Table 4.

Major factors (under-appreciated for aquatic species in general) that need to be defined, controlled, and reported to enable direct comparisons of sperm cryopreservation results for protocol standardization and application.

Step or process	Factor to be defined	Relevance to cryopreservation
Source, housing, and conditioning of fish	Strain and source	Variation among populations or mutant lines can influence results
	Size and age	Can affect gamete quality and quantity: report body weight, length, age
	Maturity	Reproductive condition: report sperm volume, or testis mass, and GSI*
	Fish culture	Water quality parameters, temperature, salinity, and light:dark cycles
	Male selection	Using best males may not represent normal variation: report selection criteria
Sample preparation and freezing process	Initial quality	Major influence on post-thaw quality: requires assessment and definition
	Sperm density	Can affect cryopreservation and fertilization: a major uncontrolled variable unless set
	Cryoprotectant	Type and final concentration is a critical factor: should be defined and reported
	Motility	Sperm motility prior to addition of cryoprotectant: should be defined and reported
	Equilibration time	Requires tight control of methods and temperature: should be defined and reported
	Packaging	Affects heat transfer: type, size, and materials should be defined and reported
	Cooling rate	Critical factor: method should be defined with start and finish temperatures
	Storage time	Duration can differentiate freezing and super-cooling: should be defined and reported
Egg collection and use of thawed sperm samples	Pooling of eggs	Should be reported if used to provide sufficient numbers for experiments
	Thawing process	Warming temperature and duration (rate): should be defined and reported
	Post-thaw motility	Necessary to estimate effects of cryopreservation: should be defined and reported
	Fertilization	Can influence gamete activation and fertilization especially for thawed sperm
	Sperm-to-egg ratio	Concentration and volume of sperm for fertilization: should be defined and reported
	Egg quality	Should report if fresh sperm was used to evaluate fertilization of eggs
	Fertilization rate	Reporting of exact definition of fertility criteria should be compulsory
	Hatching rate	Reporting of absolute or relative values should be identified (report both)

^*GSI, gonadosomatic index, the percentage of testis weight in relation to the body weight.