EXAMINATIONS WITH LIGHT MICROSCOPY
LIVE/DEAD AND ACROSOME STAINING
Kovács A. & Foote, R.H. (1992): Viability and acrosome staining of bull, boar and rabbit spermatozoa Biotechnic and Histochemistry 67:3 119-124.
Dilution of the semen with 0.9% NaCl.
Fresh bull semen: 100x
Frozen-thawed bull semen: 5-10x
Viability testing stain: 0.20% trypan blue (prepared from 0.4% trypan blue, Sigma T 8154 diluted 1:1 with 0.9% NaCl). This working solution is stable for several months at room temperature.
The fixative is composed of 86 ml of 1 N HCl plus 14 ml 37% formaldehyde solution and 0.2 g neutral red (Sigma N 2880), it is stable for about two months at room temperature and may be used repeatedly.
The acrosome stain is 7.5% Giemsa stock solution (Sigma GS 500) in distilled water prepared freshly before use.
Staining procedure:
1. Equal drops of trypan blue and diluted semen are mixed on slides with the edge of another slide and smeared.
2. Air drying near vertically at room temperature.
3. Fixing in a staining jar for 2 min.
4. Rinsing with tap, and distilled water.
5. Staining in Giemsa for 3.5 hr, or overnight.
6. Rinsing in tap, and distilled water.
7. Differentiation in distilled water for 2 min.
8. Air drying.
9. mounting with coverslip.
10. Evaluation using 40x dry, or 100x oil immersion objectives.
For live/dead assessment the posterior, for the status of the acrosome the anterior part of the head provides information (Figures).
Live spermatozoa are white to light pink,
Dead spermatozoa are black to dark violet or grey.
Intact acrosomes are purple,
Loose acrosomes are dark lavender,
Damaged acrosomes are pale lavender,
Live spermatozoa with no acrosome are white to light pink,
Dead spermatozoa with no acrosome are white to pale grey.
Spermatozoa with stained (black) tails are nonmotile.
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