Overview :

Supravital stains can help differentiate live from dead sperm. The findings depend on the integrity of the plasma membrane of viable sperm; an intact plasma membrane is able to exclude certain stains. The percent viable sperm correlate with sperm motility.

Indications for performing a supravital stain: when motility is < 40%

Stains:

(1) eosin Y (1% eosin Y in isotonic phosphate buffer)

(2) eosin Y-nigrosin (1% eosin Y and 10% nigrosin in distilled water)

A small amount of semen is mixed with the stain. A smear is then prepared and air-dryed. The smear is examined with an oil-immersion lens.

percent viable sperm =

= (number of viable sperm) / ((number of viable sperm) + (number of nonviable sperm)) * 100%

Limitations:

• Supravital stains do not correlate with motility after cryopreservation (Ochninger et al).

Adelman MM, Cahill EM. Atlas of Sperm Morphology. ASCP Press. 1989. pages 11-13 and 99-101.

Ochninger S, Duru NK, et al. Assessment of sperm cryodamage and strategies to improve outcome. Molecular and Cellular Endocrinology. 2000; 169: 3-10.

World Health Organization (WHO). WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Fourth edition. Cambridge University Press. 1999. Appendix IV, pages 68-70.