Fluorescent staining method for evaluating sperm motility of domesticated mammals

A sperm motility and fluorescent staining technology, applied in the field of fluorescent staining, can solve the problems of limited use, expensive SYBR-14 probe, spectral overlap, etc., and achieve the effect of reducing the detection cost

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

AI Technical Summary

Problems solved by technology

However, since SYBR-14 emits green fluorescence, it is impossible to simultaneously detect the integrity of its acrosome when detecting sperm motility, because most of the fluorescent probes for detecting acrosome also emit green fluorescence, which is different from SYBR-14, which also emits green fluorescence. spectral overlap
In addition, the expensive SYBR-14 probe limits the widespread use of this method

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Weigh 3.69g glucose, 0.119g sodium chloride, 0.0403g potassium chloride, 0.126g sodium bicarbonate, 0.125g EDTA and 0.005g kanamycin, dissolve them in Milli-Q ultrapure water and dilute to 100mL, and use Adjust the pH to 7.2 with hydrogen chloride or sodium hydroxide, and filter with a 0.22 μm filter membrane to prepare porcine sperm dilution solution BTS and store it in a water bath at 37 °C; take a certain volume of fresh semen and properly dilute it with BTS to make the sperm concentration reach 10 × 10 6 Individuals / mL, take 1 mL to a 2 mL centrifuge tube, add 2 μL Hoechst33342 and 8 μL PI, incubate at 37°C for 15 minutes; take 8 μL semen on a glass slide, cover with a cover slip, detect and count at least 200 sperm under a fluorescent microscope , 202 sperm were actually counted this time, and the result was 166 live sperm and 36 dead sperm, so the motility of pig sperm was 82%.

Embodiment 2

[0058] Weigh 0.76g sodium chloride, 0.03g potassium chloride, 0.252g fructose, 0.238g HEPES, 0.015g calcium chloride, 0.01g magnesium chloride, 0.1g bovine serum albumin, dissolve in Milli-Q ultrapure water and determine The volume was adjusted to 100mL, the pH was adjusted to 7.4, and the 0.22μm pore size filter was filtered to make a bovine sperm dilution and stored in a water bath at 37°C; a certain volume of fresh semen was diluted 1:8 with Hepes-0.1% BSA, and the blood count After counting the plate, adjust the sperm concentration with diluent to make it reach 30 × 10 6 Individuals / mL, take 1 mL to a 2 mL centrifuge tube, add 2 μL Hoechst33342 and 8 μL PI, incubate at 37°C for 15 minutes; take 8 μL semen on a glass slide, cover with a cover slip, detect and count at least 200 sperm under a fluorescent microscope , 212 sperm were actually counted, and the result was 187 live sperm and 25 dead sperm, so the motility of bovine sperm was 88%.

Embodiment 3

[0060] Weigh 0.76g sodium chloride, 0.03g potassium chloride, 0.252g fructose, 0.238g HEPES, 0.015g calcium chloride, 0.01g magnesium chloride, 0.1g bovine serum albumin, dissolve in Milli-Q ultrapure water and determine The volume was adjusted to 100mL, the pH was adjusted to 7.4, and the goat sperm dilution was made by filtering with a filter membrane with a pore size of 0.22 μm, and stored in a water bath at 37 °C; a certain amount of fresh semen was diluted 1:3 with Hepes-0.1% BSA, and the hemocytometer After counting on the counting plate, adjust the sperm concentration with diluent to make it reach 30 × 10 6 Individuals / mL, take 1 mL to a 2 mL centrifuge tube, add 2 μL Hoechst33342 and 8 μL PI, incubate at 37°C for 15 minutes; take 8 μL semen on a glass slide, cover with a cover slip, detect and count at least 200 sperm under a fluorescent microscope , 205 sperm were actually counted, and the result was 160 live sperm and 45 dead sperm, so the motility of sheep sperm ...

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PUM

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Abstract

The invention discloses a fluorescent staining method for evaluating sperm motility of domesticated mammals. The fluorescent staining method comprises the following step: according to the characteristic that a mammal live sperm probe Hoechst 333422 and a mammal dead sperm probe PI can be excited by the same ultraviolet light to emit fluorescent light, performing fluorescent staining on sperms of four important domesticated mammals which are pigs, cows, sheep and dogs to accurately perform sperm mobility evaluation. Compared with the conventional PI/SYBR-14 staining method, the PI/Hoechst 33342 staining method adopts Hoechst 33342 to substitute an expensive SYBR-14 probe, so that the detecting cost is greatly lowered. The fluorescent staining method can be used for successfully evaluating the sperm mobility of important domesticated mammals such as pigs, cows, sheep and dogs, and can be widely applied to evaluating the sperm motility of various domesticated mammals.

Description

technical field [0001] The invention belongs to the technical field of animal reproduction, and in particular relates to a fluorescent staining method for evaluating sperm motility of several important domesticated mammals. Background technique [0002] Sperm motility (also known as sperm plasma membrane integrity) evaluation is an essential index in the detection of sperm quality. The detection methods mainly include ordinary staining, fluorescent staining and hypotonic swelling test. Although the hypotonic swelling test is simple to operate , The price is low, but it is rarely used now because of the single detection index and the inability to analyze by flow cytometry. [0003] Dyes commonly used in common dyeing methods include eosin, eosin-nigrosin, Giemsa, typan blue, Bismarck brown Y, Rose Bengal, etc. . The dyeing methods used include single dyeing method using only one dye, and double dyeing or triple dyeing method using two or more dyes at the same tim...

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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