Prevention of high-temperature-induced chromosome damage in mouse spermatozoa freeze-dried using Ca2+ chelator-containing buffer alkalinized with NaOH or KOH

In order to protect sperm chromosomes against degradation when they are being stored for relatively high temperatures, we investigated the optimal pH of the freeze-drying solution, EGTA/Tris-HCl buffered solution alkalinized by NaOH (Na-ETBS) or KOH (K-ETBS). Mouse spermatozoa suspended in Na-ETBS or K-ETBS were freeze-dried at pH 5.0-8.4 and stored at 4 °C or 50 °C for 3 days. Some freeze-dried samples were stored at 25 °C for 3 days or 1 month. After storage, samples injected into oocytes using intracytoplasmic sperm injection were assessed for chromosome damage in resulting zygotes. Irrespective of freeze-drying solutions and storage temperatures, almost all the zygotes (97-100%) produced using the spermatozoa freeze-dried at pH 5.0 had structural chromosome aberrations of sperm origin. When freeze-drying was conducted at pH 8.0-8.4 using Na-ETBS, the incidence of chromosome damage decreased to 14-17% in 4 °C storage and 24-26% in 50 °C storage. When freeze-dried in K-ETBS, the lowest level of chromosome damage occurred at pH levels of 7.7-8.4 at 4 °C storage (13-15%) and at pH 7.7-8.0 at 50 °C storage (16-23%). Spermatozoa freeze-dried in Na-ETBS at pH 8.2 and K-ETBS at pH 7.7 showed no significant increase in chromosome damage during 25 °C storage from 3 days to 1 month (11%-20% in Na-ETBS; 13%-18% in K-ETBS). Thus, use of the solutions optimized for short-term storage at high temperature (50 °C, 3 days) permits prolonged storage (1 month) of freeze-dried spermatozoa at room temperature.