CN112471008A - Low-oxygen-resistant hybrid breeding method for epinephelus lanceolatus - Google Patents
Low-oxygen-resistant hybrid breeding method for epinephelus lanceolatus 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- 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
- Y02A40/81—Aquaculture, e.g. of fish
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- Biodiversity & Conservation Biology (AREA)
- Farming Of Fish And Shellfish (AREA)
Abstract
The invention discloses a method for cross breeding of hypoxia-resistant epinephelus lanceolatus. The invention comprises the following steps: 1) breeding parent fish of the blue grouper and obtaining sperms; 2) freezing and preserving sperms; 3) parent fish breeding of the mackerel and obtaining of ova; 4) artificial insemination is carried out to obtain the frozen semen of the large grouper, the frozen semen is unfrozen and then is evenly mixed with ovum, and seawater is added for fertilization; standing, adding high salinity seawater, and collecting floating eggs; 5) hatching for 6-10h, placing in high salinity seawater, collecting floating egg again, and placing in culture pond for hatching; 6) and (5) later-stage cultivation, namely, cultivating by using biological bait to obtain the hybrid fish fry of the mackerel. The fry obtained by the crossbreeding of the mackerel has high survival rate, high growth speed, hypoxia resistance, low temperature resistance, uniform shape and body color of filial generation, 2 n-1 sm +47t of chromosome karyotype, unique genetic characteristic, obvious hybridization advantage, high market acceptance degree and considerable economic benefit.
Description
Technical Field
The invention belongs to the technical field of fish crossbreeding, and particularly relates to a method for crossbreeding hypoxia-resistant epinephelus lanceolatus.
Background
Grouper is the major mariculture and fishing fish in the world. In 2019, the yield of grouper culture and fishing in China respectively reaches 183127 tons and 97411 tons, the yield is the second place of the marine cultured fishes, and the yield value reaches more than 200 hundred million yuan.
Epinephelus (Epinephelus awoara) is classified as Perciformes, Epinephelidae (Epinephilidae), Epinephelus (Epinephelus), colloquially known as Cladosporium. The mackerel is a high-quality precious fish which inhabits in the middle and lower layers of sea warm water, is usually distributed in the western sea area of the northern pacific, and is mostly distributed in the south sea area and the south east sea area in China.
The mackerel has high nutritive value, delicious taste and high market price. The content of unsaturated fatty acid in muscle of Mylopharyngodon Piceus reaches above 66.38%, the content of docosahexaenoic acid (DHA) reaches 6.49-14.14%, and DHA is called NAOHUANGJIN, and can be used for preventing cardiovascular diseases. The adult Mylopharyngodon Piceus is oblong and slightly flat, scales on the surface of the fish body are small comb scales, yellow brown spots are scattered on the fish body, the back of the fish body is dark brown, the abdomen of the fish body is light brown, and five brown stripes with different thicknesses are grown on the body side. The depth of the water of the moving water area of the mackerel is usually 2-30m, and the optimal growth temperature is 22-28 ℃. However, the growth speed of the mackerel is slow, the growth of the mackerel in the current year can reach more than 120g, and the total culture amount in the market is relatively low.
Disclosure of Invention
The invention aims to provide a method for cross breeding of hypoxia-resistant epinephelus malabaricus, and aims to solve the problem that the breeding benefit of the epinephelus malabaricus is low due to slow growth of the epinephelus malabaricus in the prior art.
In order to solve the technical problems, the invention is mainly realized by the following technical scheme:
the invention relates to a method for crossbreeding of hypoxia-resistant epinephelus lanceolatus, which comprises the following steps:
1) parent fish breeding and sperm obtaining for blue grouper mackerel
Selecting male large grouper with the body length of 50-70cm and the body weight of 20-30Kg, and culturing in an industrial culture environment under the following conditions: dissolved oxygen 7-10mg/L, water flow exchange capacity 3-4m3H, natural light, the cultivation time is 5-9 months, and the cultivation temperature is 18-25 ℃; the cultivation is started in 10 months of the first year, the water temperature is 23-24 ℃, the water temperature is gradually reduced to 18-20 ℃ in 12 months, and the water temperature is gradually increased to 25-26 ℃ in 3 months of the next year;
selecting mature male large grouper with large size when the water temperature reaches 25-26 deg.C in months 4-6, squeezing abdomen to collect semen;
2) sperm cryopreservation
Placing the semen obtained in the step 1) in a cryopreservation solution, balancing for 5-8min at 3-4 ℃, then placing in liquid nitrogen steam for balancing for 10-12min, and finally placing in liquid nitrogen for low-temperature preservation;
3) parent fish breeding of mackerel and obtaining of ovum
Selecting female mackerel with the body length of 20-25cm and the body weight of 0.5-1.0Kg, and culturing in an industrial culture environment under the following conditions: 5-9mg/L of dissolved oxygen, 25-31 per mill of salinity and 2-3m of water flow exchange capacity3H, the water temperature is 12-29 ℃, and the cultivation time is 5-9 months;
when the water temperature reaches 23-26 ℃ in 4 months, selecting well-developed female mackerel with swollen abdomen, anesthetizing by adopting MS-222, injecting chorionic gonadotropin and gonadotropin releasing hormone analogues into the basal part of the pectoral fin of the female mackerel, and extruding the abdomen of the female mackerel after injecting for 48-60 hours to obtain eggs;
4) artificial insemination
Taking the frozen semen of the large grouper with the blue body obtained in the step 2), unfreezing at 36-37 ℃, pouring into the ovum obtained in the step 3), and adding seawater to uniformly mix the semen and the ovum for fertilization; then, adding high salinity seawater, collecting floating eggs, cleaning and hatching;
5) hatching management
Incubating for 6-10h, placing in high salinity seawater,floating again, collecting floating fertilized eggs, placing the fertilized eggs in a culture pond for incubation and cultivation, wherein the culture conditions are as follows: the egg laying density is 30-45g/m3Salinity of 29-30 per mill, temperature of 25-27 ℃, and dissolved oxygen of 6-10 mg/L;
6) late stage cultivation
Feeding oyster fertilized eggs and chlorella when the yolk sac is completely absorbed at the age of 2-4 days; feeding the S rotifers at 3-16 days old; feeding L-shaped rotifers at the age of 13-35 days; feeding artemia nauplii at the age of 25-45 days; feeding adult artemia from 40-55 days old, and gradually transitioning to compound pellet feed; and (5) timely screening the seedlings, and culturing under the dark condition to obtain the hybrid cultured fish of the mackerel.
The invention relates to a novel hybrid cultured fish of mackerel, which is cultured by taking mackerel as a female parent and taking large mackerel of blue body as a male parent, and the fertility rate and the hatchability of the hybrid cultured fish of mackerel are similar to those of pure breed. However, the survival rate of the hybrid cultured fish of the mackerel is high, the survival rate of the pure mackerel on the 11 th day is only 5 percent under the same breeding condition, and the survival rate of the hybrid is 40 percent; the mackerel has remarkable growth advantage, the weight of the hybrid seeds is 3.16 times of that of the pure seeds when the mackerel is cultured to be 13 months old, and the weight of the hybrid seeds is 2.52 times of that of the pure seeds when the mackerel is cultured to be 17 months old; the hybrid cultured fish of the mackerel is low temperature resistant, the hybrid can be normally cultured in a factory workshop in the north in winter, and the hybrid can normally feed and grow at the temperature of about 16-18 ℃; the hybridized mackerel fish is low-oxygen resistant, the oxygen consumption rate is obviously lower than that of the pure mackerel fish, and the suffocation point is also obviously lower than that of the pure mackerel fish; the hybridized mackerel has uniform shape and body color, is similar to the female parent, has high market acceptance and considerable economic benefit.
In a preferred embodiment, in step 2), the cryopreservation solution is an ELs-3 cryopreservation solution, and the composition of the ELs-3 cryopreservation solution is as follows: each liter of distilled water contains 10g of glucose, 10g of NaCl and NaHCO30.5g and 10mL calf serum.
The blue-body giant grouper (Epinephelus tukula) is a medium-large reef habitat fish living in warm sea areas, is one of the known largest groupers, has a high growth speed, has the body length of at least 200cm and the body weight of at least 100Kg, is mostly distributed in India-Pacific areas, and is generally distributed in northern Taiwan and southern sea areas in China. The adult blue grouper is oblong and flat on the side, and scales on the surface of the fish body are comb scales; the fish body is grey white, and except the head part distributed with small black spots, the other parts are covered with brown patches with different sizes and shapes. The growth speed of the large epinephelus hybridus is high, but the production amount of male fishes is small, and female fishes are difficult to lay eggs under artificial breeding conditions, so that the artificial breeding of the large epinephelus hybridus is difficult to develop.
Due to geographical distribution, living environment, breeding water temperature and the like, the mackerel and the mackerel have reproductive isolation, so natural mating cannot be performed under natural conditions. Therefore, by combining the sperm preservation technology of the large grouper bluebeard with the crossbreeding, the combination of the heterosis characters of the two kinds of fishes can be realized, and the breeding of a new excellent variety is achieved. Therefore, the semen of the mackerel is collected and placed in the ELs-3 frozen diluent for freezing and preservation, so that the problem of low yield of the male mackerel of the mackerel is solved, the reproductive isolation of the mackerel and the mackerel is also solved, and the crossbreeding of the mackerel and the mackerel is realized.
In a preferred embodiment, in the step 2), the volume ratio of the cryopreservation liquid to the semen is 1: 1. The semen of the large-body mackerel is firstly stored in the frozen preservation solution, and the volume ratio of the semen to the frozen preservation solution is controlled; in general, the semen collection method of the large grouper with blue body comprises the following steps: anaesthetizing the large grouper bluebody by adopting an MS-222 full pool, squeezing the abdomen of the large grouper bluebody by hands, selecting a male with a large and mature body and flowing semen, and squeezing the abdomen to collect the semen. In the semen collection process, the abdomen is squeezed by hands, semen is collected through a plastic suction pipe, the semen is placed in a 2mL freezing pipe, 1.5mL semen is collected in each pipe, and urine flowing out of a drainage hole is timely wiped off by using toilet paper so as to reduce the negative influence on the subsequent semen freezing operation; under the condition of well controlling temperature and nutrition, the semen can be produced to the middle of August; seawater is prevented from being mixed in the sperm squeezing process, otherwise, the sperm is activated in advance, so that the sperm is not preserved in a frozen way; and thawing in advance to observe sperm motility, and selecting the batch of frozen sperm with strong sperm motility to transport to a destination for insemination with the ovum.
As a preferred embodiment, in the step 3), the dosage of the chorionic gonadotropin is 200-300IU/Kg, and the dosage of the gonadotropin releasing hormone analogue is 35 mug/Kg. The invention injects chorionic gonadotropin and gonadotropin releasing hormone analogue into the basal part of the pectoral fin, and controls the dosage of the chorionic gonadotropin and gonadotropin releasing hormone analogue, so that the dosage is proper and the additional injury is avoided.
As a preferred embodiment, in said step 3), chorionic gonadotropin and gonadotropin releasing hormone analogue are co-dissolved in physiological saline with a mass concentration of 0.9%. In the invention, chorionic gonadotropin and gonadotropin releasing hormone analogue can be dissolved in 0.9 percent normal saline to prepare uniform solution, and then the solution is injected into the base part of the pectoral fin at one time, so that the operation is convenient and the injection is convenient; typically, about 1mL of a mixed physiological saline solution of chorionic gonadotropin and gonadotropin releasing hormone analog is injected per fish.
In a preferred embodiment, the mass concentration of the high salinity seawater in the step 4) and the step 5) is 33-35 per mill. The invention adopts high salinity seawater to float the fertilization rate and collect the floating eggs, the grouper eggs are floating eggs, the high salinity seawater can screen the good floating fertilized eggs, and the sinking eggs are over-mature eggs and dead eggs.
In a preferred embodiment, in the step 4), the washing is performed by using high salinity seawater with salinity of 33-35% o. The invention also adopts seawater to clean the floating eggs, the purpose of cleaning is to avoid the interference of other pollutants in the hatching process, and because the blood and the excrement of the parent fish are extruded together in the egg extruding process, the pollutants are cleaned.
As a preferred embodiment, in the step 6), the feeding amount of the oyster fertilized eggs is 20/mL, and the chlorella is adoptedThe feeding amount is 50 multiplied by 104The feeding amount of the S rotifers is 12-15/mL, the feeding amount of the artemia nauplii is 3-4/mL, and the feeding amount of the adult artemia is 3-4/mL. The fertilization rate of the invention is that after the membrane is emerged, chlorella, oyster fertilized eggs, S rotifers, L rotifers, artemia larvae, artemia adults and mixed granular feeds with different specifications are fed in sequence, and the culture is carried out by utilizing biological baits; and the feeding amount and the feeding time are adjusted according to the growth condition of the groupers. When the specifications of the fries are different, the fries are screened in time to avoid self-mutilation.
The dark condition refers to indoor cultivation, and except for feeding and cleaning, all lights are turned off in other time to avoid mutual disability. The fry of the invention has the phenomenon that big fish eats small fish in the growth process, so that the big fish and the small fish die at the same time; to avoid this, when the size difference between individuals is significant, fish of different sizes are placed in different ponds; in addition, except feeding, the workshop is kept in a dark state, so that the fry grows in the dark state for a long time, the aggressiveness of the fry is reduced, and the phenomenon that big fishes eat small fishes is further avoided.
Compared with the prior art, the invention has the beneficial effects that: the invention relates to a novel hybrid variety of mackerel which is bred by taking mackerel as a female parent and blue mackerel as a male parent, wherein the fertility rate and the hatchability of the hybrid variety of mackerel are similar to those of pure breed. However, the survival rate of the hybrid of the mackerel is high, and under the same feeding condition, the survival rate of pure mackerel on the 11 th day is only 5 percent, while the survival rate of the hybrid is 40 percent; the mackerel has remarkable growth advantage, the weight of the hybrid seeds is 3.16 times of that of the pure seeds when the mackerel is cultured to be 13 months old, and the weight of the hybrid seeds reaches 2.52 times of that of the pure seeds when the mackerel is cultured to be 17 months old; the hybrid cultured fish of the mackerel is low temperature resistant, the hybrid can be normally cultured in a factory workshop in the north in winter, and the hybrid can normally feed and grow at the temperature of about 16-18 ℃; the hybrid seed of the mackerel is resistant to low oxygen, the oxygen consumption rate is obviously lower than that of the pure mackerel, and the suffocation point is also obviously lower than that of the pure mackerel; the physique characteristics of the hybridized mackerel are similar to that of the female parent mackerel, the shape and the body color of the filial generation are consistent, the market acceptance degree is high, and the economic benefit is considerable.
Drawings
FIG. 1 is a chromosome karyotype chart of a filial generation of Epinephelus brueckii and Epinephelus hybridus obtained by the present invention;
FIG. 2 is a photograph showing the appearance of the filial generation of Epinephelus brueckii and Epinephelus hybridus obtained by the present invention;
FIG. 3 is a photograph showing the appearance of pure Mylopharyngodon Piceus.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to specific embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention relates to a method for crossbreeding of hypoxia-resistant epinephelus lanceolatus, which comprises the following steps:
1) parent fish breeding and sperm obtaining for blue grouper mackerel
Selecting male large grouper with the body length of 50-70cm and the body weight of 20-30Kg, and culturing in an industrial culture environment under the following conditions: dissolved oxygen 7-10mg/L, water flow exchange capacity 3-4m3H, natural light, the cultivation time is 5-9 months, and the cultivation temperature is 18-25 ℃; the cultivation is started in 10 months of the first year, the water temperature is 23-24 ℃, the water temperature is gradually reduced to 18-20 ℃ in 12 months, and the water temperature is gradually increased to 25-26 ℃ in 3 months of the next year;
selecting mature male large grouper with large size when the water temperature reaches 25-26 deg.C in months 4-6, squeezing abdomen to collect semen;
2) sperm cryopreservation
Placing the semen obtained in the step 1) in a cryopreservation solution, balancing for 5-8min at 3-4 ℃, then placing in liquid nitrogen steam for balancing for 10-12min, and finally placing in liquid nitrogen for low-temperature preservation;
3) parent fish breeding of mackerel and obtaining of ovum
Selecting female mackerel with the body length of 20-25cm and the body weight of 0.5-1.0Kg, and culturing in an industrial culture environment under the following conditions: 5-9mg/L of dissolved oxygen, 25-31 per mill of salinity and 2-3m of water flow exchange capacity3H, the water temperature is 12-29 ℃, and the cultivation time is 5-9 months;
when the water temperature reaches 23-26 ℃ in 4 months, selecting well-developed female mackerel with swollen abdomen, anesthetizing by adopting MS-222, injecting chorionic gonadotropin and gonadotropin releasing hormone analogues into the basal part of the pectoral fin of the female mackerel, and extruding the abdomen of the female mackerel after injecting for 48-60 hours to obtain eggs;
4) artificial insemination
Taking the frozen semen of the large grouper with the blue body obtained in the step 2), unfreezing at 36-37 ℃, pouring into the ovum obtained in the step 3), and adding seawater to uniformly mix the semen and the ovum; then, adding high salinity seawater, collecting floating eggs, cleaning and hatching;
5) hatching management
Incubating for 6-10h, placing in high salinity seawater, floating upward again, collecting floating fertilized eggs, placing in a culture pond for culture under the culture conditions: the density is 30-45g/m3Salinity of 29-30 per mill, temperature of 25-27 ℃, and dissolved oxygen of 6-10 mg/L;
6) late stage cultivation
Feeding oyster fertilized eggs and chlorella when the yolk sac is completely absorbed at the age of 2-4 days; feeding the S rotifers at 3-16 days old; feeding L-shaped rotifers at the age of 13-35 days; feeding artemia nauplii at the age of 25-45 days; feeding adult artemia from 40-55 days old, and gradually transitioning to compound pellet feed; and (5) timely screening the seedlings, and culturing under the dark condition to obtain the hybrid cultured fish of the mackerel.
Preferably, in the step 2), the cryopreservation solution is an ELs-3 cryopreservation diluent, and the composition of the ELs-3 cryopreservation diluent is as follows: each liter of distilled water contains 10g of glucose, 10g of NaCl and NaHCO30.5g and 10mL calf serum.
Preferably, in the step 2), the volume ratio of the cryopreservation liquid to the semen is 1: 1.
Preferably, in the step 3), the dosage of the chorionic gonadotropin is 200-300IU/Kg, and the dosage of the gonadotropin releasing hormone analogue is 35 mu g/Kg.
Further, in the step 3), the chorionic gonadotropin and the gonadotropin releasing hormone analogue are dissolved in physiological saline with the mass concentration of 0.9%.
Preferably, in the step 4) and the step 5), the mass concentration of the high salinity seawater is 33-35 per thousand.
Further, in the step 4), the cleaning is performed by using high-salinity seawater with the mass concentration of 33-35 per mill.
Preferably, in the step 6), the feeding amount of the oyster fertilized eggs is 20/mL, and the feeding amount of the chlorella is 50 x 104The feeding amount of the S rotifers is 5-10/mL, the feeding amount of the artemia nauplii is 3-4/mL, and the feeding amount of the adult artemia is 3-4/mL.
Example A cultivation of blue grouper and Mylopharyngodon Piceus
The culture environment of the large blue grouper is industrial culture, the size of a culture pond is 6m multiplied by 3m, 30 large blue groupers are cultured in each pond, and the total length of the large blue groupers is 50-70cm and the weight of the large blue groupers is 20-30 Kg. The Dissolved Oxygen (DO) in the pool is 7-10mg/L, and the water flow exchange capacity is 3-4m3And/h, adopting natural light. The water temperature is 23-24 ℃ in 10 months of the first year, the water temperature is gradually reduced to 18-20 ℃ in 12 months, and the water temperature is gradually increased to 25-26 ℃ in 3 months of the next year; when the water temperature is stabilized at 25-26 deg.C in the months of 4-9, mature male fish can be selected for semen collection. Wherein the parent feeding bait is one or mixture of two of mackerel and mackerel at any ratio, and the feeding amount is about 2-3% of the weight of the parent fish. The pond is regularly poured in daily life to keep the water quality fresh.
The Mylopharyngodon Piceus is cultured in net cages of 5m × 5m × 3m, 50 Mylopharyngodon Piceus is cultured in each net cage, the total length is 20-25cm, and the weight is 0.5-1.0 Kg. The bait is prepared from Engraulis japonicus Temminck et Schlegel or frozen fresh trash fish as bait, the bait feeding rate is 2-3% of body weight, and the bait feeding is carried out twice daily. The dissolved oxygen is 5-9mg/L, the salinity is 25-31 per mill, the temperature is the temperature of seawater outside the sea area near Fujian, and the water temperature range is 12-29 ℃; when the water temperature reaches 23-26 ℃ in the month of 4, mature female fish are selected to collect eggs.
EXAMPLE two cryopreservation of sperm from Epinephelus macrostoma
(1) Acquisition of mature sperm
The sperm production time of the mackerel with blue body is prolonged from 4 months to 9 middle days under the environment of artificial regulation and control, at the moment, precious and less amount of the mackerel with blue body sperm can be preserved for a long time by the sperm freezing technology, and the mackerel with blue body sperm can be inseminated with mackerel roes in the next year.
Semen is collected every half month in the reproductive season, and the male fish after semen collection is put into another fishpond with fresh water. Before semen collection, MS-222 full pool anesthesia is adopted, the male fish is placed on a obstetric table, the abdomen is squeezed by hands, mature males with semen flowing out are selected, and the abdomen is squeezed to obtain semen. The seawater is prevented from being mixed in the semen extruding process, otherwise, the sperms can be activated in advance, and the urine flowing out of the excretion hole is wiped off by using toilet paper in time in the semen extruding process, so that the urine is prevented from being mixed in the sperms.
The semen collection method comprises the following steps: squeezing the abdomen with hands, collecting semen through a plastic straw, placing the semen into a 2mL freezing tube, wherein each tube contains about 1.5mL of semen, placing the freezing tube into a prepared heat preservation box, and placing crushed ice blocks in the box; meanwhile, the sperm motility is detected by a microscope, and then the split charging is carried out.
About 50mL of semen is collected in the reproductive period of 2020, and the sperm motility reaches more than 90%.
(2) Sperm cryopreservation
Diluting semen and freezing diluent ELs-3 at a ratio of 1:1, and mixing by shaking. The formula of ELs-3 is that each liter of distilled water contains 10g of glucose, 10g of NaCl and 10g of NaHCO30.5g, and 10mL of calf serum (volume ratio).
The sperm freezing process comprises three steps, firstly, balancing the freezing tube with sperm in a refrigerator at 4 deg.C for 5min, secondly, placing into 10 × 10cm cloth bags with 6-8 pieces per bag, and balancing the cloth bags in liquid nitrogen tank steam about 10cm away from the liquid nitrogen surface for 10 min; then, the cloth bag was put into liquid nitrogen (-196 ℃) to be frozen and preserved.
After the sperm is stored for 24 hours at low temperature, 2 freezing tubes are randomly drawn for unfreezing, the sperm motility is observed through a microscope, and the sperm survival rate reaches over 75-85 percent.
By utilizing the method, a mackerel sperm freezer is established, and the frozen and stored sperm reaches 100 mL. Before use, the sperm motility is observed by thawing, and frozen sperm with strong sperm motility is selected for insemination with ovum. The frozen sperms can be transported to any breeding company for a long distance in China by using a liquid nitrogen tank, and are crossbred with the female mackerel.
In the breeding season of the mackerel in 2020, the frozen sperms of the mackerel with blue body and large mackerel are transported to Fujian Zhang from a certain Shandong company by utilizing a liquid nitrogen tank, and are hybridized and seedling-cultured with the locally-cultured mackerel
Example Artificial insemination of frozen sperm of Epinephelus malabaricus and ovum of Epinephelus malabaricus
(1) Spawning induction of Mylopharyngodon Piceus
When the water temperature reaches 23-26 ℃ in the month of 4, selecting well-developed female mackerel with swollen abdomen; chorionic gonadotropin and gonadotropin releasing hormone analogue were injected into the basal part of the pectoral fin at a dosage of 200-300IU/kg chorionic gonadotropin and 35. mu.g/kg gonadotropin releasing hormone analogue, both mixed in 0.9% physiological saline, and about 1mL was injected per fish. After 48-60 hours of injection, the development state is observed by sucking out the eggs through a suction pipe, mature female fish are selected and placed on an egg laying bed, the abdomen of the fish is squeezed to collect the eggs, and then the eggs are collected in a dry plastic basin to obtain 10Kg of eggs.
(2) Insemination of frozen sperm and eggs
And extruding eggs once every 3 hours or so in 48-60 hours after the mackerel is injected with the spawning inducing needle. After the eggs are extruded, taking out the frozen mackerel sperms from the liquid nitrogen tank, unfreezing the frozen mackerel sperms in a water bath kettle at 37 ℃, and taking out the frozen mackerel sperms from the water bath kettle in time when the frozen sperms are just unfrozen so as to avoid negative influence on the sperm motility caused by overhigh temperature. Then pouring the mixture into a plastic basin containing the ovum, inseminating about 1000mL of the ovum with 1mL of semen, and then washing the semen in the freezing tube with seawater and pouring the semen into the plastic basinAdding seawater while mixing sperm and ovum uniformly to make them fully contact, placing fertilized ovum in plastic basin with high salinity seawater (salinity of 33-35 ‰) to make it float, collecting floated ovum with bolting silk, cleaning with seawater, and incubating in incubation pool with volume of 3m3The pond is continuously inflated and slightly boiled to avoid the fertilized eggs from sinking to the bottom, the temperature is 28 ℃, and the salinity is 29-30 per mill.
Example four transportation, hatching and seed rearing of hybrid fertilized eggs
(1) Fertilized egg transportation and hatching:
after hatching for about 8 hours, when the fertilized eggs develop to about the blastocyst stage, the fertilized eggs are placed in a plastic basin with high salinity seawater, and the fertilized eggs float upwards again, wherein the high salinity seawater is 33-35 per thousand. Collecting floating fertilized eggs, oxygenating by using a nylon bag, packing and air transporting to a target company, wherein the actions in the packing and transporting process are gentle, the time of the travel is reduced as much as possible, and 1Kg of each of the hybrid eggs and the pure fertilized eggs of the target company are transported back together. After arriving at the destination company, fertilized eggs were transferred to 7m3The culture pond of (1) is used for hatching eggs in about 200-300g per pond. The culture conditions of the culture pond include salinity of 29-30 ‰, temperature of 25-27 deg.C, and dissolved oxygen of 6-10 mg/L.
(2) Fry rearing
When the hatched fry is 2-4 days old and the yolk sac is about to be completely absorbed, oyster fertilized eggs (20 per mL) and chlorella (50X 10) are adopted4Per mL) feeding; feeding by using S rotifers (12-15/mL) when the feed is 3-16 days old; feeding L-shaped rotifers (12-15/mL) reinforced by a reinforcer when the seedlings are 13-35 days old; feeding artemia nauplii (3-4/mL) at the age of 25-45 days; feeding adult artemia (3-4/mL) from 40 to 55 days old, gradually changing to compound pellet feed, feeding once at 8 am and 5 pm, and culturing with biological bait to obtain the hybrid cultured fish of the mackerel. In the growth process, the phenomenon that big fishes eat small fishes exists, so that the big fishes and the small fishes die at the same time. In order to avoid the situation, when the size difference between individuals is obvious, fishes are sorted and screened in time, and the fishes with different sizes are placed into different culture ponds to avoid self-mutilation.
Example five comparison of hybrid and inbred breeding
(1) Cultivation process
Inseminating eggs of the same batch of epinephelus malabaricus with the frozen blue epinephelus malabaricus sperm and the fresh epinephelus malabaricus sperm respectively to obtain hybrid seeds and pure seeds, wherein fertilized eggs of the two are in the same external environment, and the fertilization rates and the hatchability rates of the two are counted 3 hours after fertilization and before membrane emergence; and when the hatching seedlings are cultivated for 11-20 days, carrying out preliminary statistics on the survival rate of the seedlings.
The hybrid seeds and the pure seeds are cultured in an indoor cement pond in running water before 6 months, and the water body in the culture pond is 7m36-10mg/L of dissolved oxygen, 29 per mill of salinity and 25-26 ℃; the water temperature is gradually reduced to about 20-22 deg.C with the temperature of open seawater at 5-6 months of age, and the water is transferred from the cultivation workshop to the cultivation workshop at 6 months of age, wherein the cultivation workshop is factory running water cultivation condition, and the water body in the cultivation pond is 24m3Respectively stocking 1000 hybrid seeds and pure fry, controlling the temperature at 20-24 ℃ in summer and autumn and 16-20 ℃ in winter and spring, and feeding the compound pellet feed once every morning and afternoon.
(2) Cultivation results
Under the same conditions, the fertility rate of the hybrid is 83.4 percent, and the hatchability is 75.3 percent; the fertility rate of the pure breed is 87.6%, and the hatching rate is 78.7%, so that the fertility rate and the hatching rate of the hybrid are similar to those of the pure breed.
When the hybrid seedlings are cultivated for 11-20 days, the survival rate is about 40 percent, and at the moment, the survival rate of pure seeds is only about 5 percent.
After 30-50 hybrids and pure varieties were collected at 4-month, 13-month and 17-month ages, respectively, and anesthetized with MS-222, growth data such as body weight, total length, body length, head length and body height were measured, and the measurement results are shown in Table 1.
TABLE 1 comparison of the male and female filial generations of Mylopharyngodon Piceus and pure Mylopharyngodon Piceus
As can be seen from table 1, at 4 months of age, the hybrid progeny of the mackerel and the mackerel exhibit the advantage of rapid growth, the weight of the hybrid progeny (also referred to as hybrid fish for short) begins to be greater than that of the pure mackerel, the full length of the hybrid progeny also begins to be greater than that of the pure mackerel, and the body length of the hybrid progeny is also greater than that of the pure mackerel. At 13 months of age, the weight of the filial generation of the mackerel and the blue grouper is 3.16 times of that of the pure mackerel, the total length of the filial generation is 1.42 times of that of the pure mackerel, and the length of the filial generation is 1.40 times of that of the pure mackerel. At the age of 17 months, the weight of the filial generation of the mackerel and the blue mackerel is 2.52 times of that of the pure mackerel, the total length of the filial generation is about 8cm longer than that of the pure mackerel, and the length of the filial generation is about 6cm longer than that of the pure mackerel. Therefore, the hybrid species has a significant growth advantage over the pure species.
Example comparison of six hybrids and pure species hypoxia-resistant cultures
Pure epinephelus lanceolatus and filial generation of 17 months old are selected from the fifth example, and divided into three groups of controls, each control comprises 4 fish, 12 fish of epinephelus lanceolatus, 3139g of total weight, and 12 fish of hybrid, 6229g of total weight. Oxygen consumption and asphyxia point tests were performed at 22 ℃ and the experimental procedure was as follows: putting experimental fishes into three transparent boxes, filling water into four fish in each box, sticking the experimental fishes on the water surface by using a plastic film, and covering the plastic film with a cover for sealing; and (3) taking an initial water sample, putting the initial water sample into a water sample bottle, fixing the water sample after one hour, and taking the water sample after two fishes fall off in the box out of balance and the gill cover is still. Dissolved oxygen was measured by iodometry within 24 hours, and the oxygen consumption rate and the asphyxia point were calculated, and the test results are shown in Table 2.
TABLE 2 comparison of the male and female hybrid offspring of Myxocyprinus lanceolatus with the oxygen resistance of pure Myxocyprinus lanceolatus
| Fish seeds | Oxygen consumption rate (mg/g. h) | Suffocation point (mg/mL) |
| Mylopharyngodon Piceus | 0.1438±0.0214 | 0.5269±0.0260 |
| Black grouper (male parent) x blue grouper | 0.0976±0.0064 | 0.3903±0.0125 |
As can be seen from Table 2, under the same conditions, the oxygen consumption rate of the filial generation of the epinephelus lanceolatus and the epinephelus lanceolatus is 0.0976 + -0.0064 mg/g/h, whereas the oxygen consumption rate of the pure epinephelus lanceolatus is 0.1438 + -0.0214 mg/g/h; the SPSS independent sample T test analysis shows that the oxygen consumption rates of the filial generation and the pure-breed mackerel are obviously different; therefore, the oxygen consumption rate of the filial generation is obviously lower than that of the pure-breed mackerel. In addition, under the same conditions, the suffocation point of the filial generation of the mackerel and the mackerel with blue body is 0.3903 +/-0.0125 mg/mL, while the suffocation point of the pure mackerel is 0.5269 +/-0.0260 mg/mL; the SPSS independent sample T test analysis shows that the asphyxiation points of the filial generation and the pure breed mackerel are obviously different; therefore, the suffocation point of the filial generation is obviously lower than that of the pure breed mackerel. Therefore, the hybrid offspring of the mackerel and the blue mackerel obtained by the invention is more hypoxia-resistant than the pure mackerel.
Example seven genetic test experiments
Preparing chromosomes by a head kidney-colchicine method, randomly selecting 20 pure mackerel fish with the weight of 10-19 g/tail, randomly selecting 20 female parent mackerel x blue body mackerel hybrid offspring with the weight of 10-13 g/tail. Injecting PHA solution into the thoracic cavity of the sample, wherein the dosage is 6 mug/g; after 12h, injecting colchicine solution into the same position of the thoracic cavity of the sample, wherein the dosage is 10 mu g/g; after 3 hours, the specimen was anesthetized with MS-222(180mg/L), cut obliquely upward from the base of the hip fin with scissors until bleeding occurred, and the head and kidney were removed after 10min of bleeding and washed three times with physiological saline. Completely cutting the head kidney with scissors, filtering through 260-mesh bolting silk, collecting filtrate in four graduated centrifuge tubes, and making the volume of the kidney cell sap in the centrifuge tubes equal in pairs. Then, 3mL of 0.075mol/L KCl solution was added to the cell sap, hypotonic treatment was performed at room temperature for 30min, and the precipitated cells were gently blown with a pipette, and then a newly prepared Carnoy's fixative (V (methanol): V (glacial acetic acid): 3:1) was added to the cell sap to make up to 4.5mL, and the resulting mixture was placed in a 0 ℃ ice box, pre-fixed for 10min, then fixed, and the cell suspension after pre-fixation was centrifuged for 10min (4000 r/min). Pouring out the supernatant, adding Carnoy's stationary liquid to 4.5mL, gently blowing the precipitated cells by using a pipette, and fixing for 45min at room temperature; centrifuging again, fixing for the second time, and fixing at room temperature for 30 min; and (4) centrifuging again, fixing for the third time at room temperature for 30min, and gently blowing and beating the precipitated cells by using a pipette in the fixing process. After the fourth centrifugation, about 1mL of Carnot's fixative was added to the cell pellet and placed in an ice box for future use. Dropping the slide by cold slide method, taking a clean glass slide (precooling for half an hour in a beaker filled with ethanol in a refrigerator), sucking the cell suspension, dropping 1-2 drops on the glass slide from the height of 15-20cm to spread the cells, burning the glass slide by an alcohol lamp to form flame, and naturally drying at room temperature. Finally, a 10% Giemsa staining solution is prepared by using a phosphate buffer solution, the dried glass slide is stained for 45 minutes, and then washed by using distilled water, naturally dried at room temperature, and observed and photographed under a microscope.
As can be seen from FIG. 1, the karyotype of the hybrid is 2 n-1 sm +47t, NF-49, and has an unpaired heterochromosome, which is not found in other Epinephelus malabaricus except for Pseudomalabaricus. Therefore, the filial generation of the epinephelus lanceolatus and the epinephelus lanceolatus has uniqueness in heredity, the karyotype formula of the hybrid is 2 n-1 sm +47t, and NF-49; the male parent and the female parent are both 2 n-48 t and NF-48.
As can be seen from the fig. 2 and 3, the hybrid generation of the mackerel and the mackerel of the invention has big size, fat body and much flesh; however, the pure-bred mackerel is small in size and thin in body; therefore, the hybridized offspring of the mackerel and the blue mackerel are obviously larger than the pure mackerel in body shape and body length. In addition, the body shape characteristics of the hybrid of the mackerel are similar to those of the female parent mackerel, and the shape and the body color of the filial generation are uniform.
Therefore, compared with the prior art, the invention has the beneficial effects that: the present invention relates to a new hybrid of epinephelus malabaricus which is bred by using the epinephelus malabaricus as female parent and using the blue-body large epinephelus malabaricus as male parent, and the fertility rate and hatchability of the hybrid cultured fish of the epinephelus malabaricus are similar to those of pure breed. However, the survival rate of the hybrid cultured fish of the mackerel is high, under the same breeding condition, the survival rate of pure mackerel on the 11 th day is only 5 percent, while the survival rate of the hybrid is 40 percent; the mackerel has remarkable growth advantage, the weight of the hybrid seeds is 3.16 times of that of the pure seeds when the mackerel is cultured to be 13 months old, and the weight of the hybrid seeds is 2.52 times of that of the pure seeds when the mackerel is cultured to be 17 months old; the hybrid cultured fish of the mackerel is low temperature resistant, the hybrid can be normally cultured in a factory workshop in the north in winter, and the hybrid can normally feed and grow at the temperature of about 16-18 ℃; the hybridized mackerel fish is low-oxygen resistant, the oxygen consumption rate is obviously lower than that of the pure mackerel fish, and the suffocation point is also obviously lower than that of the pure mackerel fish; the physique characteristics of the hybridized mackerel are similar to that of the female parent mackerel, the hybridized offspring has uniform shape and body color, the market acceptance degree is high, and the economic benefit is considerable.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A method for cross breeding of hypoxia-resistant epinephelus lanceolatus is characterized by comprising the following steps:
1) parent fish breeding and sperm obtaining for blue grouper mackerel
Selecting male large grouper with the body length of 50-70cm and the body weight of 20-30Kg, and culturing in an industrial culture environment under the following conditions: dissolved oxygen 7-10mg/L, water flow exchange capacity 3-4m3H, natural light, the cultivation time is 5-9 months, and the cultivation temperature is 18-25 ℃; the cultivation is started in 10 months of the first year, the water temperature is 23-24 ℃, the water temperature is gradually reduced to 18-20 ℃ in 12 months, and the water temperature is gradually increased to 25-26 ℃ in 3 months of the next year;
selecting mature male large grouper with large size when the water temperature reaches 25-26 deg.C in months 4-6, squeezing abdomen to collect semen;
2) sperm cryopreservation
Placing the semen obtained in the step 1) in a cryopreservation solution, balancing for 5-8min at 3-4 ℃, then placing in liquid nitrogen steam for balancing for 10-12min, and finally placing in liquid nitrogen for cryopreservation;
3) parent fish breeding of mackerel and obtaining of ovum
Selecting female mackerel with the body length of 20-25cm and the body weight of 0.5-1.0Kg, and culturing in an industrial culture environment under the following conditions: 5-9mg/L of dissolved oxygen, 25-31 per mill of salinity and 2-3m of water flow exchange capacity3H, the water temperature is 12-29 ℃, and the cultivation time is 5-9 months;
when the water temperature reaches 23-26 ℃ in 4 months, selecting well-developed female mackerel with swollen abdomen, anesthetizing by adopting MS-222, injecting chorionic gonadotropin and gonadotropin releasing hormone analogues into the basal part of the pectoral fin of the female mackerel, and extruding the abdomen of the female mackerel after injecting for 48-60 hours to obtain eggs;
4) artificial insemination
Taking the frozen semen of the large grouper with the blue body obtained in the step 2), unfreezing at 36-37 ℃, pouring into the ovum obtained in the step 3), adding seawater for fertilization, uniformly mixing the semen and the ovum, and standing for 5 min; then adding high salinity seawater, collecting floating eggs, cleaning and hatching;
5) hatching management
Incubating for 6-10h, placing in high salinity seawater, floating upward again, collecting floating fertilized eggs, placing in a culture pond for incubation and cultivation, wherein the culture conditions are as follows: the density is 30-45g/m3Salinity of 29-30 per mill, temperature of 25-27 ℃, and dissolved oxygen of 6-10 mg/L;
6) late stage cultivation
Feeding oyster fertilized eggs and chlorella when the yolk sac is completely absorbed at the age of 2-4 days; feeding the S rotifers at 3-16 days old; feeding L-shaped rotifers at the age of 13-35 days; feeding artemia nauplii at the age of 25-45 days; feeding adult artemia from 40-55 days old, and gradually transitioning to compound pellet feed; and (5) timely screening the seedlings, and culturing under the dark condition to obtain the hybrid cultured fish of the mackerel.
2. The method for the hybrid breeding of the low-oxygen-resistant epinephelus lanceolatus according to claim 1, wherein:
in the step 2), the frozen preservation solution is ELs-3 frozen diluent, and the ELs-3 frozen diluent comprises the following components: each liter of distilled water contains 10g of glucose, 10g of NaCl and NaHCO30.5g and 10mL calf serum.
3. The method for the hybrid breeding of the low-oxygen-resistant epinephelus lanceolatus according to claim 2, wherein:
in the step 2), the volume ratio of the frozen preservation solution to the semen is 1: 1.
4. The method for the hybrid breeding of the low-oxygen-resistant epinephelus lanceolatus according to claim 1, wherein:
in the step 3), the dosage of the chorionic gonadotropin is 200-300IU/Kg, and the dosage of the gonadotropin releasing hormone analogue is 35 mu g/Kg.
5. The method for the hybrid breeding of the hypoxia-resistant epinephelus lanceolatus according to claim 4, wherein:
in the step 3), the chorionic gonadotropin and the gonadotropin releasing hormone analogue are jointly dissolved in physiological saline with the mass concentration of 0.9%.
6. The method for the hybrid breeding of the low-oxygen-resistant epinephelus lanceolatus according to claim 1, wherein:
in the step 4) and the step 5), the mass concentration of the high-salinity seawater is 33-35 per mill.
7. The method for the hybrid breeding of the low-oxygen-resistant epinephelus lanceolatus according to claim 1, wherein:
in the step 4), the cleaning is performed by using high-salinity seawater with the mass concentration of 33-35 per mill.
8. The method for the cross breeding of low-oxygen-resistant epinephelus lanceolatus according to any one of claims 1 to 7, wherein:
in the step 6), the feeding amount of oyster fertilized eggs is 20/mL, and the feeding amount of chlorella is 50 multiplied by 104The feeding amount of the S rotifers is 12-15/mL, the feeding amount of the artemia nauplii is 3-4/mL, and the feeding amount of the adult artemia is 3-4/mL.
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