Veterinary Andrology and Reproductive Techniques

Sperm-oocyte co-incubation

SPERM OOCYTE CO-INCUBAION

• After one hour of capacitation, 100 ml of capacitated sperm drops is placed in a sterile disposable petridish to which 15-20 zona-free oocytes are added.
• Mineral oil should be layered over the sperm drop to avoid possible loss by evaporation.
• The petridishes are then incubated in air incubator at 37oC for three hours to allow sperm-oocyte coincubation.
• At the end of coincubation, the oocyte-sperm complexes are washed several times with handling medium to remove loosely attached spermatozoa.
• The oocytes are then subjected to either fluorescent staining or aceto-orcein staining to assess sperm penetration.
• Spermatozoa with decondensed heads and associated tails are considered to have penetrated the oocyte.