CN101595847B - Method for inducing Stichopus Japonicus Selenka to lay eggs in vivo - Google Patents

Abstract

The invention discloses a method for inducing Stichopus Japonicus Selenka to lay eggs in vivo, which is characterized by simple operation, short time, synchronous egg laying, large discharge quantity and good quality of eggs, and capability of greatly improving induction rate, fertility rate and hatching rate; the method is implemented as the following steps: dissolving NGLWY acid amides in ultrapure water to formulate 1 mumol/L mother liquor, then dissolving the mother liquor in 0.5mol/L KCl sea water solution to prepare 1nmol/L inducer; selecting gonad mature parent Stichopus Japonicus Selenka with the weight being at least 200g; injecting the inducer with the ratio of volume and the weight of parent Stichopus Japonicus Selenka being 0.1% ml/g but less than 0.3ml into coelom from the back of the Stichopus Japonicus Selenka to perform in vivo induction; and collecting the eggs.

Description

The method of inducing Stichopus Japonicus Selenka to lay eggs in vivo

Technical field:

The present invention relates to a kind of inducing Stichopus Japonicus Selenka to lay eggs in vivo method, especially a kind of simple to operate, weak point consuming time, synchronous spawning and the ovum discharge capacity is large, quality good can greatly improve the method for the inducing Stichopus Japonicus Selenka to lay eggs in vivo of the efficient of hastening parturition, fertilization rate and incubation rate.

Background technology:

Sea cucumber as the delicacies on the Pacific Ocean such as China and Southeast Asia and the western Indian Ocean coastwise contries people dining table beautiful hide well-known already, particularly world Chinese has the custom of eating sea cucumber " tonic " always, and nowadays sea cucumber more and more is subjected to people's favor because having abundant nutritive value.The increase of demand causes adopting catches intensity and increases, and causes stichopus japonicus stock number and the output of China's self-sow sharply to descend, and the apostichopus japonicus culture industry thereupon fast development get up.The development of Apostichopus japonicus Aquaculture causes the seed demand to increase, yet existing Stichopus Japonicus Selenka to lay eggs efficient is but contained seed output always, has directly affected the economic benefit of apostichopus japonicus culture.

At present, sea cucumber to lay eggs mainly be by natural spawning and two approach of artificial stimulation induced spawning.Natural spawning requires the parent participation gonad development fully ripe, its egg-laying time and egg laying amount can not manual controls, often because of at dusk or lay eggs night, ejaculation, egg laying amount are excessive, polyspermism etc. is former thereby cause water quality to ruin, and then cause a large amount of phenomenons of disintegrating of fertilized egg.The advantage of artificial stimulation induced spawning maximum be can the manual control stichopus japonicus egg-laying time and egg laying amount, avoid the existing water quality of natural spawning the problem such as to ruin, but existing artificial stimulation induced spawning method or the problem of various degrees still can not at utmost satisfy the needs that juvenile stichopus is produced.

Artificial stimulation induced spawning commonly used be divided into external and body in induce two kinds, external evoked is the method that stimulates by temperature differential method and the flowing water that dries in the shade etc., but induce effect unstable, sea cucumber egg laying amount few (every female ginseng egg laying amount is generally below 1,000,000), account for about 20% of total fish brood amount, often needing repeatedly hastens parturition every other day just can meet the needs of production, and labour intensity is high, seedling raise period is long, otherwise can only increase seed ginseng quantity, strengthen seedling cost.The method of inducing simple use KCl injection to hasten parturition in the body, the method is not only the same with natural spawning, the maturity of fish gonads that exists parent participation requires height, the few problem of egg laying amount, and because of injection volume requirement 0.5mol/L, 1～2ml, be easy to cause that the parent participation discharge comprises the internal organ of sexual gland, therefore can not be applied in the production of juvenile stichopus.Also having the method for inducing in a kind of body, is document (Develop-mental biology, 326 (2009): 169-175) about the report (Asn-Gly-Leu-Trp-Tyr-NH2) of pentapeptide NGIWY acid amides and derivative NGLWY acid amides thereof.Although document points out that the interior injection of body NGLWY acid amides can be induced the stichopus japonicus Spawning and be disclosed each individual injection volume is that 0.1% (v/w), optium concentration are 1nmol/L, but our experiments show that: it is few to inject separately the NGLWY egg laying amount, as average weight be 210 the gram ripe parent participations, average egg laying amount only has 430,000, survival rate only has 20～50% during the floating larva, and young ginseng distortion ratio only has 15～30%.

Summary of the invention:

The present invention is in order to solve the existing above-mentioned technical problem of prior art, provide a kind of simple to operate, weak point consuming time, synchronous spawning and the ovum discharge capacity is large, quality good can greatly improve the method for the inducing Stichopus Japonicus Selenka to lay eggs in vivo of the efficient of hastening parturition, fertilization rate and incubation rate.

Technical solution of the present invention is: a kind of method of inducing Stichopus Japonicus Selenka to lay eggs in vivo is characterized in that carrying out as follows:

A. prepare ocyodinic

The NGLWY acid amides is dissolved in the mother liquor that is configured to 1 μ mol/L in the ultra-pure water, then mother liquor is dissolved in the KCl sea water solution of 0.5mol/L, make the ocyodinic of 1nmol/L;

B. choose parent participation

Choose the gonad maturity parent participation of body weight at least 200 grams;

C. inject ocyodinic

Be 0.1%ml/g with volume and parent participation weight ratio and inject body cavity from the stichopus japonicus back less than the ocyodinic of 0.3ml and carry out inducing in the body;

D. collect smart ovum.

The present invention carries out body in injection as the artificial induced spawning agent to stichopus japonicus with the mixed solution of NGLWY acid amides and KCl to hasten parturition.The reasonable compatibility of NGLWY acid amides and KCl, interaction directly act on the stichopus japonicus sexual gland, induce the stichopus japonicus oocyte maturation and take off folliculus, and the whole individualities of basic guarantee of once hastening parturition all discharge smart ovum, and spawning rate reaches more than 90%.Its consumption can guarantee not arrange in person dirty and ovulation rate improves greatly, average egg laying amount can be brought up to 4,000,000, reached more than 70% of total fish brood amount, improved greatly the parent participation availability, namely avoided the existing amount of quoting of independent injection KCl to cause greatly the dirty problem of parent participation row, also solve the little problem of the independent use existing egg laying amount of NGLWY acid amides, had weak point simple to operate, consuming time, low cost and other advantages.Synchronously discharging of ovum and quality are good, have directly improved fertility rate and hatchability (all more than 98%), and survival rate reaches 50～90% during the floating larva, and young ginseng distortion ratio reaches 40～80%, greatly improves seed output.Ocyodinic used in the present invention have no side effect and required dosage few, the parent participation after hastening parturition still can normally cultivate, grow, and has further reduced the seed rearing cost, has increased the economic benefit of enterprise.

Embodiment:

Embodiment 1:

A. prepare ocyodinic

To be purchased from Shanghai and give birth to worker biotech firm, purity is dissolved in the mother liquor that is configured to 1 μ mol/L in the ultra-pure water greater than 98% NGLWY acid amides, and-20 ℃ of Refrigerator stores are dissolved in mother liquor in the KCl sea water solution of 0.5mol/L before the injection, make the ocyodinic of 1nmol/L.

B. choose parent participation

Gather May from 28 of the celestial parent participations of bathing gulf plant in Dalian, the parent participation body weight is between 200g～260g, and average weight is 224g, and the parent participation of collection is transported to and supports temporarily 24h in the storage pond, holding pond.

C. inject ocyodinic

With the disposable medical syringe of 1ml ocyodinic is injected into around the stichopus japonicus sexual gland, injected dose (ml) is 0.1% (0.20～0.26ml) of parent participation body weight (g), parent participation places respectively through injection that temperature is housed is 17.5 ℃ filtering sea plastic box behind the ocyodinic, male stichopus japonicus begins to creep to seawater surface along the plastics tank wall in 30min, head begins to swing, and begins to discharge sperm behind the 40min.From 60min, female ginseng crawls into the water surface and discharges ovum along tank wall rapidly, and the female ginseng that has is namely ovulated at the bottom of case, and Spawning continues about 40min.

D. collect smart ovum according to prior art.

Detect ovum density and calculate the ovulation amount with blood counting chamber.

Spawning effect: 28 whole ejaculations of parent participation are laid eggs (16 of ejaculations are laid eggs 12), and 12 female parent participation ovulation amounts are between 2,700,000 to 5,200,000, and average ovulation amount is about 3,500,000.

After detecting the ovulation amount, cultivate and the metamorphosis of the young, adhere to, cultivate and at microscopically detection fertility rate and hatchability according to prior art artificial insemination and hatching, seed selection, floating larva.Observed result shows the incubation rate of the fertilization rate of ovum and fertilized egg all more than 98%, and fertilized egg develops into the microtia young after developing into rotation blastaea, 24h behind the 12h.The survival rate of floating larva is 73%, young ginseng distortion ratio is 67.2%.

Embodiment 2:

Except the b step choose parent participation and embodiment 1 different, other steps are with embodiment 1.

Gather May from 110 of the parent participations of Pulandian, Dalian stone river, the parent participation body weight is between 240g～350g, and average weight is 274g, and 72h is supported in the parent participation of collection temporarily in storage pond, holding pond, the bait of not throwing something and feeding during supporting temporarily.The injection volume of ocyodinic is 0.24～0.3ml.

Ejaculation egg-laying time and example 1 are basically identical, have 54 parent participation ejaculations, and 52 parent participations are laid eggs, and female ginseng egg laying amount is between 2,920,000 to 6,000,000, and average egg laying amount is 495.4 ten thousand.Artificial insemination and hatching, seed selection, floating larva cultivate and the metamorphosis of the young, adhere to and cultivate all with embodiment 1, and the floating larva survival rate is 74.6%, and metamorphosis is 68%.

Claims (1)

1. the method for an inducing Stichopus Japonicus Selenka to lay eggs in vivo is characterized in that carrying out as follows:

A. prepare ocyodinic

The NGLWY acid amides is dissolved in the mother liquor that is configured to 1 μ mol/L in the ultra-pure water, then mother liquor is dissolved in the KCl sea water solution of 0.5mol/L, make the ocyodinic of 1nmol/L;

B. choose parent participation

Choose the gonad maturity parent participation of body weight at least 200 grams;

C. inject ocyodinic

To be 0.1%ml/g and volume injected inject body cavity less than the ocyodinic of 0.3ml carries out inducing in the body from the parent participation back with volume injected and parent participation weight ratio;

D. collect smart ovum.