Single ultraviolet light excited fluorescent staining method used for evaluating sperm activity of Kunming wolf dogs

A fluorescent dyeing and ultraviolet light technology, applied in the field of animal reproduction, can solve the problems of unseen and lack of data, and achieve the effects of accurate results, simple operation and high reliability.

Inactive Publication Date: 2015-04-08
YUNNAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
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  • Application Information

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Problems solved by technology

[0005] The Kunming dog is the only excellent stereotyped dog breed listed in the world's famous police dogs in China, and the only working dog breed independently bred in China. It plays an important role, and its germplasm resources are extremely valuable, but the data about the reproductive biology of Kunming dogs are still lacking, and there is no report about the fluorescent staining method of spermatozoa of Kunming dogs. The double fluorescent staining method of PI / Hoechst33342 of the present invention The evaluation of the activity of canine sperm in Kunming is the first in the world, so far, there is no report on the use of this method to detect the activity of canine sperm

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Weigh 2.4 g Tris, 1.4 g citric acid, 0.8 g glucose, 0.06 g penicillin G sodium salt and 0.1 g streptomycin sulfate into Milli-Q ultrapure water, mix well, add water to 100mL, adjust pH to 6.8 , to make TCG semen washing solution; transfer 2 mL of fresh semen collected from Kunming dogs to a sterilized 15 ml centrifuge tube, add 10 mL of TCG washing semen solution kept in a water bath at 37°C, centrifuge at 700 g for 5 min, discard the supernatant, and add 10 mL Centrifuge and wash the 37°C TCG semen once more, and resuspend the pellet with 2-3mL TCG buffer to make the sperm concentration 30×10 6 / mL; Take 1 mL of diluted semen, add 2 μL Hoechst 333422 and 8 μL PI respectively, and incubate at 37°C for 15 minutes; Take 8 μl of semen on a glass slide, cover with a cover slip, detect and count at least 200 spermatozoa under a fluorescent microscope , 213 sperm were actually counted this time, and the result was 172 live sperm and 41 dead sperm, so the sperm motility of ...

Embodiment 2

[0037]Weigh 2.4 g Tris, 1.4 g citric acid, 0.8 g glucose, 0.06 g penicillin G sodium salt and 0.1 g streptomycin sulfate into 60mL Milli-Q ultrapure water, dissolve and add 20mL egg yolk, mix well and add Milli-Q Q ultrapure water to 100mL, centrifuged at 7000g for 1 hour to remove yolk granules, took the supernatant to adjust the pH to 6.8, and made a dilution solution for cryopreservation, which is the TCG semen washing solution mentioned later; add Glycerin with a volume ratio of 10% was used to make 2× antifreeze; transfer 3 mL of fresh semen to a sterilized 15 mL centrifuge tube, add 10 mL of 37°C preheated TCG semen washing solution, and centrifuge at 700 g for 5 min to wash twice; Resuspend in 800μl TCG washing semen to make the sperm concentration 400×10 7 pcs / ml, take 50μl in a 4mL centrifuge tube, add 450μl of glycerin-free frozen diluent, and cool in an ice bath at 4°C for 2 hours; At intervals of 6 minutes, slowly add the cooled sperm, and continue to equilibrate ...

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Abstract

The invention discloses a single ultraviolet light excited fluorescent staining method used for evaluating sperm activity of Kunming wolf dogs. The method is realized by virtue of four steps of preparing a TCG sperm washing solution, washing sperms, staining and detecting with a microscope. According to the method disclosed by the invention, two types of fluorescent dyes which can be excited by ultraviolet light of a same wavelength are adopted to perform fluorescent staining on sperms of Kunming wolf dogs, and the sperm activity can be accurately identified without switching a light source; and moreover, one of the two types of fluorescent dyes emits red fluorescent light and the other one emits blue fluorescent light after laser excitation, so that the activity is detected, a dye emitting green fluorescent light can be introduced to detect acrosome integrality. The fluorescent staining method disclosed by the invention is simple to operate, accurate in result and high in reliability, and the sperm activity of the Kunming wolf dogs and the acrosome integrality can be accurately detected by using a fluorescence microscope or a flow cytometry.

Description

technical field [0001] The invention belongs to the technical field of animal reproduction, in particular to a fluorescent staining method excited by a single ultraviolet light for evaluating the sperm activity of Kunming dogs. Background technique [0002] Sperm activity is an essential criterion for evaluating sperm quality, which is mainly carried out by various staining methods. Non-fluorescent dyes such as eosin, Giemsa, and trypan blue have been used for the evaluation of canine sperm activity. This method can detect sperm activity with the help of an ordinary optical microscope, but it is cumbersome to use, and the proportion of dead sperm will be determined. The estimation is too high and the test results of different laboratories vary greatly. Therefore, the reliability of non-fluorescence staining method is far less than that of fluorescent staining method. [0003] Now, the detection of canine sperm motility usually uses propidium iodide (PI), Hoe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/06
Inventor 杨明华李亚辉
Owner YUNNAN AGRICULTURAL UNIVERSITY
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