CN111657186B - Method for cultivating natural gynogenesis megalobrama amblycephala - Google Patents
Method for cultivating natural gynogenesis megalobrama amblycephala Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a method for cultivating natural gynogenesis megalobrama amblycephala, which takes megalobrama amblycephala as a female parent and Xenocypus davidi as a male parent, and artificially injects a mixed oxytocic of LRH-A and HCG into the parent fish of the female parent for artificial oxytocic, wherein the dosage of LRH-A is 8-10 mu g/kg, and the dosage of HCG is 400-500 IU/kg; injecting female parent fish, and after 3h, manually injecting a mixed oxytocic of LRH-A and HCG into the male parent fish for manual induced spawning, wherein the injection dose is half of that of the female parent fish, and a ventral cavity one-needle injection method without scales at the base of pectoral fins is adopted; and carrying out artificial insemination, running water incubation, feeding, detection and screening to obtain the natural gynogenesis megalobrama amblycephala. The breeding method changes the activity of the ovum and the sperm of the parent, so as to obtain the natural megalobrama amblycephala capable of being bred in both sexes and developing the gynogenesis, and the technical treatment such as sperm inactivation, ovum doubling and the like is not needed in the whole breeding process, thereby greatly reducing the workload.
Description
Technical Field
The invention relates to a distant hybridization breeding method for fish, in particular to a method for preparing natural gynogenesis megalobrama amblycephala by distant hybridization between megalobrama amblycephala and xenocypris davidi subfamily.
Background
Distant crosses are crosses between species or between species that are related and allow the transfer of a genome from one species to another, resulting in an alteration of the genotype and phenotype of the offspring. The distant hybridization can also generate gynogenesis offspring, so that the natural gynogenesis offspring can show heterospermia hybridization effect in the aspects of meat quality, disease resistance, stress resistance, growth speed and the like, and therefore, the natural gynogenesis offspring which can be bred in an amphoteric way is formed through the distant hybridization, and an important germplasm resource library is provided for genetic breeding. How to form novel natural gynogenesis fish by using the prior art is a problem to be solved by the technical personnel in the field.
Megalobrama amblycephala (Megalobrama amblycephala) belonging to family Cyrinales, family Cyprinaceae, subfamily Erythroculter; megalobrama amblycephala is one of the main freshwater aquaculture fishes in China. However, as megalobrama amblycephala is a fish sensitive to hypoxia, the hypoxia content causes huge economic loss in the culture process, and in addition, the growth speed of megalobrama amblycephala is relatively slow. In the prior art, the operation process for obtaining the gynogenesis megalobrama amblycephala is complicated, the selection requirements on a sperm source parent, a sperm inactivation method and a chromosome doubling method are very strict, and the obtained gynogenesis megalobrama amblycephala is female offspring, so that the application of the gynogenesis megalobrama amblycephala in production practice is limited. Therefore, the exploration of distant hybridization between the megalobrama amblycephala and the xenocypris davidi bleekers to obtain the novel natural gynogenesis megalobrama amblycephala capable of being bred in both sexes has important significance in production practice and genetic breeding.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a cultivation method of an amphiprotic fertile natural gynogenesis megalobrama amblycephala.
In order to solve the technical problem, the invention provides a cultivation method of a natural gynogenesis megalobrama amblycephala, which comprises the following steps: taking megalobrama amblycephala as a female parent and Xenocypris davidi as a male parent, and manually injecting a mixed oxytocic of a luteinizing hormone releasing hormone analogue and chorionic gonadotropin into female parent fish with swollen belly, softness and high elasticity in a breeding season to perform artificial oxytocic, wherein the dosage of the luteinizing hormone releasing hormone analogue is 8-10 mu g/kg, and the dosage of the chorionic gonadotropin is 400-500 IU/kg; injecting female parent fish, selecting male parent fish with light extrusion of white semen on the belly after 3h, and manually injecting a mixed oxytocic of luteinizing hormone releasing hormone analogue and chorionic gonadotropin for artificial oxytocic, wherein the injection dose is half of that of the female parent fish, and an intraperitoneal one-needle injection method without scales on the base part of pectoral fins is adopted during injection; and after the injection is finished, putting the parent fish into a spawning pond, carrying out artificial insemination after the parent fish is estrualized, then carrying out flowing water incubation on fertilized eggs in the incubation pond, feeding the fish after the fry is incubated, and then detecting and screening the fed fish to obtain the natural gynogenesis megalobrama amblycephala.
Xenocypris davidodi bleker (Xenocypris davidodi Bleeker) belongs to Cyprinales, Cyprinidae, Xenocypris davidodi subfamily; xenocypris davidi bleeker has the characteristics of quick growth, disease resistance, cold resistance, hypoxia resistance, high meat content, delicious taste and the like. The xenogenic breeding catfish is used as a sperm source provider to be hybridized with the megalobrama amblycephala in a distant way to generate gynogenesis offspring, and the heterospermic hybridization effect can enable the natural gynogenesis megalobrama amblycephala to have various excellent characteristics of the xenogenic catfish, so that the natural gynogenesis megalobrama amblycephala which is high in growth speed, low-oxygen resistant, good in meat quality and attractive in body shape and can be bred in an amphotericity mode is expected to be obtained. However, according to the conventional breeding method, only diploid hybrid fish is formed in distant hybridization offspring of megalobrama amblycephala and xenocypris davidi, and no natural gynogenesis megalobrama amblycephala is found.
In the invention, the activity and quality of ova and sperms of parents are changed by changing the species and dosage of an oxytocic, so that a novel natural gynogenesis megalobrama amblycephala which can be bred in an amphotericity manner is formed in offspring, and experiments prove that chromosome fragments from Megalobrama lutescens are found in the natural gynogenesis megalobrama amblycephala, so that an ectopic 'hybridization' effect is generated, and the natural gynogenesis megalobrama amblycephala has some excellent characteristics of Megalamus lutescens and is expressed in the breeding process, which is very important for improving the germ plasm resources of the megalobrama amblycephala.
Preferably, the breeding method comprises the steps of selecting female megalobrama amblycephala and male xenocypris davidi bleekers with beautiful body types, no diseases or injuries and obvious sexual maturity characteristics as parents to carry out special pond breeding 2-3 months before the breeding season, feeding with fine baits 1 month before the breeding season, and carrying out running water stimulation every day to promote gonad maturation.
Preferably, the artificial induced spawning is carried out in a breeding season when the water temperature is stable at more than 20 ℃, and after the mixed induced spawning agent is injected, the ratio of male and female parent fishes 1: 3 is put into a spawning pond.
Preferably, the artificial insemination comprises the following steps: after the parent fish is in heat, fishing the parent fish by using a soft net, selecting a female megalobrama amblycephala which is smooth in spawning and large in spawning amount, slightly squeezing the belly, squeezing dark green ova into a clean porcelain basin, selecting xenocypris davidi bleeker which is large in sperm production, slightly squeezing the belly, squeezing white sperm into the porcelain basin, rapidly mixing the white sperm with feathers, and then rapidly pouring the mixture into a palm sheet prepared in advance for insemination.
Preferably, the raising of the fry comprises the following steps: after the fries are completely hatched, the fries are cultured in an incubation pool for 2-3 days, and are transferred to a pond fattening in advance after waist spots appear; and sprinkling soybean milk after the fry are put into the pool for 2 days, wherein the soybean milk is required to be uniform and fine.
Preferably, the detection is performed by measuring the profile, the number of chromosomes and the DNA content of the fed fish by a profilometry, a method for detecting the ploidy of chromosomes in the culture of peripheral blood cells and a DNA content measurement method by flow cytometry.
The method for cultivating the natural gynogenesis megalobrama amblycephala adopts distant hybridization of the megalobrama amblycephala and xenocypris davidi to obtain the natural gynogenesis megalobrama amblycephala which has high growth speed, low oxygen resistance and good meat quality and can be bred in an amphotericity way.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the breeding method, the megalobrama amblycephala and the xenocypris davidi bleekers are subjected to distant hybridization, the specific type and the specific dosage of the oxytocic are combined, the activities of ova and sperms of parents are changed, the natural gynogenesis megalobrama amblycephala which can be bred in a two-sex way is further obtained, the technical treatment of sperm inactivation, ovum doubling and the like is not needed in the whole breeding process, and the workload is greatly reduced.
2. The novel natural gynogenesis megalobrama amblycephala obtained by the cultivation method is similar to the megalobrama amblycephala in appearance and biological characteristics, is amphoteric and fertile, has the characteristics of high growth speed, hypoxia resistance, rich nutrition, beautiful body and the like, and has important significance in the aspects of genetic breeding, production practice and the like; the amphiprotic fertile natural gynogenesis megalobrama amblycephala obtained by the invention can obtain an improved megalobrama amblycephala strain through selfing, and provides a very good foundation for improving germ plasm resources of the megalobrama amblycephala.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a photograph of the outline of a megalobrama amblycephala;
FIG. 2 is a photograph showing the appearance of xenocypris davidi;
FIG. 3 is a photograph of the appearance of a natural gynogenesis megalobrama amblycephala;
FIG. 4 is a flow diagram of a megalobrama amblycephala;
FIG. 5 is a flow chart of xenocypris davidi;
FIG. 6 is a flow chart of a natural gynogenesis megalobrama amblycephala;
FIG. 7 is a chromosome map of megalobrama amblycephala;
FIG. 8 is a chromosome map of Xenocypris davidi;
FIG. 9 is a chromosome map of natural gynogenesis megalobrama amblycephala.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example (b):
a cultivation method of natural gynogenesis megalobrama amblycephala comprises the following steps:
(1) selecting female megalobrama amblycephala and male xenocypris davidi with beautiful body type, no disease and no injury and obvious sexual maturity characteristics as parents to carry out special pond breeding 2-3 months before a breeding season, wherein the special pond is required to have good water quality, high oxygen content and sufficient illumination; furthermore, the fine baits were carefully fed, 1 running water stimulation per day, particularly 1 month before reproduction, to promote gonad maturation;
(2) in 5-6 months of the year, when the water temperature is stabilized to be more than 20 ℃, selecting abdominal distension, soft and elastic megalobrama amblycephala as a female parent fish, gently pressing Xenocypus flavus extruded with white semen on the abdomen as a male parent fish, and injecting a mixed oxytocic of luteinizing hormone releasing hormone analogue (LRH-A) and chorionic gonadotropin (HCG) for induced spawning, wherein the dose of LRH-A injected by the female parent fish is 8-10 mu g/kg, and the dose of HCG is 400-500 IU/kg; injecting female megalobrama amblycephala for 3h, and then injecting male xenocypris davidi bleeker, wherein the injection dosage of xenocypris davidi bleeker is halved, and a peritoneal injection method without scales at the base of pectoral fins is adopted during injection; after the injection, the megalobrama amblycephala and the xenocypris davidi bleeker are injected according to the proportion of 1: 3, putting the mixture into an induced spawning pond, putting some male megalobrama amblycephala into the induced spawning pond, and flushing the induced spawning pond with water to promote the spawning of the female and male fishes;
(3) when the parent fish chases in the pond and a few eggs are on the net piece in the pond, the net is opened, and the action is light when the net is opened; selecting megalobrama amblycephala and xenocypris davidi bleekers which have large spawning amount and smooth spawning, and xenocypris davidi bleekers which have smooth spawning, then extruding the megalobrama amblycephala ova into a clean porcelain basin, simultaneously extruding the semen of xenocypris davidi bleekers, rapidly stirring by using feathers, uniformly spreading on a palm sheet prepared in advance after uniformly stirring, and putting fertilized ova into an incubation pool for incubation until the oosperm emerge;
(4) and (3) after the fries are completely hatched in the hatching pond, firstly culturing the fries in the hatching pond for 2-3 days, transferring the fries to a soil pond which is fattened in advance for culturing after the fries have waist points, transferring the fries to the pond, sprinkling soybean milk for 2 days, sprinkling for 3 times a day for one circle along the wall, and requiring uniformity and fineness until the fries start to normally eat.
After the fry are raised for 4 months, randomly sampling, measuring and analyzing the biologically relevant data, and comparing the result with the adult fish shape photos of megalobrama amblycephala and xenocypris davidi and various known data respectively, wherein the results are shown in figure 1-figure 3 and table 1:
table 1: comparison of countable characters of natural gynogenesis megalobrama amblycephala, megalobrama amblycephala and xenocypris davidi
Note: the upper-case Roman numerals in the above table represent the number of the hardwood bars and the Arabic numerals represent the number of the fin bars.
The countable-related traits of fig. 1-3, table 1 show that: the natural gynogenesis megalobrama amblycephala is similar to the megalobrama amblycephala, which indicates that a novel natural gynogenesis megalobrama amblycephala is formed through distant hybridization.
The megalobrama amblycephala is used as a reference, the DNA content of the natural gynogenesis megalobrama amblycephala is detected by a flow cytometer, and fig. 4, fig. 5 and fig. 6 are flow charts of the megalobrama amblycephala, xenocypris davidi and the natural gynogenesis megalobrama amblycephala respectively. The average DNA contents of the megalobrama amblycephala, the yellowtail catfish and the natural gynogenesis megalobrama amblycephala are 70.03, 65.70 and 67.29 respectively. The ratio of the average DNA content of the natural gynogenesis megalobrama amblycephala to the average DNA content of the natural gynogenesis megalobrama amblycephala is 0.96, and the ratio is not obviously different from the expected ratio 1. Meanwhile, tail fin cells are cultured by a tail fin cell culture method to prepare chromosomes for checking ploidy, and fig. 7, 8 and 9 are the chromosome maps of megalobrama amblycephala, xenocypris davidi bleeker and natural gynogenesis megalobrama amblycephala respectively; the detection finds that the number of chromosomes of the natural gynogenesis megalobrama amblycephala is the same as that of the chromosomes of the megalobrama amblycephala, and the result accords with the result of detecting the DNA content by using a flow cytometer, and the natural gynogenesis megalobrama amblycephala is proved to be diploid 2 n-48.
In order to prove the oxytocic effect of different doses of oxytocic, a control test is also carried out, in the process of artificial oxytocic, the dose of LRH-A injected by female parent fish of a test control group is 20-30 mu g/kg, and the dose of HCG is 600-800 IU/kg; injecting female megalobrama amblycephala for 3h, and then injecting male xenocypris davidi bleeker, wherein the injection dosage of xenocypris davidi bleeker is halved, and a peritoneal injection method without scales at the base of pectoral fins is adopted during injection; other operations are the same as those of the present invention; the results are shown in table 2:
table 2: oxytocic effect of different doses of oxytocic
| The invention | Test control group |
| LRH-A:8-10μg/kg；HCG：400-500IU/kg | LRH-A：20-30μg/kg；HCG：600-800IU/kg |
| Discovering natural gynogenesis megalobrama amblycephala | No natural gynogenesis megalobrama amblycephala is found |
As can be seen from Table 2, the invention changes the activity and quality of the ovum and sperm of the parent by reducing the injection dosage of the oxytocic, and further forms a novel natural gynogenesis megalobrama amblycephala with fertile property in the offspring.
In order to research the characteristics of the natural gynogenesis megalobrama amblycephala, the natural gynogenesis megalobrama amblycephala and the common megalobrama amblycephala are cultured under the same condition, a large number of floating heads and even death of the common megalobrama amblycephala occur in sultry summer, but the phenomenon is rarely found in the natural gynogenesis megalobrama amblycephala, and the natural gynogenesis megalobrama amblycephala is more resistant to hypoxia from the culture experience. And the meat quality of the natural gynogenesis megalobrama amblycephala is more delicious. The natural gynogenesis megalobrama amblycephala cultured by the method generates the heterospermia hybridization effect, so that the natural gynogenesis megalobrama amblycephala has some excellent characteristics of xenocypris davidi bleeker.
The molecular marker is used for detecting the natural gynogenesis megalobrama amblycephala, DNA fragments of Xenocypris davidi are found in the natural gynogenesis megalobrama amblycephala, and the DNA fragments are proved to form a 'hybridization' effect in the natural gynogenesis megalobrama amblycephala. Further proves that the megalobrama amblycephala and the xenocypris davidi bleeker are hybridized to form the natural gynogenesis megalobrama amblycephala to generate the heterospermic hybridization effect.
The chromosome ploidy detection method for peripheral blood cell culture comprises the following operation steps: 1) in superPreparing a culture medium in a clean workbench, wherein 100ml of the culture medium contains the following components: 84ml of RPMI-1640, 15ml of calf serum, 2 pieces of PHA, 1ml of 0.1% heparin sodium, 7.5% NaHCO3Adjusting the pH value to 7.2-7.4 (asepsis) or 1N HCl; 2) sucking a small amount of sterilized heparin sodium solution by using a syringe, and taking blood from the tail vein of the experimental fish after being sterilized by iodine; 3) adding anticoagulation blood into culture solution according to the standard that about 0.2ml anticoagulation blood is added into every 10ml of culture solution, culturing for 68-72 h in an incubator with 24 ℃ and 5% carbon dioxide concentration, and shaking up regularly during the culture period to ensure that cells fully contact with the culture medium; 4) 24 hours before terminating the culture, 10 mu g/ml colchicine is dripped into the culture solution by using a 1ml syringe to ensure that the final concentration is 0.05-0.07 mu g/ml. The above steps all need aseptic operation.
The chromosome preparation steps are as follows: 1) transferring all the culture into a clean 10ml centrifuge tube, centrifuging at 1000rpm for 5 minutes, and removing the supernatant; 2) adding 9ml of hypotonic solution into the centrifugal tube, uniformly mixing, and performing hypotonic for 25-30 minutes; 3) adding 1ml of stationary liquid, gently mixing uniformly, centrifuging at 1000rpm for 5 minutes, and removing supernatant; 4) adding 5ml of stationary liquid, mixing the mixture evenly and gently, standing the mixture for 20 minutes, centrifuging the mixture for 5 minutes at 1000rpm, and removing supernatant; 5) repeating the step 4 once; 6) adding a proper amount of fixing liquid according to the number of the final cells to prepare cell suspension (generally 1-2 ml), sucking the cell suspension, dropping the cell suspension from a height of 20-30 cm, slightly blowing the cell suspension, and naturally drying the cell suspension in the air; 7) and dyeing with Giemsa dye liquor for 20-40 minutes, washing the back surface of the glass slide with fine water flow to remove the dye liquor, drying in air, and performing microscopic examination and photographing.
The flow cytometry DNA content determination method comprises the following steps: blood is collected by a heparin-wetted disposable syringe from a fish tail vein by about 0.2mL, the blood is injected into an Eppendorf tube filled with 0.8% physiological saline, 1mL of cell nucleus extract DAPI-A (nuclear extraction solution, provided by Partec Gmbh in Germany) is added into the mixed solution of the blood and the physiological saline, and the treatment time is 10-15 min; the sample was filtered through a 20 μm nylon filter (supplied by Partec GmbH, germany); the DNA staining solution (DAPI-B, supplied by Partec GmbH, Germany) is stood still in the dark to stain the sample for about 5-10 min, and then the sample is tested on a machine.
According to the breeding method, the megalobrama amblycephala and the xenocypris davidi bleekers are subjected to distant hybridization, the specific type and the specific dosage of the oxytocic are combined, the activities of ova and sperms of parents are changed, the natural gynogenesis megalobrama amblycephala which can be bred in a two-sex way is further obtained, the technical treatment of sperm inactivation, ovum doubling and the like is not needed in the whole breeding process, and the workload is greatly reduced. The novel natural gynogenesis megalobrama amblycephala obtained by the cultivation method is similar to the megalobrama amblycephala in appearance and biological characteristics, is amphoteric and fertile, has the characteristics of high growth speed, hypoxia resistance, rich nutrition, beautiful body and the like, and has important significance in the aspects of genetic breeding, production practice and the like; the amphiprotic fertile natural gynogenesis megalobrama amblycephala obtained by the invention can obtain an improved megalobrama amblycephala strain through selfing, and provides a very good foundation for improving germ plasm resources of the megalobrama amblycephala.
Claims (2)
1. A cultivation method of natural gynogenesis megalobrama amblycephala is characterized by comprising the following steps: selecting female megalobrama amblycephala and male xenocypris davidi as parents with beautiful body types, no diseases and no injuries and obvious sexual maturity characteristics for special pond feeding 2-3 months before the breeding season, feeding with fine bait 1 month before the breeding season, and performing running water stimulation every day to promote gonad maturation;
taking megalobrama amblycephala as a female parent and Xenocypris davidi as a male parent, and manually injecting a mixed oxytocic of a luteinizing hormone releasing hormone analogue and chorionic gonadotropin into female parent fish with swollen belly, softness and high elasticity in a breeding season to perform artificial oxytocic, wherein the dosage of the luteinizing hormone releasing hormone analogue is 8-10 mu g/kg, and the dosage of the chorionic gonadotropin is 400-500 IU/kg; injecting female parent fish, selecting male parent fish with light extrusion of white semen on the belly after 3h, and manually injecting a mixed oxytocic of luteinizing hormone releasing hormone analogue and chorionic gonadotropin for artificial oxytocic, wherein the injection dose is half of that of the female parent fish, and an intraperitoneal one-needle injection method without scales on the base part of pectoral fins is adopted during injection; the artificial induced spawning is carried out in the breeding season when the water temperature is stable at more than 20 ℃, and after the mixed induced spawning agent is injected, the ratio of male and female parent fishes is 1: 3, putting the fish in a spawning pond, carrying out artificial insemination after the parent fish is estrualized, then carrying out flowing water incubation on fertilized eggs in an incubation pond, feeding the fish after the fry is incubated, and then detecting and screening the fed fish to obtain the natural gynogenesis megalobrama amblycephala;
the artificial insemination comprises the following steps: after the parent fish is in heat, fishing the parent fish by using a net with soft texture, selecting female megalobrama amblycephala which can lay eggs smoothly, slightly squeezing the belly, squeezing dark green ova into a clean container, selecting xenocypris davidi bleeker which can produce sperm smoothly, slightly squeezing the belly, squeezing white sperm into the container, uniformly mixing the white sperm with feather, and then quickly pouring the mixture into a palm sheet prepared in advance for insemination;
the fry rearing method comprises the following steps: after the fries are completely hatched, the fries are cultured in an incubation pool for 2-3 days, and are transferred to a pond fattening in advance after waist spots appear; and sprinkling soybean milk after the fry are put into the pool for 2 days, wherein the soybean milk is required to be uniform and fine.
2. The cultivation method as claimed in claim 1, wherein the detection is performed by measuring the shape, the number of chromosomes and the DNA content of the fed fish by a shape measuring method, a chromosome ploidy detection method in peripheral blood cell culture and a flow cytometry DNA content measuring method.
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