Separate animals were used for measurement of total CO2 (TCO2),
lactate and ammonia, and calcium and chloride ion concentrations.
The coelomic fluid samples (500 μl) were withdrawn anaerobically
and transferred to 1.5 ml Eppendorf tubes under mineral oil. They
were immediately frozen and stored at −80 °C for not more than a
week prior to analysis. The samples were allowed to thaw at room temperature
and total CO2 was measured for 50 μl samples using a Corning
965 carbon dioxide analyser (Ciba Corning Diagnostic, Halstead, 203
Essex, UK). Comparison of freshly collected and frozen samples showed
the freezing process had no effect on TCO2 levels. Coelomic fluid [HCO3
−]
and PCO2 were calculated using measured values of fluid pH and total