Ml136

The ECG electrodes were connected to bioamplifiers (ML136, AD instruments, Castle Hill, Australia; Range: 10 mV; Low-pass filter: 1 kHz; High-pass filter: 0.3 Hz; 50 Hz notch filter). The water temperature was recorded continuously using a custom-built temperature logger (EW 7221, Crn Tecnopart, Barcelona, Spain). Analog outputs from the recording equipment were relayed to a PowerLab system (AD Instruments, Sydney, Australia) connected to a computer running Labchart Pro software (v7.2.2, AD Instruments, Castle Hill, Australia).
The raw ECG signals were filtered and optimized (Band-pass digital filters in Labchart; High cut-off frequency range: 15–40 Hz; Low-pass range: 1–4 Hz) and heart rate was determined from the rate of ventricular depolarizations (i.e., R peaks) in the ECG. For each fish, we determined the highest heart rate during warming where heart rate peaked or plateaued (i.e., peak heart rate), which was typically followed by a progressive decline due to cardiac arrhythmias and/or a prolonged inter-beat interval. We also determined the temperature at which peak heart rate occurred. Fish condition factor was calculated as: $$\text{Condition factor}=(100\times \text{Body mass})/{\text{Body length}}^3$$ using body mass in grams and length in centimeters.

Animals were anesthetized using ketamine hydrochloride (100 mg/kg, i.m.) and diazepam (5 mg/kg, i.m.) in combination and mounted on a wax tray. The ECG signals were recorded using platinum hook electrodes connected to bio amplifier (ML-136) and single channel PowerLab (ML-826) (AD Instruments, Australia). The ECG signals were used to perform HRV analysis (Labchard PRO-8, AD Instruments, Australia) using the method described previously (Roy et al., 2018 (link)).