Table 3.

Summary of sources of variation in flow cytometry scatter plots and subsequent population gating of zebrafish sperm exposed to cryoprotectants reported in this study, and possible solutions to reduce their effects on data interpretation.

Source of variation	Effect on sample	Effect on population gating	Magnitude of potential error*	Potential solution
Cellular or particulate contamination	Increased non-sperm events on FSC vs. SSC	Decreased sperm count as a proportion of collected events	+++	Calculate intact percentages based on fluorescent count
			Dependent on amount of contamination
Precipitation of solutes	Increased non-sperm events on FSC vs. SSC	Decreased sperm count as a proportion of collected events	+++	Omit calcium from cryopreservation buffers
			Dependent on cryoprotectant concentration	Filter cryoprotectant solution prior to sperm addition
				Calculate intact percentages based on fluorescent count
Bulk volume effect	Reduce total solution volume	Increase relative sperm concentration	+	Unavoidable physical property of aqueous solutions containing cryoprotectants
			Dependent on cryoprotectant concentration
Change in FSC characteristics	Events fall outside sperm gating region	Reduction in gated and fluorescent event counts	++	Wash to remove cryoprotectant prior to analysis
	Events fall below threshold	Reduction in total event count	Dependent on cryoprotectant type and concentration	Calculate intact concentration based on initial sperm concentration
Change in SSC characteristics	Events fall outside sperm gating region	Reduction in gated and fluorescent event counts	++	Wash to remove cryoprotectant prior to analysis
			Dependent on cryoprotectant type and concentration	Calculate intact concentration based on initial sperm concentration

^*The magnitude of potential error is expressed as≤5% (+),≤25%, (++), or ≤50% (+++).