This document outlines the standard procedure for freezing semen, which involves:
1) Diluting the semen sample into two parts, with one part containing glycerol and the other not, then gradually combining the parts to achieve the desired final glycerol concentration of 7%.
2) Allowing the diluted semen to equilibrate at 5°C for 4-18 hours.
3) Packaging the equilibrated semen in vials, pellets or straws at 5°C.
4) Freezing the packaged semen by placing straws above liquid nitrogen vapor for 2 minutes then immersing in liquid nitrogen until use.
2. Standard technique of freezing
- Procedure for glycerolation:
-Divide diluter into part I and part II of equal
volume - Part I: no glycerol and semen will be
prediluted with part I
- Part II: glycerol added at twice the level
desired in the final mixture = 14% glycerol for
yolk-citrate or tris buffered-yolk diluter and
20-25% for milk diluter
-Both (I) and (II) solutions were kept in
refrigerator 5 cº
3. -After gradual addition of (II) to
(I) , keep the final mixture
containing 7%glycerol for
equilibrium period (at 5 cº) for 4-18
hours
-Package in either vials , pellets or
straws at the same temperature
4. Freezing
Freezing procedure: A single layer of straws
are placed on a tray at 5.5 cm above the
liquid nitrogen level; Straws will reach the
temperature of liquid nitrogen vapor in about
2 min.
Then immerse deeply inside the container
and kept until use.
