The Need for a Framework Addressing the Temporal Aspects of Fish Sperm Motility Leading to Community-Level Standardization

Motility is a widely available parameter that can be used to assess sperm quality of aquatic species. Sperm from fishes with external fertilization usually undergo a dynamic and short-lived period of motility after activation. The common practice of assigning a single value at an arbitrary peak of motility presents challenges for reproducibility, community-level standardization, and comparisons across studies. This study aimed to explore statistical approaches to standardize motility reporting, and to develop an initial framework for community-level standards. Sperm samples from 14 zebrafish (Danio rerio) with a total of 21,705 cells were analyzed by use of computer-assisted sperm analysis with data collection starting at 10 s after activation at 5-s intervals for 50 s. Four common motility variables were selected for analyses: curvilinear velocity, straight-line velocity, beat cross frequency, and amplitude of lateral head displacement. Cluster analysis was used to evaluate sperm subpopulations within and among males over time, least-square means was used to explore temporal aspects, and the first derivative of the regression equations was used to calculate the rate of change for the motility parameters. Cluster analysis proved informative, but overlapping ephemeral clusters were not valuable for providing standardization options. Analysis of temporal aspects and rate of change indicated opportunities for standardization by reporting the overall motility-time functions or reporting during stable time windows instead of peak motility or at random times. These approaches could minimize the inconsistencies caused by male-to-male variation and dynamic changes of subpopulations while providing comparable information. An overall temporal framework was identified for motility reporting along the collection-processing-cryopreservation-thawing sequence to provide a basis to support efforts of community-level standardization.