**2.3 Preparation of extender-cryoprotectant**

Two extenders were used in this study, phosphate buffered saline (PBS) and Ginzburg Fish Ringer (GFR) with pH of 7.4 and 7.6 respectively. The extenders were prepared as shown in Table 1 with Calcium-free Hanks Balanced Salt Solution (Ca-FHBSS) used in experiment 2. 14; after which they were sterilized for 20 min at 15 lbs/inch2 using a pressure cooker to avoid contamination and deterioration of the spermatozoa.
