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Disseminating mouse stocks as frozen materials offers both ethical and logistical advantages over live animal shipment, minimizing the welfare issues and avoiding some of the complex custom regulations that are associated with live animal transportation. Embryo freezing in liquid nitrogen (LN) at -196 °C has traditionally been the method of choice for archiving mouse lines. However, spermatozoa freezing is emerging as a more convenient alternative due to the application of innovative cryopreservation and recovery protocols. In addition, frozen spermatozoa are less sensitive to post-freezing temperature fluctuations. Here we demonstrated that spermatozoa frozen using standard laboratory protocols can be safely stored in dry ice (-79 °C) for at least seven days. The protocol we report here is robust and has been validated in a multi-centric study involving mouse spermatozoa samples exchanged between five European Mouse Mutant Archive (EMMA) nodes. Furthermore, following shipment on dry ice the spermatozoa can be returned to LN for long term storage without any noticeable detrimental effect. This protocol permits frozen spermatozoa to be shared and shipped in dry ice between biorepositories, networks and scientific institutions at low cost, using common courier companies, while avoiding the complexities, risks and hazards associated with using a traditional LN dry-shipper.

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D-Aspartate (D-Asp) treatment improved the fertility of young male C57BL/6N mice in vivo revealing a direct role on capacitation, acrosome reaction, and fertility in vitro in young males only. We investigated whether the positive effect of D-Asp on fertility could be extended to adult males and evaluated the efficacy of a 2- or 4-week-treatment in vivo. Therefore, 20 mM sodium D-Asp was supplied in drinking water to males of different ages so that they were 9 or 16 weeks old at the end of the experiments. After sperm freezing, the in vitro fertilization (IVF) rate, the birth rate, hormone levels (luteinizing hormone (LH), epitestosterone, and testosterone), the sperm quality (morphology, abnormalities, motility, and velocity), the capacitation rate, and the acrosome reaction were investigated. Oral D-Asp treatment improves the fertilizing capability in mice regardless of the age of the animals. Importantly, a short D-Asp treatment of 2 weeks in young males elevates sperm parameters to the levels of untreated adult animals. In vivo, D-Asp treatment highly improves sperm quality but not sperm concentration. Therefore, D-Asp plays a beneficial role in mouse male fertility and may be highly relevant for cryorepositories to improve mouse sperm biobanking.

Background We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a −80°C freezer, maintaining their ability to fertilize mouse eggs. Objectives The main objective here was to evaluate the effects of five years at −80°C compared to liquid nitrogen storage (LN2, control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. Materials and methods Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR‐14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3‐AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. Results Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at −80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2. Discussion Long‐term mouse sperm storage at −80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. Conclusion It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a −80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.