The aim of this study is to determine the usefulness of overnight incubation at 37oC in gaseous phase (6%CO2 in air) of microTESA testicular spermatozoa harvested (mTS) in HEPES-buffered flushing medium (FM).
The testicular tissue was macerated in FM and apportioned into 4 equal portions for individual treatments (Tx). The macerated microTESA tissue suspension was incubated with or without incubation gas (6%CO2 in air) and were held at either room temperature (RT) or 37oC overnight as follows: (i) Without CO2@RT; (ii) Without CO2@37oC; (iii) With CO2@RT; With CO2@37oC. After exposure to incubation gas for maximum 60 mins in centrifuge tubes, it is sealed airtight with its cap with the gases sealed inside the tube, and placed at RT or at 37oC. Statistical analyses used were paired-T test, Pearson’s correlation studies, Chi-square and 2 by 2 tables.
Significant proportion of TS (11.4% vs 3.3%, p>0.0001) became motile after Tx’s indicating both physiological temperature (37oC) and physiological pH (attained after overnight incubation in 6%CO2 in air) induced motility in and retained the viability of the mTS. The differences between Tx’s (except between control versus RT20; P>0.05) were statistically highly significant (p<0.0005). There was significant interaction between Tx’s and highly significant (p=0.0000) positive correlations.
Physiological pH and temperature induces motility in mTS, which is a safer method to initiate motility in mTS than embryotoxic pentoxyfylline or theophylline. The HEPES buffered medium drifted towards alkaline phase which makes this medium less reliable for maintaining the physiological pH of 7.3-7.4 for extended periods of time when exposed to ambient air and temperature especially in absence of CO2 gas mixture. In conclusion, physiological temperature and pH is critical to retain the viability and to initiate motility in mTS. The HEPES medium must be equilibrated in 6%CO2 in air to maintain the pH close to physiological level.