Principles of Genetics and Breeding

9.1.3 Methods followed in cryopreservation

Sperm collection

A. Stripping

1. First step is to anaesthetize brood fish with MS 222 or suitable anaesthetic.
2. Blot fish dry with towel to prevent water from mixing with sperm.
3. Gently massage abdomen to expel sperm, being careful not to contaminate sperm with urine or faeces.
4. Collect sperm in microhematocrit tubes, syringes, or centrifuge tubes depending on the amount of sperm to be collected.
5. Measure volume of sperm and dilute with extender solution (1:3,1:5,1:10.) depending on species, concentration.

In some species, pressure applied under the pectoral fins can cause release of sperm. This technique minimizes contamination of sperm with urine or faeces, which can cause activation of sperm or bacterial contamination. Dilution of samples in extender at the time of collection can counteract the effects of contamination by urine or water.

B. Surgical removal of testis (for fishes from which sperm cannot be stripped, catfishes and murrels)

1. Anaesthetize fish with MS 222 or suitable anaesthetic.
2. Blot fish dry with towel to prevent water from mixing with sperm.
3. Surgically remove testis from fish.
4. Remove excess blood and tissue from testis using cotton or tissue paper.
5. Weigh testis.
6. Measure extender solution (10 to 20 ml per g of testis).
7. Crush or slice testis to release sperm in a dry clean dish.