FIG. 4.

ZIRC test thaws suggest that successive implementation of protocol changes resulted in a stepwise increase in post-thaw fertilization rates. The cryopreservation and in vitro fertilization protocols utilized were: Draper and Moens (2004–2008, n = 1475); ZIRC 1 (2009, n = 591): Draper–Moens freeze protocol with reduced IVF volume and no mixing of sperm and eggs; ZIRC 2 (2012, n = 334): cryopreserved using E400, RMMB, and optimized freeze rate, same IVF protocol as ZIRC 1; and ZIRC 3 (2014, n = 173): same as ZIRC 2, but osmolality of fertilization solution was raised (Table 2). Post-thaw fertilization rates were obtained and averaged, and an ordinary post hoc one-way ANOVA was performed using Tukey's multiple test to compare the mean of each column against the average of every other column. 0.0001 > p > 0.00001; the averages of every column were significantly different from every other column. Cohen's d (size effect) was determined for all comparisons: a versus b, d = 1.29, b versus c, d = 1.0; c versus d, d = 0.93; a versus d, d = 3.59; a versus c, d = 2.53; a versus d, d = 3.59). IVF, in vitro fertilization; ZIRC, Zebrafish International Resource Center.