(A) Representation of the zebrafish in the chambers, the Oroboros Oxygraph, and the information output for each age of the zebrafish. The zebrafish are placed in the Oroboros oxygraph chamber with 2 mL of E3 media with the stirrer speed set to 26 rpm. The chambers are closed to accurately measure the oxygen concentration in the chamber. If additional reagents are added, they are added through the standpipes via a Hamilton syringe. An example of the output in embryos is on the right side of the figure. The blue line is oxygen concentration in the chamber. The red line is the tangent of the blue line. (B) Mitochondrial respiration measurements in 5 days post fertilization embryos using the Oroboros stir bar set at a speed of 26 rpm. Basal respiration was measured 5 min after the embryos acclimated to the chamber (circles). Oligomycin was used to initiate leak respiration (25 μM; squares). Maximum respiration was achieved by adding FCCP (2.5 µM, triangles). Rotenone (1.3 µM) and antimycin A (1.8 µM) were used to determine non-mitochondrial respiration (upside down triangles). Complex IV activity (diamonds) was measured by the addition of ascorbate (10 mM) and TMPD (0.3 mM). Each point equates to one experiment and each experiment contains 60 embryos.

(A) Mitochondrial respiration measurements in larvae 0.6–0.9 cm in length using the thinner stir bar (mentioned in methods) set at a speed of 26 rpm. Basal respiration was measured 5 min after the embryos acclimated to the chamber (circles). Oligomycin was used to initiate leak respiration (25 μM; squares). Maximum respiration was achieved by adding FCCP (2.5 µM, triangles). Rotenone (1.3 µM) and antimycin A (1.8 µM) were used to determine non-mitochondrial respiration (upside down triangles). Complex IV activity (diamonds) was measured by the addition of ascorbate (10 mM) and TMPD (0.3 mM). Each point equates to one experiment and each experiment contains 5 larvae ranging in size from 0.6–0.9 cm. (B) Comparison of the different respiration rates from Figure 1 and Figure 2 for embryos (black) and larvae (grey). The rates were normalized to the basal respiration rates for each experiment. (**** indicates a p-value < 0.00001).

(A) Basal respiration rates in young (3-month-old) adult male and female zebrafish. Using the thin stir bar (see methods) set at 26 rpm, basal respiration was measured on males and females. (* indicates a p-value < 0.5) (B) Basal respiration rates in old (12-month-old) adult male and female zebrafish. Using the thin stir bar (see methods) set at 26 rpm, basal respiration was measured on males and females. (** indicates a p-value ≤0.01).

(A) Basal respiration rates in young (3-month old) compared to old (12-month-old) adult males. Using the thin stir bar (see methods) set at 26 rpm, basal respiration was measured on males and females. (B) Basal respiration rates in young (3-month old) compared to old (12-month-old) adult females. Using the thin stir bar (see methods) set at 26 rpm, basal respiration was measured on males and females. (**** indicates a p-value < 0.00001).

Individual basal respiration rates in young (3-month old) adult males (A) and females (B). Using the thin stir bar (see methods) measurements were made initially at 26 rpm (blue), then at 100 rpm (green), then at 200 rpm (yellow), and after return to 26 rpm (red).