Table 2.

Summary of Zebrafish International Resource Center Sperm Cryopreservation, Thawing, and In Vitro Fertilization Protocol Changes from 2004 to 2014

Cryopreservation and IVF protocols	Thawing and IVF steps	Volume added (μL)	Total vol. (μL)	Osmolality (mmol/kg)
Draper–Moens Cryo	1. Thaw sample at 33°C	10		603
Draper–Moens IVF11,12 (2004–2008)	2. Add 70 μL HBSS	70		347
	3. Transfer sperm to eggs
	4. Activate with 750 μL fish water	750		44
	5. Swirl to mix		830
ZIRC 1
Draper–Moens Cryo ZIRC IVF Version 1 (2009)	1. Thaw sample at 38°C	10		603
	2. Add 40 μL HBSS	40		367
	3. Transfer sperm to eggs
	4. Activate with 320 μL dH2O	320		52
	5. No mixing		370
ZIRC 2
E400/RMMB Cryo ZIRC IVF Version 1 (2012)	1. Thaw sample at 38°C	20		547
	2. Add 40 μL HBSS	40		378
	3. Transfer sperm to eggs
	4. Activate with 320 μL dH2O	320		54
	5. No mixing		380
ZIRC 3 (current)
E400/RMMB Cryo ZIRC IVF Version 2 (2014)	1. Thaw sample at 38°C	20		547
	2. Add 150 μL SS300	150		335
	3. Activate with 200 μL dH2O	200		142
	4. Transfer sperm to eggs
	5. No mixing		370

Key changes made to the sperm thawing and IVF procedures are detailed. Solution volumes and resulting osmolality at each step are shown. See also Figure 4.

HBSS, Hanks' balanced salt solution; IVF, in vitro fertilization; ZIRC, Zebrafish International Resource Center.