DNA fragmentation of epididymal freeze-dried ram spermatozoa impairs embryo development

Sperm freeze-drying is a revolutionary technique that resolves many of the drawback of long term storage under liquid nitrogen. The first significant result of this method was provided by Wakayama and Yanagimachi in 1998, demonstrating for the first time the birth of healthy offspring from epididymal freeze-dried (mouse) spermatozoa. Since then, all animal born from ejaculated sperm. In this work, aiming to repeat the strong result of Wakayama and Yanagimachi, we tried to apply this technique to epididymal spermatozoa from a large mammal (ram). Moreover, we checked the correlation between freeze-dried spermatozoa DNA integrity and embryo development. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, Trolox media. To evaluate DNA damage and fragmentation, at rehydration part of the sperm was processed for Sperm Chromatin Dispersion test (SCD) and Two-Tailed Comet Assay and the rest was used for Intracytoplasmic Sperm Injection (ICSI). When compared to rams 1 and 3, rams 2 and 4 had higher rate of spermatozoa with intact DNA (the median is 3.3 % vs 16.5 % respectively), lower rate of Single Strand Break (SSBs) (the median is 94.2% vs. 81.5% respectively) and lower rate of Double Strand Break (DSBs) (2.5% vs. 2% respectively). Embryo development following ICSI displayed that blastocyst stage was reached only from rams that had sperm with more intact DNA - ram 2 (4.8 %, n= 83) and ram 4 (6.3 %, n=64). Spermatozoa from ram 1 and 3 produced no blastocysts. This can be explained that the ram 2 and 4 had the higher rate of spermatozoa with intact DNA than the ram 1 and 3. Definitively, the implication of sperm DNA damage in embryonic development should depend on the balance between the extent of sperm DNA fragmentation, the type of fragmentation (SSBs or DSBs), and the oocyte’s repair capacity. Therefore, rams 2 and 4 were the only rams that produced blastocyst because they had considerably more sperm with normal DNA and so it is important to select the spermatozoa with the best quality to do a good ICSI. To conclude, oocytes injected with epididymal freeze-dried ram spermatozoa can reach the blastocyst stage. DNA fragmentation due to the lyophilization process impairs embryonic development. These are preliminary results. More conclusive outcome will be given following embryo transfer experiments, now in progress.