CN112262794A - Method for sustainable large-scale production of all-female mandarin fish - Google Patents
Method for sustainable large-scale production of all-female mandarin fish Download PDFInfo
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- A01K61/00—Culture of aquatic animals
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Abstract
The invention discloses a method for sustainable large-scale production of full-female mandarin fish, which comprises the following steps: firstly, selecting 2-5-year old siniperca chuatsi female parent with excellent characters, good development, no diseases and no injuries, and producing pure line all-female filial generation XX female parent by using a gynogenesis technology; after the mandarin fish is cultured to 2.0cm under intensive conditions such as a net cage or a cement pond and the like, domesticating the live fish, domesticating the fresh fish and domesticating the soft pellet feed in sequence for 7-10 days to obtain mandarin fish all-female fingerlings capable of completely ingesting the soft pellet feed; continuously feeding pure-line all-female siniperca chuatsi with fadrozole hormone for 60 days to cultivate physiological male siniperca chuatsi (XX); after the good growth of the XX male-female rotary mandarin fish is discharged from a pond, the mandarin fish is raised by live baits until the next year, and the XX male-female mandarin fish can be established in a large scale; the female parent system (XX male parent) of the all-female mandarin fish and the female parent system (XX female parent) of the common mandarin fish are bred in a matching mode, and the all-female mandarin fish can be produced continuously in a large scale.
Description
Technical Field
The invention belongs to the technical field of genetic breeding of fish cell engineering, and particularly relates to a method for sustainable large-scale production of all-female mandarin fish.
Background
Mandarin fish (Siniperca chuatsi) belongs to Perciformes (Perciformes), Percoidei (Percoidei), Perciferaceae (Percichthyoidae) and Mandarin fish (Siniperca) and is widely distributed in several water systems in China. The mandarin fish is fed by live fishes and shrimps for a whole life from the feeding of the fish fries, has the characteristics of quick growth, delicious taste, no muscle thorns and the like, is one of important and rare fresh water culture varieties in China, and has high economic value. In recent years, the mandarin fish farming industry has been greatly developed, main farming areas are Hubei, Guangdong, Jiangsu, Zhejiang, Jiangxi and other provinces, and the statistical yearbook data of the Chinese fishery show that the annual output of the mandarin fish farming in 2018 reaches 31.59 ten thousand tons. The growth difference of male and female mandarin fish individuals is large, and the growth speed of female mandarin fish is obviously faster than that of male mandarin fish[1][2]Therefore, the Siniperca chuatsi all-female strain is cultivated by the sex control technology, and the cultivation yield and the economic benefit can be effectively improved.
Production of parthenocarpic females generally involves methods such as artificial selection, gynogenesis, direct estrogen-induced reversal, and the like. However, the labor intensity of manual selection is high, and the difficulty in accurately distinguishing male and female fishes is high, so that the method cannot be widely applied to actual production. The remnant keen etc. obtains the pure filial generation of mandarin fish diploid through the induction of artificial gynogenesis, but the gynogenesis survival rate is only 4.72%, and the growth and development of the gynogenesis diploid larval fish is obviously slower than that of the normally developed diploid larval fish[3]The method only depends on the development of the artificial gynogenesis, and the large-scale production of the all-female mandarin fish cannot be met. In addition, as the Siniperca chuatsi fish takes live fishes and shrimps as food for the whole life from the beginning of eating, the research of inducing the sex reversal of the Siniperca chuatsi fish by utilizing exogenous hormone at present is that artificial feed is firstly fed to bait fish for hormone enrichment, and then the Siniperca chuatsi fry before gonad differentiation is subjected to the sex reversal by catching the bait fish containing the exogenous hormone. However, the efficiency of the bait fish for enriching exogenous hormones is very easily influenced by endogenous factors such as food intake, metabolism level and the like, andthe method cannot accurately control the dosage of exogenous hormone due to the influence of environmental factors such as water temperature, water quality and the like, not only causes the cost to rise and the efficiency to be reduced, but also has the risk of sex hormone residue, and is banned by many countries. Varadaraj and the like produce the super-male Morganbike tilapia by utilizing hormonal reversion and combining gynogenesis technology[4]The defects of the method are effectively overcome. However, the technology for producing the full-female fish in a large scale by combining gynogenesis and hormonal reversion technology still needs to be broken through so far.
Disclosure of Invention
The invention aims to perform technical fusion and optimization aiming at the defects of the prior art, provide a method for continuously producing the full-female mandarin fish in a large scale, effectively improve the culture yield and economic benefit and realize the sustainable development of the mandarin fish culture industry.
The technical scheme of the invention is as follows:
1. and (3) obtaining pure line all-female filial generation through artificial gynogenesis: selecting female siniperca chuatsi and male siniperca scherzeri with no disease, no injury, good character and good gonad development, adopting an artificial induced spawning method to obtain allogenic sperms of siniperca chuatsi ovum and siniperca scherzeri, irradiating the sperms by ultraviolet rays of 90mJ/cm for 25min to completely inactivate, combining the sperms with the ovum to complete fertilization, starting cold shock treatment 5min after fertilization, wherein the treatment temperature is 3 ℃, the duration is 30min, and finally obtaining pure line all-female offspring XX female.
2. Domesticating pure line full-female filial generation: selecting individuals with the body length of 2.0cm from pure line full-female offspring obtained from gynogenesis, and firstly carrying out colony feeding training; after the ingestion rhythm is formed, controlling the conditions such as illumination and the like, and feeding live baited fishes and fresh baited fishes, wherein the occupation ratio of the live baited fishes is gradually reduced, and the occupation ratio of the fresh baited fishes is gradually improved; feeding soft pellet feed after the fresh bait fish ingests stably, wherein the method comprises the steps of feeding the soft pellet feed after a small amount of fresh bait fish is matched with sound (water) to induce clustering, and canceling a feeding link of the fresh bait fish after the clustering is stable; finally obtaining pure line full female filial generation capable of completely eating soft pellet feed.
3. Fadrozole induction: the gonads of the pure-line full-female filial generations which succeed in domestication are already differentiated into ovaries, and at the moment, the feed mixed with fadrozole hormone is continuously fed. The preparation method of the feed comprises the following steps: fadrozole hormone is dissolved in absolute ethyl alcohol to prepare a stock solution with the concentration of 10mg/mL, and the stock solution is diluted by 10 times with ethyl alcohol when preparing a feed each time, and then is mixed into the feed to obtain fadrozole soft granular feed for freezing storage, wherein the content of the fadrozole hormone is about 500 mg/kg. After the continuous feeding for 60 days, checking whether differentiated ovaries are converted into functional spermary through gonad histology observation and microscopic semen microscopic examination, and feeding more than 95% of well domesticated mandarin fish containing hormone feed to convert into XX male-female mandarin fish; after tail seedlings are removed, XX male rotation mandarin fish is planted in the soil pond and cultivated until sexual maturity.
4. Continuously producing full-female mandarin fish fries: and (3) taking the XX male parent-male turning mandarin fish as a male parent, carrying out artificial propagation with the XX male parent of the normal female mandarin fish, and carrying out large-scale production of pure-line all-female mandarin fish fries by adopting an artificial insemination mode.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the pure line full-female filial generation is obtained by the gynogenesis method, compared with the methods such as manual selection, selective breeding or crossbreeding, the breeding time for obtaining the pure line is effectively shortened, the accuracy is improved, and the labor intensity is reduced.
(2) Compared with the technology of directly performing sex reversal on normal offspring, the method does not need to eliminate XY type male individuals in the sex reversed population by technical identification such as sex molecular marking and the like, is convenient and efficient to operate, has high accuracy and is suitable for industrial production.
(3) According to the method, after the Mandarin fish domesticates successfully, the pure-line all-female mandarin fish differentiated ovary is induced to be converted into the functional spermary by directly feeding the medicinal feed containing fadrozole, compared with the technology of feeding the exogenous hormone-enriched live bait, the method can accurately control the dosage of exogenous hormone, reduce the cost, improve the production efficiency, and is safer and more environment-friendly; and the gonads of individuals have been developed and differentiated during sexual reversion, the anti-stress capability is improved, and the survival rate of the castrated individuals is improved.
(4) According to the method, XX male parent rotating mandarin fish and XX female parent normal female mandarin fish are subjected to matched propagation, full-female mandarin fish fries can be produced in a sustainable large-scale mode, the yield and the efficiency are remarkably improved compared with a method for reversing estrogen inductivity directly, and the problem of batch production of the parthenogenetic population of the full-female mandarin fish father line can be well solved. In addition, the method has no hormone residue risk, and effectively ensures the food safety and the breeding environment safety.
Drawings
FIG. 1 is a technical route diagram of a method for sustainable large-scale production of full-female mandarin fish.
FIG. 2 shows the ovarian tissue section microstructure of pure filial generation of gynogenesis of the hologynic mandarin fish.
Fig. 3, the spermary tissue section microstructure of XX male-male turning mandarin fish.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the following detailed description of a preferred embodiment of the present invention will be provided with reference to the accompanying drawings, which are not intended to limit the present invention, and those skilled in the art can make modifications according to circumstances, such as time, person, and place.
1. Obtaining pure line full-female filial generation by artificial gynogenesis
The pure line is constructed according to a selective breeding or crossbreeding method, 8-10 generations of breeding are needed, the gene loci can be completely homozygous only by inhibiting the first cleavage through artificially inducing gynogenesis, and the generation of the offspring is pure line full-female seedlings because the sex-determining mechanism of siniperca chuatsi is the same type of female gametes. The specific operation steps are as follows:
(1) collecting ovum and semen: the method comprises the steps of injecting chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone analogue (LRH-A2) into abdominal cavities by a needle injection method for artificial spawning induction, wherein the dosage of female fish is 1800IU HCG/kg +15 mu g/kg LRH-A2, the dosage of male fish is halved, and after the effect time is reached, eggs and semen are manually extruded out, and the eggs are placed in a cold dark place and kept in a dark place for later use;
(2) sperm inactivation: taking 1mL of the fresh siniperca scherzeri heterologous semen, diluting the fresh siniperca scherzeri heterologous semen with Hank's solution according to the volume ratio of 1: 4, flatly paving the diluted solution in a precooled culture dish, wherein the thickness of the diluted solution is about 1-2 mm, and irradiating the siniperca scherzeri heterologous semen by using an ultraviolet cross-linking instrument, wherein the ultraviolet irradiation dose is 90mJ/cm, and the irradiation time is 25min, so that the genetic substances of sperms are completely inactivated;
(3) artificial insemination: after the sperm is inactivated, the mixture is immediately mixed with the mandarin fish eggs, and physiological saline is added to activate the sperm and stimulate the development of the fish eggs;
(4) cold shock treatment: 5min after insemination, placing the ovum into water with the temperature of 3 ℃ for cold shock treatment for 30min, blocking the second polar body of the meiosis of the ovum from discharging, and recovering the gynogenesis diploid;
(5) artificial incubation: and after cold shock treatment, putting the eggs into water at 28 ℃ for hatching to finally obtain pure-line full-female filial generation XX (male parent).
2. Domestication of pure line full-female filial generation
After pure line full-female filial generation XX is obtained, domesticating and feeding are firstly carried out on the fry so that the fry can be completely adapted to soft pellet feed, and the specific method is as follows:
(1) cluster training: and selecting about 2000 individuals with more consistent specifications and 2.0cm body length from pure line full-female filial generation obtained by gynogenesis to carry out cluster training. The cluster training is specifically operated as follows: feeding live baits to the mandarin fish regularly, at fixed points and quantitatively when the light intensity is not high in the morning and at night every day, inducing the mandarin fish to crowd for food robbing, and continuing the crowd training for 3-5 days;
(2) domesticating fresh bait fish: feeding live and fresh baited fishes regularly, at fixed points and quantitatively when the early and late illumination intensity is not high every day after the colony training is finished, wherein the occupation ratio of the live baited fishes is gradually reduced, the occupation ratio of the fresh baited fishes is gradually increased, and only feeding the fresh baited fishes after the continuous 2-3 days;
(3) domesticating soft pellet feed: a small amount of fresh bait fish is matched with sound (water) to induce the colony, then soft pellet feed is fed, after the colony is stable, the link of the fresh bait fish is cancelled, and only the soft pellet feed is fed;
3. fadrozole induced castration: the gonad of the pure-line all-female offspring successfully domesticated becomes an ovary after being blind, and the feed mixed with fadrozole hormone is continuously fed for castration at the moment, wherein the specific method comprises the following steps:
(1) preparation of a pharmaceutical feed containing fadrozole: fadrozole hormone is dissolved in absolute ethyl alcohol to prepare a stock solution with the concentration of 10mg/mL, the stock solution is diluted by 10 times with ethyl alcohol each time when a feed is prepared, the feed is mixed, the mixture is uniformly mixed to prepare a soft pellet feed with the diameter of 3-5 mm and the length of 10-20 mm, and the soft pellet feed is subpackaged, frozen and stored and unfrozen half an hour before use. The concentration of fadrozole hormone in the feed is 500 mg/kg.
(2) Continuously feeding the medicinal feed to induce the ovary to be transformed into the spermary: continuously feeding the pure-line all-female filial generation successfully domesticated with a medicinal feed containing fadrozole for 60 d.
(3) Identifying the gonads of the trans-male fishes: after the induction test is finished, judging whether differentiated ovaries are converted into functional spermary nests or not through gonad histology observation and microscopic semen microscopic examination, selecting 896 total XX male trans-male mandarin fish which are successfully converted and well developed, transferring the obtained product to a special pond for live fish culture, enhancing culture management, culturing until sexual maturity, and using the obtained product to construct a trans-male mandarin fish germplasm library.
4. Continuously producing full-female mandarin fish fries: after the XX male trans-male mandarin fish stored in the next-year germplasm bank is mature, the normal female siniperca chuatsi matched with the sex mature in the breeding season can be bred. Still adopting a needle method to inject HCG and LRH-A2 into abdominal cavity for artificial induced spawning, under the condition that parent fish is well cultivated, semen generated by XX turning male and mouth tilting mandarin fish can be inseminated with 100-300 ten thousand normal female mouth tilting mandarin fish eggs, and the fertilization rate and the hatching rate can reach more than 96%.
Therefore, a breeding system and a production process for sustainable large-scale production of the all-female mandarin fish fries are formed and are used for supplying farmers in various regions.
Reference to the literature
[1] A Collection of aquatic biology of the Siniperca Chuatsi in Jiana Liangzi lake, 1959(3) 375 and 384.
[2] Lida, Yangchun, Poyang lake mandarin fish biology [ J ]. Jiangxi agro-Proc., 1998,10(4):14-22.
[3] Yuanrui, Liangxu, Zhang, etc. preliminary study on artificial gynogenesis of siniperca chuatsi [ J ] freshwater fishery, 2012, (3):84-87.
[4]Varadaraj K,Pandian TJ.First report on production of supermale tilapia by integrating endocrine sex reversal with gynogenetic technique[J].Current Science,1989,58(8):434-441.
Claims (6)
1. A method for sustainable large-scale production of full-female mandarin fish is characterized by comprising the following steps:
A. and (3) obtaining pure line all-female filial generation through artificial gynogenesis: selecting mature female Siniperca chuatsi (Siniperca chuatsi) 2-5-year-old mandarin fish with excellent properties, good development, no diseases and no injuries, and artificially hastening parturition to obtain ova; selecting mature male fishes of siniperca scherzeri of 3-5 ages with excellent characters, good development, no diseases and no injuries, and obtaining sperms through artificial spawning induction; inducing the gynogenesis of the mandarin fish egg by adopting an ultraviolet inactivated Siniperca scherzeri (Siniperca scherzeri) heterogenous sperm to produce pure line full-female filial generation XX female;
B. domesticating pure line full-female filial generation: selecting individuals with the body length of 2.0cm and good growth from pure-line all-female offspring, controlling feeding conditions and feeding rhythm, sequentially domesticating and feeding live fish clusters, domesticating and feeding fresh fish and domesticating soft pellet feed for 7-10 days to obtain 3.0-4.0 cm mandarin fish domesticated offspring seeds;
C. fadrozole induction: continuously feeding the mandarin fish fries successfully domesticated with the feed mixed with fadrozole hormone, inducing the differentiated ovary of the pure-line all-female mandarin fish to be converted into a functional spermary, obtaining XX male-trans-male mandarin fish, and culturing until sexual maturity;
D. continuously producing full-female mandarin fish fries: and (3) taking the XX male parent-male turning mandarin fish as a male parent fish, and carrying out pairing propagation with XX male parent of normal female mandarin fish to produce pure-line all-female mandarin fish fries in a large scale.
2. The method for sustainable large-scale production of hologynic mandarin fish according to claim 1, wherein the method comprises the following steps: the mandarin fish is Siniperca chuatsi.
3. The method of claim 1, wherein: and B, when pure line all-female filial generation XX is produced by an artificial gynogenesis induction method in the step A, the ultraviolet dose for inactivating the siniperca scherzeri heterogenous sperms is 90mJ/cm, the irradiation time is 25min, cold shock treatment is started 5min after eggs are combined with the inactivated sperms, the treatment temperature is 3 ℃, and the duration is 30 min.
4. The method of claim 1, wherein: the preparation method of the feed containing fadrozole hormone in the step C comprises the following steps: fadrozole hormone is dissolved in 95% alcohol to prepare a stock solution, and the stock solution is diluted by alcohol and mixed into the mandarin fish culture feed to obtain fadrozole soft granular feed, wherein the fadrozole soft granular feed contains the fadrozole hormone with the amount of about 500 mg/kg.
5. The method of claim 1, wherein: and C, feeding the feed containing fadrozole hormone to the all-female mandarin fish seedlings of which the gonads are differentiated into ovaries and domesticated successfully, wherein the continuous feeding time is 60 days.
6. The method of claim 1, wherein: and D, matching the XX male parent of the rotary mandarin fish and the XX female parent of the rotary mandarin fish which are well developed in the step C, and producing the all-female mandarin fish with pure germplasm and excellent characters in a large scale.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114592076A (en) * | 2022-05-10 | 2022-06-07 | 中山大学 | Siniperca scherzeri male molecular marker primer and application thereof |
| CN115349496A (en) * | 2022-08-19 | 2022-11-18 | 湖南师范大学 | Method for inducing gynogenesis of micropterus salmoides by siniperca chuatsi sperms |
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| CN115349496B (en) * | 2022-08-19 | 2024-01-16 | 湖南师范大学 | Method for inducing gynogenesis of micropterus salmoides by siniperca chuatsi sperms |
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