Thermostability Studies of Desiccated Murine Spermatozoa Nuclear DNA to Predict the Beneficial Effect of Trehalose in Long Term Storage

Long term preservation of mouse sperm in a desiccated state using sugars like trehalose may offer attractive economic benefits in the management of rapidly increasing transgenic mouse strains. The goal of the current study was to evaluate the protective effect of intracellular trehalose on sperm nucleus by predicting the long-term nuclear degradation kinetics of desiccated spermatozoa using an Arrhenius model whose parameters are obtained from high temperature-short time storage studies. B6D2F1 sperm isolated in an EGTA supplemented tris-HCl buffer (with or without 0.5M intracellular trehalose) were convectively dried with inert nitrogen gas in a controlled manner to moisture content >5%. The samples were then vacuum packed and stored at 22, 37, 45, 60 and 90°C for 1, 3 or 7 days. Following rehydration, the sperm sample was assayed for DNA damage using the sperm chromatin structure assay (SCSA). Results indicate significantly (p>0.05) lower DNA degradation for cells dried with intracellular trehalose at 45, 60 and 90°C for 1, 3 or 7 days compared to cells dried without trehalose. Based on a 10% increase in the index of injury, the calculated activation energy and frequency factors were 10.33 kcal/mole and 5.4×10⁵ hr⁻¹ respectively for cells dried in EGTA solution only. The corresponding numbers for cells dried in EGTA solution supplemented with 0.5M trehalose were 5.7 kcal/mole and 43.73 hr⁻¹. Based on these parameters the time required for 10% DNA degradation are 279 and 759 hours for samples desiccated in plain EGTA vs. trehalose supplemented EGTA. These results indicate the beneficial effect of intracellular trehalose for the long-term storage of desiccated sperm.