Normal semen is a mixture composed of secretions from the testes and epididymis with suspended spermatozoa, combined with secretions from the prostate, seminal vesicles, and bulbourethral glands upon ejaculation. The final ejaculated mixture is a viscous liquid. There are numerous methods for sperm analysis, among which culture-based detection is applicable.
The Stock Solution of BWW Medium for Sperm Cells is mainly composed of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and phenol red. It does not contain glucose, sodium pyruvate, BSA, or antibiotics. As a common nutrient solution designed for capacitation treatment of sperm cells from a wide range of animals and humans, it can be formulated into BWW Medium for Sperm Cells (also called capacitation medium) by adding glucose, sodium pyruvate, BSA, and antibiotics. Its standardized composition is based on the research published by the Biggers-Whitten-Whittingham (BWW) group in 1971, making it suitable for processing sperm cells from various animals and humans. This reagent is subjected to sterile treatment. For research use only. Not intended for clinical diagnosis or other purposes.
Materials to Be Prepared by Users
Sterile centrifuge tubes, Centrifuge, Cell culture incubator
Operating Procedures (For Reference Only)
1. Place the clean semen sample at room temperature for 30-60 minutes to allow complete liquefaction.
2. Sperm cell preparation (for reference only, non-mandatory step)
(1) Swim-up method
Take a sterile 15 mL conical-bottom centrifuge tube and add 1 mL of liquefied semen. Gently overlay 1.2 mL of Earle’s medium on top of the semen. Incline the tube at a 45° angle and incubate at 37°C for 1 hour. Slowly erect the tube and aspirate the top 1 mL of the liquid. Dilute the aspirated liquid with 8 mL of supplemented Earle’s medium, centrifuge at 500×g for 5 minutes, and discard the supernatant. Resuspend the cells with 0.5 mL of Earle’s medium for the evaluation of sperm density or function.
(2) Discontinuous density gradient method
Take a sterile 15 mL conical-bottom centrifuge tube and add 3 mL of 80% Percoll. Gently overlay 3 mL of 40% Percoll on top of the 80% Percoll layer; handle with care to avoid disturbing the interface between the two solutions. Gently overlay 1-2 mL of semen on top of the gradient solution, centrifuge at 500×g for 20 minutes, and discard the supernatant. Resuspend the sperm pellet at the bottom of the tube in 5-10 mL of Earle’s medium, centrifuge at 500×g for 5 minutes, and discard the supernatant. Resuspend the cells with 1 mL of Earle’s medium.
3. Incubate the centrifuge tube containing sperm cells in a cell culture incubator with 5% CO₂ and 95% air at 37°C for 1 hour. If such an incubator is unavailable, seal the tube with a cap and incubate in a conventional incubator at 37°C. During incubation, most motile sperm will swim out of the seminal plasma into the overlying culture medium.
4. Centrifuge the sperm suspension at 500×g for 5 minutes to adjust the sperm density to approximately 10×10⁶ cells/mL. Resuspend the sperm in 0.5-1 mL of BWW Medium for Sperm Cells (the medium supplemented with glucose, sodium pyruvate, BSA, and antibiotics is defined as capacitation medium). Incubate the suspension in a cell culture incubator with 5% CO₂ and 95% air at 37°C for 18-24 hours. If such an incubator is unavailable, seal the tube with a cap and incubate in a conventional incubator at 37°C. (Note: Incline the tube at a 20° angle during incubation.)
Precautions
1. Ensure aseptic operation and minimize the risk of contamination.
2. If Earle’s medium and supplemented Earle’s medium are unavailable, they can be replaced with the Stock Solution of BWW Medium for Sperm Cells.
3. Use the reagent as soon as possible after opening to prevent adverse effects on subsequent experimental results.
4. For your safety and health, wear a lab coat and disposable gloves during operation.
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