CN111879686A - Method for detecting semen quality of mammal by flow cytometry - Google Patents
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Abstract
The invention discloses a method for detecting the semen quality of mammals by adopting flow cytometry, which is characterized by comprising the following steps: (1) diluting semen to 1mL by DPBS, centrifuging for 5min, discarding supernatant, and repeating for 1 time; (2) mix well with 1ml DPBS to a final sperm concentration of 1X106Per mL; (3) acrosome integrity was checked with 1 ug/uL FITC-PNA staining; (4) PI/RH123 combined staining is used for detecting the completeness of the model and the activity of mitochondria; (5) detecting a stained specimen by a flow cytometer; (6) 200 sperm were detected and recorded by fluorescence microscopy. Compared with the existing pig semen quality detection method, the method has the advantages of simpler, more convenient, comprehensive, rapid, efficient, accurate and strong specificity, and obtains a large number of parameters,Effectively solves the problems of detecting the quality of the semen in a short time and the like. The invention can provide reference for deeply researching the reproduction performance of Guizhou fragrant pigs and improving the quality of fragrant pig semen, and is beneficial to the development and utilization of fragrant pigs from Jiang.
Description
Technical Field
The invention belongs to the technical field of bioscience, and particularly relates to a method for detecting the semen quality of mammals by adopting flow cytometry.
Background
Mammalian semen quality is an important factor affecting animal fertility, and sperm with good fertilization ability are the primary conditions for successful fertilization. Sperm are produced in the testicular seminiferous epithelium, which forms sperm through a complex series of activities such as self-proliferation, meiosis and sperm cell deformation, followed by maturation in the epididymis. After ejaculation, the sperm enters the female reproductive tract to acquire energy, and finally the fertilization process is completed. In order to maximize the breeding value of the male livestock, artificial insemination technology is increasingly adopted in modern farms. Compared with natural mating, the artificial insemination technology has the advantages of saving production cost, improving the utilization of breeding boars, improving the breeding benefit, preventing cross contamination and the like, and is watched by people. The quality of boar semen is related to success or failure and actual utilization effect of artificial insemination. The semen quality detection and semen cryopreservation technology plays an irreplaceable role in establishing a good variety resource library and storing good varieties.
Flow CytoMetry (FCM) is a high-tech biological technique for realizing high-speed, one-by-one quantitative analysis and sorting of cells by detecting labeled fluorescent signals of single cells or other biological particles in suspension. This technique can be used to perform a continuous multi-parameter analysis of individual cells flowing past an optical or electronic detector. Compared with the conventional sperm conventional detection technology, the flow cytometry effectively solves the limitation of the conventional detection, realizes quantitative automatic analysis, comprehensively and efficiently and quickly collects standard parameters for evaluating the sperm quality, and combines a fluorescence microscope to collect parameters in various aspects of sperm function detection, such as the integrity of the plasma membrane, the mitochondrial function, the acrosome state and the like of the sperm. The cells or particles stained by the fluorescent pigment in the fast flowing state are irradiated by laser, and the intensity of scattered light and emitted light generated by the cells or particles is measured, so that the qualitative or quantitative detection of the physicochemical properties, immunity, molecular biological properties, functional states and the like of the sperm cells is carried out. Previous studies have shown that flow cytometry separates cells of different properties according to collected parameters to obtain pure cell populations for biological and medical research. Therefore, the flow detection of the quality of the sperm of the Xiangjiang pigs in Guizhou has irreplaceable effect on the etalon for evaluating the quality of the sperm.
From Jiangxiang pigs, Guizhou southeast, Guizhou, from Jiangxu county, national geographical signs of agricultural products. The Jiangxiang pigs have the characteristics of short body type, fragrant and tender meat quality, homozygous gene, purity, no pollution and the like. The pig feed is characterized in that the pig feed can be slaughtered and eaten in different growth periods, suckling piglets and weaning piglets are free from milk fishy smell or other peculiar smell when eaten, the broth after boiling is not turbid, transparent, clear, sweet, thin in skin, tender in meat, fragrant and delicious in meat, good in taste, succulent, glutinous, mellow in taste, free of residue texture, fragrant and thick in meat taste, deep and long in aftertaste, and not greasy when eaten, and the pig feed is named as ' one cooked meat is four-o-fragrant ', and the remaining fragrance of buccal teeth is three days long ', and is the most remarkable characteristic compared with other pig species when eaten by Jiangxiang pigs.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for detecting the semen quality of mammals by adopting flow cytometry, which has the characteristics of simple and convenient operation, comprehensiveness, rapidness, high efficiency, accuracy, strong specificity, acquisition of a large number of parameters, effective solution of the problems of detecting the semen quality in a short time, realization of automatic analysis of the quantity and the like, and has stronger adaptability and better effect compared with other methods.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for detecting the quality of mammalian semen using flow cytometry, comprising the steps of:
(1) preparing a reagent: PI is diluted to 1mg/ml by DPBS, RH123 and FITC-PNA are diluted to 1mg/ml by DMSO, and the diluted reagent is stored at-20 ℃ in the dark;
(2) collecting fresh semen of mammal by hand method, performing routine semen detection on one part of the semen, fixing the other part of the semen with a 37 deg.C vacuum cup, and taking the fixed semen back to the laboratory using a Malan full-automatic sperm analyzer CASA and a flow cytometer for detection and analysis;
(3) treating a semen sample:
detecting sperm density according to sperm full-automatic analysis system (CASA), placing appropriate amount of semen into EP tube, centrifuging (2000 g, 5 min), discarding supernatant, adding DPBS (0.01 mol/L, PH 7.4), mixing, centrifuging, discarding supernatant, adding DPBS (0.01 mol/L, PH 7.4), mixing to obtain sperm with final concentration of 1x106/mL；
(4) FITC-PNA staining for acrosomal integrity:
adding 1mL of the treated sperm suspension into an EP tube, adding 10uL of FITC-PN dye solution (1 mg/mL) dye solution, dyeing for 15 min in a dark place, centrifuging (2000 g, 5 min), discarding supernatant, repeatedly washing with DPBS (0.01 mol/L, pH 7.4) and centrifuging twice, mixing with 1mL of DPBS (0.01 mol/L, pH 7.4) to obtain sperm with concentration of 1x106/mL；
(5) PI staining to check plasma membrane integrity:
adding 1mL of the treated sperm suspension into an EP tube, adding 10uL of PI (1 mg/mL) dye solution, dyeing for 15 min in a dark place, centrifuging (2000 g, 5 min), discarding the supernatant, repeatedly washing with DPBS (0.01 mol/L, pH 7.4) and centrifuging twice, and mixing with 1mL of DPBS (0.01 mol/L, pH 7.4) to obtain a sperm concentration of 1x106/mL；
(6) The method for detecting the mitochondrial activity by RH123 dyeing is the same as the above;
(7) and PI/RH123 combined staining for detecting plasma membrane integrity and mitochondrial activity:
placing 1mL of the treated sperm suspension in an EP tube, adding 10uL of RH123 (1 mg/mL) dye solution, dyeing for 15 min in a dark place, adding 10uL of PI (1 mg/mL) dye solution, dyeing for 5min in a dark place, centrifuging (2000 g, 5 min), discarding the supernatant, washing with DPBS (0.01 mol/L, pH 7.4) repeatedly and centrifuging twice, mixing with 1mL of DPBS (0.01 mol/L, pH 7.4) to obtain a sperm concentration of 1x106/mL；
(8) Fluorescence microscopy:
and (3) taking 10uL of the stained sperm to a clean glass slide, covering the glass slide, and observing whether the sperm is stained by using a fluorescence microscope:
(9) detecting the stained specimen by a flow cytometer:
after starting the device, adding the prepared semen sample into a flow cytometer sample chamber, starting the device, collecting all data after 1 min, setting parameters of a fluorescence channel FL1 and FL2 as logarithmic amplification, and collecting RH123 and FITC-PNA fluorescence signals by a fluorescence channel green FL 1; the fluorescence channel red FL2 can collect PI fluorescence signals; side scatter SSC and forward scatter FSC are set to linear amplification; using FSC as abscissa and SSC as ordinate to make scatter diagram, and delineating target cell group (setting gate); when PI/RH123 is jointly dyed, PI and RH123 dyeing is firstly detected, fluorescence compensation is carried out, and then double-dyed sample detection is carried out;
(10) detecting biochemical parameters of sperms:
SOD (superoxide dismutase) kit, GSH-Px kit and MDA kit of Nanjing institute of bioengineering are used for respectively detecting the activities of thawed sperm SOD, GSH-Px and MDA, the specific steps are described with reference to the kit, and the specific judgment method comprises the following steps:
the head of the sperm shows red fluorescence, namely damaged sperms of plasma membrane with PI staining (PI +) and undyed sperms (PI-) which are intact sperms of plasma membrane, 200 sperms are observed, and the percentage of the undyed sperms (intact plasma membrane) is calculated;
the sperm mitochondria has active sperm with bright green fluorescence RH123 staining (RH 123 +), while the undyed or shaded green fluorescence (RH 123-) is poor mitochondria activity or inactive sperm. 200 sperm cells were observed and the percentage of bright green fluorescent sperm cells (mitochondria active) was calculated.
the acrosome-incomplete sperm is obtained by that the acrosome area has no or only dark green fluorescence. 200 sperm were observed and the percentage of acrosomal zone with no or only faint green fluorescence (acrosomal integrity) was calculated.
PI-/RH123+ (mitochondria has active sperm) is the sperm with bright green fluorescence in the mitochondria part; PI-/RH123- (mitochondria inactive live sperm) is undyed or dim green fluorescent sperm at mitochondria part; PI +/RH123+ (mitochondria have active dead sperm) are red-green sperm with red fluorescence on the head and bright green fluorescence on the mitochondria; PI +/RH123- (mitochondria dead sperm without activity) is the sperm with red fluorescence on the head. The number of sperm cells was counted at 200, and the percentage of each type of sperm cell to the total number of sperm cells was calculated.
The treatment of the final concentration of the sperms in the step (3) mainly comprises the following steps: after the semen is diluted according to a certain proportion, the diluted semen is dripped into a blood cell counting plate, and five middle squares are selected for counting until the final concentration of the semen is determined.
The sperm teratogenesis rate in the step (8) is mainly determined as follows: sperm smears were made and fixed with methanol for 10min after greenhouse drying. Eosin staining, randomly observing 500 sperms under a microscope, and counting the proportion and the type of the malformation of the sperms.
The method for detecting the integrity of the sperm mitochondrial membrane in the step (10) comprises the following steps: the method comprises the following steps of (1) detecting the activity of sperm mitochondria by using rhodamine (RH 123) and Propidium Iodide (PI) as a fluorescent dye in a combined dyeing manner, and then detecting the integrity of the sperm mitochondrial membrane by using flow cytometry, wherein the specific operation is as follows:
(1) preparation of main dye liquor:
PI: when in use, the extract is diluted to 1mg/mL by DPBS and is stored at-20 ℃ in a dark place;
PH123, FITC-PNA: when in use, the mixture is diluted to 1mg/ml by DMSO and stored at-20 ℃ in the dark.
(2) Routine examination of semen:
(3) Processing a semen sample:
detecting sperm density according to sperm full-automatic analysis system (CASA), placing appropriate amount of semen into EP tube, centrifuging (2000 g, 5 min), discarding supernatant, adding DPBS (0.01 mol/L, PH 7.4), mixing, centrifuging, discarding supernatant, adding DPBS (0.01 mol/L, PH 7.4), mixing to obtain sperm with final concentration of 1x106/ml。
(4) FITC-PNA staining for acrosomal integrity:
1mL of the treated sperm suspension was placed in an EP tube,adding 10uL FITC-PN dye solution (1 mg/mL), dyeing for 15 min in dark, centrifuging (2000 g, 5 min), discarding supernatant, washing with DPBS (0.01 mol/L, pH 7.4) repeatedly and centrifuging twice, mixing with 1mL DPBS (0.01 mol/L, pH 7.4) to obtain sperm concentration of 1 × 106/mL。
(5) PI staining for plasma membrane integrity
Adding 1mL of the treated sperm suspension into an EP tube, adding 10ul of PI (1 mg/mL) dye solution, dyeing for 15 min in a dark place, centrifuging (2000 g, 5 min), discarding the supernatant, repeatedly washing with DPBS (0.01 mol/L, pH 7.4) and centrifuging twice, and mixing with 1mL of DPBS (0.01 mol/L, pH 7.4) to obtain a sperm concentration of 1x106/mL。
(6) The method for detecting mitochondrial activity by RH123 staining is as above
(7) PI/RH123 combined staining for detecting plasma membrane integrity and mitochondrial activity
Placing 1mL of the treated sperm suspension in an EP tube, adding 10uL of RH123 (1 mg/mL) dye solution, dyeing for 15 min in a dark place, adding 10uL of PI (1 mg/mL) dye solution, dyeing for 5min in a dark place, centrifuging (2000 g, 5 min), discarding the supernatant, washing with DPBS (0.01 mol/L, pH 7.4) repeatedly and centrifuging twice, mixing with 1mL of DPBS (0.01 mol/L, pH 7.4) to obtain a sperm concentration of 1x106/mL。
(8) Microscopic examination of fluorescence microscope
10ul of stained sperm was placed on a clean slide, covered with a cover slip, and observed for staining using a fluorescence microscope.
(9) Flow cytometer for detecting stained specimen
And after starting the device, adding the prepared semen sample into a flow cytometer sample chamber. The instrument is started, and data can be collected completely after 1 min. The fluorescent channel FL1 and FL2 parameters were set to logarithmic amplification, and the fluorescent channel green FL1 could collect RH123, FITC-PNA fluorescent signals; the fluorescence channel red FL2 can collect PI fluorescence signals. The side scatter SSC and forward scatter FSC are set to linear amplification. A scatter plot was made with FSC as abscissa and SSC as ordinate, and the target cell population was circled (gated). And when the PI/RH123 is subjected to combined staining, the PI and RH123 are firstly detected and stained, fluorescence compensation is carried out, and then double-staining specimen detection is carried out.
(10) Sperm biochemical parameter detection
And respectively detecting the activities of the thawed sperm SOD, GSH-Px and MDA by using an SOD kit, a GSH-Px kit and an MDA kit of Nanjing institute of bioengineering. The specific steps are described by referring to a kit, and the specific determination method comprises the following steps:
The head of the sperm shows red fluorescence, and the head shows damaged sperms of plasma membrane stained by PI (PI +), while the head shows intact sperms of plasma membrane without PI (PI-). 200 sperm cells were observed and the percentage of unstained sperm cells (plasma membrane intact) was calculated
The sperm mitochondria has active sperm with bright green fluorescence RH123 staining (RH 123 +), while the undyed or shaded green fluorescence (RH 123-) is poor mitochondria activity or inactive sperm. 200 sperm cells were observed and the percentage of bright green fluorescent sperm cells (mitochondria active) was calculated.
The acrosome-incomplete sperm is obtained by that the acrosome area has no or only dark green fluorescence. 200 sperm were observed and the percentage of acrosomal zone with no or only faint green fluorescence (acrosomal integrity) was calculated.
PI-/RH123+ (mitochondria has active sperm) is the sperm with bright green fluorescence in the mitochondria part; PI-/RH123- (mitochondria inactive live sperm) is undyed or dim green fluorescent sperm at mitochondria part; PI +/RH123+ (mitochondria have active dead sperm) are red-green sperm with red fluorescence on the head and bright green fluorescence on the mitochondria; PI +/RH123- (mitochondria dead sperm without activity) is the sperm with red fluorescence on the head. The number of sperm cells was counted at 200, and the percentage of each type of sperm cell to the total number of sperm cells was calculated.
Compared with the prior art, the method has the characteristics of simple and comprehensive operation, rapidness, high efficiency, accuracy, strong specificity, capability of obtaining a large number of parameters, effectively solving the problems of semen quality detection in a short time and the like, realizing automatic analysis of the quantity and the like, and has stronger adaptability and better effect compared with other methods. The invention is simple and easy to understand and convenient to operate.
Detailed Description
Example (b): the method for detecting the quality of the sperm of the Xiandan pig by adopting the flow cytometry comprises the following steps:
the sperm full-automatic analysis system is purchased from south Ningsong Crassulaceae Biotech limited; flow cytometry was purchased from beckmann coulter co; inverted fluorescence microscope purchased from nikon, japan; centrifuges were purchased from Thermo Fisher ltd, usa; slides and coverslips were purchased from Jiangsu Shitai laboratory instruments, Inc.; propidium Iodide (PI) was purchased from shanghai bi yunnan biotechnology limited; mingdalo (RH 123) was purchased from Shanghai Biyuntian biotechnology, Inc.; fluorescein isothiocyanate labeled peanut lectin (FITC-PNA) was purchased from Thermo Fisher, Inc., USA; du's Phosphate Buffer (DPBS) was purchased from Solebao biology, Inc., Shanghai; methyl Sulfoxide (DMSO) was purchased from Beijing Soilebao Tech.
Firstly, reagent preparation: PI is diluted to 1mg/ml by DPBS, RH123 and FITC-PNA are diluted to 1mg/ml by DMSO, and the diluted reagent is stored at-20 ℃ in the dark.
Secondly, collecting fresh boar semen by a hand-held method, then carrying out conventional semen detection on one part on the spot, and fixing the other part by a heat preservation cup (about 37 ℃) and taking the part back to a laboratory Malan full-automatic sperm analyzer (CASA) and a flow cytometer for detection and analysis;
treating semen sample
Detecting sperm density according to sperm full-automatic analysis system (CASA), placing appropriate amount of semen into EP tube, centrifuging (2000 g, 5 min), discarding supernatant, adding DPBS (0.01 mol/L, PH 7.4), mixing, centrifuging, discarding supernatant, adding DPBS (0.01 mol/L, PH 7.4), mixing to obtain sperm with final concentration of 1x106/ml。
Fourthly, dyeing
(1) FITC-PNA staining for acrosome integrity
And (3) putting 1ml of the processed sperm suspension into an EP tube, adding 10ul of FITC-PN dye liquor (1 mg/ml) dye liquor, dyeing for 15 min in a dark place, centrifuging (2000 g, 5 min), discarding supernatant, repeatedly washing and centrifuging twice by using DPBS (0.01 mol/L, pH 7.4), and uniformly mixing by using 1ml of DPBS (0.01 mol/L, pH 7.4) to ensure that the sperm concentration is 1x 106/ml.
(2) PI staining for plasma membrane integrity
Adding 1ml of the treated sperm suspension into an EP tube, adding 10ul of PI (1 mg/ml) dye solution, dyeing for 15 min in a dark place, centrifuging (2000 g, 5 min), discarding the supernatant, repeatedly washing with DPBS (0.01 mol/L, pH 7.4) and centrifuging twice, and mixing with 1ml of DPBS (0.01 mol/L, pH 7.4) to obtain a sperm suspension with a sperm concentration of 1x106/ml。
(3) The method for detecting mitochondrial activity by RH123 staining is as above
(4) PI/RH123 combined staining for detecting plasma membrane integrity and mitochondrial activity
Putting 1ml of the processed sperm suspension into an EP tube, adding 10ul of RH123 (1 mg/ml) dye solution, dyeing for 15 min in a dark place, adding 10ul of PI (1 mg/ml) dye solution, dyeing for 5min in a dark place, centrifuging (2000 g, 5 min), discarding supernatant, washing with DPBS (0.01 mol/L, pH 7.4) repeatedly and centrifuging twice, and mixing with 1ml of DPBS (0.01 mol/L, pH 7.4) to obtain a sperm concentration of 1x106/ml。
Fifth, fluorescence microscopy
10ul of stained sperm was placed on a clean slide, covered with a cover slip, and observed for staining using a fluorescence microscope.
Sixthly, detecting the stained specimen by a flow cytometer
And after starting the device, adding the prepared semen sample into a flow cytometer sample chamber. The instrument is started, and data can be collected completely after 1 min. The fluorescent channel FL1 and FL2 parameters were set to logarithmic amplification, and the fluorescent channel green FL1 could collect RH123, FITC-PNA fluorescent signals; the fluorescence channel red FL2 can collect PI fluorescence signals. The side scatter SSC and forward scatter FSC are set to linear amplification. A scatter plot was made with FSC as abscissa and SSC as ordinate, and the target cell population was circled (gated). And when the PI/RH123 is subjected to combined staining, the PI and RH123 are firstly detected and stained, fluorescence compensation is carried out, and then double-staining specimen detection is carried out.
Seventhly, detecting the biochemical parameters of sperms
And respectively detecting the activities of the thawed sperm SOD, GSH-Px and MDA by using an SOD kit, a GSH-Px kit and an MDA kit of Nanjing institute of bioengineering. The specific steps are described by referring to a kit, and the specific determination method comprises the following steps:
(1) plasma membrane integrity
The head of the sperm shows red fluorescence, and the head shows damaged sperms of plasma membrane stained by PI (PI +), while the head shows intact sperms of plasma membrane without PI (PI-). 200 sperm cells were observed and the percentage of unstained sperm cells (plasma membrane intact) was calculated
(2) Mitochondrial Activity
The sperm mitochondria has active sperm with bright green fluorescence RH123 staining (RH 123 +), while the undyed or shaded green fluorescence (RH 123-) is poor mitochondria activity or inactive sperm. 200 sperm cells were observed and the percentage of bright green fluorescent sperm cells (mitochondria active) was calculated.
(3) Integrity of roof
The acrosome-incomplete sperm is obtained by that the acrosome area has no or only dark green fluorescence. 200 sperm were observed and the percentage of acrosomal zone with no or only faint green fluorescence (acrosomal integrity) was calculated.
(4) PI/RH123 combined staining for simultaneous detection of plasma membrane integrity and mitochondrial activity
PI-/RH123+ (mitochondria has active sperm) is the sperm with bright green fluorescence in the mitochondria part; PI-/RH123- (mitochondria inactive live sperm) is undyed or dim green fluorescent sperm at mitochondria part; PI +/RH123+ (mitochondria have active dead sperm) are red-green sperm with red fluorescence on the head and bright green fluorescence on the mitochondria; PI +/RH123- (mitochondria dead sperm without activity) is the sperm with red fluorescence on the head. The number of sperm cells was counted at 200, and the percentage of each type of sperm cell to the total number of sperm cells was calculated.
Eighth, experimental results
Flow cytometric staining is divided into a single staining group and a double staining group, and the results of the flow cytometric staining of the Yangtze fragrant pigs and the large white pigs are compared by using t test, and the acrosome integrity difference is obvious (P<0.05), the integrity of the top body of the fragrant pig is (68.72 +/-2.70)%, the integrity of the top body of the large white pig is (76.83 +/-2.03)%, the integrity of the top body of the fragrant pig is lower than that of the large white pig, and the mitochondrial activity and the plasma membrane integrity have no obvious difference (P)>0.05). The flow cytometric double-staining method is used for detecting the sperm quality of the Xiandan pigs and the white pigs by using t test, and the test result shows that: the PI-/RH123+ difference is significant (P)<0.05) the active sperm with the pig mitochondria activity (PI-/RH 123 +) is (70.27 +/-1.76)%, the active sperm with the pig mitochondria activity (PI-/RH 123 +) is (61.32 +/-0.86)%, the pig is lower than the pig, the reason is probably that the pig sperm death is more caused by stress factors in transportation, and the PI +/RH123+ has no obvious statistical difference (P) with the PI-/RH 123-)>0.05). Compared with the single-staining (PI and RH123 staining) result and the double-staining (PI/RH 123 staining) result of the Xiangjiang pigs and the big white pigs, no obvious difference exists, and the feasibility of staining and detecting the sperm quality by using a double-staining method is proved. Meanwhile, the conventional analysis result is combined with flow cytometry for correlation analysis, and the forward motion activity rate of Jiangxiang pigs and white pigs has no obvious correlation with the integrity of plasma membranes, the activity of mitochondria and the integrity of acrosomes.
At present, five semen conventional inspection methods, namely an appearance inspection method, a microscopy inspection method, a biochemical inspection method, a sperm motility inspection method, a computer aided analysis system inspection method and the like, which are adopted for the semen quality determination and evaluation of mammals, have the standard that the semen quality can not be completely evaluated by the results determined by the methods reported in documents, and the invention finds that the semen parameters obtained by the methods are less, are greatly influenced by subjective factors, waste time and labor and cause a great deal of sperm motility reduction due to long-time observation when the invention passes the preliminary experiments of the methods in the earlier stage. However, the method can realize quantitative automatic analysis, comprehensive, high-efficiency and rapid collection of standard parameters for evaluating the sperm quality, has strong specificity and accurate data, and combines a fluorescence microscope to collect parameters in various aspects of sperm function detection, such as the integrity of the sperm plasma membrane, the mitochondrial function, the acrosome state and the like. Therefore, the flow detection of the quality of the fragrant pig semen has an irreplaceable effect on the etalon for evaluating the quality of the semen.
The present invention is not limited to the above-described preferred embodiments, and other forms of products can be obtained by anyone in light of the present invention. However, any changes in the preparation of semen sample processing reagent, semen dilution ratio, semen weight concentration and sequence of operations are within the scope of the present invention, and all technical solutions identical or similar to the present application are included.
Claims (5)
1. A method for detecting the quality of the semen of a mammal by adopting flow cytometry is characterized in that: the method comprises the following steps:
(1) routine examination of semen: namely sperm appearance inspection: collecting fresh semen with a clean test tube, placing in a 37 deg.C vacuum flask, and observing semen color, smell, ejaculation amount, and cloud state;
preparation of main dye liquor: PI: when in use, the extract is diluted to 1mg/mL by DPBS and is stored at-20 ℃ in a dark place; PH123, FITC-PNA: when in use, the mixture is diluted to 1mg/mL by DMSO and is stored at-20 ℃ in a dark place;
(2) processing a semen sample: detecting sperm density according to CASA of sperm full-automatic analysis system, placing appropriate amount of semen in EP tube, centrifuging for 5min, discarding supernatant, adding 0.01mol/L, PH 7.4.4 DPBS, mixing, centrifuging, discarding supernatant, adding 0.01mol/L, PH 7.4.4 DPBS, mixing to obtain sperm with final concentration of 1x106/mL；
(3) FITC-PNA staining detection roofIntegrity of the body: adding 1mL of the treated sperm suspension into an EP tube, adding 10 uLFITC-PN dye solution with the proportion of 1mg/mL, dyeing for 15 min in a dark place, 2000g, centrifuging for 5min, then discarding supernatant, repeatedly washing and centrifuging twice by using 0.01mol/L, pH 7.4.4 DPBS, uniformly mixing by using 1mL of DPBS with the concentration of 1x10, wherein the DPBS is 0.01mol/L and the pH is 7.46/mL；
(4) PI staining to check plasma membrane integrity: adding 1mL of the treated sperm suspension into an EP tube, adding 10uL of PI staining solution with the proportion of 1mg/mL, carrying out dark staining for 15 min and 2000g, centrifuging for 5min, then discarding supernatant, repeatedly washing and centrifuging twice by using 0.01mol/L, pH 7.4.4 DPBS (0.01 mol/L, pH 7.4.4), uniformly mixing by using 1mL of DPBS with the concentration of 1x10 to ensure that the sperm concentration is 1x106/mL；
(5) The method for detecting the mitochondrial activity by RH123 dyeing is the same as the above;
(6) and PI/RH123 combined staining for detecting plasma membrane integrity and mitochondrial activity: putting 1mL of the treated sperm suspension into an EP tube, adding 10uL of 1mg/mL RH123 dye solution, dyeing for 15 min in a dark place, adding 10uL of 1mg/mL PI dye solution, dyeing for 5min in a dark place, 2000g, centrifuging for 5min, discarding the supernatant, repeatedly washing and centrifuging twice by using 0.01mol/L DPBS, uniformly mixing by using 1mL of 0.01mol/L DPBS to ensure that the sperm concentration is 1x106/mL；
(7) Fluorescence microscopy: taking 10uL of the stained sperm to a clean glass slide, covering a cover glass, and observing whether the sperm is stained by using a fluorescence microscope;
(8) detecting the stained specimen by a flow cytometer: after starting up, adding the prepared semen sample into a flow cytometer sample chamber, starting up the instrument, and collecting all data after 1 min; the fluorescent channel FL1 and FL2 parameters were set to logarithmic amplification, and the fluorescent channel green FL1 could collect RH123, FITC-PNA fluorescent signals; the fluorescence channel red FL2 can collect PI fluorescence signals; side scatter SSC and forward scatter FSC are set to linear amplification; using FSC as abscissa and SSC as ordinate to make scatter diagram, and delineating target cell group (setting gate); when PI/RH123 is jointly dyed, PI and RH123 dyeing is firstly detected, fluorescence compensation is carried out, and then double-dyed sample detection is carried out;
(9) detecting biochemical parameters of sperms: SOD (superoxide dismutase) kit, GSH-Px kit and MDA kit of Nanjing institute of bioengineering are used for respectively detecting the activities of thawed sperm SOD, GSH-Px and MDA, the specific steps are described with reference to the kit, and the specific judgment method comprises the following steps:
(10) integrity of plasma membrane: the head of the sperm shows red fluorescence and is plasma membrane damaged sperm with PI staining (PI +) and undyed (PI-) is plasma membrane intact sperm, 200 sperms are observed, and the percentage of the plasma membrane intact undyed sperms is calculated;
(11) mitochondrial activity: the sperm mitochondria part presents bright green fluorescence and is mitochondria with active sperm stained by RH123 (RH 123 +), and undyed or stained with dark green fluorescence (RH 123-) is mitochondria with poor activity or inactive sperm; observing 200 sperms, and calculating the percentage of bright green fluorescent sperms with mitochondrial activity;
(12) integrity of the top body: the acrosome-incomplete sperm has no or only dark green fluorescence in the acrosome area; observing 200 sperms, and calculating the percentage of sperms with complete acrosomes without or with dim green fluorescence in the acrosomal region;
(13) PI/RH123 combined staining simultaneously detects plasma membrane integrity and mitochondrial activity: PI-/RH123+ is the active sperm with mitochondria activity, and is the sperm with bright green fluorescence in the mitochondria part; PI-/RH 123-is mitochondria inactive live sperm, is undyed or mitochondria part dull green fluorescent sperm; PI +/RH123+ is the dead sperm with mitochondria activity, which is the red and green sperm with red fluorescence at the head and bright green fluorescence at the mitochondria; PI +/RH 123-is dead sperm with no activity of mitochondria, which is the sperm with red fluorescence at the head, and the percentage of each type of sperm in the total number of the sperm is calculated by calculating 200 sperms.
2. A method for detecting the quality of mammalian semen using flow cytometry as claimed in claim 1 wherein: the treatment of the final concentration of the sperms in the step (3) mainly comprises the following steps: after the semen is diluted according to a certain proportion, the diluted semen is dripped into a blood cell counting plate, and five middle squares are selected for counting until the final concentration of the semen is determined.
3. A method for detecting the quality of mammalian semen using flow cytometry as claimed in claim 1 wherein: the sperm teratogenesis rate in the step (7) is mainly determined as follows: preparing a sperm smear, drying in a greenhouse, and fixing for 10min by using methanol; eosin staining, randomly observing 500 sperms under a microscope, and counting the proportion and the type of the malformation of the sperms.
4. A method for detecting the quality of mammalian semen using flow cytometry as claimed in claim 1 wherein: the method for detecting the integrity of the sperm mitochondrial membrane in the step (10) comprises the following steps: the method comprises the following specific operations of utilizing rhodamine RH123 and a fluorescent dye propidium iodide PI to jointly dye and detect the activity of sperm mitochondria, and then adopting flow cytometry to detect the integrity of sperm mitochondrial membranes:
(1) preparation of main dye liquor:
PI: when in use, the extract is diluted to 1mg/mL by DPBS and is stored at-20 ℃ in a dark place;
PH123, FITC-PNA: when in use, the mixture is diluted to 1mg/mL by DMSO and is stored at-20 ℃ in a dark place;
(2) routine examination of semen:
(3) processing a semen sample:
detecting sperm density according to CASA of sperm full-automatic analysis system, placing appropriate amount of semen in EP tube, centrifuging for 5min, discarding supernatant, adding 0.01mol/L DPBS, mixing, centrifuging, discarding supernatant, adding 0.01mol/L DPBS, mixing to obtain sperm with final concentration of 1x106/mL；
(4) FITC-PNA staining for acrosomal integrity:
adding 1mL of the processed sperm suspension into an EP tube, adding 10ul of FITC-PN dye solution with the concentration of 1mg/mL, dyeing for 15 min and 2000g in a dark place, centrifuging for 5min, discarding supernatant, repeatedly washing and centrifuging twice by using 0.01mol/L DPBS, uniformly mixing by using 1 mL0.01 mol/L DPBS to ensure that the sperm concentration is 1x106/ml；
(5) PI staining to check plasma membrane integrity:
adding 1mL of the treated sperm suspension into an EP tube, adding 10uL of 1mg/mL PI staining solution, staining for 15 min and 2000g in a dark place, centrifuging for 5min, and discarding the supernatantWashing with 0.01mol/L DPBS and centrifuging twice, mixing with 1mL of 0.01mol/L DPBS to obtain sperm concentration of 1 × 106/ml；
(6) The method for detecting the mitochondrial activity by RH123 dyeing is the same as the above;
(7) and PI/RH123 combined staining for detecting plasma membrane integrity and mitochondrial activity:
putting 1mL of the treated sperm suspension into an EP tube, adding 10uL of 1mg/mL RH123 dye solution, dyeing for 15 min in a dark place, adding 10uL of 1mg/mL PI dye solution, dyeing for 5min in a dark place, 2000g, centrifuging for 5min, discarding the supernatant, repeatedly washing with 0.01 mol/LDPBS and centrifuging twice, uniformly mixing with 1mL of 0.01mol/L DPBS to ensure that the sperm concentration is 1x106/mL；
(8) Fluorescence microscopy:
taking 10ul of the stained sperm to a clean glass slide, covering a cover glass, and observing whether the sperm is stained by using a fluorescence microscope;
(9) detecting the stained specimen by a flow cytometer:
after starting up, adding the prepared semen sample into a flow cytometer sample chamber, starting up the instrument, and collecting all data after 1 min; the fluorescent channel FL1 and FL2 parameters were set to logarithmic amplification, and the fluorescent channel green FL1 collected RH123, FITC-PNA fluorescent signals; the fluorescence channel red FL2 collects PI fluorescence signals; side scatter SSC and forward scatter FSC are set to linear amplification; using FSC as abscissa and SSC as ordinate to make scatter diagram, and delineating target cell group (setting gate); when PI/RH123 is jointly dyed, PI and RH123 dyeing is firstly detected, fluorescence compensation is carried out, and then double-dyed sample detection is carried out;
(10) detecting biochemical parameters of sperms:
SOD (superoxide dismutase) kit, GSH-Px kit and MDA kit of Nanjing institute of bioengineering are used for respectively detecting the activities of thawed sperm SOD, GSH-Px and MDA, the specific steps are described with reference to the kit, and the specific judgment method comprises the following steps:
the head of the sperm shows red fluorescence, namely PI & lt + & gt stained plasma membrane damaged sperm, and undyed PI & lt- & gt is plasma membrane intact sperm; observing 200 sperms, and calculating the intact percentage of plasma membrane of the unstained sperms;
the sperm mitochondria have active sperm with bright green fluorescence RH123+ stained mitochondria, and have poor activity or inactive sperm without staining or with dark green fluorescence RH 123-; observing 200 sperms, and calculating the percentage of mitochondrial activity of bright green fluorescent sperms;
the acrosome-incomplete sperm has no or only dark green fluorescence in the acrosome area; observing 200 sperms, and calculating that the acrosome area has no or only dim green fluorescence, which is the percentage of the complete acrosome;
PI-/RH123+ is live sperm with active mitochondria, and the mitochondria part is bright green fluorescence sperm; PI-/RH 123-is mitochondria inactive live sperm, non-staining or mitochondria part dull green fluorescent sperm; PI +/RH123+ is the dead sperm with mitochondria activity, the head of the sperm is red fluorescence, and the mitochondria part is bright green fluorescence 'red green' sperm; PI +/RH 123-is dead sperm without activity of mitochondria, and the head of the sperm is red fluorescent sperm; the number of sperm cells was counted at 200, and the percentage of each type of sperm cell to the total number of sperm cells was calculated.
5. A method for detecting the quality of mammalian semen using flow cytometry as claimed in claim 1 wherein: the mammal is a Yuanjiang fragrant pig.
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