CN103875569A

CN103875569A - Method for obtaining high-proportion male loaches through interspecies cross

Info

Publication number: CN103875569A
Application number: CN201410111329.5A
Authority: CN
Inventors: 曹小娟; 王卫民; 黄松钱; 高坚; 田先畅
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2014-03-24
Filing date: 2014-03-24
Publication date: 2014-06-25
Anticipated expiration: 2034-03-24
Also published as: CN103875569B

Abstract

The invention discloses a method for obtaining high-proportion male loaches through interspecies cross. The method includes the steps that female paramisgurnus dabryanus and male tetraploid loaches serve as parents for cross; oxytocic medicine is injected into abdominal cavities of the female paramisgurnus dabryanus and abdominal cavities of the male tetraploid loaches, after hasten parturition is carried out for 12-16 hours, testes of the male tetraploid loaches and egg grains of the female paramisgurnus dabryanus 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 male loaches are obtained from the hybrid loach fries obtained through breeding and incubation. In triploid hybrid loach groups obtained through the method, the proportion of the male hybrid loaches reaches up to 87.64% to 98.53%, and the ovaries of the female loaches are in a hypoplasia state.

Description

A kind ofly obtain the method for male loach at high proportion by interspecific cross

Technical field

The present invention relates to fish unisexuality mating system, more particularly, it relates to and a kind ofly obtains the method for male 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 male loach at high proportion by interspecific cross, can obtain a high proportion of male hybridization loach by the method.

Above-mentioned purpose realizes by the following technical solutions:

Obtain a method for male loach at high proportion by interspecific cross, the method is: hybridize take the secondary loach of female large squama and male Tetraploid Loach, Misgurnus as parent.

Preferably, parent's ratio of the secondary loach of described female large squama and male Tetraploid Loach, Misgurnus is about 4-8:3.

Further preferably; the method of described hybridization is: to the secondary loach of female large squama, male Tetraploid Loach, Misgurnus lumbar injection oxytocic drug; hasten parturition after 12-16 hour; gather the ovum grain of spermary and the secondary loach of female large squama of male 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 male 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 the secondary loach injection ovulation induction element of female large squama, male Tetraploid Loach, Misgurnus 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 male hybridization loach is up to 87.64%-98.53%, raun ovarian dysgenesis.

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 male 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 the secondary loach of large squama, Tetraploid Loach, Misgurnus and hybridization loach, and reference is dliploid cock blood;

Fig. 2 is hybridization loach male and female adult sexual gland outward appearance and sexual gland slice map, in figure: A: male hybridization loach adult fish, black arrow is pointed to spermary, and white arrow is pointed to pectoral fin; B: the spermary of male hybridization loach adult fish; C: hybridization loach spermary slice map; D: the ovary of female hybridization loach adult fish.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in detail.

Embodiment 1

First secure good health the 8 good tail secondary loach of wild female large squama and the male Tetraploid Loach, Misgurnus of 6 tails from Ezhou, Hubei for parent (utilizing flow cytometer evaluation parent ploidy) (seeing accompanying drawing 1), male and female loach parent ratio is 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 178 tail hybridization loach (total length is 13-18cm), 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 178 tails hybridization loach, having 156 tails is male (male ratio is 87.64%), and the pectoral fin fin ray of male hybridization loach extends and is sharp knife shape, and the second fin ray base portion exists projection hone lamella, testis development well in a tubular form, the visible spermatogonium of spermary sections observation; 22 tails are female, and the pectoral fin of female hybridization loach is round and smooth is fan-shaped, and the second fin ray base portion is without hone lamella, ovarian dysgenesis, and quality is transparent, tiny, the ovule grain of the developmental immaturity of accidental disperse.

The sex ratio of the secondary loach ♀ × Tetraploid Loach, Misgurnus of the large squama of table 1 ♂ hybrid generation

Embodiment 2

Select the secondary loach of the female large squama of well-developed 8 tail and the male Tetraploid Loach, Misgurnus of 3 tails to hybridize, random acquisition 136 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 2.

The sex ratio of the secondary loach ♀ × Tetraploid Loach, Misgurnus of the large squama of table 2 ♂ hybrid generation

Can prove from embodiment 1,2, the present invention is hybridized take the secondary loach of female large squama and male Tetraploid Loach, Misgurnus as parent, obtain male at high proportion triploid hydrid loach colony, 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

1. obtain a method for male loach at high proportion by interspecific cross, it is characterized in that: hybridize take the secondary loach of female large squama and male Tetraploid Loach, Misgurnus as parent.

2. according to claim 1ly obtain the method for male loach at high proportion by interspecific cross, it is characterized in that: parent's ratio of the secondary loach of described female large squama and male Tetraploid Loach, Misgurnus is 4-8:3.

3. according to claim 1 and 2ly obtain the method for male loach at high proportion by interspecific cross; it is characterized in that: the method for described hybridization is: to the secondary loach of female large squama, male Tetraploid Loach, Misgurnus lumbar injection oxytocic drug; hasten parturition after 12-16 hour; gather the ovum grain of spermary and the secondary loach of female large squama of male 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 male loach.

4. according to claim 3ly obtain the method for male 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 male 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 the secondary loach injection ovulation induction element of female large squama, male Tetraploid Loach, Misgurnus reduces by half.

6. according to claim 3ly obtain the method for male 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.