Freeze-dried sperm: an alternative biobanking solution for endangered farm species

Introduction An ever-increasing number of domestic species are threatened with extinction. Biobanking of spermatozoa could represent a feasible and efficient way for preserving genetic heritage and to maintain biodiversity. Given the published evidence that lyophilized spermatozoa retain their fertilizing capacity, we have collected semen from an Italian endangered sheep breed (Pagliarola) and created a biobank of cryopreserved and freeze-dried spermatozoa. Material and Methods The fertilizing capacity of the all stored semen, cryopreserved and freeze-dried, was evaluated by IVF and ICSI, respectively. To evaluate the activating 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 ionomycin (ICSI-FDSa) and 52 were un-activated (ICSI-FDSna). Pronuclear formation (2PN) was investigated at 14-16 hours after ICSI in fixed presumptive zygotes. Results and Discussion As expected, the fertilizing capacity of cryopreserved Pagliarola’s spermatozoa was comparable to commercial semen stocks (31.8% vs 29%, respectively). Two PN were observed in 83.3% of ICSI-FDSa while only in 14.3% of ICSI-FDSna. Likewise, only artificially activated oocytes were able to develop to blastocyst after ICSI (10.2% compare to 0% with ICSI-FDSna). Oocytes injected with frozen spermatozoa (ICSI-FS) and left un-activated, served as control (81.4% of 2PN; 31% of blastocyst). In this work, we have demonstrated for the first time that freeze-dried ram spermatozoa could drive blastocyst development following ICSI. Although the developmental potential of embryos derived from lyophilized spermatozoa was significantly lower than cryopreserved ones, sperm lyophilization may be an alternative, low cost storage option, susceptible of improvement of course, to save biodiversity in domestic species.