Respirometry: Correcting for Diffusion and Validating the Use of Plastic Multiwell Plates with Integrated Optodes

Abstract

Polystyrene multiwell plates with integrated optodes act as multiple closed-system respirometers that enable the simultaneous measurement of oxygen consumption in small animals. However, the diffusion of oxygen through polystyrene needs to be taken into consideration. Here we provide an equation that accounts for the empirically determined rate of oxygen through a polystyrene well when calculating the instantaneous rate of oxygen consumption. Furthermore, we describe a novel method of calibrating a small respirometer for accuracy using micro-osmotic pumps containing an oxygen scavenger, which is delivered at a constant rate and therefore yields a constant rate of oxygen consumption in an airtight system.

Growth hormone transgenesis and polyploidy increase metabolic rate, alter the cardiorespiratory response and influence HSP expression to acute hypoxia in Atlantic salmon (Salmo salar) yolk-sac alevins

“…Measurements of metabolic rate (Ṁ O 2 ) were performed at 8°C on diploid (2n) and triploid (3n) transgenic (2nTx and 3nTx) as well as 2n and 3n non-transgenic (2nNt and 3nNt) Atlantic salmon yolk-sac alevins [Series I, as described in Polymeropoulos et al (Polymeropoulos et al, 2013)]. Subsequent measurements were conducted of Ṁ O 2 , heart rate (f H ), as well as ventilation rate (f V ; see Series II).…”

“…In parallel, 10 alevins per genotype were maintained under identical experimental conditions in normoxia (control group). For this purpose, the alevins were placed in multiwell plates [similar to that described in Polymeropoulos et al (Polymeropoulos et al, 2013)], submerged in well-aerated water with only a thin mesh separating the alevins from escaping into the surrounding water. The control group was maintained within the same multiwell plate for a duration that matched that of the experimental animals (measurement + 24 h); this included a quick removal from the wells to mimic the handling stress to which the experimental animals were exposed.…”

“…Each alevin was placed in an individual well in a polystyrene multiwell plate (24 wells×3.3 ml, Oxodish ® , PreSens, Regensburg, Germany) that was filled with air-equilibrated water from the rearing trays (partial pressure of O 2 , P O2 =21 kPa). Each well contained an optode to monitor O 2 levels and was fitted with a stainless steel mesh positioned 3 mm from the bottom to ensure the alevin did not contact the optode [as described in Polymeropoulos et al (Polymeropoulos et al, 2013)]. Four wells remained empty of animals for calibration purposes: two were filled with normoxic water from the rearing trays and two with a zero solution (1% sodium sulphite solution, which acted as an O 2 scavenger).…”

“…The Ṁ O2 (μmol O 2 min −1 ) for each egg or alevin was calculated from the decline in P O2 over time in each well, after taking into account the diffusive flux of O 2 that occurs through the polystyrene Oxodish ® well plate (see Polymeropoulos et al, 2013).…”