1) Sperm Concentration
The sperm concentration was determined using spectrophotometer (Jenway 6310, Bibby Scientific, England)
using the following formula (Dilution factor (201) × [25.97 × (Absorbance) − 0.30] for buck sperm concentration.
A total amount of 3 mL (2.9% of Sodium citrate) was placed in a microcuvette (HemoCue AB®, Angelholm,
Sweden) and calibrated using a spectrophotometer for 30 seconds. After calibration, Sodium citrate was
removed from the spectrophotometer. Thereafter, 15 μL of fresh undiluted buck semen was pipetted into a microcuvette
and then placed back in the spectrophotometer. The reading was recorded when the absorbance
number stops. Thereafter, spectrophotometer formula was used to calculate the sperm concentration for all samples.
The buck sperm concentration was recorded as (×106 sperm/mL).