Loss of fertilization potential of desiccated rhesus macaque spermatozoa following prolonged storage

Desiccation provides a novel spermatozoal preservation technique because it eliminates the need to store spermatozoa in liquid nitrogen, resulting in decreased opportunities for cross-contamination of samples and lower costs of spermatozoal banking, storage, and shipping. The objective of this study was to desiccate rhesus macaque spermatozoa and to evaluate the fertility following storage. Semen from four male rhesus macaques (Macaca mulatta) were collected using electroejaculation, washed through a Percoll gradient, and resuspended to 100 × 10⁶ spermatozoa/mL in a simple vacuum drying buffer containing the disaccharide trehalose (10 mM HEPES, 5 mM KCl, 65 mMNaCl, 150 mMtrehalose, 5.7% bovine serum albumin, BSA). Cells were desiccated in 50 μl drops under vacuum (22 inHg) at ambient temperature until the water content was less than 1 g H₂O/g dry weight. Initial motility was high (90–70%) and was reduced by desiccation (0%). Membrane integrity was investigated using the two fluorochromes, SYBR 14 and propidium iodide (PI, Molecular Probes, Inc.), with flow cytometry. After desiccation, 100% of the spermatozoa were stained red with PI indicating plasma membrane compromise. Samples were stored in air-tight polyvinyl plastic bags purged with N₂ gas for 10 s and vacuum sealed. The samples were protected from light and either stored at room temperature (treatment 1) or at −80 °C (treatment 2). Samples were rehydrated 7–10 days post desiccation in 150 μl BWW containing 0.5% BSA and used for intracytoplasmic spermatozoa injection (ICSI) to compare fertilization and embryo development to freshly collected samples. For control embryos, one freshly ejaculated motile sperm with normal morphology was immobilized by scoring a small incision in the plasma membrane over the sperm tail before injecting into an oocyte. For the dried sperm treatments, one sperm with normal morphology was selected and scored before injecting into an oocyte. After injection, the embryos were individually cultured in CMRL medium with 10% fetal bovine serum (FBS) media on pre-plated buffalo rat liver (BRL) cells at 37 °C and 5% CO₂. Fertilization was assessed at 14, 16, 22, and 24 h. Embryo development was evaluated daily from day 3 to day 11. The fertilization rate was 68%, 73%, and 45% for the control, treatment 1, and treatment 2 groups, respectively. The blastocyst rate was 40%, 5%, and 0% for control, treatments 1, and 2, respectively. Treatment group 1 had comparable fertilization rates with control group (73% vs. 68%) and was not significantly different (P < 0.05), but as development progressed, fewer embryos developed beyond the morula stage. Treatment 2 had a lower fertilization rate than control (45% vs. 68%), although not significantly different, and embryos did not develop past the morula stage. This study demonstrates that while the vacuum dried spermatozoa were immotile and had compromised plasma membrane integrity, they were capable of fertilization using ICSI and could support embryo development to the morula stage.