns in Fugu hexamaculatus
Individuals with raised abdomen and reddish and protruding genital pore were selected from the experimental group of example 1, examined on an operation platform, and pushed by hand from the abdomen of the fish toward the genital pore, and the fish were divided into two groups, and were placed in the pond for cultivation, respectively, assuming that sexual maturity had been reached. A group of induction methods used in the present invention for induction of labor; the other group allowed natural spawning as a control group.
The oxytocin for the oxytocin group is composed of ovulation-promoting hormone No. 2 and chorionic gonadotropin I, and is injected according to an international unit dosage of 5 micrograms of ovulation-promoting hormone No. 2 per kilogram of fish and 350 units of fish per kilogram, the oxytocin time is 10: 00-11: 00, and the effect time is 40-46 h. After the effect time, fertilized eggs were collected from the parent fish pond with a scoop net, and the fertilization rate and hatchability were checked. And (4) selecting high-quality fertilized eggs to cultivate seedlings.
The results of this example show that parent fish without induced spawning eventually obtained an egg diameter of 1.5 ± 0.2mm (fig. 4); the diameter of the ovum after induced spawning is 1.8 plus or minus 0.1mm (figure 5), and the fertilized ovum embryo develops well (figure 6).
The results of the experimental group and the control group of this example were statistically compared, as shown in table 1. By adopting the nutrition strengthening method and the variety and dosage of the oxytocic, the relative fecundity is improved by 18 percent (p is less than 0.05) compared with a control group; the individual spawning synchronous rate after nutrition enhancement and spawning induction is 80 percent and is obviously higher than 30 percent of that of a control group; the fertilization rate of the experimental group (51 +/-5.1%) is obviously higher than that of the control group (38 +/-2.9%); hatchability the experimental group (62. + -. 8.4%) was not significantly different from the control group (59. + -. 6.1%) (p > 0.05).
Therefore, after the nutrient enrichment and spawning promotion treatment by the method, the spawning quality of the parent fish is obviously improved, and the spawning time is controllable, so the method can be applied to the nutrient enrichment and spawning promotion in the tetraodon elatus gonad development process.
Table 1 comparison of the effects of experimental group and control group on the egg laying amount, fertility rate and hatching rate of Fugu hexamaculatus
| Experimental group | Control group | |
| Relative amount of eggs (g) | 4.62±0.34 | 3.79±0.74 |
| Spawning synchronization rate (%) | 80 | 30 |
| Fertilization Rate (%) | 51±5.1 | 38±2.9 |
| Hatching rate (%) | 62±8.4 | 59±6.1 |
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.
Claims (5)
1. A method for gonadotropic development and artificial spawning of parent fish of takifugu hexamaculatus is characterized by comprising the following steps:
(1) parent fish selection
Selecting high-quality Fugu hexapetalus with body mass of over 1000g, body length of over 18cm, no injury on body surface, bright body color and good health activity as parent, culturing in specific culture environment with culture density of 5-7 tails/m 3 ；
(2) Parent fish fortification
Carrying out parent gonadotropic development nutrition enhancement within 1-2 months, and carrying out nutrition enhancement within an artificial breeding stage within 3-9 months;
the nutrition-enhanced baits comprise two groups of A group of enhanced baits and B group of enhanced baits, and the formula of the A group of enhanced baits is as follows: 50% of fresh live shrimps, 40% of live mixed crabs, 4% of a propagation enhancer, 3% of fish oil powder, 1.5% of vitamin C, 1.2% of vitamin E and 0.3% of taurine; the formula of the B group reinforced bait is as follows: 50% of fresh and alive razor clams, 40% of squids, 4% of propagation enhancer, 3% of fish oil powder, 1.5% of vitamin C, 1.2% of vitamin E and 0.3% of taurine;
before feeding each time, dissolving the propagation enhancer, fish oil powder, vitamin C, vitamin E and taurine in 0.6% physiological saline water, and injecting into shrimp or razor clam to be fed;
the feeding mode is that the group A and the group B reinforced baits are fed alternately, the group A reinforced baits are fed at regular time every morning, and the group B reinforced baits are fed at regular time in the noon, and are all fed after being fed with enough food; meanwhile, culture management and disease prevention and control are carried out in the strengthening process;
(3) artificial ripening and spawning
After intensive culturing of parent fish for 35 days, uniformly ripening the parent fish in a parent fish pond once, selecting 65 units/kg chorionic gonadotropin I +1 microgram/kg luteinizing hormone releasing hormone A2 as ripening medicine, checking the parent fish once per week after ripening, selecting gonad mature, and hastening the parturition of the parent fish which reaches the parturition induction period,
the oxytocic drug is 5 mu g/kg of ovulation promoting hormone No. 2 and 350 units/kg of chorionic gonadotropin I, the oxytocic time is 10: 00-11: 00, and the effect time is 40-46 h; after the parent fish reaches the induced spawning effect time, the parent fish is self-spermiated, spawned and fertilized in the water body, fertilized eggs are collected after the parent fish spawns, the fertilization rate is checked, and high-quality fertilized eggs are selected for seedling cultivation.
2. The method for gonadotropic development and artificial induced spawning of parent fish of fugu hexamaculatus according to claim 1, characterized in that:
wherein, in the step (1), the conditions for parent fish cultivation are as follows: the diameter of the culture pond is 5m, and the water depth is 1.2 m; the water quality requirement of the culture pond is as follows: salinity is 28-32, the temperature of water is maintained at 24-26 ℃ for 1-2 months, the temperature of water is maintained at 26-28 ℃ for 3-9 months, the pH value is 7.8-8.5, the dissolved oxygen in the water body is maintained to be more than 5mg/L, and the illumination of the water surface layer is 500 lux.
3. The method for gonadotropic development and artificial spawning of parent fish of takifugu hexamaculatus according to claim 1, characterized in that:
wherein, in the step (2), the group A baits are fed at 7 points in the morning every day in the timing feeding time; feeding the group B baits at 12 noon, and feeding after full feeding.
4. The method for gonadotropic development and artificial spawning of parent fish of takifugu hexamaculatus according to claim 1, characterized in that:
in the step (2), in the parent fish strengthening process, the breeding management mode is as follows:
detecting water temperature, pH, dissolved oxygen and illumination every morning, and adjusting by a constant temperature machine, changing water, an inflator pump and shading cloth; draining water 30% at 16 pm every day, cleaning the garbage at the bottom of the pool, adding fresh water with the water temperature of 24-26 ℃ after cleaning, and after filling with the fresh water, flowing water in a trace amount, wherein the water temperature is maintained at 24-26 ℃;
the disease control method is as follows: soaking parent fish with 25ppm florfenicol for 12 hours every 6 days, changing water for 100 percent after 12 hours, and continuously using for 2 days; 0.45ppm copper sulfate is used for sprinkling once every 10 days; the pond is poured once every month.
5. The method for gonadotropic development and artificial spawning of parent fish of takifugu hexamaculatus according to claim 1, characterized in that:
wherein, in the step (3), the method for checking the parent fish comprises the following steps: selecting individuals with raised abdomen and reddish and prominent genital pores from parent fish groups; pushing with hand from fish belly to genital pore direction, calculating that white semen or transparent ovum has reached sexual maturity, and breeding in another pool for induced spawning.
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