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Bivalve mollusc semen quality detection method

A technology for sperm quality and bivalve molluscs, applied in measuring devices, individual particle analysis, scientific instruments, etc., can solve the problems of plasma membrane integrity and mitochondrial activity, the inability to detect changes in sperm structure, and the impact of sperm quality, etc. problems, to avoid low accuracy, ensure accuracy, and improve accuracy

Inactive Publication Date: 2018-10-23
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Although CASA can measure parameters such as sperm density and motility, it has certain limitations and cannot detect changes in the internal structure of sperm, especially sperm that have been cryopreserved or stressed, because of their plasma membrane integrity and mitochondrial activity. Injured, significantly affecting the quality of sperm
In the study of the quality of shellfish sperm, the sperm are stained with two fluorescent dyes to make the sperm release fluorescence of different colors, and then randomly counted under a fluorescent microscope to count the survival rate of the sperm. Although this method can detect changes in the internal structure of the sperm , but there is a large error, and it is time-consuming and labor-intensive

Method used

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  • Bivalve mollusc semen quality detection method

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Effect test

Embodiment 1

[0023] A bivalve mollusk quality detection method and its application, the specific operation steps are as follows:

[0024] 1. Semen preparation: select well-developed oysters, select male individuals with well-developed gonads through anatomical microscopy, and use a straw to absorb sperm with better vitality as the sperm stock solution.

[0025] 2. Gradient seawater configuration: Use sterilized seawater, sea crystals and aerated freshwater to prepare gradient seawater with a salinity of 10, 15, 20, 25, 30, and 35, add the sperm stock solution to the gradient seawater, and adjust the sperm concentration to 10 7 ~10 8 individual / mL.

[0026] 3. Preparation of fluorescent dye solution: Dissolve PI and Rh123 in ultrapure water respectively to make a storage solution with a concentration of 1mg / mL, and store them at -20°C and -4°C respectively according to the reagent storage requirements.

[0027] 4. Semen staining: Dilute the two storage solutions to 100 μg / mL, take 160 μL...

Embodiment 2

[0032] A bivalve mollusk quality detection method and its application, the specific operation steps are as follows:

[0033] 1. Semen preparation: Anatomical microscopy, select the male oysters with well-developed glands, use a straw to absorb the sperm with better vitality as the sperm stock solution.

[0034] 2. Environmental temperature setting: Use heating rods, ice cubes, and refrigerators to set the temperature in the water bath environment at -20°C, 4°C, 14°C, 24°C, 34°C, 44°C, and 54°C, and adjust the sperm concentration to 10°C. 7 ~10 8 individual / mL, and then put the sperm into water baths at different temperatures.

[0035] 3. Preparation of fluorescent dye solution: Dissolve PI and Rh123 in ultrapure water respectively to make a storage solution with a concentration of 1mg / mL, and store them at -20°C and -4°C respectively according to the reagent storage requirements.

[0036]4. Sperm staining: Dilute the two storage solutions to 100 μg / mL, take 240 μL of sperm a...

Embodiment 3

[0040] A bivalve mollusk quality detection method and its application, the specific operation steps are as follows:

[0041] 1. Semen preparation: Microscopically examine the male scallops with well-developed selective glands, and use a straw to absorb the sperm with better vitality as the sperm stock solution.

[0042] 2. Gradient seawater configuration: Use sterilized seawater, sea crystals and aerated freshwater to prepare gradient seawater with a salinity of 5, 10, 15, 20, 25, 30, 35, and 40, and add the sperm stock solution to the gradient seawater. Adjust sperm concentration to 10 7 ~10 8 individual / mL.

[0043] 3. Preparation of fluorescent dye solution: Dissolve PI and Rh123 in ultrapure water respectively to make a storage solution with a concentration of 1mg / mL, and store them at -20°C and -4°C respectively according to the reagent storage requirements.

[0044] 4. Sperm staining: dilute the two storage solutions to 100 μg / mL, take 160 μL of the spermatozoa subjec...

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Abstract

The invention provides a bivalve mollusc semen quality detection method, in which fluorochrome double staining and flow cytometry are combined to detect the bivalve mollusc semen quality. The method includes steps of: adjusting concentration of semen, staining the to-be-detected semen with a membrane-permeable fluorochrome and a membrane-impermeable fluorochrome, and performing room temperature incubation and flow cytometry detection to obtain a detection result. The membrane-permeable fluorochrome can be combined with mitochondrial membrane while the membrane-impermeable fluorochrome can penetrate damaged cell membrane and be specifically combined with intracellular nucleic acids. Through one detection operation, a plurality of parameter results can be obtained. The method not only can clearly distinguish semen in different status and display ratio thereof, and obtain death rate, survival rate, plasma membrane damage rate and ratio of mitochondrial activity, but also can avoid low accuracy due to subjective factors and guarantee the accuracy of the detection results.

Description

technical field [0001] The invention relates to a method for detecting the quality of bivalve mollusk sperm, in particular to a method and application for detecting sperm survival rate, plasma membrane integrity and mitochondrial activity after the sperm is stressed. Background technique [0002] At present, the methods for testing the quality of bivalve mollusk sperm are all microscopic examination. Place the sperm under an optical microscope to observe the movement time, movement ratio and intensity of the movement. However, due to the different experience and judgment standards of each person, and the long time interval for observing the survival of sperm, this type of method often has greater subjectivity and inaccuracy, which is easy to affect the accuracy of the experiment and is easy to obtain. draw wrong conclusions. The application of computer-aided sperm analysis system (CASA) has greatly improved the research on sperm quality. Through the analysis of sperm traje...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N15/14
CPCG01N15/14
Inventor 王昭萍杜俊鹏于瑞海马培振
Owner OCEAN UNIV OF CHINA
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