This work aims to ascertain the usefulness of incubating overnight TESA spermatozoa (TS) in HEPES-buffered flushing medium (FM) at 37°C in the gaseous phase (6% CO2 in air).
After maceration in FM, the testicular tissue was divided into four equal portions for individual treatments (Tx). The suspensions are held overnight at either room temperature (RT) or 37°C with or without incubation gas (6% CO2 in air) as follows: (i) without CO2@RT; (ii) without CO2@37°C; (iii) with CO2@RT; with CO2@37°C. The tubes are capped tightly with incubation gases sealed within the tube, and kept at RT or 37°C (in the incubator) overnight. Statistical analyses performed were Chi-square, Pearson's correlation studies, paired-T test, and two-by-two tables.
A significant proportion of TS (21.4% vs 5.3%, p<0.001) became motile after Tx’s, indicating that physiological temperature (37 °C) and physiological pH (attained by overnight incubation in 6%CO2 in air) induced motility in and retained the viability of the TS. The differences between Tx’s were statistically highly significant (p<0.001), indicating the critical impact of both physiological temperature and physiological pH on TS viability and motility induction. There was a significant strong interaction (p=0.0000) between Tx’s and positive correlations between the Tx’s (p<0.001; highly significant).
Physiological temperature and pH appear critical for retaining the viability of and for initiating motility in TS. It is safer to induce motility with physiological temperature and pH than with embryotoxic pentoxyfylline or theophylline. When exposed to ambient temperature and air, the HEPES medium drifted toward the alkaline phase, making it less dependable for sustaining physiological pH levels between 7.3 and 7.4 for prolonged periods of time, particularly when there was no CO2 incubation gas present. In conclusion, physiological temperature and pH is critical to maintain the motility/viability of TS. HEPES medium must be equilibrated in 6%CO2 in air to maintain pH.