Challenges in Development of Sperm Repositories for Biomedical Fishes: Quality Control in Small-Bodied Species

Abstract

Quality control (QC) is essential for reproducible and efficient functioning of germplasm repositories. However, many biomedical fish models present significant QC challenges due to small body sizes (<5 cm) and miniscule sperm volumes (<5 μL). Using minimal volumes of sperm, we used Zebrafish to evaluate common QC endpoints as surrogates for fertilization success along sequential steps of cryopreservation. First, concentrations of calibration bead suspensions were evaluated with a Makler^® counting chamber by using different sample volumes and mixing methods. For sperm analysis, samples were initially diluted at a 1:30 ratio with Hanks' balanced salt solution (HBSS). Motility was evaluated by using different ratios of sperm and activation medium, and membrane integrity was analyzed with flow cytometry at different concentrations. Concentration and sperm motility could be confidently estimated by using volumes as small as 1 μL, whereas membrane integrity required a minimum of 2 μL (at 1 × 10⁶ cells/mL). Thus, <5 μL of sperm suspension (after dilution to 30-150 μL with HBSS) was required to evaluate sperm quality by using three endpoints. Sperm quality assessment using a combination of complementary endpoints enhances QC efforts during cryopreservation, increasing reliability and reproducibility, and reducing waste of time and resources.

Keywords: biomedical model fishes; quality control; reproducibility; sperm volume.

FIG. 1.

Concentrations (beads/mL) of a calibration bead suspension estimated by using different volumes in a Makler^® counting chamber. Significant differences were not observed among the tested volumes of bead suspension placed on the chamber. The gray area represents the known concentration range of the calibration bead suspensions (mean ± SEM). SEM, standard error of the mean.

FIG. 2.

Concentrations (beads/mL) of a calibration bead suspension estimated by using different dilution methods with dilution performed directly on a Makler chamber (M), or dilution in a microcentrifuge tube (T) before placement in the chamber. Significant differences were observed between the two mixing methods. The gray area represents the known concentration range of the calibration bead suspension (mean ± SEM).

FIG. 3.

Percent total motility (proportion of swimming sperm) after activation across a range of osmolalities (N = 22) of Hanks' balanced salt solution.

FIG. 4.

Sperm concentration and the percentage of cells with intact membranes were analyzed and compared among five different sperm volumes (y-axis). Individual bars represent the means for each sperm volume (±SEM). The total number of cells (left axis, black bars), and percent of cells with intact membranes (right axis, white bars) are depicted side by side. Sperm volumes showing common letters were not significantly different (p < 0.0001).

FIG. 5.

Schematic of sperm suspension volumes (μL) potentially needed to address sperm concentration, motility, and membrane integrity at each step of the cryopreservation process for use in protocol development and validation (left) or in a production freezing scenario (right). In each scenario, based on this study, the total volume of sperm used to analyze the three endpoints was 4 μL. For protocol validation, three treatments and three replicates (3 treatments × 3 replicates × 4 μL = 36 μL sperm suspension) were proposed for comprehensive analysis of: fresh and refrigerated stored sperm, sperm exposed to cryoprotectants (equilibration), sperm frozen at different cooling rates, and frozen sperm thawed at different temperatures. All treatments included three replicates, leading to 144 μL of total sperm suspension. Based on the conservative assumption that only 2 μL of sperm was obtained per fish, and after a 1:30 initial dilution (which leads to 60 μL of sperm suspension at an approximate concentration of 2 × 10⁸ sperm cells/mL), three fish would be needed to obtain the necessary volume of sperm. In the production freezing scenario, four quality control check points, each with three replicates (3 × 4 μL = 12 μL sperm suspension), were proposed after sperm collection, storage, cryoprotectant addition, and sperm thawing. A total of 48 μL of sperm suspension would be needed (27% of the total sample if stripping three fish), indicating that (regardless of the number of fish needed in a particular cryopreservation assay) the sperm from one extra fish (60 μL after dilution) would be enough to address sperm quality along the cryopreservation process of pooled samples following the assumption of obtaining 2 μL of sperm per fish.