Beyond swimming: emerging parameters for predicting the fertility of mouse spermatozoa

Cryopreservation of spermatozoa can be used as a cost-effective way of preserving the ever-increasing number of genetically modified mouse lines. Nevertheless, discontinuing the breeding of a line or strain is only warranted after the quality control of cryopreserved sperm; the ability of sperm to survive the freezing-thawing process and produce well-developed embryos needs to be confirmed. In animal research husbandries, the fertility of frozen-thawed sperm is routinely tested using in vitro fertilization. However, this procedure requires euthanizing a considerable number of females to acquire a sufficient number of oocytes, contradicting the 'reduction' principle of the 3Rs (replacement, reduction and refinement). Therefore, the research community has an interest in replacing in vitro fertilization tests with proxies that can collectively characterize the fertilizing potential of mouse sperm. Methods such as computer-assisted sperm analysis and flow cytometry enable a precise and multiparametric approach to evaluate sperm quality, encompassing 'traditional' traits and the functional status of subcellular structures of sperm. Moreover, single-cell data can be processed with machine learning algorithms, offering a deeper insight into sperm physiology and functional heterogeneity. Despite the advancements made, many of these assays are still far from being used in mouse sperm quality control owing to their limited time- and cost-efficiency, the insufficiency of fertility validation studies, and the complex data analysis needed to identify fertility markers. The genetic and phenotypic diversity of different mouse strains and lines makes the establishment of a robust methodology for fertility prognostics even more challenging. Thus, this Review summarizes the available methods for assessing sperm functional characteristics in laboratory mice and discusses their contribution to fertility prognosis.