CN104498583B - Fluorescent staining method to domestic mammals evaluating sperm activity - Google Patents
Fluorescent staining method to domestic mammals evaluating sperm activity Download PDFInfo
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Abstract
A kind of fluorescent staining method to domestic mammals evaluating sperm activity of disclosure, do you utilize mammal sperm probe Hoechst alive? 333422 and Necrospermia probe PI can be gone out the feature of fluorescence by same ultraviolet excitation, pig, cattle, sheep, the important domestic mammals sperm of dog 4 kinds are carried out fluorescence staining, carries out evaluating sperm activity exactly. With traditional PI/? is SYBR-14 staining compared, PI/? Hoechst? 33342 staining Hoechst? 33342? instead of the SYBR-14 probe of costliness, make testing cost be substantially reduced. The present invention is used successfully to the evaluation of the important domestic mammals motility of sperm such as pig, cattle, sheep, dog, can be widely applied to the evaluating sperm activity of various domestic mammals.
Description
Technical field
The invention belongs to technical field of animal reproduction, particularly relate to the fluorescent staining method to several important domestic mammals evaluating sperm activities.
Background technology
It is a requisite index in sperm quality detection that motility of sperm (also referred to as plasmalemmae of sperms integrity) is evaluated, its detection method mainly has common dyeing method, fluorescent staining method and hypotonic swelling experiment etc., although hypotonic swelling experiment is simple to operate, cheap, but because Testing index is single, cannot be rarely employed now through reasons such as flow cytometry analysis.
The dyestuff that common dyeing method is commonly used has eosin (eosin), eosin-nigrosine (eosin-nigrosin), Jim Sa, platform to expect orchid (typanblue), bismarck brown (BismarckbrownY), bengal rose (RoseBengal) etc. Colouring method used has only single stain with a kind of dyestuff, also has and uses the double; two of two or more dyestuff to contaminate or three stainings simultaneously. The double-staining that wheat palm fibre and bengal rose combination use like that can be used for the detection of human spermatogoa; Jim Sa and platform expect blue combine for cattle, pig, rabbit sperm dyeing; Eosin-nigrosine staining is expected in the detection for animal sperms such as people, mice, horse, sheep, pigs of three stainings of blue combination for detection and bismarck brown, bengal rose and the platform of human spermatogoa. Plasmalemmae of sperms can be made detection by ordinary optical microscope by above-mentioned staining, but these methods use comparatively laborious, Necrospermia is more too high than regular meeting is estimated, and the testing result of different experiments room differs greatly, therefore, the credibility of non fluorescent stain method can not show a candle to fluorescent staining method.
The fluorescent probe that detection plasmalemmae of sperms integrity is conventional has iodate the third ingot (propidiumiodide, PI), Hoechst33258, ethidium bromide (EB), ethidium dimer (ethidiumhomodimer, EH), Yo-Pro-1, SYBR-14, CF 5(6)-Carboxyfluorescein diacetate (carboxyfluoresceindiacetate, CFDA), carboxyl dimethyl fluorescein diacetate (carboxydimethylfluorescceindiacetate, CMFDA), Carboxy-SNARF-1, SYTO-17 etc. Wherein, PI, Hoechst33258, EB, EH and Yo-Pro-1 are the specific fluorogenic probe of Necrospermia, and the sperm that they can only enter plasma membrane damaged is combined with DNA, and therefore the sperm of only plasma membrane breakage just can be colored.SYBR-14, CFDA, CMFDA, Carboxy-SNARF-1 and SYTO-17 are the specific fluorogenic probe of sperm of living, and they can enter the sperm that cell membrane is complete. Carboxy-SNARF-1 is pH indicator in a kind of born of the same parents, sends out fluorescent red-orange living in sperm, the nucleus of SYTO-17 labelling sperm alive, sends out red fluorescence. CFDA and CMFDA is the substrate of various nonspecific esterase, it themselves it is a class non-fluorescence material, cell can be entered and be easily hydrolyzed by intracellular nonspecific esterase, form hyperfluorescence product, fluoresced green, but extracellular of can not overflowing, therefore has the sperm fluoresced green of intact cell film. Want the considered critical time when evaluating plasmalemmae of sperms integrity with CFDA and CMFDA, constantly strengthen because intracellular Fluorescence elapses over time. SYBR-14 is then very stable, and it can reach balance at short notice with nucleic acid. Therefore, with compared with the staining of esterase hydrolyzed, SYBR-14 staining has dyeing and is not subject to time effects and the advantage being absent from background stainings.
At present, mammalian sperm fluorescent staining method has only single stain with a kind of dyestuff, also have and use two kinds of dyestuffs (such as PI/SYBR-14 simultaneously, PI/CFDA coupling) double fluorescent staining, it is generally believed that in all methods of motility of sperm detection, the specific fluorogenic probe PI of Necrospermia is best with the use in conjunction effect of the specific fluorogenic probe SYBR-14 of sperm of living, the life or death of sperm can be detected easily simultaneously, clearly Necrospermia is separated with sperm zone of living. PI/SYBR-14 staining is suitable for the detection of various animal sperm plasma membrane, classical report has in the sperm detection using it for the species such as cattle, pig, sheep, rabbit, mice, people, under fluorescence microscope, the sperm that plasma membrane is complete is green, the sperm of plasma membrane breakage takes on a red color, and is in and is then sent out two kinds of fluorescence by the sperm of dead transitive state of living simultaneously. But due to SYBR-14 fluoresced green, just cannot take into account to detect its acrosomal integnity when detecting spermatozoon activity simultaneously, because the most also fluoresced green of the fluorescent probe of detection acrosome, with the SYBR-14 spectra overlapping of same fluoresced green. Additionally, SYBR-14 probe is expensive, limit widely using of this method.
Hoechst33342 be a kind of can the blue fluorescent dyes of permeates cell membranes, the toxicity of cell is relatively low, also it is a kind of membrane permeability vital stain, but, with other vital stains the difference is that Hoechst33342 energy labelling sperm alive and Necrospermia, send out blue-fluorescence, on this basis, we develop PI/Hoechst33342 plasmalemmae of sperms double fluorescent staining before, blue-fluorescence is sent out the difference is that sperm of living by institute, Necrospermia sends out red fluorescence, use it for macaque, machin, rat, in the plasmalemmae of sperms detection of the common experimental animals such as mice, acquired results is consistent with the result of PI/SYBR-14 staining, but owing to instead of SYBR-14 with Hoechst33342, experimental cost is made to be substantially reduced, and, while detection membrane integrity, also can introduce the acrosome probe acrosomal integnity to sperm make evaluation.
Summary of the invention
It is an object of the invention to for the deficiency existed in above-mentioned art methods, it is provided that the fluorescent staining method to several important domestic mammals evaluating sperm activities, can be widely applied to the evaluating sperm activity of various domestic mammals. Through test, the present invention can be used successfully to the evaluation of the important domestic mammals motility of sperm such as pig, cattle, sheep, dog, and, the work animal sperm vigor such as pig, cattle, sheep, dog being evaluated with PI/Hoechst33342 dual staining has no report.
The technical solution used in the present invention is as follows:
Fluorescent staining method to domestic mammals evaluating sperm activity, step is as follows:
(1) Boar spermatozoa diluent BTS preparation
Weigh 3.69g glucose, 0.119g sodium chloride, 0.0403g potassium chloride, 0.126g sodium bicarbonate, 0.125gEDTA and 0.005g kanamycin, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, regulate pH to 7.2, with 0.22 ��m of aperture membrane filtration, make Boar spermatozoa diluent BTS;
(2) pig semen processes
Take the fresh semen of certain volume, dilute with Boar spermatozoa diluent BTS, make sperm concentration reach 10 �� 106/mL, take 1 ~ 2mL and dye in centrifuge tube;
(3) staining of sperm
In seminal fluid after above-mentioned 1 ~ 2mL dilutes, add 2 �� L blue fluorescent dyes Hoechst33342 and 8 �� L propidium iodide PI, 37 DEG C of temperature bath 15min;
(4) microscopic examination
Take the seminal fluid after 8 �� L dyeing on microscope slide, covered, detects under fluorescence microscope and counts.
Fluorescent staining method to domestic mammals evaluating sperm activity, step is as follows:
(1) cattle spermatozoa diluent preparation
Cattle sperm and sheep spermatozoa diluent Hepes-0.1%BSA preparation
Weigh 0.76g sodium chloride, 0.03g potassium chloride, 0.252g fructose, 0.238gHEPES, 0.015g calcium chloride, 0.01g magnesium chloride and 0.1g bovine serum albumin, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, regulate pH to 7.4, with 0.22 ��m of aperture membrane filtration, make cattle spermatozoa diluent Hepes-0.1%BSA;
(2) cattle liquefacient duration
Take the fresh semen of certain volume, it is diluted with cattle spermatozoa diluent Hepes-0.1%BSA 1:8 in mass ratio, adjust sperm concentration with Hepes-0.1%BSA again after blood counting chamber counting, make sperm concentration reach 30 �� 106/mL, take 1 ~ 2mL and dye in centrifuge tube;
(3) staining of sperm
In seminal fluid after above-mentioned 1 ~ 2mL dilutes, add 2 �� L blue fluorescent dyes Hoechst33342 and 8 �� L propidium iodide PI, 37 DEG C of temperature bath 15min;
(4) microscopic examination
Take the seminal fluid after 8 �� L dyeing on microscope slide, covered, detects under fluorescence microscope and counts.
Fluorescent staining method to domestic mammals evaluating sperm activity, step is as follows:
(1) sheep spermatozoa diluent preparation
Weigh 0.76g sodium chloride, 0.03g potassium chloride, 0.252g fructose, 0.238gHEPES, 0.015g calcium chloride, 0.01g magnesium chloride and 0.1g bovine serum albumin, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, regulate pH to 7.4, with 0.22 ��m of aperture membrane filtration, make sheep spermatozoa diluent Hepes-0.1%BSA;
(2) sheep liquefacient duration
Take the fresh semen of certain volume, be diluted with sheep spermatozoa diluent Hepes-0.1%BSA 1:3 in mass ratio, adjust sperm concentration with Hepes-0.1%BSA again after blood counting chamber counting, make sperm concentration reach 30 �� 106Individual/mL, takes 1 ~ 2mL and dyes in centrifuge tube;
(3) staining of sperm
In seminal fluid after above-mentioned 1 ~ 2mL dilutes, add 2 �� L blue fluorescent dyes Hoechst33342 and 8 �� L propidium iodide PI, 37 DEG C of temperature bath 15min;
(4) microscopic examination
Take the seminal fluid after 8 �� L dyeing on microscope slide, covered, detects under fluorescence microscope and counts.
Fluorescent staining method to domestic mammals evaluating sperm activity, step is as follows:
(1) dog spermatozoa diluent preparation
Weigh 2.4gTris, 1.4g citric acid, 0.8g glucose, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate, dissolve in Milli-Q ultra-pure water, add water and be settled to 100mL after mix homogeneously, regulate pH to 6.8, with 0.22 ��m of aperture membrane filtration, make dog spermatozoa diluent TCG;
(2) liquefacient duration
Take the fresh semen of certain volume, be diluted with dog spermatozoa diluent TCG 1:1 in mass ratio, adjust sperm concentration with diluent again after blood counting chamber counting, make to reach 30 �� 106Individual/mL, takes 1 ~ 2mL and dyes in centrifuge tube;
(3) staining of sperm
In seminal fluid after above-mentioned 1 ~ 2mL dilutes, add 2 �� L blue fluorescent dyes Hoechst33342 and 8 �� L propidium iodide PI, 37 DEG C of temperature bath 15min;
(4) microscopic examination
Take the seminal fluid after 8 �� L dyeing on microscope slide, covered, detects under fluorescence microscope and counts.
Preferably, in above-described staining of sperm step, blue fluorescent dyes Hoechst33342 and propidium iodide PI is the stock solution of concentration 1mg/mL, in advance with the preparation of Milli-Q ultra-pure water and subpackage be stored in-20 DEG C of refrigerators.
Described EDTA is the english abbreviation of disodiumedetate.
Described Tris is the english abbreviation of trishydroxymethylaminomethane.
Described PI is the english abbreviation of propidium iodide.
The invention have the advantage that and utilize mammal sperm probe Hoechst333422 and the Necrospermia probe PI alive feature that can be gone out fluorescence by same ultraviolet excitation, pig, cattle, sheep, the important domestic mammals sperm of dog 4 kinds are carried out fluorescence staining, carries out evaluating sperm activity exactly.
Owing to these two kinds of dyestuffs turn blue respectively normal complexion red fluorescence after being optically excited, therefore the dyestuff that also can introduce fluoresced green while detection vigor carries out the detection of perforatorium integrity, by fluorescence microscope or flow cytometer, 4 kinds of important domestic mammals motility of sperm and acrosomal integnity is made accurate detection.
Additionally, compared with traditional PI/SYBR-14 staining, PI/Hoechst33342 staining Hoechst33342 instead of the SYBR-14 probe of costliness, makes testing cost be substantially reduced.
Detailed description of the invention
By the following examples the present invention is described further.
Embodiment 1:
Weigh 3.69g glucose, 0.119g sodium chloride, 0.0403g potassium chloride, 0.126g sodium bicarbonate, 0.125gEDTA and 0.005g kanamycin, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, pH to 7.2 is regulated with hydrogen chloride or sodium hydroxide, 0.22 ��m of aperture membrane filtration, makes Boar spermatozoa diluent BTS and preserves in 37 DEG C of water-baths; Take the fresh semen of certain volume, suitably dilute with BTS, make sperm concentration reach 10 �� 106Individual/mL, takes in 1mL to 2mL centrifuge tube, adds 2 �� LHoechst33342 and 8 �� LPI, 37 DEG C of temperature bath 15min; Taking 8 �� L seminal fluid on microscope slide, covered, detection count at least 200 sperms under fluorescence microscope, 202 sperms of this actual count, result is sperm 166 of living, Necrospermia 36, therefore Boar spermatozoa vigor is 82%.
Embodiment 2:
Weigh 0.76g sodium chloride, 0.03g potassium chloride, 0.252g fructose, 0.238gHEPES, 0.015g calcium chloride, 0.01g magnesium chloride, 0.1g bovine serum albumin, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, regulate pH to 7.4,0.22 ��m of aperture membrane filtration, makes cattle spermatozoa diluent and preserves in 37 DEG C of water-baths; Take the fresh semen of certain volume, carry out 1:8 dilution with Hepes-0.1%BSA, adjust sperm concentration with diluent again after blood counting chamber counting, make to reach 30 �� 106Individual/mL, takes in 1mL to 2mL centrifuge tube, adds 2 �� LHoechst33342 and 8 �� LPI, 37 DEG C of temperature bath 15min;Taking 8 �� L seminal fluid on microscope slide, covered, detection count at least 200 sperms under fluorescence microscope, 212 sperms of actual count, result is sperm 187 of living, Necrospermia 25, therefore cattle motility of sperm is 88%.
Embodiment 3:
Weigh 0.76g sodium chloride, 0.03g potassium chloride, 0.252g fructose, 0.238gHEPES, 0.015g calcium chloride, 0.01g magnesium chloride, 0.1g bovine serum albumin, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, regulate pH to 7.4, with 0.22 ��m of aperture membrane filtration, make sheep spermatozoa diluent and preserve in 37 DEG C of water-baths; Take a certain amount of fresh semen, carry out 1:3 dilution with Hepes-0.1%BSA, adjust sperm concentration with diluent again after blood counting chamber counting, make to reach 30 �� 106Individual/mL, takes in 1mL to 2mL centrifuge tube, adds 2 �� LHoechst33342 and 8 �� LPI, 37 DEG C of temperature bath 15min; Taking 8 �� L seminal fluid on microscope slide, covered, detection count at least 200 sperms under fluorescence microscope, 205 sperms of actual count, result is sperm 160 of living, Necrospermia 45, therefore sheep motility of sperm is 78%.
Embodiment 4:
Weigh 2.4gTris, 1.4g citric acid, 0.8g glucose, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate add in Milli-Q ultra-pure water, add water after mix homogeneously and be settled to 100mL, regulate pH to 6.8, with 0.22 ��m of aperture membrane filtration, make dog spermatozoa diluent and preserve in 37 DEG C of water-baths; Take the fresh semen of certain volume, carry out 1:1 dilution with TCG diluent, adjust sperm concentration with diluent again after blood counting chamber counting, make to reach 30 �� 106Individual/mL, takes 1mL to 2mL in centrifuge tube, adds 2 �� LHoechst33342 and 8 �� LPI, 37 DEG C of temperature bath 15min; Taking 8 �� L seminal fluid on microscope slide, covered, detection count at least 200 sperms under fluorescence microscope, 211 sperms of actual count, result is sperm 179 of living, Necrospermia 32, therefore dog motility of sperm is 85%.
Claims (5)
1. the fluorescent staining method of pair domestic mammals evaluating sperm activity, it is characterised in that step is as follows:
(1) 3.69g glucose, 0.119g sodium chloride, 0.0403g potassium chloride, 0.126g sodium bicarbonate, 0.125gEDTA and 0.005g kanamycin are weighed, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, regulate pH to 7.2, with 0.22 ��m of aperture membrane filtration, make Boar spermatozoa diluent BTS and preserve in 37 DEG C of water-baths;
(2) take the fresh pig seminal fluid of certain volume, dilute with Boar spermatozoa diluent BTS, make sperm concentration reach 10 �� 106Individual/mL, takes 1 ~ 2mL in centrifuge tube, adds 2 �� L blue fluorescent dyes Hoechst33342 and 8 �� L propidium iodide PI, 37 DEG C of temperature bath 15min, dyes;
(3) pig semen after 8 �� L dyeing is taken on microscope slide, covered, under fluorescence microscope, detect and count and count at least 200 sperms, 202 sperms of actual count, result is sperm 166 of living, Necrospermia 36, therefore Boar spermatozoa vigor is 82%.
2. the fluorescent staining method of pair domestic mammals evaluating sperm activity, it is characterised in that step is as follows:
(1) 0.76g sodium chloride, 0.03g potassium chloride, 0.252g fructose, 0.238gHEPES, 0.015g calcium chloride, 0.01g magnesium chloride and 0.1g bovine serum albumin are weighed, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, regulate pH to 7.4, with 0.22 ��m of aperture membrane filtration, make cattle spermatozoa diluent Hepes-0.1%BSA and preserve in 37 DEG C of water-baths;
(2) take the fresh cattle seminal fluid of certain volume, be diluted with cattle spermatozoa diluent Hepes-0.1%BSA 1:8 in mass ratio, adjust sperm concentration with Hepes-0.1%BSA again after blood counting chamber counting, make sperm concentration reach 30 �� 106Individual/mL, takes 1 ~ 2mL in centrifuge tube, adds 2 �� L blue fluorescent dyes Hoechst33342 and 8 �� L propidium iodide PI, and 37 DEG C of temperature bath 15min dye;
(3) taking the cattle seminal fluid after 8 �� L dyeing on microscope slide, covered, detection at least 200 sperms of counting, 212 sperms of actual count under fluorescence microscope, result is sperm 187 of living, Necrospermia 25, therefore cattle motility of sperm is 88%.
3. the fluorescent staining method of pair domestic mammals evaluating sperm activity, it is characterised in that step is as follows:
(1) 0.76g sodium chloride, 0.03g potassium chloride, 0.252g fructose, 0.238gHEPES, 0.015g calcium chloride, 0.01g magnesium chloride and 0.1g bovine serum albumin are weighed, it is dissolved in Milli-Q ultra-pure water and is settled to 100mL, regulate pH to 7.4, with 0.22 ��m of aperture membrane filtration, make sheep spermatozoa diluent Hepes-0.1%BSA;
(2) take the fresh sheep seminal fluid of certain volume, be diluted with sheep spermatozoa diluent Hepes-0.1%BSA 1:3 in mass ratio, adjust sperm concentration with Hepes-0.1%BSA again after blood counting chamber counting, make sperm concentration reach 30 �� 106Individual/mL, takes 1 ~ 2mL in centrifuge tube, adds 2 �� L blue fluorescent dyes Hoechst33342 and 8 �� L propidium iodide PI, and 37 DEG C of temperature bath 15min dye;
(3) taking the sheep seminal fluid after 8 �� L dyeing on microscope slide, covered, detection at least 200 sperms of counting, 205 sperms of actual count under fluorescence microscope, result is sperm 160 of living, Necrospermia 45, therefore sheep motility of sperm is 78%.
4. the fluorescent staining method of pair domestic mammals evaluating sperm activity, it is characterised in that step is as follows:
(1) 2.4gTris, 1.4g citric acid, 0.8g glucose are weighed, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate, dissolve in Milli-Q ultra-pure water, add water and be settled to 100mL after mix homogeneously, regulate pH to 6.8, with 0.22 ��m of aperture membrane filtration, make dog spermatozoa diluent TCG;
(2) take the fresh dog sperm of certain volume, be diluted with dog spermatozoa diluent TCG 1:1 in mass ratio, adjust sperm concentration with diluent again after blood counting chamber counting, make to reach 30 �� 106Individual/mL, takes 1 ~ 2mL in centrifuge tube, adds 2 �� L blue fluorescent dyes Hoechst33342 and 8 �� L propidium iodide PI, and 37 DEG C of temperature bath 15min dye;
(3) taking the dog sperm after 8 �� L dyeing on microscope slide, covered, detection at least 200 sperms of counting, 211 sperms of actual count under fluorescence microscope, result is sperm 179 of living, Necrospermia 32, therefore dog motility of sperm is 85%.
5. the fluorescent staining method of the domestic mammals evaluating sperm activity according to any one of Claims 1-4, it is characterized in that, described blue fluorescent dyes Hoechst33342 and propidium iodide PI is the stock solution of concentration 1mg/mL, in advance with the preparation of Milli-Q ultra-pure water and subpackage be stored in-20 DEG C of refrigerators.
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| CN104789684B (en) * | 2015-05-04 | 2018-03-30 | 四川大学华西第二医院 | A kind of kit and application method for embryonic development quality evaluation |
| CN104897630B (en) * | 2015-05-18 | 2017-12-08 | 四川新生命干细胞科技股份有限公司 | A kind of method for detecting human spermatogoa vigor |
| CN112629982A (en) * | 2020-12-14 | 2021-04-09 | 四川农业大学 | Sperm plasma membrane protective agent TRIS/BSA, staining solution and method |
| CN112649268A (en) * | 2020-12-14 | 2021-04-13 | 四川农业大学 | Sperm plasma membrane protective agent HEPES/BSA, staining solution and staining method |
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