CN102579475B - Medicament applicable to intervention shrimp ovary ripening and preparation method as well as application thereof - Google Patents
Medicament applicable to intervention shrimp ovary ripening and preparation method as well as application thereof Download PDFInfo
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- CN102579475B CN102579475B CN2011100048223A CN201110004822A CN102579475B CN 102579475 B CN102579475 B CN 102579475B CN 2011100048223 A CN2011100048223 A CN 2011100048223A CN 201110004822 A CN201110004822 A CN 201110004822A CN 102579475 B CN102579475 B CN 102579475B
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention belongs to the technical field of aquaculture, and particularly relates to a medicament applicable to intervention shrimp ovary ripening and a preparation method as well as application thereof. The preparation method is characterized by comprising the following three steps of: extracting bletilla striata polysaccharide as a carrier; preparing bletilla striata polysaccharide sol of a shrimp blood lymph characteristic; and preparing a ripening intervention medicament. Experiments show that the brooding rate is not less than that of an eyestalk ablation group when the medicament is applied to red swamp crayfishes at the early stage of vitellogenesis under the same breeding condition. The death rate tends to be zero after the shrimps are treated for a week. Comparatively, the death rate for bilateral eyestalk ablation under the same condition in a week is 71.67 percent, and the death rate for a unilateral eyestalk ablation group under the same condition in a week is 57.5 percent. After the medicament is used for treating the red swamp crayfishs during vitellogenesis for 45 days, the brooding rate is 84.82 percent, the death rate in a week is only 1.67 percent, the stability is guaranteed and the death rate is obviously reduced, P is less than 0.01, and synchronous development and maturing of ovary are promoted. The medicament has a stable material source; and the method is simple and practical and has the prospect of being applied as an alternative method for an eyestalk ablation shrimp ovary ripening method.
Description
Technical field
The invention belongs to technical field of aquaculture, be specifically related to a kind of be applicable to medicine that the intervention shrimp ovary accelerates the ripening and preparation method thereof and application.Utilize intervention medicament of the present invention to remove optic stalk to the neuroendocrine inhibitory action of During Ovarian Maturation in certain period, reach the purpose that promotes the better growth of shrimps ovary and artificial repening.
Background technology
Generally taking be used to inducing the economic crustacean development of ovary and sexually matured method in production practices is to remove optic stalk and deriving technology thereof.These technology comprise: (1) Eyestalk (2) burns optic stalk; (3) ligation or constriction optic stalk base portion (T.subramoniam, 1999).Remove optic stalk and have on the one hand the effect of accelerating the ripening significantly, also can't avoid on the other hand following negative impacts: the individuality that (1) removes the rear parent of optic stalk (even by ligation or constriction optic stalk base portion) very high mortality rate, survive and lay eggs by sheer good luck also can produce permanent injury, after some precious parent's resource can't reuse (2) removal optic stalk, gonad continue to be grown, the quality of the ovum of output and the filial generation of hatching obviously descend (Laufer etc. 1999).So a kind of method of seeking low actual promotes the healthy mature of gonad and lay eggs to become a kind of objective demand, the neuroendocrine regulation mechanism of crustacean breeding always is study hotspot, and (Fingerman 1995; Subramoniam 1999; Nagaraju 2007).
MO is the crayfish development of ovary and the important endocrine organ of middle performance of laying eggs, and secretes a kind of sesquiterpenoids hormone MF (Nagaraju2007).Closely related (Zhao Weixin etc., 2001) occur with promoting yolk in the activity of the synthetic MF of MO.The synthetic MF of MO accepts the adjusting of the neurosecretion factor in optic stalk.At present, isolation identification has gone out in cultivation MO experiment, to two kinds of inhibited CHH neuropeptide MOIH-I MOIH-II of family of MO from optic stalk.
DA and 5-HT are also brought into play important and sure effect (Richardson etc., 1991 by increasing evidence prove in breeding; Kulkarni etc., 1992; Sarojini etc. 1995; Vaca etc., 2000; Cai Shengli etc., 2000; Ye Haihui etc., 2003; The .2005 such as Cui Qingman; The .2006 such as Ye Haihui; The .2008 such as Tinikul; The .2009 such as Santhoshi).Wherein, 5-HT is to the growth of ovary and lay eggs and have facilitation, and DA has inhibitory action to the growth of ovary and the function of 5-HT.Wherein, optic stalk is that most important DA produces and the secretion position.
The intervention method is the medical approaches that a kind of forward position physiological function is disturbed.The present invention exports by XO-SG secretory product MOIH in the drug moiety sealer blocking-up optic stalk of the special intervention of employing, and the inhibit feature of the DA of slow release DOM releasing simultaneously reaches the purpose that promotes the better growth of shrimps ovary and artificial repening.This processing method is easy to implement, and the close shrimp survival rate after processing is significantly higher than and removes optic stalk, has to substitute the application prospect that removes optic stalk.
Summary of the invention
The object of the invention is to overcome the defective of prior art, utilize the short blood clotting of extract of bletilla and the characteristic that can slowly be absorbed by organism, as the sealer and the slow releasing agent that are difficult for diffusion, XO-SG secretory product big jaw device inhibitory hormone (MOIH) output in certain period internal interference optic stalk, slow release domperidone (DOM) is removed the inhibit feature of dopamine (DA) simultaneously, reach that to promote the shrimps ovary better to reach maturity ripe, and obtain the purpose of high survival rate.
Realize that technical scheme of the present invention is as follows:
A kind ofly be applicable to the medicine that the intervention shrimp ovary accelerates the ripening, it comprises the steps:
(1) Pseudobulbus Bletillae polysaccharose extracts: with the Pseudobulbus Bletillae (Rhizoma Bletillae) peeling of drying, be cut into the 1.2mm fritter, by volume/mass ratio adds the tri-distilled water of 20 times, soaks under 4 ℃ 36 hours, then is placed in 70 ℃ of water-baths and decocts 4-8h, use the 1mm strainer filtering, filtrate is placed under 4 ℃ standing, filtering residue by volume/mass ratio adds 5 times of volume tri-distilled waters to be placed in 70 ℃ of water-bath 2h, repeats to decoct 3 times, use the 1mm strainer filtering, merging filtrate; With ultrasonic wave concussion 30min, then the centrifugal 20min of 6000g, collect supernatant with above-mentioned filtrate, and check whether supernatant can pass through the 30G syringe needle; The dehydrated alcohol that adds 10 times of volumes in the supernatant of gained is stirred to the white floccule of whole generation exquisitenesses, standing 48h, and sucking filtration is collected white floccule, extracts with 500 order Steel nets; With the dehydrated alcohol repeated washing of 2 times of volumes 3 times, sucking filtration is collected white floccule, extracts, and rubs to be broken to the 0.1mm powder, in 70 ℃ of lower hot air dryings, obtains the Pseudobulbus Bletillae polysaccharose extract, puts under 4 ℃ and preserves, and is standby;
(2) Pseudobulbus Bletillae polysaccharose salt sol preparation: by mass, get analytical pure NaCl32g, analytical pure KCl0.1g, analytical pure MgCl26H2O 0.5g, analytical pure and HEPES0.12g are dissolved in the 1L tri-distilled water, obtain solution 1, add the Pseudobulbus Bletillae polysaccharose extract 80g of step (1) preparation in the described solution 1, obtain the Pseudobulbus Bletillae polysaccharose salt sol;
(3) ovary accelerates the ripening with the preparation that gets involved medicament: be to add domperidone injection at 1: 50 by volume before each the use in the described Pseudobulbus Bletillae polysaccharose salt sol of step (2), obtain accelerating the ripening with the intervention medicament with the ultrasound wave mixing;
Wherein: step (1) and (3) described Ultrasonic Conditions are as follows: Φ 25mm static probe, and working time 10s, gap 20s, amplitude 30um, container is put in mixture of ice and water.
A kind of accelerate the ripening preparation method of medicine of intervention shrimp ovary that is applicable to, it comprises the steps:
(1) Pseudobulbus Bletillae polysaccharose extracts: with the Pseudobulbus Bletillae (Rhizoma Bletillae) peeling of drying, be cut into the 1.2mm fritter, by volume/mass ratio adds the tri-distilled water of 20 times, soaks under 4 ℃ 36 hours, then is placed in 70 ℃ of water-baths and decocts 4-8h, use the 1mm strainer filtering, filtrate is placed under 4 ℃ standing, filtering residue by volume/mass ratio adds 5 times of volume tri-distilled waters to be placed in 70 ℃ of water-bath 2h, repeats to decoct 3 times, use the 1mm strainer filtering, merging filtrate; With ultrasonic wave concussion 30min, then the centrifugal 20min of 6000g, collect supernatant with above-mentioned filtrate, and check whether supernatant can pass through the 30G syringe needle; The dehydrated alcohol that adds 10 times of volumes in the supernatant of gained is stirred to the white floccule of whole generation exquisitenesses, standing 48h, and sucking filtration is collected white floccule, extracts with 500 order Steel nets; With the dehydrated alcohol repeated washing of 2 times of volumes 3 times, sucking filtration is collected white floccule, extracts, and rubs to be broken to the 0.1mm powder, in 70 ℃ of lower hot air dryings, obtains the Pseudobulbus Bletillae polysaccharose extract, puts under 4 ℃ and preserves, and is standby;
(2) Pseudobulbus Bletillae polysaccharose salt sol preparation: by mass, get analytical pure NaCl32g, analytical pure KCl0.1g, analytical pure MgCl26H2O 0.5g, analytical pure and HEPES0.12g are dissolved in the 1L tri-distilled water, obtain solution 1, add the Pseudobulbus Bletillae polysaccharose extract 80g of step (1) preparation in the described solution 1, obtain the Pseudobulbus Bletillae polysaccharose salt sol;
(3) ovary accelerates the ripening with the preparation that gets involved medicament: be to add domperidone injection at 1: 50 by volume before each the use in the described Pseudobulbus Bletillae polysaccharose salt sol of step (2), obtain accelerating the ripening with the intervention medicament with the ultrasound wave mixing;
Wherein: step (1) and (3) described Ultrasonic Conditions are as follows: Φ 25mm static probe, and working time 10s, gap 20s, amplitude 30um, container is put in mixture of ice and water.
The accelerate the ripening medication of medicine of shrimps ovary of the present invention:
With the syringe needle of 27G-30G, with common or high pressure injector accelerating the ripening with getting involved drug injection to optic stalk base portion sinus gland place the steps A gained.
Compared with prior art, good effect of the present invention is:
(1) method of the present invention's foundation is stable significantly promotes ovary synchronously to reach maturity.
(2) method set up of the present invention is with respect to the cultivation survival rate that removes optic stalk and can the utmost point significantly improve close shrimp.
(3) raw material steady sources, method is simple, can use as the alternative method that removes optic stalk.
The specific embodiment
Below in conjunction with example, the present invention is described in further detail, but embodiment is not limited to this.
Embodiment 1: yolk is accelerating the ripening of emergence period Procambrus clarkii ovary not
A, medicine preparation:
1, with dry Pseudobulbus Bletillae (Rhizoma Bletillae) 20g peeling, be cut into the 1.2mm fritter, add 20 times of (V/M) tri-distilled waters, after 4 ℃ of environment soak 36 hours, be placed in 70 ℃ of water-baths and decocted 4-8 hour, with the 1mm strainer filtering, it is standing that filtrate is placed in 4 ℃ of environment, and filtering residue repeats to decoct 3 times adding 5 times of (V/M) tri-distilled waters to be placed in 70 ℃ of water-baths 2 hours, with 1mm strainer filtering, merging filtrate.
2, (Ultrasonic Conditions is as follows: Φ 25mm static probe with ultrasound wave with above-mentioned filtrate, working time 10s, gap 20s, amplitude 30um, container is put in mixture of ice and water) shook 30 minutes, then 6000g is centrifugal 20 minutes, collects supernatant, and checks whether supernatant can pass through the 30G syringe needle.
3, add 10 times of volume dehydrated alcohol in supernatant, be stirred to the white floccule of whole generation exquisitenesses, standing 48 hours, sucking filtration was collected white floccule, extracts.
4, with 2 times of volume dehydrated alcohol repeated washings 3 times, sucking filtration is collected white floccule, extracts, and rubs to be broken into the little end of 0.1mm, and 70 ℃ of hot air dryings are placed in 4 ℃ of long preservation standby.
5, according to the quality meter, get analytical pure NaCl32g, analytical pure KCl0.1g, analytical pure MgCl26H2O 0.5g, analytical pure and HEPES0.12g (available from Sigma company) are dissolved in the 1L tri-distilled water, and obtaining solution, to be placed in 4 ℃ of preservations stand-by.
6, the solution 50ML of employing above-mentioned 5 dissolving Pseudobulbus Bletillae polysaccharose extract 4g, preparation Pseudobulbus Bletillae polysaccharose salt sol.
7, be to add domperidone injection (Ningbo second hormone factory product) at 1: 50 by volume before each the use, with ultrasonic Treatment 10 seconds (condition is with 1), the medicine mixing obtained accelerating the ripening with getting involved medicament;
B, close shrimp are selected: selecting body weight is 25 ± 5g, body length is that the body colour green grass or young crops of 7.5 ± 0.5cm is brown, the body limb is complete, anosis energy is strong without wound, and body constitution is healthy and strong, gonad development the is in yolk Procambrus clarkii of emergence period not, be preset in 1-2d in 50cm * 60cm * 40cm plastic cysle boxes before processing, density is 50/case, adds the 2cm breeding water to keep the cheek silk of processing Procambrus clarkii moistening at the bottom of case.
C, close shrimp are processed: use 30G syringe needle, ordinary syringe to accelerate the ripening with getting involved drug injection to optic stalk base portion sinus gland place of the present invention.
D, cultivation: close shrimp is put in a suitable place to breed, and adjusting breeding density is 36 tails/m2, and female, male sex ratio is 2: 1; Throw something and feed by regularly fixing a point every day, is satiated with food, and animal food accounts for 70%, and plant food accounts for 30%, changes fresh water 30% (volume) every day, makes cultivation water meet State Standard of the People's Republic of China's " water quality standard for fishery ", standard No.: GB11607-89.
E, result: under equal cultivating condition, in the present invention 45 days, the survival rate of Procambrus clarkii parent shrimp and egg laying rate of red swamp (84.82%) are apparently higher than excision optic stalk processed group (66.67%); It is 1.67% that method of the present invention is processed all interior mortality rates, mortality rate 71.67% and interior mortality rate 57.5% of one-sided one week of optic stalk of excision in all with respect to excision bilateral optic stalk one under equal conditions, stable promoted Procambrus clarkii parent shrimp ovary synchronously to reach maturity, P＜0.01.
Table 1 insertion type ovary accelerates the ripening method and the excision optic stalk ovary method of accelerating the ripening embraces the fourth of the twelve Earthly Branches and survival in 45 days to Procambrus clarkii effect relatively
The main reference document
1. Zhao Wei letter etc., (2001). the research of synthesis of methyl farnesoate by mandibular organ in Procambarus clarkii, aquatic product journal 25 (003): 193-196.
2. leaf Hai Hui etc., (2003). biogenic amine is to the neuroendocrine regulating and controlling effect of female Scylla serrata (Forskal). reproduction, Oceanologia et Limnologia Sinica.
3. Cai Shengli etc., (2000). the impact of hormone hormone on the Chinese prawn development of ovary, Zhongshan University's journal: natural science edition (0z1): 91-95.
4. Cui Qing is graceful etc., (2005). the exsomatize regulating and controlling effect of the development of ovary of Eriocheir sinensis, Chinese aquatic science 12 (004): 487-491.
5.Fingerman，M.(1995).″Endocrine?mechanisms?in?crayfish，with?emphasis?on?reproduction?and?neurotransmitter?regulation?of?hormone?release.″
Integrative?and?Comparative?Biology?35(1)：68.
6.Kulkarni，G.，R.Nagabhushanam，et?al.(1992).″In?vivo?stimulation?of?ovarian?development?in?the?red?swamp?crayfish，Procambarus?clarkii(Girard)，by?5-hydroxytryptamine.″Invertebrate?reproduction?&?development?21(3)：231-239.
7.Laufer，H.(1992).Method?for?increasing?crustacean?larval?production，Google?Patents.
8.Nagaraju，G.P.C.(2007).″Is?methyl?farnesoate?a?crustacean?hormone？″Aquaculture?272(1-4)：39-54.
9.Richardson，H.，M.Deecaraman，et?al.(1991).″The?effect?of?biogenic?amines?on?ovarian?development?in?the?fiddler?crab，Uca?pugilator.″Comparative?Biochemistry?and?Physiology?Part?C：Comparative?Pharmacology?99(1-2)：53-56.
10.Sarojini，R.，R.Nagabhushanam，et?al.(1995).″Dopaminergic?inhibition?of?5-hydroxytryptamine-stimulated?testicular?maturation?in?the?fiddler?crab，Uca?pugilator.″Comparative?Biochemistry?and?Physiology?Part?C：Pharmacology，Toxicology?and?Endocrinology?111(2)：287-292.
11.Subramoniam，T.(1999).″Endocrine?regulation?of?egg?production?in?economically?important?crustaceans.″CURRENT?SCIENCE-BANGALORE-76：350-360.
12.Tinikul，Y.，A.Joffre?Mercier，et?al.(2008).″Changes?in?the?levels?of?serotonin?and?dopamine?in?the?central?nervous?system?and?ovary，and?their?possible?roles?in?the?ovarian?development?in?the?giant?freshwater?prawn，Macrobrachium?rosenbergii.″General?and?Comparative?Endocrinology?158(3)：250-258.
13.Santhoshi，S.，V.Sugumar，et?al.(2009).″Serotonergic?stimulation?of?ovarian?maturation?and?hemolymph?vitellogenin?in?the?Indian?white?shrimp，Fenneropenaeus?indicus.″Aquaculture?291(3-4)：192-199.
14.Vaca，A.and?J.Alfaro(2000).″Ovarian?maturation?and?spawning?in?the?white?shrimp，Penaeus?vannamei，by?serotonin?injection.″Aquaculture?182(3-4)：373-385.
Claims (3)
1. one kind is applicable to the medicine that the intervention shrimp ovary accelerates the ripening, and the preparation method of described medicine comprises the steps:
(1) Pseudobulbus Bletillae polysaccharose extracts: with the Pseudobulbus Bletillae (Rhizoma Bletillae) peeling of drying, be cut into the 1.2mm fritter, by volume/mass ratio adds the tri-distilled water of 20 times, soaks under 4 ℃ 36 hours, then is placed in 70 ℃ of water-baths and decocts 4-8h, use the 1mm strainer filtering, filtrate is placed under 4 ℃ standing, filtering residue by volume/mass ratio adds 5 times of volume tri-distilled waters to be placed in 70 ℃ of water-bath 2h, repeats to decoct 3 times, use the 1mm strainer filtering, merging filtrate; With ultrasonic wave concussion 30min, then the centrifugal 20min of 6000g, collect supernatant with above-mentioned filtrate, and check whether supernatant can pass through the 30G syringe needle; The dehydrated alcohol that adds 10 times of volumes in the supernatant of gained is stirred to the white floccule of whole generation exquisitenesses, standing 48h, and sucking filtration is collected white floccule, extracts with 500 order Steel nets; With the dehydrated alcohol repeated washing of 2 times of volumes 3 times, sucking filtration is collected white floccule, extracts, and rubs to be broken to the 0.1mm powder, in 70 ℃ of lower hot air dryings, obtains the Pseudobulbus Bletillae polysaccharose extract, puts under 4 ℃ and preserves, and is standby;
(2) Pseudobulbus Bletillae polysaccharose salt sol preparation: by mass, get analytical pure NaC l32g, analytical pure KCl0.1g, analytical pure MgCl
26H
2O0.5g and analytical pure HEPES0.12g are dissolved in the 1L tri-distilled water, obtain solution 1, add the Pseudobulbus Bletillae polysaccharose extract 80g of step (1) preparation in the described solution 1, obtain the Pseudobulbus Bletillae polysaccharose salt sol;
(3) ovary accelerates the ripening with the preparation that gets involved medicament: be to add domperidone injection at 1: 50 by volume before each the use in the described Pseudobulbus Bletillae polysaccharose salt sol of step (2), obtain accelerating the ripening with the intervention medicament with the ultrasound wave mixing;
Wherein: step (1) and (3) described Ultrasonic Conditions are as follows: Φ 25mm static probe, and working time 10s, gap 20s, amplitude 30 μ m, container is put in mixture of ice and water.
2. one kind is applicable to the accelerate the ripening preparation method of medicine of intervention shrimp ovary, and its feature comprises the steps:
(1) Pseudobulbus Bletillae polysaccharose extracts: with the Pseudobulbus Bletillae (Rhizoma Bletillae) peeling of drying, be cut into the 1.2mm fritter, by volume/mass ratio adds the tri-distilled water of 20 times, soaks under 4 ℃ 36 hours, then is placed in 70 ℃ of water-baths and decocts 4-8h, use the 1mm strainer filtering, filtrate is placed under 4 ℃ standing, filtering residue by volume/mass ratio adds 5 times of volume tri-distilled waters to be placed in 70 ℃ of water-bath 2h, repeats to decoct 3 times, use the 1mm strainer filtering, merging filtrate; With ultrasonic wave concussion 30min, then the centrifugal 20min of 6000g, collect supernatant with above-mentioned filtrate, and check whether supernatant can pass through the 30G syringe needle; The dehydrated alcohol that adds 10 times of volumes in the supernatant of gained is stirred to the white floccule of whole generation exquisitenesses, standing 48h, and sucking filtration is collected white floccule, extracts with 500 order Steel nets; With the dehydrated alcohol repeated washing of 2 times of volumes 3 times, sucking filtration is collected white floccule, extracts, and rubs to be broken to the 0.1mm powder, in 70 ℃ of lower hot air dryings, obtains from the splendid achnatherum polyoses extract, puts under 4 ℃ and preserves, and is standby;
(2) Pseudobulbus Bletillae polysaccharose salt sol preparation: by mass, get analytical pure NaCl32g, analytical pure KCl0.1g, analytical pure MgCl
26H
2O0.5g and analytical pure HEPES0.12g are dissolved in the 1L tri-distilled water, obtain solution 1, add the Pseudobulbus Bletillae polysaccharose extract 80g of step (1) preparation in the described solution 1, obtain the Pseudobulbus Bletillae polysaccharose salt sol;
(3) ovary accelerates the ripening with the preparation that gets involved medicament: be to add domperidone injection at 1: 50 by volume before each the use in the described Pseudobulbus Bletillae polysaccharose salt sol of step (2), obtain accelerating the ripening with the intervention medicament with the ultrasound wave mixing;
Wherein: step (1) and (3) described Ultrasonic Conditions are as follows: Φ 25mm static probe, and working time 10s, gap 20s, amplitude 30 μ m, container is put in mixture of ice and water.
3. the application of medicine claimed in claim 1 in close shrimp ovary accelerates the ripening.
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| CN105368765A (en) * | 2015-10-15 | 2016-03-02 | 中国水产科学研究院淡水渔业研究中心 | Method for recombinant production of procambarus clarkii ATP synthase F0 subunit 6 protein |
| CN107821263B (en) * | 2017-12-04 | 2023-02-17 | 中国水产科学研究院南海水产研究所 | Ripening strip for penaeus vannamei eyestalk pinching and light control and ripening method |
| CN109673556A (en) * | 2019-01-30 | 2019-04-26 | 盐城工学院 | A method of improving freshwater shrimp egg laying rate of red swamp |
| CN115088657B (en) * | 2022-07-29 | 2023-06-13 | 杭州师范大学 | Method for improving fertility rate and hatching rate of older litopenaeus vannamei |
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| US5161481A (en) * | 1991-11-14 | 1992-11-10 | Hans Laufer | Method for increasing crustacean larval production |
| US6740794B1 (en) * | 1999-01-28 | 2004-05-25 | University Of Hawaii | Methods of isolating the androgenic sex hormone from crustacean prawn and marine shrimp and methods of use |
| CN1299695C (en) * | 2004-11-16 | 2007-02-14 | 南京大学 | Liquid embolism agent for intracranial aneurysm and its preparing process |
| CN101720707A (en) * | 2009-12-11 | 2010-06-09 | 华中农业大学 | Method for improving egg laying rate of red swamp crayfish |
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