Figure 2:

Identification of best performing internal CPAs cocktail capable of retaining larvae (Tg(cmlc2:nGFP, mKate-CAAX)^fb18Tg) viability after 22 mins of storage at −10°C when injected through the duct of Cuvier and immersed in CPAs cocktail 33⁺ (A). Larvae viability was determined by quantifying heart rate, dehydration and blood circulation and reported as the internal composite viability score (CVS_Int, n = 4 animals per treatment). (B) Thirteen CPAs were arranged in a L25 Taguchi experimental design and tested at different concentrations (6) yielding 24 CPAs cocktails. Pre-freeze toxicity was determined after 10 mins of incubation with CPAs cocktails 1, 2, 6, 7 and 8 resulted in the best larvae outcomes as determined by above threshold (>1.5) CVS_Int (1.78, 1.75, 1.93, 2.3 and 2.3 respectively) post-thaw. (C) Heartbeat retention was seen in all larvae (4 per group) treated with CPAs cocktails 6, 7 and 8, while heartbeat retention was only seen 3 out of the 4 larvae treated with CPAs cocktails 1 and 2. (D) The best performing cocktails were re-tested in larger sample sizes (n = 8 per cocktail). Heart rate analysis of larvae treated with CPAs 6, 7 and 8 demonstrated the presence of approximately half the heart rate post-thaw when compared to pre-freeze rates. (E) Representative microscopic images of larvae GFP-labeled heart. High GFP signal intensity revealed the presence of viable cardiomyocytes in the hearts of larvae treated with CPAs 6, 7 and 8, while decreased signal intensity in hearts frozen without CPAs (Frozen^CON) indicate a reduction of cardiomyocyte viability. All statistical data is expressed as mean ± standard deviation and analyzed via ordinary one-way ANOVA with Tukey’s multiple comparison test. **** = p < 0.001.