CN101543191B - Method for forecasting laying season of portunus trituberculatus - Google Patents
Method for forecasting laying season of portunus trituberculatus Download PDFInfo
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- CN101543191B CN101543191B CN2009100498459A CN200910049845A CN101543191B CN 101543191 B CN101543191 B CN 101543191B CN 2009100498459 A CN2009100498459 A CN 2009100498459A CN 200910049845 A CN200910049845 A CN 200910049845A CN 101543191 B CN101543191 B CN 101543191B
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Abstract
The invention relates to a method for forecasting the laying season of portunus trituberculatus. The invention samples a small quantity of ovarian tissue through drilling under the condition of not killing a parent strain and can forecast the approximate laying season of female portunus trituberculatus by the combination of visual inspection and histological observation. The key points of the method are to acquire the ovarian tissue by drilling the position which is close to the rear parts of eyes and is on the anterior border of the carapace, seal the sampling hole and adopt a modified tissue fixation liquid to fix the ovary. The method is suitable to be used by scientific research and production units and forecasts the laying season by knowing the development condition of the ovary of the parent strain in vivo. Moreover, the forecast of the laying season of the female portunus trituberculatus is favorable for the preparation of seedling facilities and baits in artificial seedling and has important significance in artificial seedling of the portunus trituberculatus.
Description
Technical field
The present invention relates to a kind of method of forecasting laying season of portunus trituberculatus, belong to the mariculture technology field.
Background technology
Portunus trituberculatus Miers (Portunus trituberculatus) is the important seawater economic crab of China; Since the eighties in 20th century; Owing to reasons such as overfishing and coastal ocean environmental deteriorations, the natural resources amount of China's Portunus trituberculatus Miers sharply descending (Xue Junzeng etc., 1997; Xie Zhongming etc., 2002), therefore, the artificial breeding of Portunus trituberculatus Miers and pond culture develop (Wu Xugan etc., 2007) rapidly in that China is coastal since the nineties in 20th century.Yet in actual production; Brought very big difficulty for the production of growing seedlings owing to can't accurately predict its egg-laying time; So the prediction egg-laying period is of great practical significance for the Portunus trituberculatus Miers artificial breeding; Can avoid growing seedlings produce in blindness, in time prepare culture facility and young bait etc., can reduce so to a certain extent and grow seedlings risk and alleviate seedling cost.
Portunus trituberculatus Miers belongs to the crab class that first post-coitum carries out the development of ovary, in case under suitable temperature and illumination condition, just can lay eggs after the development of ovary maturation, therefore accurately understands parent's the development of ovary stage and just possibly predict egg-laying time.But; The research of relevant laying season of portunus trituberculatus forecast is less both at home and abroad; Only the Hamasaki et al (2004) of Japan has carried out Primary Study to the Portunus trituberculatus Miers development of ovary with laying eggs to temperature and illumination under the indoor culture condition, finds that water temperature is lower than 12 ℃, the slow perhaps stasi of the Portunus trituberculatus Miers development of ovary; Water temperature and periodicity of illumination surpass 12 ℃ and 12h respectively, and the ripe female body of Portunus trituberculatus Miers of the development of ovary just possibly laid eggs.But they unpredictable each parent's egg-laying time; The natural spawning peak of only finding Japanese sea area Portunus trituberculatus Miers is in mid-April; In November or December wild crab is put into 21 ± 0.5 ℃ of tanks of water temperature under the artificial condition, these parents' egg production peak time can be ahead of time at the beginning of 2 months.Domestic; The forecast that relevant Portunus trituberculatus Miers is laid eggs mainly rests on the experience stage, relies on the ovary of observing the live body parent under certain illumination condition usually and whether is full of carapace, if ovary is full of carapace; Explain that then the parent then possibly will lay eggs very soon; But this method error is big (Xie Zhongming etc., 2002) very, and its main cause is the development of ovary state that this method can not accurately be understood the parent.In addition; The ovary maturity of Portunus trituberculatus Miers is divided into growth and maturity and physiological ripening; The ovary of Portunus trituberculatus Miers promptly is full of carapace after the growth and maturity; The ovary volume just no longer increases, and still needs and wants a period of time (Zhu Dongfa etc., 2005) but carry out the transition to fertilizable mature egg (physiological ripening) from the egg mother cell of growth and maturity.Obviously, by virtue of experience observe ovary and whether be full of carapace, can't judge the physiological ripening and the growth and maturity of ovary, so can't accurately predict its egg-laying time.Judge the growth and maturity or the physiological ripening of ovary; Whether method is the female body parent of vivisection more accurately, obtains ovary, observe ovum and be free in the ovarian cavity; Further can confirm the maturity state of ovum through histologic section; But must need like this to put to death expensive parent, yet unpredictable each female body parent's egg-laying time does not have feasibility in actual production.
, forecast that therefore the egg-laying period at first need confirm development of ovary situation under certain condition because but the just development of ovary of the female body post-coitum of Portunus trituberculatus Miers just can lay eggs after the development of ovary maturation.Obtain ovary tissue research through putting to death the parent, can't forecast live body parent's egg-laying period.
Given this; The inventor is on the basis of carrying out Portunus trituberculatus Miers artificial propagation research for many years; Through studying repeatedly and verifying; Invent a kind of method of easy, forecasting laying season of portunus trituberculatus that be prone to learn, under the situation of not putting to death female body parent, taken a sample, can accurately predict the method for laying season of portunus trituberculatus.
Summary of the invention
The present invention is exactly in order to address the above problem; Overcome the existing forecasting procedure whether ovary is full of carapace of by virtue of experience observing; Can't judge the physiological ripening and the growth and maturity of ovary,, overcome existing forecasting procedure in addition and obtained ovary tissue research through putting to death the parent so can't accurately predict the problem of its egg-laying time; The problem that can't forecast live body parent's egg-laying period provides a kind of method of forecasting laying season of portunus trituberculatus.This method is applicable to the female body of Portunus trituberculatus Miers.This invention is applicable to R&D institution and production unit, also uses applicable to the peasant household of growing seedlings.
The technical problem that will solve required for the present invention, can realize through following technical scheme:
1. the method for a forecasting laying season of portunus trituberculatus may further comprise the steps,
(1) under the condition of not dissecting the female body parent of Portunus trituberculatus Miers, adopt sampling instrument drilling the sampling holes on the crust of the female body of Portunus trituberculatus Miers, to take a sample, the ovary tissue that takes a morsel after the sampling seals thief hole;
(2) inspection development of ovary situation can or utilize microscope that development of ovary situation is carried out Histological research through perusal after the sampling;
(3) according to egg-laying period of the histological observation of step (2) prediction Portunus trituberculatus Miers.
Sampling instrument uses drill bit and tweezers in the step (1); Said drill bit and tweezers must prevent the courses of infection parent through strict sterilization before use, and bit diameter is the 0.8-1.2 millimeter; Drill bit is excessive to cause bigger injury to the parent easily, thereby reduces parent survival rate.
Thief hole is positioned at the carapace leading edge on the crust described in the step (1), near the position behind the eyes, takes a sample from said thief hole; Can get the ovary sample; And can not cause damage to other tissues such as hearts, the crust of said thief hole is more smooth, and the sampling back is the sealing thief hole easily.
Step (1) thief hole is sealed into the sampling back and adopts the thief hole on the plastic plate sealing crust of 502 glue and 0.5 millimeters thick; Prevent that seawater entering or courses of infection etc. from causing the parent dead; Spend 2-3 minute then and treat after 502 adhesive curings parent to be put into seawater immediately, reduce the harmful effect of sampling process the parent.
Take a sample and seal thief hole in back 5 minutes.
The said inspection development of ovary of step (2) situation further comprises and adopts fixer that ovary tissue is fixed, and carries out histological observation.
Said fixer is the BouinShi liquid of improvement or 3% formalin solution of improvement, otherwise the egg mother cell in slicing processes of the ovary sample after fixing is broken easily, causes yolk to come off, and influences histological observation.
The prescription of the BouinShi liquid of said improvement is 2.4 milliliters of saturated picric acid solutions, 64.5 milliliters of formalin, 12.5 milliliters of glacial acetic acid solutions and 190 ml distilled waters.
3% formalin solution of said improvement is that 3% formalin is dissolved in the standard artificial seawater and prepares.
The index of histological observation described in the step (3) is size and the homogenising degree etc. of shape, ovum footpath, the yolk particle of egg mother cell, judges the development of ovary situation of Portunus trituberculatus Miers then according to index, thereby predicts the egg-laying period of female body.
With the naked eye directly observe the ovary sample; Parent's egg mother cell has been free in the ovarian cavity; Egg mother cell is rounded or oval, and prediction is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, will lay eggs in most of individual 4 days.
Said parent's egg mother cell still is not free in the ovarian cavity; Then measure the ovum footpath and observe the egg mother cell shape at microscopically; If average ovum footpath about 350 microns, and egg mother cell still mutually extruding be polygon, can predict; Is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, just may lay eggs after most of individual 7 days.
Said parent's egg mother cell still is not free in the ovarian cavity; Then measure the ovum footpath and observe the egg mother cell shape at microscopically; Parent's egg mother cell still is a polygon; Average ovum footpath is less than 300 microns, is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, may lay eggs after most of individual 10 days.
The egg-laying time of the forecast of this method, must satisfy day illumination is the condition of 24-30ppt greater than 12h, water temperature 16-19 ℃, salinity.
Principle of the present invention:
Will accurately forecast the female body parent's of Portunus trituberculatus Miers egg-laying period, key is to confirm parent's the development of ovary stage, if ovary and egg mother cell have reached physiological ripening, the parent will be laid eggs very soon under suitable illumination, temperature and salinity condition.Usually adopt in the scientific research and put to death the parent; Open crust, obtain ovary then, observe egg mother cell (ovum) then and whether be free in the ovarian cavity; Further can confirm the maturity state of ovum through histologic section; This method not only need be put to death expensive parent, and yet unpredictable each female body parent's egg-laying time does not have feasibility in actual production.
The present invention referring to Fig. 1-Figure 10, is used for somatoscopy and Histological research through taking out a small amount of ovary tissue in the boring of Portunus trituberculatus Miers crust according to the years of researches result, seals aperture with little plastic plate (0.5 millimeter of thickness) and 502 glue rapidly then.So not only can accurately understand the situation that ovary and egg mother cell are grown, thereby forecast the egg-laying time of female body, and need not dissect expensive female body parent, so can save great amount of cost.
Concrete outcome is:
(1) if parent's egg mother cell (ovum) has been free in the ovarian cavity; Histological observation shows that egg mother cell (ovum) has broken away from follicular cavity, and is rounded or oval, can predict that be under the condition of 24-30ppt in illumination greater than 12h, water temperature 16-19 ℃, salinity; Can lay eggs in most of individual 4 days; Forecast accuracy reaches 80.95% (n=63), if but the parent lives in water temperature to be lower than under 15 ℃ of situation, and egg-laying time can be postponed greatly; Be lower than 12 ℃ if the parent lives in water temperature always, do not find to lay eggs individuality.
(2) if parent's egg mother cell (that is, ovum) still is not free in the ovarian cavity, average ovum footpath is about 350 microns; Histology demonstration egg mother cell still pushes mutually and is polygon; Is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, just may lay eggs after most of individual 7 days, and forecast accuracy reaches 84.62% (n=52); If water temperature is lower than 15 ℃, egg-laying time also will prolong.If parent's egg mother cell still is a polygon; Average ovum footpath is less than 300 microns; And intracellular yolk material does not still homogenize; Is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, just may lay eggs after most of individual 10 days, and forecast accuracy reaches 90.57% (n=53).
Owing to adopted technique scheme, beneficial effect of the present invention:
1, forecast accuracy is brought up to more than 80%;
2, for R&D institution; Obtain a small amount of ovary sample through this method live body; Can further be engaged in histology, biochemical component and molecular biological further investigation; This not only can practice thrift a large amount of sampling expenses, and helps carrying out animal welfare, reduces the kill demand of scientific research activity to the live body Portunus trituberculatus Miers.
3, method is simple, for most Portunus trituberculatus Miers grow seedlings enterprise with grow seedlings for the peasant household, only need one dissection tweezers and small-sized electric drill, in the condition of not killing the parent, just can accurately predict the egg-laying time of live body Portunus trituberculatus Miers.
Description of drawings
In the position of Fig. 1 adult Portunus trituberculatus Miers ovary, red line is the position of mature ovarian, the zone of yellow area for holing.
Fig. 2 adopts the sketch map of miniature electric drill boring.Miniature electric drill is the miniature electric drill pictorial diagram of the special QT-35 of the application of force among the figure, and bit diameter is 1 millimeter.
The take a sample tweezers of usefulness of Fig. 3, A antimagnetic type tweezer (GOOI-TS-14, Guangzhou leads to brightness electronic tool Co., Ltd), B ophthalmic forceps (6 6 53072, Suzhou six or six visual science limited companies).
The Portunus trituberculatus Miers of the sealing of hole back of the body after Fig. 4 takes a sample (, preventing that seawater from getting into body cavity and bacterial infection) with 502 glue and green plastic plate sealing thief hole.
The ovary outward appearance of nearly maturation of Fig. 5 or growth and maturity, ovary does not expand loose, and egg mother cell does not still dissociate out.
Fig. 6 is the enlarged drawing of egg mother cell shown in Figure 5, the histology pictures that egg mother cell presses together, and egg mother cell does not still break away from follicular cavity.
The nearly ripe perhaps histology of the egg mother cell of growth and maturity of Fig. 7, egg mother cell is polygon because of extruding.
The ovary outward appearance of Fig. 8 physiological ripening (being about to ovulation), ovary expands loose.
Fig. 9 is the enlarged drawing that is about to the egg mother cell of ovulation shown in Figure 8.
The egg mother cell histology of Figure 10 physiological ripening state, egg mother cell are circular or subcircular, and the yolk particle size is more even.
Embodiment
In order to make technological means of the present invention, creation characteristic, to reach purpose and effect and be easy to understand and understand,, further set forth the present invention below in conjunction with concrete diagram.
Embodiment 1
Shown in Fig. 1-10.Instrument and reagent are prepared: miniature electric drill (the special QT-35 of the application of force; The special tools factory of the Ningbo high bridge application of force) referring to Fig. 2, antimagnetic type tweezer (GOOI-TS-14; Guangzhou leads to brightness electronic tool Co., Ltd) referring to Fig. 3, south of the River electric light source microscope (XS-212-201; Nanjing Jiangnan Yongxin Optical Co., Ltd.), C1 type order micrometer (C1 type, Shanghai are got over magnetoelectricity scarabaeidae skill Co., Ltd), 75% alcohol (Shanghai Ling Feng chemical reagent Co., Ltd), 502 glue (Guangdong Ai Bida adhesive Co., Ltd), dry towel and cotton balls.
Living body sampling: take out the female body parent of stock Portunus trituberculatus Miers, dry the moisture on the crust with dry towel, with cotton ball soaked in alcohol to being about to the part of boring, promptly the carapace leading edge, carry out disinfection near the position behind the eyes.(diameter is 1 millimeter a drill bit to adopt miniature electric drill then; Drill bit need disinfect in alcohol in advance) in carapace leading edge on the crust, bore the 0.8-1.2mm thief hole near the position behind the eyes; The thief hole of 1mm is good; Referring to Fig. 2, the antimagnetic type tweezer that disinfected in alcohol then takes out a small amount of ovary sample to observe development of ovary situation.The rapid plastic plate of 502 glue and 0.5 millimeters thick that adopts in back of taking a sample seals the aperture of taking a sample on the crust, referring to Fig. 4, prevents that seawater entering or courses of infection etc. from causing parent's death.
Sample is observed: with the naked eye directly observe the ovary sample; If parent's egg mother cell (ovum) has been free in the ovarian cavity; Egg mother cell is rounded or oval; Can predict that be under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, will lay eggs in most of individual 4 days, referring to Fig. 8-10; If be lower than under 15 ℃ of situation but the parent lives in water temperature, egg-laying time can be postponed greatly.If parent's egg mother cell (ovum) still is not free in the ovarian cavity; Then measure the ovum footpath and observe the egg mother cell shape at microscopically, if average ovum footpath about 350 microns, and egg mother cell still mutually extruding be polygon; Can predict; Is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, just may lay eggs after most of individual 7 days, referring to Fig. 7; If parent's egg mother cell still is a polygon, average ovum footpath is less than 300 microns, is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, just may lay eggs after most of individual 10 days, referring to Fig. 5 and Fig. 6.
Present embodiment is applicable to grow seedlings peasant household and the general enterprise of growing seedlings.
Embodiment 2
Shown in Fig. 1-10.Instrument and reagent are prepared: miniature electric drill (the special QT-35 of the application of force; The special tools factory of the Ningbo high bridge application of force); Referring to Fig. 2, antimagnetic type tweezer (GOOI-TS-14, Guangzhou leads to brightness electronic tool Co., Ltd) or ophthalmic forceps (6 6 53072, Suzhou six or six visual science limited companies) referring to Fig. 3, electric light source anatomical lens (Leica Zoom-2000; Meyer Instruments; Inc, Houston, TX 77084), electric light source microscope (south of the River XS-212-201; Nanjing Jiangnan Yongxin Optical Co., Ltd.), C1 type order micrometer (C1 type; Magnetoelectricity scarabaeidae skill Co., Ltd is got in Shanghai), 75% alcohol (Shanghai Ling Feng chemical reagent Co., Ltd), 502 glue (Guangdong Ai Bida adhesive Co., Ltd), histotome (Leica RM 2016, Leica Microsystems Inc., Bannockburn; IL, USA), the BouinShi fixer of improvement (2.4 milliliters of saturated picric acid solutions, 64.5 milliliters of formalin, 12.5 milliliters of glacial acetic acid solutions and 190 ml distilled waters) or 3% formalin solution (3% formalin is dissolved in the standard artificial seawater), dry towel and cotton balls.
Living body sampling: take out the female body parent of stock Portunus trituberculatus Miers, dry the moisture on the crust with dry towel, with cotton ball soaked in alcohol to being about to the part of boring, promptly the carapace leading edge, carry out disinfection near the position behind the eyes.Adopt miniature electric drill (diameter is 1 millimeter a drill bit) in carapace leading edge on the crust, bore the 0.8-1.2mm thief hole then near the position behind the eyes; The thief hole of 1mm is good; Referring to Fig. 2, the antimagnetic type tweezer that disinfected in alcohol then takes out a small amount of ovary sample and is used for Histological research with after observing development of ovary situation and fixing.The rapid plastic plate of 502 glue and 0.5 millimeters thick that adopts in back of taking a sample seals the aperture of taking a sample on the crust, referring to Fig. 4, prevents that seawater entering or courses of infection etc. from causing parent's death.
Sample is observed: under anatomical lens, directly observe the ovary sample; If parent's egg mother cell (ovum) has been free in the ovarian cavity; Egg mother cell is rounded or oval; Can predict that be under the condition of 24-30ppt in illumination greater than 12h, water temperature 16-19 ℃, salinity, will lay eggs in most of individual 4 days, referring to Fig. 8-10; If be lower than under 15 ℃ the condition but the parent lives in water temperature, egg-laying time can be postponed greatly.If parent's egg mother cell (ovum) still is not free in the ovarian cavity; The histology of then observing egg mother cell through routine paraffin wax section back is measured the ovum footpath at microscopically, if average ovum footpath is about 350 microns; Egg mother cell still pushes mutually and is polygon; Yolk even particle size and be full of whole egg mother cell in the egg mother cell can be predicted, is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity; Just may lay eggs after most of individual 7 days, referring to Fig. 7; If parent's egg mother cell still is a polygon; Average ovum footpath is less than 300 microns; And the yolk particle size in the egg mother cell is inhomogeneous; Can predict that be under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, just may lay eggs after most of individual 10 days, referring to Fig. 5 and Fig. 6.
Present embodiment is applicable to R&D institution and the Portunus trituberculatus Miers that the satisfies the requirements enterprise of growing seedlings.
More than show and described basic principle of the present invention, principal character and advantage of the present invention.The technical staff of the industry should understand; The present invention is not restricted to the described embodiments; That describes in the foregoing description and the specification just explains principle of the present invention; The present invention also has various changes and modifications under the prerequisite that does not break away from spirit and scope of the invention, and these variations and improvement all fall in the scope of the invention that requires protection.The present invention requires protection domain to be defined by appending claims and equivalent thereof.
Claims (3)
1. the method for a forecasting laying season of portunus trituberculatus may further comprise the steps,
(1) under the condition of not dissecting the female body parent of Portunus trituberculatus Miers, adopt sampling instrument drilling the sampling holes on the crust of the female body of Portunus trituberculatus Miers, to take a sample, the ovary tissue that takes a morsel after the sampling seals thief hole;
(2) inspection development of ovary situation, the sampling back is observed development of ovary situation through naked eyes or microscope;
(3) according to egg-laying period of the index prediction Portunus trituberculatus Miers of the histological observation of step (2);
Sampling instrument uses drill bit and tweezers in the step (1), and said drill bit and tweezers must prevent the courses of infection parent through strict sterilization before use, and bit diameter is the 0.8-1.2 millimeter;
Thief hole is positioned at the carapace leading edge on the crust described in the step (1), and near the position behind the eyes, the crust of said thief hole is more smooth, and the sampling back is the sealing thief hole easily;
Step (1) thief hole is sealed into the sampling back and adopts the thief hole on the plastic plate sealing crust of 502 glue and 0.5 millimeters thick, spends 2-3 minute then and treats after 502 adhesive curings parent to be put into seawater immediately;
The method in the egg-laying period of said step (3) prediction Portunus trituberculatus Miers is: 1. with the naked eye directly observe the ovary sample; If parent's egg mother cell has been free in the ovarian cavity; Egg mother cell is rounded or oval; Can predict that be under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, will lay eggs in most of individual 4 days; If parent's egg mother cell still is not free in the ovarian cavity; Then measure the ovum footpath and observe the egg mother cell shape at microscopically; If average ovum footpath is at 350 microns, and egg mother cell still mutually extruding be polygon, can predict; Is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, just may lay eggs after most of individual 7 days; If parent's egg mother cell still is a polygon, average ovum footpath is less than 300 microns, is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, may lay eggs after most of individual 10 days; Perhaps: 2. under anatomical lens, directly observe the ovary sample; If parent's egg mother cell has been free in the ovarian cavity; Egg mother cell is rounded or oval; Can predict that be under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, will lay eggs in most of individual 4 days; If parent's egg mother cell still is not free in the ovarian cavity, the histology of then observing egg mother cell through routine paraffin wax section back is measured the ovum footpath at microscopically; If average ovum footpath is at 350 microns; And egg mother cell still mutually extruding be polygon, yolk even particle size and be full of whole egg mother cell in the egg mother cell can be predicted; Is under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, just may lay eggs after most of individual 7 days; If parent's egg mother cell still is a polygon; Average ovum footpath is less than 300 microns; And the yolk particle size in the egg mother cell is inhomogeneous, can predict that be under the condition of 24-30ppt in day illumination greater than 12h, water temperature 16-19 ℃, salinity, may lay eggs after most of individual 10 days.
2. method according to claim 1 is characterized in that: the said observation development of ovary of step (2) situation, further inspection comprises and adopts fixer that ovary tissue is fixed, and carries out histological observation.
3. method according to claim 2 is characterized in that: said fixer is the BouinShi liquid of improvement or 3% formalin solution of improvement;
The prescription of the BouinShi liquid of said improvement is 2.4 milliliters of saturated picric acid solutions, 64.5 milliliters of formalin, 12.5 milliliters of glacial acetic acid solutions and 190 ml distilled waters;
3% formalin solution of described improvement is that 3% formalin is dissolved in the standard artificial seawater and prepares.
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| CN111149747B (en) * | 2020-02-23 | 2021-07-27 | 中国水产科学研究院黄海水产研究所 | Classified overwintering cultivation method for portunus trituberculatus |
| CN114128661B (en) * | 2021-11-17 | 2023-04-07 | 江苏大学 | Pond river crab accurate bait casting method based on river crab growth model |
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| CN101543191A (en) | 2009-09-30 |
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