CN104498582B - The fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation - Google Patents
The fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation Download PDFInfo
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Abstract
The fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation, the preparation of seminal fluid, semen washing, dyeing, four steps realizations of microscopic examination are washed by TCG, the present invention adopts and by two kinds of fluorescent dyes that Same Wavelength ultraviolet light excites, Kunming dog sperm can be carried out fluorescence staining, just can differentiate spermatozoon activity exactly without toggle lights; And owing to these two kinds of dyestuffs send out red fluorescence, another kind of blue-fluorescence of sending out in laser excitation latter, therefore carry out the detection of perforatorium integrity at the active dyestuff that also can introduce fluoresced green of detection simultaneously. The present invention is simple to operate, and result is accurate, with a high credibility, it is possible to fluorescence microscope or flow cytometer, Kunming dog spermatozoon activity and acrosomal integnity to be made accurate detection.
Description
Technical field
The invention belongs to technical field of animal reproduction, specifically the fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation.
Background technology
Spermatozoon activity is the requisite standard evaluating sperm quality, and it carries out mainly through various stainings. The dyeing of dog spermatozoon activity evaluation once used eosin, Jim Sa, platform to expect the non-fluorescence dyestuffs such as orchid, spermatozoon activity can be made detection by ordinary optical microscope by this kind of method, but use relatively complicated, Necrospermia is estimated too high and different experiments room testing result than regular meeting and differs greatly, therefore, the credibility of non fluorescent stain method is nothing like fluorescent staining method.
Now, the detection of dog spermatozoon activity is generally adopted iodate the third ingot (propidiumiodide, PI), Hoechst33258, CF 5(6)-Carboxyfluorescein diacetate (carboxyfluoresceindiacetate, CFDA), the fluorescent probe such as carboxyl dimethyl fluorescein diacetate (carboxydimethylfluorescceindiacetate, CMFDA) carries out under fluorescence microscope. PI and Hoechst33258 is the specific fluorogenic probe of Necrospermia, and the sperm that they can only enter plasma membrane damaged is combined with DNA, and the sperm therefore only having plasma membrane damaged just can be colored. CFDA and CMFDA is the specific fluorogenic probe of sperm alive, they can enter the sperm that cell membrane is complete, CFDA and CMFDA itself is a class non-fluorescence material, cell can be entered and be easily hydrolyzed by intracellular nonspecific esterase, form hyperfluorescence product, fluoresced green, 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.
At present, the method adopted has only single stain with a kind of dyestuff, also there is the dual staining simultaneously using two kinds of dyestuffs (such as PI+CFDA), but the double-staining of PI+CFDA coupling excites the light wave difference of fluorescence due to two kinds of dyestuffs, therefore need during dyeing to switch different light sources, operate relatively complicated. In addition, CFDA is fluoresced green under laser excitation, other indexs to detect its acrosomal integnity or sperm when detecting spermatozoon activity simultaneously just cannot be taken into account, because the most also fluoresced green of the fluorescent probe of detection acrosome, with the CFDA spectra overlapping of same fluoresced green.The present invention develops PI/Hoechst33342 double fluorescent staining, with it, Kunming dog sperm is carried out Activity determination, sperm of living sends out blue-fluorescence, the rubescent color of Necrospermia or pink fluorescence, it is direct, objective not only to detect, the more important thing is that two kinds of fluorescent probes only need the ultraviolet excitation of Same Wavelength, it is possible to detect the life or death of sperm simultaneously, clearly Necrospermia is separated with sperm zone of living. Dye not by time effects, the problem being absent from background stainings simultaneously, therefore operate extremely convenient, and, this staining also can introduce the perforatorium probe of fluoresced green and detect multiple index simultaneously.
Kunming dog is the excellent sizing kind of dog that China uniquely lists world-famous police dog in, it it is the kind of dog that uniquely works of Chinese independent cultivation, following the trail of, differentiate, search poison, search quick-fried, patrol, wait for, warning, fire-fighting, the fields such as detecting a mine play an important role, its germ plasm resource is extremely valuable, but the data about the research of Kunming dog biology of reproduction also lack very much, have not yet to see the report about Kunming dog sperm fluorescent dyeing method, the activity evaluating Kunming dog sperm with the PI/Hoechst33342 double fluorescent staining of the present invention is international pioneering, up to now, also there are no the report detecting dog spermatozoon activity in this way.
Summary of the invention
The present invention provides by the fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation, Kunming dog sperm is carried out double staining by the redness of the method Same Wavelength laser excitation and blue fluorescent dyes simultaneously, through fluorescence microscope or flow cytomery, the life or death of sperm just can be directly judged without toggle lights, easy and simple to handle, result is accurate, and, the present invention also can with the dyestuff coupling of the third fluoresced green, as with detection perforatorium integrity dyestuff coupling, spermatozoon activity and acrosomal integnity can be made simultaneously evaluate accurately.
The technical solution used in the present invention is as follows:
The fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation, is realized by following steps:
(1) buffer, namely TCG washes the preparation of seminal fluid
Weigh 2.4gTris, 1.4g citric acid, 0.8g glucose, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate add mixing in Milli-Q ultra-pure water, add Milli-Q ultra-pure water after mix homogeneously and be settled to 100mL, regulate pH to 6.8 with 1NNaOH or 1NHCl, make TCG and wash seminal fluid;
(2) semen washing
Being gone to by the fresh semen 2-3mL of collection in the 15ml centrifuge tube of sterilizing, the TCG adding the pre-thermic of 10ml water-bath 37 DEG C washes seminal fluid, and 700g is centrifuged 5min, abandon supernatant, the TCG adding 10ml37 DEG C washes seminal fluid centrifuge washing more once, and it is resuspended that precipitation washes seminal fluid with appropriate TCG, and making sperm concentration is 30 �� 106/ mL;
(3) dyeing
Take the seminal fluid after 1ml cleans, be separately added into 2 �� LH342 and 8 �� LPI, 37 DEG C of temperature bath 15min;
(4) microscopic examination
Take 8 �� L seminal fluid on microscope slide, covered, detects under fluorescence microscope and counts.
When freezing sperm is detected, realize as follows:
(1) TCG washes the preparation of seminal fluid, anti-icing fluid
Weigh 2.4gTris, 1.4g citric acid, 0.8g glucose, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate add in 60mLMilli-Q ultra-pure water, add 20mL yolk, add Milli-Q ultra-pure water and be settled to 100mL after mix homogeneously after dissolving, yolk protein is removed in the centrifugal 1h of 7000g, take supernatant 1NNaOH or 1NHCl and regulate pH to 6.8, make the diluent of freezen protective, that is TCG washes seminal fluid;In diluent, add the glycerol that volume ratio is 10% make 2 �� anti-icing fluid;
(2) washing of seminal fluid, preservation and recovery
2-3mL fresh semen being transferred in sterilized 15ml centrifuge tube, the TCG adding 10mL37 DEG C of preheating washes seminal fluid, 700g centrifuge washing 2 times, each 5min; It is resuspended that gained precipitation washes seminal fluid with 800 �� lTCG, and making sperm concentration is 400 �� 107Individual/ml; Seminal fluid after resuspended takes 50 �� l in 4mL centrifuge tube, adds 450 �� lTCG and washes seminal fluid, lowers the temperature 2 hours in 4 DEG C of ice baths; By equal-volume, being cooled to the 2 �� anti-icing fluid of 4 DEG C in advance and divide 5 times, every minor tick 6min is slowly added in the sperm after cooling, continues to balance 30 minutes at 4 DEG C, and the sperm obtained is respectively charged into 0.25mL freezing straw and adds hot-seal; Put in liquid nitrogen after straw being placed in 5cm place above liquid nitrogen surface stifling freezing 10 minutes and preserve; Straw is put into rapidly in 37 DEG C of water-baths during recovery and be gently agitated for 2 minutes; Seminal fluid after the recovery TCG of 37 DEG C of preheatings washes seminal fluid in 700g, centrifuge washing twice, each 5min, and precipitation TCG washes semen dilution makes sperm count be 30 �� 106Individual/mL;
(3) dyeing
Take the seminal fluid after the above-mentioned recovery of 1ml is cleaned, add 2 �� lH342 and 8 �� lPI, in 37 DEG C of temperature bath 15min;
(4) microscopic examination
Take 8 �� l seminal fluid on microscope slide, covered, detects under fluorescence microscope and counts.
TCG used by described step (2) centrifuge washing washes seminal fluid with water bath heat preservation in 37 DEG C.
In described step (3), H342 represents Hoechst33342.
In described step (3), H342 and PI is the stock solution of concentration 1mg/mL, in advance with deionized water prepare, subpackage be stored in-20 DEG C of refrigerators.
Described Tris is the english abbreviation of trishydroxymethylaminomethane.
Described TCG represents the English acronym of Tris, citric acid, glucose.
The invention have the advantage that
Kunming dog sperm can be carried out fluorescence staining by two kinds of fluorescent dyes that Same Wavelength ultraviolet light excites by employing, just can differentiate spermatozoon activity exactly without toggle lights; And owing to these two kinds of dyestuffs send out red fluorescence, another kind of blue-fluorescence of sending out in laser excitation latter, therefore carry out the detection of perforatorium integrity at the active dyestuff that also can introduce fluoresced green of detection simultaneously. The present invention is simple to operate, and result is accurate, with a high credibility, it is possible to fluorescence microscope or flow cytometer, Kunming dog spermatozoon activity and acrosomal integnity to be made accurate detection.
Detailed description of the invention
By the following examples the present invention is described further.
Embodiment 1:
Weigh 2.4gTris, 1.4g citric acid, 0.8g glucose, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate to add in Milli-Q ultra-pure water, add water after mix homogeneously and be settled to 100mL, regulate pH to 6.8, make TCG and wash seminal fluid; The fresh semen 2mL picking up from Kunming dog is gone in the 15ml centrifuge tube of sterilizing, add 10mL and wash seminal fluid in the TCG of 37 DEG C of water bath heat preservations, 700g is centrifuged 5min, abandon supernatant, the TCG adding 10mL37 DEG C washes seminal fluid centrifuge washing more once, precipitation 2-3mLTCG buffer is resuspended, and making sperm concentration is 30 �� 106/ mL; Take the seminal fluid after 1mL dilution, be separately added into 2 �� LHoechst333422 and 8 �� LPI, in 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, 213 sperms of this actual count, result is sperm 172 of living, Necrospermia 41, therefore Kunming dog motility of sperm is 81%.
Embodiment 2:
Weigh 2.4gTris, 1.4g citric acid, 0.8g glucose, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate add in 60mLMilli-Q ultra-pure water, add 20mL yolk, add Milli-Q ultra-pure water and be settled to 100mL after mix homogeneously after dissolving, yolk protein is removed in the centrifugal 1h of 7000g, take supernatant and regulate pH to 6.8, make the diluent of freezen protective, that is TCG described below washes seminal fluid; In diluent, add the glycerol that volume ratio is 10% make 2 �� anti-icing fluid; 3mL fresh semen being transferred in sterilized 15ml centrifuge tube, the TCG adding 10mL37 DEG C of preheating washes seminal fluid, and the centrifugal 5min of 700g washs 2 times; It is resuspended that gained precipitation washes seminal fluid with 800 �� lTCG, and making sperm concentration is 400 �� 107Individual/ml, takes 50 �� l in 4mL centrifuge tube, adds the 450 not glycerinated freeze-extenders of �� l, lowers the temperature 2 hours in 4 DEG C of ice baths; By equal-volume, it is cooled to the 2 �� anti-icing fluid of 4 DEG C in advance and divides 5 times, every minor tick 6min, it is slowly added in the sperm after cooling, continues to balance 30 minutes at 4 DEG C; Sperm is respectively charged into 0.25mL freezing straw and adds hot-seal with sealing machine; Put in liquid nitrogen after straw being placed in 5cm place above liquid nitrogen surface stifling freezing 10 minutes and preserve. Straw is put into rapidly in 37 DEG C of water-baths during recovery and be gently agitated for 2 minutes; Seminal fluid after the recovery TCG of 37 DEG C of preheatings washes seminal fluid in 700g, centrifuge washing twice, each 5min, and precipitation TCG washes semen dilution makes sperm count be 30 �� 106/ ml, takes 1ml and adds 2 �� lH342 and 8 �� lPI, in 37 DEG C of temperature bath 15min; Taking 8 �� l seminal fluid on microscope slide, covered, detection at least 200 sperms of counting, 216 sperms of this actual count under fluorescence microscope, sperm of living is 117, and Necrospermia is 99, therefore the Kunming dog motility of sperm after Cryopreservation is 54%.
Examples detailed above is only for example of the present invention is clearly described, for the those of ordinary skill in described field, the apparent change thus amplified out or variation still among the protection domain of the invention.
Claims (5)
1. the fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation, it is characterised in that realize as follows:
(1) buffer, that is TCG washes the preparation of seminal fluid
Weigh 2.4gTris, 1.4g citric acid, 0.8g glucose, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate and add mixing in Milli-Q ultra-pure water, add Milli-Q ultra-pure water after mix homogeneously and be settled to 100mL, regulate pH to 6.8, make TCG and wash seminal fluid;
(2) semen washing
The Kunming dog fresh semen 2-3mL of collection is gone in the 15ml centrifuge tube of sterilizing, the TCG adding the pre-thermic of 10ml water-bath 37 DEG C washes seminal fluid, 700g is centrifuged 5min, abandon supernatant, the TCG adding 10ml37 DEG C washes seminal fluid centrifuge washing more once, it is resuspended that precipitation 2-3mLTCG washes seminal fluid, and making sperm concentration is 30 �� 106Individual/mL;
(3) dyeing
Take the seminal fluid after 1ml cleans, be separately added into 2 �� LH342 and 8 �� LPI, in 37 DEG C of temperature bath 15min;
(4) microscopic examination
Taking 8 �� L seminal fluid on microscope slide, covered, detection at least 200 sperms of counting, 213 sperms of actual count under fluorescence microscope, result is sperm 172 of living, Necrospermia 41, therefore Kunming dog motility of sperm is 81%.
2. the fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation, it is characterised in that realize as follows:
(1) TCG washes the preparation of seminal fluid, anti-icing fluid
Weigh 2.4gTris, 1.4g citric acid, 0.8g glucose, 0.06g penicillin G sodium salt and 0.1g streptomycin sulfate add in 60mLMilli-Q ultra-pure water, add 20mL yolk, add Milli-Q ultra-pure water and be settled to 100mL after mix homogeneously after dissolving, yolk protein is removed in the centrifugal 1h of 7000g, take supernatant and regulate pH to 6.8, make the diluent of freezen protective, that is TCG washes seminal fluid;In diluent, add the glycerol that volume ratio is 10% make 2 �� anti-icing fluid;
(2) washing, frozen with recovery of seminal fluid
Being transferred to by Kunming dog sperm fresh for 2-3mL in sterilized 15ml centrifuge tube, the TCG adding 10mL37 DEG C of preheating washes seminal fluid, 700g centrifuge washing 2 times, each 5min; It is resuspended that gained precipitation washes seminal fluid with 800 �� lTCG, and making sperm concentration is 400 �� 107Individual/ml; Seminal fluid after resuspended takes 50 �� l in 4mL centrifuge tube, adds 450 �� lTCG and washes seminal fluid, lowers the temperature 2 hours in 4 DEG C of ice baths; By equal-volume, being cooled to the 2 �� anti-icing fluid of 4 DEG C in advance and divide 5 times, every minor tick 6min is slowly added in the sperm after cooling, continues to balance 30 minutes at 4 DEG C, and the sperm obtained is respectively charged into 0.25mL freezing straw and adds hot-seal; Put in liquid nitrogen after straw being placed in 5cm place above liquid nitrogen surface stifling freezing 10 minutes and preserve; Straw is put into rapidly in 37 DEG C of water-baths during recovery and be gently agitated for 2 minutes; Seminal fluid after the recovery TCG of 37 DEG C of preheatings washes seminal fluid in 700g, centrifuge washing twice, each 5min, and precipitation TCG washes semen dilution makes sperm count be 30 �� 106Individual/mL;
(3) dyeing
Take the seminal fluid after the above-mentioned recovery of 1ml is cleaned, add 2 �� lH342 and 8 �� lPI, in 37 DEG C of temperature bath 15min;
(4) microscopic examination
Taking 8 �� l seminal fluid on microscope slide, covered, detection at least 200 sperms of counting, 216 sperms of actual count under fluorescence microscope, sperm of living is 117, and Necrospermia is 99, therefore the Kunming dog motility of sperm after Cryopreservation is 54%.
3. the fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation according to claim 1 or claim 2, it is characterised in that in described step (3), H342 represents Hoechst33342.
4. the fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation according to claim 1 or claim 2, it is characterized in that, in described step (3), H342 and PI is the stock solution of concentration 1mg/mL, in advance with Milli-Q ultra-pure water prepare, subpackage be stored in-20 DEG C of refrigerators.
5. the fluorescent staining method evaluating Kunming dog spermatozoon activity of single ultraviolet excitation according to claim 1 or claim 2, it is characterised in that described TCG represents the English acronym of Tris, citric acid, glucose.
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