The objectives of this experiment are (1) to master the method of sperm collection from the testis, epididymis and vas deferens of mice; (2) to master the method of sperm counting and motility test by the hemocyte counting plate method; (3) to recognize the process of maturation of sperms in the epididymis after being produced in the testis by the motility test of sperms from the testis, epididymis and vas deferens; (4) to master the method of sperm smearing; and (5) to master the method of staining for the structural observation of sperms and the acrosome structure observation methods, and recognize the normal structure of spermatozoa.
Operation method
hemocytometer
Principle
Mammalian spermatozoa must undergo a series of morphological, physiological and biochemical changes in the epididymis to reach maturity before they can acquire the ability to move forward, and this forward movement is an important indicator of the spermatozoa's ability to fertilize. Mammalian spermatozoa consist of two main parts, the head and the tail, the neck is between the head and the tail, forming the connection between the head and the tail. The head of the sperm consists mainly of the nucleus, with the acrosome at the front of the head, which surrounds the front of the nucleus to form a cap. The neck of the spermatozoon thins and forms the connection between the head and the tail. The tail is the motile organ of the spermatozoon and can be divided into the middle, main and end segments.
Materials and Instruments
Mouse Move I. Sperm Collection Caveat In a table glass dish containing culture fluid, the tunica and fat around the testis, epididymis and vas deferens were removed with ophthalmic scissors and rinsed out so that blood or fat globules would not mix with the fluid to hinder sperm observation. For more product details, please visit Aladdin Scientific website.
Gentian violet Alcohol Formalin phosphate buffer Kiemsa's solution
Absorbent paper Drip bottles Dropper tubes Slides Coverslips Scissors Tape Markers Watertight incubators CZB liquid KSOM liquid Syringes Instrument trays Tweezers Surgical scissors Ophthalmic scissors Ophthalmic forceps Ophthalmic foreign body needles Table slides Test tubes Microscope Scrubbing paper Constant-temperature water baths Hematocrit plates
The mice were executed by pinching the tail in the left hand and holding forceps in the right hand, or by dislocating the neck in the method shown in Figure 4-1. The executed mice were placed on their backs, and the hair and skin of the abdominal opening were sterilized with a 75 % alcohol cotton ball. The abdominal wall was cut open to expose the reproductive system. The testes, epididymis and vas deferens were isolated aseptically. In a table glass dish containing culture fluid, the tunica and fat around the testis, epididymis and vas deferens were removed with ophthalmic scissors and rinsed well so that blood or fat globules would not be mixed with the fluid to prevent spermatozoa observation.
The separated and cleaned testis, epididymis and vas deferens were further separated with ophthalmic scissors into three parts: testis, epididymal tail and the attached small section of vas deferens, and the rest of the vas deferens, and the epididymal head was discarded. To collect testicular sperm, the testis was transversely cut into several segments or tissue blocks in a surface dish with 1 mL of culture medium, and the testicular tissue blocks were gently squeezed with ophthalmic forceps to squeeze the sperm into the culture medium, and the tissue blocks were removed. Incubate at 37 ℃, 5% CO2, and saturated humidity for 20 minutes to allow the spermatozoa to disperse on their own.
To collect epididymal tail spermatozoa, cut them into several segments, gently squeeze the epididymis and vas deferens with ophthalmic forceps, squeeze the spermatozoa into the culture medium, and remove the epididymis and vas deferens. The spermatozoa were incubated at 37 ℃, 5% CO2 and saturated humidity for 20 minutes to allow the spermatozoa to disperse on their own. When collecting vas deferens spermatozoa, since the mouse vas deferens is very fine and the lumen cannot be flushed directly, the vas deferens was placed into a surface dish containing 1 mL of culture medium, and under a solid microscope, the vas deferens was fixed with an ophthalmic foreign body needle, and the vas deferens was torn longitudinally in the opposite direction with another foreign body needle, and the spermatozoa would float into diluted solution. The large piece of vas deferens tissue was dislodged, and the liquid portion was sucked out with a pipette together with the spermatozoa and stored in a 2 mL stoppered test tube.
Sperm motility test
The semen was aspirated with a dropper and placed in the counting chamber of the hemocyte counting plate in contact with the coverslip, so that the semen naturally flowed into the counting chamber. Count the number of sperm in 80 small squares in the middle 5 middle squares (diagonal 5 or four corners and the middle), and the count value is X. When counting, first count the X1 value of dead sperm, put the counting plate in the enameled plate in the water bath at 50 ℃, and kill the sperm at 50 ℃ for 10 min, and then count the total number of sperm, the X2 value, and the ( X2-X1 ) /X2 value is the motility of the sperm. The counting was repeated three times with three counting plates for each sperm, and the average value was taken.
III.Observation of overall structure of sperm
One small drop of preserved semen was taken on a slide and the sample drop was made into a smear in the form of a pull. Stain with 0.5 % gentian violet alcohol for 3 min, dry naturally, wash with water and then examine microscopically. Most of the spermatozoa were structurally normal and some of them were malformed spermatozoa, such as head malformations (e.g., huge, thin, elongated, rounded, inconspicuous, wrinkled, defective, double head, etc.), neck malformations (enlarged, slender, flexed, incomplete, double neck, etc.), tail middle malformations (enlarged, slender, curved, flexed, incomplete, double body, etc.), and tail main malformations (curved, flexed, gyrated, short, grown, double tail, etc.). , short, large, double tail, etc.) were categorized and counted. Some spermatozoa with incomplete tail development are immature spermatozoa and can be regarded as malformed spermatozoa.
Sperm acrosome structure observation
Semen smears were dried naturally for 2～20 min, fixed in 1～2mL formalin phosphate buffer for 15 min, washed and dried naturally. Stained with Jimsa's working solution for 90 min, washed with water and air-dried. The abnormal spermatozoa of acrosome were observed and counted by oil microscope under 1000x microscope. Most of the spermatozoa with normal acrosomal structure and some with abnormal acrosomes were observed under the microscope, and the spermatozoa with abnormal acrosomes were mainly characterized by swelling, defects, partial or complete detachment. 