CN101305701B - Fish family establishing and disease-resistant high yield fine breeding method - Google Patents
Fish family establishing and disease-resistant high yield fine breeding method Download PDFInfo
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Abstract
The invention relates to a method for the fish genealogy establishment and the disease resistance and high yield improved type selection. By adopting the technologies, such as group selection, genealogy selection, hybridization and artificial infection, as well as the sieving of the quick growth genealogy and disease resistance genealogy, the genealogy which has the advantages of strong flounder disease resistance and quick growth is selected, and the selection method for the disease resistance and high yield high quality improved type fish is established. The technology has the advantages of strong pertinence, high selection efficiency, obvious effect, etc. and the survival percent of the disease resistance flounder fish fry is more than 80 percent, which is increased by 30 percent more than the survival percent of the common flounder fish fry; the growth speed of the disease resistance flounder fish fry is also increased by more than 10 percent. The method in the invention opens up a new technical approach for culturing the disease resistance fish variety. As a disease resistance high yielding variety selection method applied to the improved fish type selection, the method is suitable for the popularization and the application on all the cultured fishes, and has important significance and application value in culturing the cultured fish of disease resistance and high yield improved type.
Description
Affiliated technical field:
The invention belongs to the selection of the fish disease-resistant high yield kind in the aquaculture field, is a kind of disease-resistant high yield fine breeding method that can use in the fish fine-variety breeding.
Background technology:
Fish are important aquaculture kinds, also are human important food sources.China is the highest country of aquaculture output in the world.The annual production of China's cultured fishes at present reaches more than 2,000 ten thousand tons, except that satisfying China domestic market, has every year 300000 tons of fishery-ies products to export to foreign countries approximately, is one of main farm produce of China's foreign exchange earning.But, in recent years, along with the fast development of fish culture industry, disease takes place frequently, germplasm is degenerated, weed survival rate is low, and economic loss every year of giving that fish culture already causes is up to more than 150 hundred million yuans, thereby has greatly limited China's fish culture development already.
The main disease of harm fish is bacillary and viral disease at present, and the harm that the former causes is bigger, and wherein the maximum a kind of bacteriosis of harm is the vibriosis (Zhou Li etc., 1997) that is caused by Vibrio anguillarum.Although take traditional prophylactic measures such as antibiotic class medicine, but still can't fundamentally solve the disease problem of fish cultures such as lefteye flounder; And antibiotic class medicine has easily at the fish cylinder accumulation, consumer's health is had potential hazard, pathogen developed immunity to drugs and problem such as severe contamination breeding environment easily, thereby the application in the fish culture industry more and more is restricted; The use of antibiotic simultaneously can not be satisfied the growing demand to the drug residue free green aquatic product of people.
Therefore, employing modern biotechnology cultivation premunition is strong, the cultured fishes new varieties of fast growth, has become the key subjects that China's aquatic science and technology must solve, also is the fundamental way that solves the fish diseases problem.Aspect the fish genetic breeding, domesticly begin the eighties from twentieth century, on the fresh water cyprinid fish, carried out the research work of some crossbreeding, cultivate the clever carp (Pan Guangbi etc. of quick growth in succession by the hybridization between the different carps, 1989), jian carp new varieties such as (Zhang Jiansen etc., 1994).Aspect the research of seawater fish genetic breeding, research work (Wang Xincheng etc., 1997 that artificial gynogenesis is cultivated the unisexuality seed mainly on lefteye flounder, large yellow Crocker and turbot, have been carried out; Wang Xiaoqing etc., 2006; Su Pengzhi etc., 2008), simultaneously, also carried out the research work of aspects such as some molecular marker screenings and genetic diversity evaluation, Germplasm Identification and disease-resistant related molecular marker screening, the check and analysis of adopting the RAPD technology that lefteye flounder and turbot cultured population genetic structure are carried out as (2000) such as Zou Shuming; The analysis that You Feng etc. (2001) adopt isoenzyme technique that coastal waters, Shandong lefteye flounder and the genetic diversity of culturing lefteye flounder are carried out; Liuetal. (2005a b) utilizes the genetic structure of microsatellite marker and AFLP technology assessment lefteye flounder cultured population and wild population, and the genetic diversity that shows cultured population has decline to a certain degree; Chen Wei etc. (2005) have carried out the molecular studies of screening of lefteye flounder microsatellite marker and genetic diversity; Zhang Yuxi etc. (2006) have analyzed disease-resistant fish of lefteye flounder and the mhc gene polymorphism of disease-resistant fish not, and Preliminary screening is to disease-resistant relevant mhc gene type; But the disease-resistant family of relevant fish is set up and the seed selection of disease-resistant high yield fine, has not yet to see report.
Summary of the invention:
The objective of the invention is for the technical method of fish family foundation and disease-resistant high yield fine breeding is provided, for the breed of variety of fish disease-resistant high yield provides technological means.
Its technology contents of the present invention is as follows: comprise that fish family is set up and the technical method of disease-resistant high yield fine breeding;
One, fish family method for building up; Comprise: the preparation of (), disease-resistance population; (2), the foundation of fish family; (3), the labeling method of different familys.
(1), the preparation of disease-resistance population: adopt natural selection and artificial infection's approach to obtain disease-resistance population (RS).
Natural selection: in the morbidity busy season, collect the fry and the fingerling individuality of vibriosis morbidity back survival, cultivate;
Artificial infection's approach: infect common fry with the pathogen Vibrio anguillarum:
Fry is the common fry of lefteye flounder Huanghai Sea colony, and body weight is a 15-20 gram/tail;
The Vibrio anguillarum bacterial strain that Vibrio anguillarum preserves for this laboratory, the medium of Vibrio anguillarum is 2216E, Vibrio anguillarum is seeded in 2216E medium (peptone 5g, yeast extract 1g, the FePO of sterilization
44H
2O0.1g, Chen Haishui 1000mL) in, placing velocity of vibration is on the shaking table of 200r/m, cultivates under 28 ℃, measures the OD600 value after 20 hours, centrifugal collection thalline is diluted to the bacterium liquid that concentration is 10OD with original bacteria liquid with the 0.9%NaCl physiological saline of sterilizing.
Infect experiment in advance with above-mentioned bacterium liquid: earlier the bacterium liquid of 10OD concentration is diluted to different concentration (diluting 50,100,120,150,240 and 300 times) respectively and carries out preliminary experiment with physiological saline, each concentration is injected the fry of 10 tail 15-20 grammes per square metres, and injection volume is 0.3-0.5mL.The concentration that will cause the fry death of 50-60% in the fry of injection is defined as half lethal concentration.The half lethal concentration of the fry of above-mentioned specification is the bacterial solution of 240 times of the dilutions of 0.3-0.5mL.
Infection experiment: formal infection experiment is with lefteye flounder tail more than totally 800, the about 15-20g of body weight, and lefteye flounder individuality lumbar injection Vibrio anguillarum, injected dose is controlled at half lethal concentration, and the injection volume of Vibrio anguillarum solution is 0.02ml bacterium liquid/gram fish.Water temperature is controlled at 21-23 ℃ during infection.
Disease-resistance population (RS) is the individuality that long-term health survives behind natural selection or the artificial infection Vibrio anguillarum.
(2), the foundation of fish family; Fish are lefteye flounder day in-group (JS) and lefteye flounder Huanghai Sea colony parent population (YS):
1, the cultivation of lefteye flounder day in-group (JS) and Huanghai Sea colony parent population (YS):
Introduce lefteye flounder fry 50 tails from Japan, long about 5 centimetres of body, through the cultivation of 3-4, fry reaches sexual maturity, is used to set up the lefteye flounder family; Huanghai Sea wild population parent population is the wild individuality of fishing for, and adopts conventional method to raise and train and breed.
Employing electronic marker method enantiopathy colony (RS), day in-group (JS) and 3 colony parent populations of Huanghai Sea wild population (YS) carry out individual mark, and every tail parent population is given a mark mark, so that match identification parent population source when breeding.
2. the collection and the artificial insemination of essence, ovum:
Utilize manual compression belly method to gather mature egg respectively, extruding is also drawn mature sperm in the vial of drying with suction pipe.The fertilization of employing dry method, the seminal fluid that is about to extrude is added in the container of containing ovum, shakes mixing, add then 15-16 ℃ sea-water activated, finish the insemination process, add 10 times of seawater again, left standstill 5-10 minute, above good ovum swims in to the ovum amount, unfertilized dead ovum then is sunken to the bottom, the ovum that will float moves in the hatching cylinder hatches, and embryonic development to primitive gut is added up fertilization rate in early days, grows to membrane and adds up incubation rate in earlier stage.
3. the foundation of family:
In lefteye flounder mating season, adopt disease-resistance population (RS), day in-group (JS) and 3 reproductive populations of Huanghai Sea wild population (YS) to match breeding as the parent.Adopt the disease-resistant lefteye flounder ♀ of disease-resistant lefteye flounder ♂ x, disease-resistant lefteye flounder ♂ x Japan lefteye flounder ♀; The disease-resistant lefteye flounder ♀ of Japan lefteye flounder ♂ x, Japanese lefteye flounder ♂ x Japan lefteye flounder ♀; The disease-resistant lefteye flounder ♀ of the wild lefteye flounder ♂ of Huanghai Sea x, the wild lefteye flounder ♂ of Huanghai Sea x Japan lefteye flounder ♀, respectively with the mating pattern structure family of 1 tail milter to 1 tail raun, sometimes be 1 tail milter respectively to 2-4 tail raun, or 1 tail raun makes up family half sibs to the mating pattern of 2-5 tail milter respectively.Essence, ovum collection and hatching are carried out as stated above.The fertilized egg that will produce from a tail milter and a tail raun mating, i.e. family is placed in the tank of 3 cubic metres of volumes and hatches, and breeding density is 20-30 milliliter fertilized egg, and tank is carried out 24 hours miniflow water, inflation.The work of setting up of family should be finished in 10-20 days.
(3), the labeling method of different familys:
Adopt the different parts of the fluorescent dye injection fry of different colours, it is subcutaneous that the fluorescent dye of the different colours for preparing is injected the fish body surface, and every needle injection amount is 3-5 μ l.Choose yellow, red, 3 kinds of fluorescent pigments of orange, before on fish body belly, having chosen the back of the body, behind, 3 positions behind the abdomen, utilize the permutation and combination method to design marker combination, by the a kind of color in 1 position, 3 kinds of colors of the 2 kinds of colors in 1 position and 1 position are carried out permutation and combination, 3375 permutation and combination are arranged altogether, promptly utilize 3 kinds of colors to inject at 3 different parts, can 3375 familys of mark.
Two, disease-resistant high yield fine breeding method; Comprise: (one), the grow screening of family fast; (2), the screening of disease-resistant family.
(1), grow the fast screening of family:
Set up the back to fluorescence labeling in family, different familys are raised in different breed tanks, and it is identical that the stocking density of different familys should keep, and culture 5000-10000 tail newly hatched larvae in 3 cubic metres the tank.After fry grew 20 days, the density of fry was adjusted into the 3000-5000 tail in the tank; After fry grew to 45 days, its density was adjusted into the 1000-1500 tail; When fry grows to 8-10 centimetre, from each family, get 200 fishes at random, carry out fluorescence labeling according to the method described above, the fish of the different familys behind the mark can be placed in the same cement pit and raise, and the cement pit of 40 square meters can be cultured 4000-5000 bar fry.Carry out one-shot measurement every half a year behind the mixed breed, utilize the data of measuring 3 times to carry out the evaluation of growth performance.Measurement index is mainly total length (holding tail fin end from kiss), body wide (health the widest part does not contain the back of the body, abdomeinal fin length), 3 indexs of body weight, and each family is measured 40-60 tail fish.Calculate that each family body is long, body is wide, the mean of body weight.Employing is set up mid-term in family and is organized in contrast by the family that the wild lefteye flounder of the Huanghai Sea (YS) mating produces.More different familys are long with contrast family body, body is wide, the difference of body weight, body weight extremely obviously greater than the family of the contrast family family (p＜0.01) that is defined as growing fast, obviously is defined as faster growing family (p＜0.05) greater than the family of contrast family with body weight; The family that the body weight utmost point is significantly less than the contrast family is defined as slow growth family (p＜0.01); Body weight is significantly less than the family family (P＜0.05) that is defined as growing more slowly of contrast family.The family of will growing the fast fry of cultivating according to a conventional method, raise up seed promptly can be used as high yield fine and cultures and promote.
(2), the screening of disease-resistant family:
Carry out infection experiment when fry grows to when body is long to be the 9-11cm left and right sides, estimate the disease-resistant performance of each family.About 100 tails of each family random sampling infect about 50 tails at every turn, repeated experiments 1 time.Each family infects independently cultures at 0.5m with fish
3The fiberglass tank in.Infecting with bacterial strain is the Vibrio anguillarum bacterial strain, with 2216E medium (peptone 5g, yeast extract 1g, the FePO of Vibrio anguillarum in sterilization
44H
2O0.1g, Chen Haishui 1000mL) in, placing velocity of vibration is on the shaking table of 200r/m, cultivates under 28 ℃, incubation time is about 18-20h; Measure OD600 value, bacterium liquid at first is diluted to the concentration of 10OD with 0.9% NaCl physiological saline of sterilization, the Vibrio anguillarum that the employing lumbar injection dilutes infects experiment in advance.During pre-the infection, again the bacterium liquid of 10OD concentration is diluted to variable concentrations with physiological saline, every group of pre-infection of carrying out different bacterial concentrations with 10-15 tail fish tested, and gropes the half lethal concentration of lefteye flounder fry, and definite lefteye flounder half lethal concentration is 49 * 10
7CFU/ml when formally infecting, injects according to half lethal concentration, and injected dose is 0.02ml bacterium liquid/gram fish, and water temperature is controlled at 21-23 ℃ during infection, keeps normally feeding between cycle water, infection period and inflating.
The survival condition of fry is regularly observed in injection back, about 12h behind the injection Vibrio anguillarum, symptoms such as that fry begins to occur moving about is slow, energy difference; 20h begins to occur a small amount of death, and 30h begins to occur dead peak, and death toll accounts for the overwhelming majority of dead sum in the whole infection in the 48h, and is basicly stable behind the 96h.Infection experiment is set up control group, and control group is for adopting the lefteye flounder fry of disease-resistance population (RS) and Huanghai Sea colony (YS) mating.According to metainfective fingerling survival rate, in conjunction with the significance of control group survival rate test of difference, we are defined as the strong family of premunition with survival rate in the family more than 60%, survival rate is defined as the more intense family of premunition in the family of 39-60%, survival rate is defined as the general family of premunition in the family of 17-39%, survival rate is defined as the family of premunition difference in the family below 17%.
The family that premunition is strong is cultivated according to a conventional method, the fry that raises up seed, and promptly can be used as disease-resistant breeding and cultures and promote.
The present invention and prior art contrast are characterized in:
The present invention adopts technology such as colony's seed selection, family selective breeding, hybridization and artificial infection, with the lefteye flounder is the technical method that material has been set up China's cultured fishes disease-resistance population and family foundation first, tentatively set up lefteye flounder disease-resistant variety breeding technique, obtained the lefteye flounder family that growth is fast, premunition is strong.
Characteristics such as pointed strong, the seed selection efficient height of this technology, effect are obvious, the survival rate of disease-resistant lefteye flounder fry is more than 80%, survival rate has improved more than 30% than common lefteye flounder fry; The growth rate of disease-resistant lefteye flounder has also improved more than 10%.Fully show thus, the disease-resistant fast growing variety selection of lefteye flounder that we set up is highly effective, opened up new technological approaches for the cultivation of fish disease-resistant variety, suited in all cultured fishes, to apply, the disease-resistant speed of cultured fishes has been given birth to the significant and using value of prevalent variety cultivation.
Description of drawings:
Fig. 1: the mating pattern that the lefteye flounder family is set up;
Fig. 2: the comparison of different parent's mating assembled growth performances;
The mating combination is expressed as respectively: 1, and day in-group ♂ * day in-group ♀; 2, disease-resistance population ♂ * disease-resistance population ♀; 3, day in-group ♂ * disease-resistance population ♀; 4, disease-resistance population ♂ * day in-group ♀; 5, the ♂ of Huanghai Sea colony * disease-resistance population ♀; 6, the disease-resistance population ♂ * ♀ of Huanghai Sea colony
Fig. 3: three basic population hybrid combination of lefteye flounder offspring premunition relatively.
JS: day in-group; RS: disease-resistance population; YS: Huanghai Sea wild population.
Embodiment:
Set up with lefteye flounder disease-resistance population and family below and disease-resistant high yield fine breeding is an example, in conjunction with the accompanying drawings, technology contents of the present invention be described in detail:
Its technology contents of the present invention is as follows: comprise that the lefteye flounder family is set up and the technical method of disease-resistant high yield fine breeding;
One, lefteye flounder method for establishing stemma; Comprise: the preparation of (), disease-resistance population; (2), the foundation of family; (3), the labeling method of different familys.
(1), the preparation of lefteye flounder disease-resistance population (RS): adopt natural selection and artificial infection's approach to obtain disease-resistance population (RS).
Natural selection: in the busy season of 2003-2004 lefteye flounder in summer morbidity, arrive the lefteye flounder plant on ground such as Laizhou, sunshine, Jimo, collect the fry and the fingerling individuality of vibriosis morbidity back survival, cultivate, lefteye flounder fingerling 250 tails (specification is a 20-200 gram/tail) of collecting the survival of morbidity back altogether are as disease-resistance population (RS) material.
Artificial infection's approach: infect the common fry of lefteye flounder with the pathogen Vibrio anguillarum.Fry is the common fry of lefteye flounder Huanghai Sea colony that Huanghai Sea aquatic products Co., Ltd produces, and body weight is a 15-20 gram/tail.The Vibrio anguillarum bacterial strain that Vibrio anguillarum preserves for this laboratory, the medium of Vibrio anguillarum is 2216E, Vibrio anguillarum is seeded in 2216E medium (peptone 5g, yeast extract 1g, the FePO of sterilization
44H
2O0.1g, Chen Haishui 1000mL) in, placing velocity of vibration is on the shaking table of 200r/m, under 28 ℃, cultivate, cultivate and measure the OD600 value after 20 hours, centrifugal collection thalline, original bacteria liquid is mixed with the bacterium liquid that concentration is 10OD with 0.9% the NaCl physiological saline dilution bacterium liquid of sterilization. infect experiment in advance with above-mentioned bacterium liquid: with physiological saline the bacterium liquid of 10OD concentration is diluted to different concentration respectively earlier and (dilutes 50,100,120,150,240 and 300 times) carry out preliminary experiment, each concentration is injected the lefteye flounder of 10 tail 15-20 grammes per square metres, and injection volume is 0.3-0.5mL.The concentration that will cause the fry death of 50-60% in the fry of injection is defined as half lethal concentration.The half lethal concentration of the fry of above-mentioned specification is the bacterial solution of 240 times of the dilutions of 0.3-0.5mL.
Infection experiment: formal infection experiment is with lefteye flounder tail more than totally 800, the about 15-20g of body weight, and lefteye flounder individuality lumbar injection Vibrio anguillarum, dosage is controlled at half lethal concentration, and the injection volume of Vibrio anguillarum solution is 0.02ml bacterium liquid/gram fish, and water temperature is controlled at 21-23 ℃ during infection; The survival of 200 tail fries is arranged after the infection, according to conventional method these fries are cultivated into parent population, as disease-resistance population (RS) material.
Disease-resistance population (RS) is the lefteye flounder individuality that long-term health survives behind natural selection or the artificial infection Vibrio anguillarum.
(2), the foundation of lefteye flounder family;
1, the cultivation of lefteye flounder day in-group (JS) and Huanghai Sea colony (YS):
In October, 2003, introduce lefteye flounder day in-group fry 50 tails from Japan, body length is about 5 centimetres during introduction.Adopt conventional method, to lefteye flounder day the in-group fry cultivate, through the cultivation of 3-4, these fries had the small part fish to reach sexual maturity in 2006, wherein the minority fish has carried out breeding and has laid eggs.2007, most of fishes reached sexual maturity, and were used to set up the lefteye flounder family.Adopt the lefteye flounder microsatellite marker of having delivered that this batch lefteye flounder fry of introducing from Japan is analyzed according to conventional method, the analysis showed that this batch fishing gear has higher genetic diversity, be a plurality of parents' offspring (Shao Changwei etc., 2008).The parent population that Huanghai Sea wild population parent population forms for the wild individual cultivation of fishing for from the Huanghai Sea, quantity is about 60 tails.
Employing is carried out individual mark from the electronic marker rifle and the notice plate of U.S. West-North Marine Ltd. import to lefteye flounder disease-resistance population, day in-group (JS) and Huanghai Sea wild population 3 colony parent populations such as (YS), the mark bullet is beaten in muscle of back, every tail parent population has a mark mark, during detection, can the mark-sense number with the portable type scanner scans muscle of back place that the said firm produces.The parent population of separate sources can be raised in same pond like this, so that genital regulating and the operation of adopting ovum.
2, the collection and the artificial insemination of essence, ovum:
Utilize manual compression belly method to gather mature egg respectively in the measuring cup of drying, extruding is also drawn mature sperm in the vial of drying with suction pipe.The fertilization of employing dry method, the seminal fluid that is about to extrude is added in the container of containing ovum, shake mixing, add then 15-16 ℃ sea-water activated, finish the insemination process, add 10 times of seawater again to the ovum amount, left standstill 5-10 minute, above good ovum swam in, unfertilized dead ovum then was sunken to the bottom, the ovum that will float moves in the hatching cylinder hatches, and the management of hatching is undertaken by plant's conventional method.Embryonic development to primitive gut is added up fertilization rate in early days, grows to membrane and adds up incubation rate in earlier stage.
3, the foundation of lefteye flounder family:
The foundation of lefteye flounder family is mainly carried out in Shandong Province Huanghai Aquatic Products Co.,Ltd., Haiyang City.In lefteye flounder mating season, adopt disease-resistance population (RS), day in-group (JS) and 3 reproductive populations of Huanghai Sea wild population (YS) to match breeding as the parent, adopt the Japanese lefteye flounder ♀ of the disease-resistant lefteye flounder ♀ of disease-resistant lefteye flounder ♂ x, disease-resistant lefteye flounder ♂ x; The disease-resistant lefteye flounder ♀ of Japan lefteye flounder ♂ x, Japanese lefteye flounder ♂ x Japan lefteye flounder ♀; The disease-resistant lefteye flounder ♀ of the wild lefteye flounder ♂ of Huanghai Sea x, the wild lefteye flounder ♂ of Huanghai Sea x Japan lefteye flounder ♀, adopt 1 tail milter that the mating pattern of 1 tail raun is made up family (Fig. 1), sometimes be 1 tail milter respectively to 2-4 tail raun, or 1 tail raun makes up family half sibs to the mating pattern of 2-5 tail milter respectively.Essence, ovum collection and hatching are carried out as stated above.The fertilized egg that will produce from a tail milter and a tail raun mating, i.e. family is placed in the tank of 3 cubic metres of volumes and hatches, and breeding density is 20-30 milliliter fertilized egg, and tank is carried out 24 hours miniflow water, inflation.The work of setting up of family should be finished in 10-20 days, the family growth rate was calculated the influence that causes to reduce the difference of family on Time Created as far as possible.
The current male disease-resistant lefteye flounder that participates in family foundation is that 13 tails, male Japanese lefteye flounder are that 4 tails, the wild male Japanese flounders of the Huanghai Sea are 4 tails; Female disease-resistant lefteye flounder is 21 tails, and female Japanese lefteye flounder is 7 tails, and the wild lefteye flounder of the female Huanghai Sea is 2 tails.The present invention has set up half sibs in 15-20 days and family full-sibs amounts to 63.
(3), the labeling method of different familys:
Employing is injected the different parts of fry from the fluorescent dye of the different colours that U.S. fluorescence labeling company buys, and different familys are carried out mark, and the preparation of fluorescent pigment and injection are carried out according to the operating instruction of manufacturer.The preparation of fluorescent pigment and injection are with reference to " Manual Elastomer InjectionSystems Instructions for Use ".It is subcutaneous that the fluorescent pigment for preparing is injected the fish body surface, and every needle injection amount is 3-5 μ l.Choose yellow, red, 3 kinds of fluorescent pigments of orange, before on fish body belly, having chosen the back of the body, behind, 3 positions behind the abdomen, utilize the permutation and combination method to design marker combination, by the a kind of color in 1 position, 3 kinds of colors of the 2 kinds of colors in 1 position and 1 position are carried out permutation and combination, 3375 permutation and combination are arranged altogether, and computing formula is
[C(3，0)+P(3，1)+P(3，2)+P(3，3)]
3＝3375
C (3,0): mark not;
P (3,1): the a kind of color in 1 position;
P (3,2): the 2 kinds of colors in 1 position;
P (3,3): the 3 kinds of colors in 1 position;
3 powers: all permutation and combination at 3 positions of expression can have 3375 permutation and combination altogether.
Promptly utilize 3 kinds of colors to inject at 3 different parts, can 3375 familys of mark.Only used wherein 63 combinations that 63 familys have been carried out mark among the present invention.
Two, lefteye flounder disease-resistant high yield fine breeding method; Comprise: grow the fast screening of family of (), lefteye flounder; (2), the screening of the disease-resistant family of lefteye flounder.
(1), the lefteye flounder screening of family of growing fast:
The work of setting up of lefteye flounder family should be finished in 10-20 days, to reduce the influence that different family causes growth rate Time Created as far as possible.Set up the back to fluorescence labeling in family, different familys are raised in different breed tanks, and it is identical that the stocking density of different familys should keep, and culture 5000-10000 tail newly hatched larvae in 3 cubic metres the tank.After fry grew 20 days, the density of fry was adjusted into the 3000-5000 tail in the tank; After fry grew to 45 days, its density was adjusted into the 1000-1500 tail; When fry grows to 8-10 centimetre, from each family, get 200 fishes at random, carry out fluorescence labeling according to the method described above, the fish of the different familys behind the mark can be placed in the same cement pit and raise, and the cement pit of 40 square meters can be cultured 4000-5000 bar fry.The aquaculture management of fry carries out according to a conventional method.Carry out one-shot measurement every half a year behind the mixed breed, utilize the data of measuring 3 times to carry out the evaluation of growth performance.Measurement index is mainly body long (holding tail fin end from kiss), body wide (health the widest part does not contain the back of the body, abdomeinal fin length), 3 indexs of body weight, and each family is measured 40-60 tail fish.Calculate that each family body is long, body is wide, the mean of body weight.Employing is set up mid-term in family and is organized in contrast by the family that the wild lefteye flounder of the Huanghai Sea (YS) mating produces.Be not quite similar the Time Created of considering family, still get with control group and carry out the growth performance analysis simultaneously or in its family of setting up later.The difference of more different familys and contrast family fry body weight extremely obviously greater than the family of the contrast family family (p＜0.01) that is defined as growing fast, obviously greater than the family of contrast family is defined as faster growing family (p＜0.05) with body weight with body weight; The family that the body weight utmost point is significantly less than the contrast family is defined as slow growth family (p＜0.01); Body weight is significantly less than the family family (P＜0.05) (table 1) that is defined as growing more slowly of contrast family.
According to above-mentioned standard, we have carried out the growth performance assessment to 63 familys.Wherein, the body weight that 8 family fries (being respectively family 2,4,5,19,36,42,43 and No. 57) are arranged is obviously greater than the control group body weight, its differences is extremely remarkable, because be not quite similar the Time Created of family, still will be defined as growing fast family simultaneously or in the family that it is set up later with control group, 4 familys (i.e. 36,42,43 and No. 57 familys) are arranged altogether; The body weight that 12 family fries (being respectively family 3,12,21,27,28,35,39,41,51,60,65 and No. 76) are arranged is also obviously greater than control group fry body weight, significant difference therebetween, equally, consider the time factor of building family, we are called the very fast family of growth with familys such as No. 27,28,35,39,41,51,60,65 and 76, familys; Family is obviously grown slowly than control group for No. 75 in addition, is referred to as the slow growth family; The growth and the control group difference of remaining family are remarkable inadequately, are referred to as the general family of growth rate, amount to 49 familys.The body length of 4 familys of growing fast that we filter out is than the high 22-28% of control group, and the weight ratio control group is high more than 99%.
Table 1: the different family fry of lefteye flounder growth performance compares:
Annotate: * represents that body weight compares significant difference (P＜0.05) with control group; * represents that body weight compares difference extremely significantly (P＜0.01) with control group
Utilize the measurement data that obtains that Different Cross Combinations offspring's growth indexes has been carried out preliminary comparison, as can be seen from Figure 2 as be distributed in two different geographic regions day in-group and disease-resistance population the growth performance of filial generation embodied tangible heterosis, hybrid vigor, the offspring of the selfing of same geographical population inside (Huanghai Sea colony also belongs to same geographic area with disease-resistance population) generation compares with filial generation and does not then embody growth vigor.
(2), the screening of the disease-resistant family of lefteye flounder:
Carry out infection experiment when fry grows to when body is long to be the 9-11cm left and right sides, estimate the disease-resistant performance of each family.About 100 tails of each family random sampling infect about 50 tails at every turn, repeated experiments 1 time.Each family infects independently cultures at 0.5m with fish
3The fiberglass tank in.Infect with bacterial strain and be this laboratory preservation and Vibrio anguillarum bacterial strain through identifying.With 2216E medium (peptone 5g, yeast extract 1g, the FePO of Vibrio anguillarum in sterilization
44H
2O0.1g, Chen Haishui 1000mL) in 28 ℃, 200r/m cultivates about 18h.Measure OD600 value, bacterium liquid at first is diluted to the concentration of 10OD with 0.9% NaCl physiological saline of sterilization, the Vibrio anguillarum that the employing lumbar injection dilutes infects experiment in advance.During pre-the infection, again the bacterium liquid of 10OD concentration is diluted to variable concentrations (concentration range is waited to replenish) with physiological saline, every group of pre-infection of carrying out different bacterial concentrations with 10-15 tail fish tested, and gropes the half lethal concentration of lefteye flounder fry.During formal the infection, inject according to half lethal concentration.The lefteye flounder half lethal concentration that the present invention determines is 4.9 * 10
7The bacterial solution of the above-mentioned concentration of CFU/ml.Injected dose is 0.02ml bacterium liquid/gram fish; Water temperature is controlled at 21-23 ℃ during infection, keeps normally feeding between cycle water, infection period and inflating.
Should regularly observe the survival condition of fry after the injection, general about 12h behind the injection Vibrio anguillarum, symptoms such as that fry begins to occur moving about is slow, energy difference; 20h begins to occur a small amount of death, and the fish of being at death's door presents the symptom of body colour blackout, the belly enlargement, and epidermal mucus increases, and internal organ expose, and phenomenon appears just moping in vain in the part individuality.The fish body is stiff in the dead back short time.30h begins to occur dead peak, and death toll accounts for the overwhelming majority of dead sum in the whole infection in the 48h, and is basicly stable behind the 96h.Infection experiment is set up control group.Control group is for to set up the lefteye flounder fry of adopting disease-resistance population and the Huanghai Sea mass mating mid-term in family.According to metainfective fingerling survival rate, in conjunction with the significance of control group survival rate test of difference, we are defined as the strong family of premunition with survival rate in the family more than 60%, survival rate is defined as the more intense family of premunition in the family of 39-60%, survival rate is defined as the general family of premunition in the family of 17-39%, survival rate is defined as the family of premunition difference in the family below 17%.By this standard, we identify 3 of the strong familys of premunition (family 50,61 and No. 68), 17 (familys 21 of family that premunition is more intense from 59 lefteye flounder familys, 22,28,45,49,51,52,53,57,58,59,60,63,64,66,70 and 79), 33 of the general familys of premunition, 6 of the familys (table 2) of premunition difference.The survival rate comparison of the family that premunition is strong is according to the high 36.9-40.5% of group difference.
Its average survival rate of family that 3 lefteye flounder basic population hybrid combination is set up relatively see Fig. 3, its average survival rate of family that two kinds of hybrid combinations of disease-resistance population ♀ * day in-group ♂ (SR=0.4144 ± 0.1217) and the ♀ of Huanghai Sea colony * disease-resistance population ♂ (SR=0.4601 ± 0.0173) are set up is significantly higher than the average survival rate of other hybrid combinations, is the lefteye flounder family of premunition raising.
The sick ability of the different family fry of table 2. lefteye flounder anti-vibrio anguillarum compares:
Annotate: * is expressed as motility rate and compares significant difference (P＜0.05) with control group.
In August, 2007, we were generalized to big fish island, Rongcheng City, Shandong plant with long disease-resistant lefteye flounder fry 40000 tails for 8-10 centimetre of the body of cultivating, show through nearly 1 year breed, the survival rate of the disease-resistant lefteye flounder fry that we develop is more than 80%, has improved more than 30% than the survival rate (50%) of common lefteye flounder fry; The growth rate of disease-resistant lefteye flounder is also fast more than 10% than common lefteye flounder fry simultaneously.Show fully that thus it is highly effective that the disease-resistant speed of the lefteye flounder that we set up is given birth to fine-variety breeding method.
Claims (2)
1. a fish family method for building up is characterized in that its technology contents comprises: the preparation of (1), disease-resistance population; (2), the foundation of fish family;
(1), the preparation of disease-resistance population: adopt the natural selection approach to obtain disease-resistance population (RS);
Natural selection: in the morbidity busy season, collect the fry and the fingerling individuality of vibriosis morbidity back survival, cultivate;
Disease-resistance population is the lefteye flounder individuality that long-term health survives behind the natural selection infection Vibrio anguillarum;
(2), the foundation of fish family; Fish are lefteye flounder day in-group and lefteye flounder Huanghai Sea colony:
A, lefteye flounder day in-group and the cultivation of Huanghai Sea colony parent population:
Introduce the lefteye flounder fry from Japan, long about 5 centimetres of body, through the cultivation of 3-4, fry reaches sexual maturity, is used to set up the lefteye flounder family; Huanghai Sea wild population parent population is the wild individuality of fishing for, and adopts conventional method to raise and train and breed;
Employing electronic marker method enantiopathy colony, day in-group and 3 colony parent populations of Huanghai Sea wild population carry out individual mark, and every tail parent population is given a mark mark, so that match identification parent population source when breeding;
The collection of b, essence, ovum and artificial insemination:
Utilize manual compression belly method to gather mature egg respectively, extruding is also drawn mature sperm in the vial of drying with suction pipe; The fertilization of employing dry method, the seminal fluid that is about to extrude is added in the container of containing ovum, shakes mixing, adds sea-water activated then, finish the insemination process, add 10 times of seawater again, left standstill 5-10 minute, above good ovum swims in to the ovum amount, unfertilized dead ovum then is sunken to the bottom, the ovum that will float moves in the hatching cylinder hatches, and embryonic development to primitive gut is added up fertilization rate in early days, grows to membrane and adds up incubation rate in earlier stage;
The foundation of c, lefteye flounder family:
In lefteye flounder mating season, adopt disease-resistance population, day in-group and 3 reproductive populations of Huanghai Sea wild population to match breeding as the parent; Adopt the disease-resistant lefteye flounder ♀ of disease-resistant lefteye flounder ♂ x, disease-resistant lefteye flounder ♂ x Japan lefteye flounder ♀; The disease-resistant lefteye flounder ♀ of Japan lefteye flounder ♂ x, Japanese lefteye flounder ♂ x Japan lefteye flounder ♀; The disease-resistant lefteye flounder ♀ of the wild lefteye flounder ♂ of Huanghai Sea x, the wild lefteye flounder ♂ of Huanghai Sea x Japan lefteye flounder ♀, respectively with the mating pattern structure family of 1 tail milter to 1 tail raun, or 1 the tail milter respectively to 2-4 tail raun, or 1 tail raun makes up family half sibs to the mating pattern of 2-5 tail milter respectively; Essence, ovum collection and hatching are carried out as stated above; The fertilized egg that will produce from a tail milter and a tail raun mating, i.e. family is placed in the tank of 3 cubic metres of volumes and hatches, and breeding density is 20-30 milliliter fertilized egg, and tank is carried out 24 hours miniflow water, inflation; The work of setting up of family should be finished in 10-20 days.
2. fish disease-resistant high yield fine breeding method is characterized in that its technology contents comprises:
The screening of growth family fast:
After family was set up, different familys were raised in different breed tanks, and it is identical that the stocking density of different familys should keep, and cultured 5000-10000 tail newly hatched larvae in 3 cubic metres the tank; After fry grew 20 days, the density of fry was adjusted into the 3000-5000 tail in the tank; After fry grew to 45 days, its density was adjusted into the 1000-1500 tail; When fry grows to 8-10 centimetre, from each family, get 200 fishes at random, be placed in the cement pit and raise, culture the back and carry out one-shot measurement every half a year, utilize the data of measuring 3 times to carry out the evaluation of growth performance; Measurement index is mainly total length, wide, 3 indexs of body weight of body, and each family is measured 40-60 tail fish; Calculate that each family body is long, body is wide, the mean of body weight; Employing is set up mid-term in family and is organized in contrast by the family that the wild lefteye flounder mating of the Huanghai Sea produces; More different familys are long with contrast family body, body is wide, the difference of body weight, body weight extremely obviously is defined as growing fast family greater than the family of contrast family, be p＜0.01, body weight obviously be defined as the faster growing family greater than the family of contrast family, i.e. p＜0.05; The family that the body weight utmost point is significantly less than the contrast family is defined as the slow growth family, i.e. p＜0.01; The family that body weight is significantly less than the contrast family is defined as growing more slowly family, i.e. P＜0.05;
The family of will growing the fast fry of cultivating according to a conventional method, raise up seed is the high yield fine that can culture and promote.
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| CN102047851B (en) * | 2009-10-29 | 2012-11-21 | 上海海洋大学 | Construction and stock breeding method for cultured grass carp families |
| CN101699998B (en) * | 2009-11-03 | 2011-10-12 | 中国水产科学研究院黄海水产研究所 | Selection and culture method of excellent breed large-tooth flounder featuring fast growth and high survival rate |
| CN101911918B (en) * | 2010-03-18 | 2011-06-15 | 中国水产科学研究院黄海水产研究所 | Selective breeding method for disease-resistant superior strains of paralichthys olivaceus |
| CN101810148B (en) * | 2010-04-28 | 2011-06-15 | 中国水产科学研究院黄海水产研究所 | Cross breeding method of fine variety of paralichthysolivaceus |
| CN102017918B (en) * | 2010-09-27 | 2012-07-04 | 大连海洋大学 | Method for feeding broad-spectrum disease-resistant sea farming through immunopolysaccharide means |
| CN102715115A (en) * | 2012-07-09 | 2012-10-10 | 中国水产科学研究院黑龙江水产研究所 | Phoxinus lagowskii fry breeding method |
| CN103947607B (en) * | 2014-05-08 | 2015-09-23 | 上海海洋大学 | A kind of construction method of grass carp bacteria resistance septicemia strain |
| CN104304096B (en) * | 2014-08-26 | 2016-01-27 | 中国水产科学研究院黄海水产研究所 | The selection of the sick excellent strain of the anti-Edwardsiella tarda of a kind of lefteye flounder |
| CN104651472A (en) * | 2015-03-18 | 2015-05-27 | 广西壮族自治区水产科学研究院 | Evaluation method based on tilapia breeding disease resistance |
| CN106912408B (en) * | 2017-03-03 | 2020-04-21 | 中国水产科学研究院黑龙江水产研究所 | Construction method of novel salmon and trout anti-saprolegniasis strain |
| CN112042573A (en) * | 2020-09-21 | 2020-12-08 | 四川润兆渔业有限公司 | Sturgeon breeding method |
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