Artificial activation of ovine oocytes is required after ICSI with freeze-dried spermatozoa.

Objectives. Freeze-drying allows to store the biological samples in a dry state and represents an interesting alternative low-cost strategy of semen biobanking to save the endangered species. Here, we have established a dry sperm biobank from an endangered Italian sheep breed (Pagliarola) and tested its efficiency through ICSI. Materials and methods. The motile spermatozoa from ram ejaculates collected with artificial vagina was selected by swim-up in TRIS-based medium (2.42g TRIS, 1.36g citric acid, 1.00g fructose, 100.000 U.I. penicillin G, 100mg streptomycin, in 67.20ml bidistilled water (ddH2O); pH was adjusted to 6.7) for 20 minutes at 38.5°C. The motile spermatozoa were frozen in freeze-drying medium (10mM EGTA and 50mM NaCl in 10mM Tris–HCl buffer; pH was adjusted to 8.4) in a -80°C freezer for 75 minutes and subsequently lyophilized by the freeze-drying apparatus SP Scientific-VirTis, Freeze-dryer 2.0 BenchTop, 20 hours with a condenser temperature of -58°C and vacuum of 20 mTorr). The vials were sealed in glass vials under vacuum and stored in the dark at 4°C for 1-2 months. Just before the ICSI, the freeze-dried spermatozoa were rehydrated by adding 100µl ddH2O. To evaluate the fertilizing capability of freeze-dried spermatozoa, 108 MII sheep oocytes were subjected to ICSI and allocated to two groups: 56 oocytes were activated by incubation with 5μM ionomycin (ICSI-FDSa); 52 were left un-activated (ICSI-FDSna). Forty-four oocytes injected with frozen spermatozoa (ICSI-FS) and left un-activated, served as control. Pronuclear formation (2PN) and blastocyst development were investigated at 14-16 hours and 7-8 days after ICSI, respectively. Differences were considered statistically significant for p<0.05 (Chi-square test). Data were analyzed using PRISM, software version 5.0; GraphPad. Results. The freeze-dried spermatozoa were completely immotile after rehydration, however they maintained the capacity to fecund oocytes after ICSI. Two PN were found in 83.3% of ICSI-FDSa, 81.4% of ICSI-FS while only in 14.3% of ICSI-FDSna (p<0.05 ICSI-FDSna vs ICSI-FDSa; p<0.01 ICSI-FDSna vs ICSI-FS). Likewise, the ICSI freeze-dried spermatozoa yielded blastocysts only following artificial activation (ICSI-FDSa: 10.2%; ICSI-FS: 31%; ICSI-FDSna: 0%; p<0.05 ICSI-FDSa vs ICSI-FDSna andICSI-FS; p<0.0001 ICSI-FDSna vs ICSI-FS). Conclusions. Our finding show that freeze-dried spermatozoa have lost the capacity to trigger oocyte activation but maintained their nuclear viability, whose developmental potential was fully released following artificial activation. Our results support the evidence that freeze-drying effective approach of spermatozoa storage to save endangered species.