CN103875568A - Method for obtaining high-proportion female loaches through interspecies cross - Google Patents
Method for obtaining high-proportion female loaches through interspecies cross Download PDFInfo
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- CN103875568A CN103875568A CN201410110587.1A CN201410110587A CN103875568A CN 103875568 A CN103875568 A CN 103875568A CN 201410110587 A CN201410110587 A CN 201410110587A CN 103875568 A CN103875568 A CN 103875568A
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- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
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Abstract
The invention discloses a method for obtaining high-proportion female loaches through interspecies cross. The method includes the steps that female tetraploid loaches and male paramisgurnus dabryanus serve as parents for cross; oxytocic medicine is injected into abdominal cavities of the female tetraploid loaches and abdominal cavities of the male paramisgurnus dabryanus, after hasten parturition is carried out for 12-16 hours, testes of the male paramisgurnus dabryanus and egg grains of the female tetraploid loaches are collected, the testes and preserving liquid are grinded and mixed evenly and then mixed with the egg grains evenly, fertilized eggs are obtained and then incubated, and the high-proportion female loaches are obtained from the hybrid loach fries obtained through breeding and incubation. In triploid hybrid loach groups obtained according to the method, the proportion of the female hybrid loaches reaches up to 78.76% to 90.30%, the female loaches are fertile and can produce two kinds of eggs of different ploidies, and male loaches are infertile.
Description
Technical field
The present invention relates to fish unisexuality mating system, more particularly, it relates to and a kind ofly obtains the method for female loach at high proportion by interspecific cross.
Background technology
Many research shows that there is being obvious advantage in fish unisexuality colony (Ci Huoquanxiong colony entirely) aspect the ecotope of growth rate or maintaining healthy.The method of producing at present both at home and abroad fish unisexuality colony mainly contains following several: sex hormone is is directly thrown something and fed, three series mating method, gynogenesis and interspecific cross.Sex hormone is is directly thrown something and fed and is realized the former use of reverse many of fish sex, often can more successfully obtain full Ci Huoquanxiong colony by the female or androgenic hormone of the high concentration of throwing something and feeding for a long time.But the fish unisexuality colony obtaining by the method exists very large food-safety problem, a large amount of sex hormone can flow in human body by the sex reversal fish that ingest, and the mankind are caused to huge harm.Given this, a kind of new method, i.e. three series mating method, is invented and is obtained and approve widely and promote.Fish take Sex Determination Mechanism as XX-XY type are as example, if need to obtain Quan Xiong colony, first can normal XY piscinity be reversed as physiological raun by the female sex hormone of throwing something and feeding, and then carry out mating with normal XY milter, obtain YY supermale fish, this supermale fish need to or utilize Sex-linked marker to screen and determine by the method for offspring's test cross.There are some researches show, utilize three series mating method can successfully obtain supermale yellow cartfish.In addition, there is scholar also the method to be improved, by adopting hormonal to reverse in conjunction with gynogenesis, can greatly shorten the time that obtains supermale yellow cartfish.Gynogenesis is also often applied in the production of fish unisexuality colony now, and for example method in conjunction with chromosome doubling by gynogenesis just can low weightly be changed places and be obtained the Quan Ci colony of XX-XY type fish.Than sex hormone directly throw something and feed, the method such as three series mating method and gynogenesis, neither there is food-safety problem in the method that obtains fish unisexuality colony by interspecific cross, simultaneously also easy and simple to handle, efficiency is high.But utilize the example of interspecific cross acquisition fish unisexuality colony more rare, the most successful case is to utilize female bolti (XX) and male Ao Liya Tilapia mossambica (ZZ) to hybridize to obtain the Tilapia mossambica colony (Sarotherodon sp) (male than 83%-100%) that is close to full hero.
Loach and the secondary loach of large squama have higher edible and medical value, are the most important two kinds of economic bullheads of China.In recent years because the market price of these two kinds of bullheads climbs up and up, domesticly start a huge loach breeding upsurge, market is huge to good loach germ plasm resource demand.The chromosome of occurring in nature loach exists Karyotype polymorphism widely, and the loach of China is take Diploid and Tetraploid as main.Infer that according to gynogenesis result and cytogenetics evidence the Sex Determination of dliploid loach, Tetraploid Loach, Misgurnus and the large secondary loach of squama (dliploid) is respectively XX-XY type, XXXX-XXXY type and ZW-ZZ type.
Summary of the invention
The object of this invention is to provide and a kind ofly obtain the method for female loach at high proportion by interspecific cross, can obtain a high proportion of female hybridization loach by the method.
Above-mentioned purpose realizes by the following technical solutions:
Obtain a method for female loach at high proportion by interspecific cross, the method is: hybridize take female Tetraploid Loach, Misgurnus and the secondary loach of male large squama as parent.
Preferably, parent's ratio of described female Tetraploid Loach, Misgurnus and the secondary loach of male large squama is 4-8:3.
Further preferably; the method of described hybridization is: to female Tetraploid Loach, Misgurnus, the secondary loach lumbar injection of male large squama oxytocic drug; hasten parturition after 12-16 days; gather the secondary spermary of loach of male large squama and the ovum grain of female Tetraploid Loach, Misgurnus; after spermary and preservation liquid are ground, mix with ovum grain again, obtain fertilized egg, fertilized egg is hatched; raise the hybridization loach seedling obtaining after hatching, obtain a high proportion of female loach.
Further preferably, described oxytocic drug is ovulation induction element No. 2 and DOM.
Further preferably, the injected dose of described oxytocic drug is: No. 2 35 μ g/kg+ DOM 4mg/kg of female Tetraploid Loach, Misgurnus injection ovulation induction element, the secondary loach of male large squama reduces by half.
Further preferably, the formula of described preservation liquid is: sodium chloride 7.5g, and potassium chloride 0.3g, calcium chloride 0.3g, glucose 3.0g, distilled water constant volume is to 1000ml.
The invention has the beneficial effects as follows:
1. the triploid hydrid loach colony that adopts the present invention to obtain, wherein the ratio of female hybridization loach is up to 78.76%-90.30%, and raun can educate and can produce the ovum of two kinds of Different Ploidy (monoploid and triploid see accompanying drawing 3) simultaneously, and milter is sterile.
2. compared with existing method (as three series mating method), the breeding generation number of wanting required for the present invention is few, can obtain a high proportion of female loach by first cross, and while method is simple, efficient, repeatable strong; The present invention's technology such as sex reversal technology, gynogenesis and chromosome doubling of need to not throwing something and feeding by hormone, also needn't be by test cross, backcross and the means such as selfing, does not have food-safety problem.
Accompanying drawing explanation
Fig. 1 utilizes the Ploidy Identification collection of illustrative plates of flow cytometer to Tetraploid Loach, Misgurnus, the secondary loach of large squama and hybridization loach, and reference is dliploid cock blood;
Fig. 2 is hybridization loach male and female adult mode of appearance, sexual gland outward appearance and sexual gland slice map, in figure: A: hybridization loach adult, black arrow is pointed to female hybridization loach, and white arrow is pointed to male hybridization loach; B: hybridization loach adult fish sexual gland, the left side is ovary, the right is spermary; C: hybridization loach ovary internal anatomy; D: hybridization loach ovarian sections figure.
Filial generation (B) and filial generation Ploidy Identification collection of illustrative plates (C, D) that Fig. 3 is hybridized the large ovule (A) of loach raun generation and produced with male dliploid loach hybridization.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
First secure good health the secondary loach of the male large squama of the good wild female Tetraploid Loach, Misgurnus of 8 tail and 6 tails from Ezhou, Hubei for parent (utilizing flow cytometer to identify parent's ploidy) (seeing accompanying drawing 1), male and female loach parent ratio is about 4:3.Parent is injected to No. 2 (LRH-A of oxytocic drug ovulation induction element
2) and DOM (DOM), the injected dose of raun is LRH-A
235 μ g/kg+DOM4mg/kg, milter dosage reduces by half, and injecting method is abdominal cavity shot method.After oxytocic drug injection, female milter is separately supported in different water tanks temporarily to water temperature 24-26 ℃.After injection oxytocic drug 12-16h, manually gather spermary, be placed in mortar, add a small amount of loach spermatozoa preservative fluid (preservation formula of liquid: sodium chloride 7.5g, potassium chloride 0.3g, calcium chloride 0.3g, glucose 3.0g, distilled water constant volume is to 1000ml) fully grind.When preparing seminal fluid, in the clean little basin of drying, clamp-on ovum grain, squeeze ovum while add seminal fluid, fully mix sperm and ovum grain, clear water activation fertilized egg is after 1-2 minute, wash fertilized egg 2 times with clear water again, then fertilized egg uniform spreading is spilt at the bottom of water tank case to aeration stone oxygenation, the depth of water is 15-20cm, hatch for water temperature 25-26 ℃, through about 36h, the complete membrane of hybridization loach seedling.About 48h(hybridization loach seedling opening after hybridization loach membrane), the halogen worm that starts to throw something and feed is raised, and halogen worm was raised after 4 days, bring into use feed to raise (6d), after hybridization loach seedling opening 10d, get at random 1000 tail hybridization loach seedlings and proceed to continuation feed raising in miniflow water, until loach grows up.
Random acquisition 113 tail hybridization loach (total length is 13.6-22.1cm), it is triploid (seeing accompanying drawing 1) that flow cytometer detects hybridization loach.The sex of hybridization loach utilizes the methods such as mode of appearance observation, internal anatomy and sexual gland sections observation comprehensively to identify (in table 1 and accompanying drawing 2).In 113 tail hybridization loach, having 89 tails is female (female ratio is 78.76%), the pectoral fin of female hybridization loach is round and smooth is fan-shaped, the second fin ray base portion is without hone lamella, and high-visible big or small two kinds of ovum grains in ovary, carry out paraffin section to ovary and observe visible well-developed egg mother cell; 24 tails are male, and the pectoral fin fin ray prolongation of male hybridization loach is sharp knife shape, and the second fin ray base portion exists projection hone lamella, and testis development is bad, tiny, quality is transparent.
The sex ratio of the secondary loach ♂ of table 1 Tetraploid Loach, Misgurnus ♀ × large squama hybrid generation
The wild diploid loach of Wuhan City, Hubei Province, wild Tetraploid Loach, Misgurnus and the secondary loach of wild large squama are picked up from utilization and hybridization loach (6 tail rauns and 5 tail milters) hybridizes, hybridize loach selfing (2 tail rauns and 2 tail milters) simultaneously, and with the selfing of wild diploid loach, wild Tetraploid Loach, Misgurnus selfing and the secondary loach selfing of wild large squama as a control group.The artificial propagation test that hybridization loach milter participates in does not obtain the fertilized egg of survival, thereby can infer that male hybridization loach is sterile.Hybridization loach raun and dliploid loach milter, Tetraploid Loach, Misgurnus milter and the secondary loach milter of large squama hybridize and have all produced filial generation, and its average fertilization rate is respectively 18.2%, 15.3% and 22.2%, and on average incubation rate is respectively 96.6%, 98.1% and 98.8%.The fertilization rate of control group is that 61.73%-85.94%, incubation rate are 95.1%-98.6%.Female hybridization loach produces the ovum (average ovum footpath is respectively 1.54mm and 1.04mm) of two kinds of specifications of size.The ploidy of hybridization loach first filial generation, after flow cytometer detects, infers that the ploidy of the large ovule of hybridization loach raun generation is respectively triploid and monoploid (in table 2 and accompanying drawing 3).
Table 2 is for hybridizing hybridization/selfing combination of loach fertility research
Note: Δ, contrast combination; √, has produced the filial generation of surviving; Χ, without survival filial generation;-, there is no this combination.
Embodiment 2
Select the secondary loach of the male large squama of the female Tetraploid Loach, Misgurnus of well-developed 8 tail and 3 tails to hybridize, random acquisition 134 tail hybridization loach, flow cytometer identifies that its ploidy is triploid.The sex of identifying hybridization loach by methods such as mode of appearance observation, internal anatomy and sexual gland sections observation, qualification result is in table 3.
The sex ratio of the secondary loach ♂ of table 3 Tetraploid Loach, Misgurnus ♀ × large squama hybrid generation
Can find out from embodiment 1,2, the present invention is hybridized take female Tetraploid Loach, Misgurnus and the secondary loach of male large squama as parent, obtain female at high proportion triploid hydrid loach colony, and raun can produce the ovum of two kinds of Different Ploidy (monoploid and triploid) simultaneously, milter is sterile, show larger growth vigor and very poor fertility, its popularization will promote the development of loach breeding industry to a great extent, creates good economic benefit.
Claims (6)
1. obtain a method for female loach at high proportion by interspecific cross, it is characterized in that: hybridize take female Tetraploid Loach, Misgurnus and the secondary loach of male large squama as parent.
2. according to claim 1ly obtain the method for female loach at high proportion by interspecific cross, it is characterized in that: parent's ratio of described female Tetraploid Loach, Misgurnus and the secondary loach of male large squama is 4-8:3.
3. according to claim 1 and 2ly obtain the method for female loach at high proportion by interspecific cross; it is characterized in that: the method for described hybridization is: to female Tetraploid Loach, Misgurnus, the secondary loach lumbar injection of male large squama oxytocic drug; hasten parturition after 12-16 hour; gather the secondary spermary of loach of male large squama and the ovum grain of female Tetraploid Loach, Misgurnus; after being ground, spermary and preservation liquid mixes with ovum grain again; obtain fertilized egg; fertilized egg is hatched; raise the hybridization loach seedling obtaining after hatching, obtain a high proportion of female loach.
4. according to claim 3ly obtain the method for female loach at high proportion by interspecific cross, it is characterized in that: described oxytocic drug is ovulation induction element No. 2 and DOM.
5. according to claim 4ly obtain the method for female loach at high proportion by interspecific cross, it is characterized in that: the injected dose of described oxytocic drug is: No. 2 35 μ g/kg+ DOM 4mg/kg of female Tetraploid Loach, Misgurnus injection ovulation induction element, the secondary loach of male large squama reduces by half.
6. according to claim 3ly obtain the method for female loach at high proportion by interspecific cross, it is characterized in that: the formula of described preservation liquid is: sodium chloride 7.5g, potassium chloride 0.3g, calcium chloride 0.3g, glucose 3.0g, distilled water constant volume is to 1000ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106957888A (en) * | 2017-04-11 | 2017-07-18 | 武汉百科金典生物科技有限公司 | A kind of method based on the ploidy analyser Rapid identification prelarva ploidy |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106957888A (en) * | 2017-04-11 | 2017-07-18 | 武汉百科金典生物科技有限公司 | A kind of method based on the ploidy analyser Rapid identification prelarva ploidy |
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