The impact of five years storage/biobanking at −80°C on mouse spermatozoa fertility, physiology, and function

Abstract

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.

Catechin Versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability—An In Vitro Study in a Swine Model

“…Compensation was assessed using CompBeads and FACSuite FC Beads (BD Biosciences) and single stained fluorescent samples. Data were analyzed first using FACSuite v 1.0.5 (BD Biosciences) software, and then FcsWizard Software was used to convert .fcs data to .csv format in order to perform an exhaustive analysis of the fluorescence emitted from every single spermatozoon for the various fluorescence probes [66]. To that, the columns "FCS", "SSC", "Fluo-4AM", "PI", "DilC12", "Mito" and "NIR" with the data from the 100,000 events acquired, were selected and filtered following these criteria: forward scatter (FCS) between 35,000 and 135,000 arbitrary units (a.u); side scatter (SSC) between 20,000 and 145,000 a.u; Fluo 3-AM, M540 and Mitotracker Red >0 a.u; PI between 0 and 30,000 a.u; and near infrared between 0 and 20,000 a.u.…”