CN104818314A - Transparent liquid for rapidly identifying oocyte nuclei of shrimps and crabs - Google Patents
Transparent liquid for rapidly identifying oocyte nuclei of shrimps and crabs Download PDFInfo
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- CN104818314A CN104818314A CN201510241280.XA CN201510241280A CN104818314A CN 104818314 A CN104818314 A CN 104818314A CN 201510241280 A CN201510241280 A CN 201510241280A CN 104818314 A CN104818314 A CN 104818314A
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Abstract
The invention relates to the field of aquaculture, and provides a transparent liquid which can be used for rapidly transparentizing oocytes of shrimps and crabs and a method for treating and detecting maturity conditions of the oocytes of the shrimps and crabs by using the transparent liquid. The transparent liquid is a mixture which is prepared from anhydrous ethyl alcohol, glacial acetic acid and formaldehyde at a volume ratio of 60:1:30. The transparent liquid provided by the invention can be used for treating the oocytes of the shrimps and crabs, can be used for effectively transparentizing the oocytes of the shrimps and crabs, and can be used for rapidly identifying germinal vesicles of the oocytes of the shrimps and crabs.
Description
Technical field
The present invention relates to aquaculture field, be specially a kind of method processing shrimp crab ovum, and for the transparent liquid of rapid examination shrimp crab ovum germinal vesicle.
Background technology
Shrimp waste crustacean is important aquaculture kind, and the generation of ovocyte mainly comprises three periods: proliferation period, vegetative period and ripening stage.Growth period residing for accurate judgement ovocyte, for grasping the time of carrying out artificial induction's oocyte maturation on producing in time, there is important practical value, can shorten the cycle of shrimp crab human assistance nursery, be also the last ripe machine-processed requisite step of research Shrimp waste ovum simultaneously.
Similar to other polylecithal egg species, shrimp crab ovocyte is in synthesis in vegetative period and lay in a large amount of yolk, for the use of the fetal development of after fertilization.Its ovocyte is after completing yolk accumulation vegetative period, enter the last meiotic maturation stage, this stage oocytes germinal vesicle (egg nucleus, nucleus) position first move around mediad ovocyte, then nuclear membrane disintegrates disappearance, form reduction division and comprise spindle body, chromosomal formation etc. mutually, finally rest on I meiosis metaphase, could ovulate afterwards, be fertilized, therefore, can developmental stage residing for the presence or absence of ovocyte germinal vesicle or position judgment ovocyte.
Due to the accumulation of a large amount of Yolk, make the light penetrability of shrimp crab ovocyte more weak, therefore, with the naked eye be all difficult to observe directly germinal vesicle with anatomical lens, usually prawn crab ovary tissue is only had to carry out prior paraffin section at present, observed the germinal vesicle of ovum by hematoxylin-eosin staining (HE dyeing) afterwards, thus determine the developmental stage of ovum.But a series of loaded down with trivial details testing sequences such as paraffin section method needs to be fixed ovary sample, rinsing, dehydration, waxdip, embedding, section, dyeing, for the experimenter of a skilled operation, whole experiment flow also will spend the time of about one week.In addition, because ovocyte yolk is more and the existence of oocyte membrane, also often there will be in the process making section because waxdip does not thoroughly cause film-making failure.In addition, because ovocyte volume after experiencing vegetative period increases, nuclear-cytoplasmic ratio is little, when cutting into slices because tangent plane easily causes the illusion of ovocyte germinal vesicle breakdown without nucleus, causes misjudgment, thus affects to production and scientific research.
Forefathers are when studying zebra fish ovum, process with the ovocyte of transparent liquid (methyl alcohol: Glacial acetic acid=3:1) to accumulation yolk, germinal vesicle position (Lee's book goose feather can be observed clearly, Korean, Sun Zhiyuan, Yan Wei, Chen Huiping, sternly continue the maturation in vitro of chin or cheek .. zebra fish ovocyte and the fertilization growth of mature egg. biotechnology journal .1993; 04:314-9+96).To in the Oocyte Development maturation research of other economic fish, other two kinds of transparent liquid are used again respectively: fast transparent liquid and turpentine alcohol transparent liquid.Wherein the formula of fast transparent liquid is: 95% ethanol 85 parts, 10 parts, the formaldehyde of 40%, 5 parts, Glacial acetic acid.It can be transparent by ovocyte in 2-3 minute, and to observe its germinal vesicle, but treatment time time-out will make germinal vesicle also transparent.And turpentine alcohol transparent liquid also can keep germinal vesicle not transparent for a long time while fast transparent ovocyte, its formula is: turpentine alcohol 25 parts, 50% alcohol 50 parts, 25 parts, Glacial acetic acid (white victory etc. of losing is write. and freshwater aquiculture 500 is asked. Beijing: Golden Shield press .2006.06).
By observe the ovocyte germinal vesicle of transparent liquid process position or with or without, determine that the method in Oocyte Development period is quick, easy, and more accurate compared with the result of HE dyeing.But, because the yolk accumulated in ovocyte contains the different material such as multiple proteins, lipid, carbohydrate, and the composition of different species yolk is very different, thus can the transparent liquid of effective transparent fish ovocyte but can not the ovocyte of transparent Shrimp waste biology.Therefore need to explore a kind of formula being applicable to the transparent liquid of transparent Shrimp waste ovocyte.
Summary of the invention
The present invention aim to provide a kind of can be used in fast transparent shrimp crab ovocyte transparent liquid and by this transparent liquid process and the method checking shrimp crab shrimp crab ovocyte germinal vesicle.
Technical scheme of the present invention is, a kind of transparent liquid for the treatment of Shrimp waste ovocyte, and be dehydrated alcohol, Glacial acetic acid and formaldehyde mixture, its volume ratio is 60:1:30.
Above-mentioned transparent liquid can be applicable to process Shrimp waste ovocyte, the ovocyte of the effective transparent shrimp crab of energy, and clearly can observe an opaque speckle in the ovocyte that germinal vesicle does not break.Detect through paraffin section HE, be confirmed that it is the germinal vesicle (i.e. nucleus) of ovum.Therefore, above-mentioned transparent liquid can be used for Rapid identification Shrimp waste ovocyte germinal vesicle (nucleus).A kind of method of Rapid identification Shrimp waste ovocyte germinal vesicle, step is: the ovary tissue of Shrimp waste is placed in described transparent liquid, vibration makes ovocyte be unbound state, leaves standstill 1 ~ 60 minute, checks the state of the free ovocyte germinal vesicle being placed in transparent liquid.
Preferably, hunting speed is 50 ~ 800rpm, and duration of oscillation is 2 ~ 20min; It is preferred, hunting speed is 100 ~ 500rpm, and duration of oscillation is 5 ~ 15min.
Preferably, time of repose is 1 ~ 5 minute.
Preferably, described Shrimp waste is mitten crab or Macrobrachium rosenbergii.When Shrimp waste is mitten crab, in step (1), mitten crab ovocyte can become safran from purple or black.
Due to a large amount of yolk of ovocyte accumulation, cause the germinal vesicle that cannot observe directly Shrimp waste ovocyte, and the method required time of routine paraffin wax section HE dyeing is long, complex operation, and production and scientific research have certain limitation.The present invention by selecting the composition of transparent liquid, and adjusts the ratio of dehydrated alcohol, glacial acetic acid and formaldehyde, obtains the transparent liquid of the effective transparent Shrimp waste ovocyte of a kind of energy.The Shrimp waste ovary of this transparent liquid process maturation, can obtain the ovocyte of transparence, and in ovocyte, find that there is little, an opaque speckle, detects, be confirmed that it is the germinal vesicle of ovocyte with paraffin section HE.This illustrate this transparent liquid can fast and effectively transparent shrimp crab ovocyte thus convenient observe its germinal vesicle.Transparent liquid of the present invention is easy to use, and authentication method is simple and quick, and qualification result is accurate, provides important help by for determining shrimp crab egg cell development mature condition, shortening the shrimp crab artificial breeding time and studying the last ripe molecular mechanism of its ovocyte.
Accompanying drawing explanation
Fig. 1 is mitten crab and Macrobrachium rosenbergii without transparent liquid process, ovocyte through transparent liquid process, and the paraffin section result of mitten crab and Macrobrachium rosenbergii ovary.In each picture, the germinal vesicle arrow of ovocyte is pointed out.
Wherein A and D is respectively mitten crab and the Macrobrachium rosenbergii ovocyte without transparent liquid process; B and E is respectively mitten crab and the Macrobrachium rosenbergii ovocyte through transparent liquid process; C and F is respectively the paraffin section result of mitten crab and Macrobrachium rosenbergii ovary.
Embodiment
Material agents: mitten crab and Macrobrachium rosenbergii are purchased from the market of farm produce, orchard, Shanghai Pudong New Area, and wherein, mitten crab body weight is 90-100g, and Macrobrachium rosenbergii body weight is 15-20g.The chemical reagent such as dehydrated alcohol, glacial acetic acid, formaldehyde, dimethylbenzene, methyl alcohol, purchased from Chemical Reagent Co., Ltd., Sinopharm Group (Shanghai).4% paraformaldehyde stationary liquid, phenodin, Yihong dyestuff are purchased from Sangon Biotech (Shanghai) Co., Ltd..
Embodiment 1 transparent liquid process mitten crab ovocyte
I. transparent liquid treatment process:
1, the preparation of transparent liquid: get dehydrated alcohol, glacial acetic acid and the formaldehyde volume ratio preparation transparent liquid according to 60:1:30, now with the current.
2, ovocyte is transparent: dissected by mitten crab, the ovary tissue getting 100 milligrams is placed in the centrifuge tube of 15 milliliters, add the transparent liquid of about 3 milliliters, rapid concuss (100 ~ 500rpm), make ovocyte be unbound state, the color of mitten crab ovocyte can become safran from black or purple.
3, leave standstill 1 ~ 2 minute (or the longer time, be no more than 60 minutes).
4, ovocyte and transparent liquid are transferred on culture dish or slide glass, are placed in the dissection Microscopic observation that background color is black.Result, as Figure 1B, finds to have in one end of ovocyte the spot of a little White-opalescent.Ovocyte continues to soak more than 1hr in transparent liquid, and germinal vesicle is not transparent yet.
II. conventional organization section (HE method) is verified the result of transparent liquid process, and method is as follows:
1, the ovary tissue getting same mitten crab is placed in paraformaldehyde stationary liquid and fixes, and fixes 24 hours for 4 DEG C.
2, the ovary tissue 1XPBS damping fluid fixed washes 3 times, each 30 minutes.After having rinsed, sample is placed in methyl alcohol ,-20 DEG C save backup.
3, the sample preserved, through the ethanol dehydration of different concns gradient, the steps include: 70% ethanol, 1 hour; 80% ethanol, 1 hour; 95% ethanol I, 45 minutes; 95% ethanol II, 45 minutes; Dehydrated alcohol I, 45 minutes; Dehydrated alcohol II, 45 minutes.
4, dimethylbenzene is transparent, the steps include: the alcohol of 1:1, dimethylbenzene, 40 minutes; Dimethylbenzene I, 15 minutes; Dimethylbenzene II, 15 minutes.
5, waxdip: the dimethylbenzene of 1:1, paraffin and paraffin were positioned in 60 degree of baking ovens in 1 hour in advance, make it fully dissolve.The step of sample waxdip is: the dimethylbenzene of 1:1, paraffin, 45 minutes; Paraffin I, 1 hour; Paraffin II, 2 hours.
6, embed: in the papery capsule folded, fill paraffin, sample picks up by rapid tweezers from paraffin, puts into the carton filling paraffin, is placed in smooth ice chest by carton.Be transferred to after the paraffin in carton in 4 degree of refrigerators, spend the night.
7, wax is repaiied: with the paraffin around scalper resection organization, accomplished prism-frustum-shaped, be then fixed on wood particle.
8, cut into slices: with rotary microtom, paraffin-embedded ovary tissue is cut into 6 microns of slabs.
9, paster, baking sheet: drip on several clear water and slide glass, then the section tweezers cut are picked up, be put on slide glass, pave.Finally slide is placed in the baking oven of 37 degrees Celsius, dries sheet more than 24 hours.
10, HE dyeing, concrete steps are: dimethylbenzene I, 10 minutes; Dimethylbenzene II, 10 minutes; The ethanol of 1:1, dimethylbenzene, 5 minutes; Dehydrated alcohol, 2 minutes; The ethanol of 95%, 2 minutes; The ethanol of 90%, 2 minutes; The ethanol of 80%, 2 minutes; The ethanol of 70%, 2 minutes; Distilled water, 2 minutes; Phenodin, 20 minutes; Running water, 30 minutes; The hydrochloric acid of 1:99, ethanol, 2 seconds; Running water, 20 minutes; The ethanol of 70%, 2 minutes; The ethanol of 80%, 2 minutes; The ethanol of 90%, 2 minutes; Yihong of 0.5%, 30 seconds; The ethanol of 95%, 2 minutes; Dehydrated alcohol, 2 minutes; The ethanol of 1:1, dimethylbenzene, 5 minutes; Dimethylbenzene I, 5 minutes; Dimethylbenzene II, 2 minutes.Result is as the C of Fig. 1.
Result: dissect and find, the Mitten Hand Crab Ovaries of growth and maturity is intense violet color or black, is almost full of whole abdominal cavity.Dissect Microscopic observation without the ovocyte of transparent liquid process, the shape such as rounded, oval, is full of yolk (Figure 1A) in cell; Free ovocyte through transparent liquid process is placed in the dissection Microscopic observation that background color is black, the transparent state of cell, and find to have in one end of ovocyte the spot (Figure 1B) of a little White-opalescent, confirm through HE method, for the germinal vesicle (Fig. 1 C) of skew occurs ovocyte.Consistent by the result of transparent liquid and the qualification of HE method.
The ovocyte of embodiment 2 transparent liquid process Macrobrachium rosenbergii
Along with the accumulation of yolk, Macrobrachium rosenbergii ovary is grown gradually and is become large, in last whole carapace, be almost filled with orange-yellow ovary.The shapes such as the ovocyte of dissecting Microscopic observation free is rounded, oval, are full of yolk (Fig. 1 D) in cell.The method that transparent liquid process and HE detect is with embodiment 1.
The formula of transparent liquid is with embodiment 1, and the ovary tissue getting 100 milligrams is placed in the centrifuge tube of 15 milliliters, adds the transparent liquid of about 3 milliliters, rapid concuss (100 ~ 500rpm), makes ovocyte be unbound state; And leave standstill 1 ~ 2 minute (or the longer time, be no more than 60 minutes).After process, the ovum color of Macrobrachium rosenbergii can be thin out.
The process of Oocytes of Macrobrachium Rosenbergii transparent liquid is placed on the dissection Microscopic observation that background color is white, the transparent state of cell, and find there is an opaque black splotch (Fig. 1 E) in ovocyte, HE is confirmed that it is the germinal vesicle (Fig. 1 F) of Oocytes of Macrobrachium Rosenbergii.Consistent by the result of transparent liquid and the qualification of HE method.
Ovocyte continues to soak more than 1hr in transparent liquid, and germinal vesicle is not transparent yet.
Reference examples
With dehydrated alcohol, glacial acetic acid and formaldehyde according to the mixing of 60:5:20,50:1:20,65:1:30,60:1:35 equal-volume ratio, or mix by 3:1 volume ratio with Glacial acetic acid with methyl alcohol, by the method process mitten crab of embodiment 1 and 2 and the ovary of Macrobrachium rosenbergii, can not there is vitreous state in the ovocyte after free.
Claims (10)
1. for a transparent liquid for Rapid identification shrimp crab ovocyte germinal vesicle, it is characterized in that, be made up of the dehydrated alcohol of volume ratio 60:1:30, Glacial acetic acid and formaldehyde.
2. transparent liquid according to claim 1 make shrimp crab ovocyte transparent in application.
3. the application of transparent liquid according to claim 1 in qualification shrimp crab ovocyte germinal vesicle.
4. the application described in Claims 2 or 3, is characterized in that, described shrimp crab is mitten crab or Macrobrachium rosenbergii.
5. a method for Rapid identification shrimp crab oocyte nuclei, it is characterized in that, step comprises:
(1) mixed with transparent liquid according to claim 1 by the ovary tissue of shrimp crab, vibration makes ovocyte be unbound state;
(2) leave standstill 1 ~ 60 minute, check the state of the free ovocyte germinal vesicle being placed in transparent liquid.
6. method according to claim 5, is characterized in that, in step (1), the amount ratio of described ovary tissue and transparent liquid is 100mg:1 ~ 10ml; Hunting speed is 50 ~ 800rpm, and duration of oscillation is 2 ~ 20min.
7. method according to claim 5, is characterized in that, in step (1), hunting speed is 100 ~ 500rpm, and duration of oscillation is 5 ~ 15min.
8. method according to claim 5, is characterized in that, in step (2), the time left standstill is 1 ~ 5 minute.
9. method according to claim 5, is characterized in that, described shrimp crab is mitten crab or Macrobrachium rosenbergii.
10. the method described in claim 5 or 9, is characterized in that, in step (1), vibration makes ovocyte be unbound state and becomes safran.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106982766A (en) * | 2017-04-01 | 2017-07-28 | 浙江海洋大学 | A kind of method for predicting laying season of portunus trituberculatus |
| CN110398405A (en) * | 2019-08-26 | 2019-11-01 | 贵州大学 | The efficiently method of production goat uterine tissue paraffin embedding |
| WO2022001825A1 (en) * | 2020-07-01 | 2022-01-06 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Kit for detecting e-cadherin expression of peripheral blood circulating tumor cells of pancreatic cancer patient and detection method |
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| CN102127521A (en) * | 2010-12-25 | 2011-07-20 | 河南科技大学 | Method for removing mouse oocyte nuclei by adopting zona pellucida solution cavity method |
| CN102627687A (en) * | 2012-03-23 | 2012-08-08 | 上海海洋大学 | Eriocheir sinensis GnRH analogue, and preparation method and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106982766A (en) * | 2017-04-01 | 2017-07-28 | 浙江海洋大学 | A kind of method for predicting laying season of portunus trituberculatus |
| CN106982766B (en) * | 2017-04-01 | 2020-12-18 | 浙江海洋大学 | Method for predicting egg laying period of portunus trituberculatus |
| CN110398405A (en) * | 2019-08-26 | 2019-11-01 | 贵州大学 | The efficiently method of production goat uterine tissue paraffin embedding |
| WO2022001825A1 (en) * | 2020-07-01 | 2022-01-06 | 天津市肿瘤医院（天津医科大学肿瘤医院） | Kit for detecting e-cadherin expression of peripheral blood circulating tumor cells of pancreatic cancer patient and detection method |
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