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Method for detecting injury of mitochondria of sperms of fishes

A technology for fish sperm and mitochondria, which is applied to measurement devices, material analysis by optical means, instruments, etc., can solve the problems of cumbersome operation, inability to quantitative analysis and statistics, and expensive instruments, and achieve the effect of intuitive results.

Inactive Publication Date: 2010-09-29
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

The current method for detecting mitochondrial damage in fish sperm is to observe through scanning electron microscope and transmission electron microscope, but this method can only be used for qualitative observation from the appearance, not quantitative analysis and statistics, and the equipment is expensive and the operation is cumbersome

Method used

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Embodiment Construction

[0006] During the breeding season of fish, a certain amount of fresh fish sperm and sperm treated with low temperature were collected, and the two semen samples were washed twice with PBS (0.01mol / L, pH 7.4) and then centrifuged at 300×g for 5 min. Suspend and precipitate sperm in PBS, adjust the sperm density to 1×10 6 / ml, add Rhl23 (final concentration 10ug / ml), incubate in a water bath at 37°C for 10min, centrifuge to discard the supernatant, wash and centrifuge twice with PBS, suspend in PBS, add PI (final concentration 10ug / ml) for staining for 5min, use FACSCalibur flow Type cytometer (BD Company, USA) detection, instrument status setting: forward scatter FSC and side scatter SSC are linear amplification, fluorescence channels FL1 (green) and FL2 (red) are logarithmic amplification, measure the fluorescence intensity of each sperm, 10,000 spermatozoa were detected for each suspension, and the data were analyzed with Cell Quest software. The results of fresh semen and p...

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Abstract

The invention provides a method for detecting the injury of the mitochondria of the sperms of fishes, which relates to an aquaculture technology and needs to provide a method for detecting the injury of the mitochondria of the sperms of fishes. In the invention, the sperms of the fishes with a certain number are collected. The method is characterized by comprising the following steps of: after washing a sperm specimen by using PBS, centrifuging by 300*g for 5min; and suspending by the PBS, precipitating the sperms, regulating the density of the sperms to be 1*106 / ml, adding Rhl23, hatching for 10min in a water bath with the temperature of 37 DEG C, centrifuging, removing supernatant, washing by the PBS repeatedly, centrifuging, suspending by the PBS, adding PI and dyeing for 5min. The sperms with the normal mitochondria and the injured mitochondria are dyed by adopting a fluorescent double-dyeing method and are directly detected by using a flow type cell analyzer after being preprocessed, and the sperms with favorable mitochondrial membrane functions, the sperms with lost mitochondrial membrane functions and the sperms with necrosis can be quantitatively distinguished.

Description

technical field [0001] The invention belongs to aquaculture technology and relates to a method for detecting mitochondrial damage of fish sperm Background technique [0002] In aquaculture production, it is often necessary to induce spawning and breeding of fish, and the quality of fish sperm is a very important link in breeding, which directly affects the fertilization rate, hatching rate and survival rate. After fish sperm are excreted from the body, their vitality is easily affected by various external environmental factors. Different environmental conditions (temperature, light, etc.) will cause certain damage to the sperm, thereby affecting the quality of the sperm, reducing the fertilization rate, hatching rate and survival. rate, causing great losses to aquaculture production. Sperm mitochondria are the energy source to maintain the normal physiological functions of sperm. Mitochondria contain a variety of enzymes involved in energy metabolism, which provide the ener...

Claims

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Application Information

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IPC IPC(8): G01N21/952
Inventor 黄晓荣章龙珍庄平江琪姚志峰刘鉴毅张涛冯广朋赵峰闫文罡
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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