Effects Of Exogenous Acetazolamide On Growth And Calcium Flux In Alligator Embryos

For normal development and growth, archosaur embryos must rely on mobilisation of calcium from the calcareous eggshell. Dissolution of calcium depends on the enzyme carbonic anhydrase (CA) expressed in the chorioallantoic membrane (CAM). Earlier work on CA function in embryos has been limited to the domestic fowl (Gallus gallus), and shown that administration of the CA-inhibitor, acetazolamide (AZA), significantly decreased total calcium deposited into the yolk and embryo. We tested the effects of topical AZA administration on embryonic growth in the American alligator (Alligator mississippiensis), and compared its effects to that of physical eggshell removal from eggs. Over two seasons, we used six clutches (190 eggs) and administered topical solution of AZA (0, 150, 300 and 600 µg) in DMSO (20 µl) daily. AZA-treated eggs were compared to manually peeled eggs and sham-handled (unpeeled) controls. Eggs were incubated at 30°C and treatment lasted 40 days. Embryos were harvested at Ferguson stages 25-28. Like the eggshell-less group, AZA-treated groups resulted in a significant decrease (20%) in alligator embryonic growth compared to the control group, and growth restriction varied in a dosedependent relationship. In contrast to the eggshellless group, however, AZA-treated embryos had a significantly higher proportion of dry yolk mass and a greater fraction of yolk ash mineral. Equatorial eggshell thickness showed no significant difference between AZA and control groups. While these results suggest that topical administration of AZA has deleterious effects on embryonic growth of crocodilians, it does not appear to significantly reduce calcium transfer from the eggshell to the embryo and yolk, as observed in the domestic fowl. We conclude that AZA administration (at doses tested) does not effectively block calcium mobilisation in crocodilian eggs, and cannot be used in studies of calcium restriction on musculoskeletal development.