Sperm freeze-drying is a revolutionary technique, which has been gaining prominence in recent years. The first related significant result was Wakayama and Yanagimachi’s demonstration in 1998 of the birth of healthy mouse offspring by Intracytoplasmic Sperm Injection (ICSI), using epididymal freeze-dried spermatozoa. Mouse, rat, and hamster models were the first small mammals born from lyophilized epididymal spermatozoa, whereas most other studies in this field used ejaculated spermatozoa. In this work, we applied this technique to ram epididymal spermatozoa, checking the correlation between DNA integrity and embryo development following ICSI. To do this, epididymal sperm from four rams was lyophilized in a trehalose, glucose, KCl, HEPES, and Trolox media. To evaluate DNA damage and fragmentation after rehydration, samples were processed for Sperm Chromatin Dispersion test (SCD), Two-Tailed Comet Assay, and were used for ICSI. Ram #2 had a higher rate of spermatozoa with intact DNA compared with rams #1, #3, and #4 (28% vs. 3.8%, 2.8%, and 5%, respectively) and the lowest rate of Single-Strand Breaks (SSBs) (70% vs. 95.9%, 92.6%, and 93% respectively). Ram #3 had a higher level of Double-Strand Breaks (DSBs) compared to Ram #1 (4.6% vs. 0.33%, respectively). Embryo development to the blastocyst stage following ICSI was only reached from rams whose sperm had higher level of intact DNA – Rams #2 and #4 (6%, 5/147 and 6.3%, 4/64, respectively). Definitively, the impact of sperm DNA damage on embryonic development depends on the balance between sperm DNA fragmentation extent, fragmentation type (SSBs or DSBs), and the oocyte’s repair capacity.

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Dry biobanking has many advantages over the current paradigm of storing cryopreserved cells under liquid nitrogen. During drying, however, the cells become damaged. The highly condensed spermatozoa DNA has been shown in many desiccation studies to generally maintain its integrity. Using ram freeze-dried epididymal spermatozoa as a model, Palazzese et al. were the first to evaluate both single- and double-strand DNA breaks (SSBs and DSBs, respectively), showing that drying causes minimal DSBs but extensive SSBs (Palazzese L et al., 2018. DNA fragmentation in epididymal freeze-dried ram spermatozoa impairs embryo development, pp. 393–400). Furthermore, the authors also demonstrated that spermatozoa capable of directing embryo development to the blastocyst stage in vitro originated from rams with the least DNA damage Overall, the impact of sperm DNA damage on embryonic development depends on a balance between the extent of sperm DNA fragmentation, fragmentation type, and the oocyte’s repair capacity.