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Evaluation method for survival rate and quality of prawn sperms

An evaluation method and technology of survival rate, applied in the field of biological cell viability analysis, can solve the problems of large error, poor evaluation effect, limited evaluation information, etc., and achieve the effects of easy reading, fast and accurate results, and reduced interference.

Inactive Publication Date: 2014-02-05
GUANGXI ACADEMY OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention: to overcome the shortcomings of the existing prawn sperm quality and survival rate evaluation methods, such as large errors, poor evaluation effects, and limited evaluation information, to provide an evaluation of the prawn sperm survival rate and quality based on biological staining and nucleic acid staining method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Step 1. First use the trypsin digestion method to separate the free prawn sperm to obtain the sperm suspension, that is, use 1g / L trypsin to digest the free sperm. The specific operation steps are: add 3mL trypsin solution and 3 Sperm pods were digested in a water bath at 37°C for 5 min, then 1 mL of 15% (V / V) fetal bovine serum was added to terminate the digestion reaction, centrifuged at 500 r / min for 10 min, and the collected supernatant was evenly distributed among three 1.5 mL centrifuge tubes at 2000 r / min Centrifuge for 5 minutes, discard the supernatant, add 1mL sterilized natural seawater to each tube and blow gently to obtain free prawn sperm suspension, and then use chemical methods to treat the sperm suspension to obtain prawn sperm with different degrees of damage.

[0035] Step 2, then carry out biological staining and observation statistics: use sterilized natural seawater to prepare a staining solution, the staining solution contains 5g / L eosin and 100g / L...

Embodiment 2

[0046] Step 1, first use the trypsin digestion method to separate free prawn sperm, and then use chemical methods to obtain sperm suspensions with different degrees of damage. The operation steps are the same as in Example 1.

[0047] Step 2, then carry out biological staining and observation statistics: the staining solution of this embodiment contains eosin of 6g / L and nigrosin of 95g / L, the staining solution is fully mixed, and the volume ratio of staining solution:sperm suspension is 1ul : Take 9ul of the two solutions respectively and place them on a glass slide to mix well, let stand at room temperature for 4min, and use Example 1 for the remaining steps.

[0048] Step 3, followed by single-cell gel electrophoresis, the specific operation includes steps:

[0049] a. Dilute, lyse and digest protein: take 50ul of sperm suspension, centrifuge and wash the sperm suspension twice with pH=7.4 phosphate buffer PBS, then dilute the free sperm with pH=7.4 phosphate buffer PBS to ...

Embodiment 3

[0057] Step 1, first use the trypsin digestion method to separate free prawn sperm, and then use chemical methods to treat the sperm to obtain prawn sperm suspensions with different damage degrees. The operation steps are the same as in Example 1.

[0058] Step 2, then carry out biological staining and observation and statistics: the dyeing solution in this example contains 4g / L eosin and 90g / L nigrosin, and the dyeing solution is fully mixed before use, and the volume ratio of the sperm suspension is 1.5ul : 8.5ul of the two solutions were respectively placed on a glass slide and mixed evenly, left standing at room temperature for 1min, and the remaining steps were performed according to Example 1.

[0059] Step 3, followed by single-cell gel electrophoresis, the specific operation includes steps:

[0060] a. Dilute, lyse and digest protein: Take 80ul of sperm suspension, centrifuge and wash the sperm suspension 3 times with pH=7.4 phosphate buffer PBS, then dilute the free s...

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Abstract

The invention discloses an evaluation method for the survival rate and the quality of prawn sperms. The evaluation method is applicable to evaluating the survival rate of prawns treated by a chemical method and the damaging degree of the sperms of the prawns; a mixed biological dye of eosin and nigrosine is used for clearly distinguishing dead and live sperms under an optical microscope and the survival rate of the sperms is counted; a single sperm dyed by GelRed is fixed by using single cell gel and sperm DNA (Deoxyribonucleic Acid) is dispersed by electrophoresis; a migration track of the DNA of the damaged sperm can be observed under a luminescence microscope and the comet rate and the damaging coefficient are counted by CASP (Computer Automated Stowage Planning) analysis software so as to further evaluate the damaging degree and the quality of the prawn sperms. According to the evaluation method, a suitable mixed biological dyeing solution is adopted and cells are clearly distinguished after being dyed; a background color has small interferences on observation and a dyeing result is easy and rapid to read; the result is accurate and specific; a single cell gel electrophoresis method can further identify the damaging condition of the sperms and can effectively and accurately obtain evaluation information of the survival rate and the quality of the prawn sperms.

Description

technical field [0001] The invention belongs to the technical field of biological cell viability analysis, and relates to a method for evaluating the survival rate and quality of shrimp sperm based on biological staining and nucleic acid staining. Background technique [0002] Prawns belong to Arthropoda, branchial subphylum, crustacea, soft armor subclass, decapod order, swimming suborder, prawn family, and genus prawns. Litopenaeus vannamei is one of the main species of the crustacean genus Penaeus. The sperm of Litopenaeus vannamei has no flagella and cannot move. Due to its characteristics, the evaluation of its quality and vitality has become a difficult problem. For this reason, many researchers are actively improving and developing reliable crustacean sperm quality and Methods for assessing survival rates. These methods include: sperm count and morphology, biological staining, hypotonic exhalation, sperm-egg interaction, biochemical analysis, sperm acrosome reac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N27/447G01N1/30
Inventor 杨春玲陈秀荔赵永贞陈晓汉李咏梅彭敏
Owner GUANGXI ACADEMY OF FISHERY SCI
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