Physiological and Behavioral Indicators to Measure Crustacean Welfare

Abstract

This project determined how neural circuits are affected during warming by examining sensory neurons, the neuromuscular junction, and the cardiac function and behavior of the commercially important crustacean species, the red swamp crayfish (Procambarus clarkii). Rapid inactivation of neural function in crustaceans prior to slaughter is important to limit exposure to noxious stimuli, thus improving animal welfare. This study demonstrated that as a crayfish is warmed at 1 °C/min, the heart beat stops at 44 °C. When temperature is rapidly increased, at 44 °C synaptic transmission at the neuromuscular junction ceases and primary sensory neurons stop functioning. Even though animals do not respond to stimuli after being warmed to 44 °C, if sensory neurons are returned to 20 °C saline after two minutes, they may regain function. Conversely, the neuromuscular junction does not regain function after two minutes in 44 °C saline. Examining behavior and heart rate while warming at 1 °C/min, 12 °C/min, or 46 °C/min to 80 °C indicated that at approximately 40 °C the heart rate is altered. Within 10 s at 80 °C, the heart stops with the highest heating rate. Directly placing crayfish in boiling water stopped the heart quickest, within 10 s, which likely represents denaturing of the tissue by heat. Using an impedance measure to detect a heartbeat may also be influenced by movements in the denaturing process of the tissue. A rapid increase in the temperature of the crayfish above 44 °C is key to limit its exposure to noxious stimuli.

Keywords: crayfish; heating; slaughter.

Figure 1

Placement of recording leads for measuring heart rate in an electrocardiogram (ECG) for a crayfish. The ECG leads are placed in an anterior-posterior arrangement for obtaining the best signals.

Figure 2

Recroding set up for ECGs while changing the water at various temperatures. The ECG leads were fed into the impedance converter which in turn was fed to an A/D board to record on a computer. The temperature probe was fed to a Vernier A/D converter to then record on a computer. The plastic chamber had insulation around it and a wire mesh on one side placed in the chamber to disseminate the water flow. A drain tube allowed the water to be removed as the new water was added at different temperatures.

Figure 3

Location of the muscle receptor organ (MRO). (A) A hemi-section of the crayfish abdomen viewed from ventral to dorsal after removal of the ventral muscle. The DEM muscle is cut on the right side of figure and pulled to left. The segmental nerve containing the nerves associated with the MRO was taken up by a suction electrode (the blue line outline is the segmental nerve). The deep extensor muscles (DEL1, DEL2, and DEM) and the superficial extensor medial muscle (SEM) are shown. The MRO organ is beneath the DEL1 muscle alongside to the DEM. (B) The superficial extensor medial muscle (SEM) was cut on the right side in the figure to expose the two MRO muscles. (C) The two MRO muscle fibers are shown with the nerve bundle pulling the fiber toward the SEM muscle. Scale bar: A and C is 2.5 mm; B is 5 mm.

Figure 4

Opener muscle preparation. (A) A view of the crayfish walking leg opener muscle and excitatory axon bundle for the muscle. (B) The muscle excitatory junction potentials were recorded from the distal muscles of the preparation.

Figure 5

Heart rate measures at various water temperatures starting at 21 °C and increasing at ~1 °C per minute. The ECG traces are shown at the various time points while the temperature was increasing. The letters in the top trace correspond to the traces shown below in alphabetical order. Each trace is 10 s in duration.

Figure 6

Heart rate measures at various water temperatures starting at 10 °C and increasing at ~12 °C per minute after conditioning to 10 °C for 24 h. The ECG traces are shown for crayfish after 24 h of being maintained at 10 °C prior to the heating and at each subsequent 12 °C interval during the heating to 60 °C. The flat line in the recording occurred at 58 °C. The sequence of events is in alphabetical order. Each trace is 10 s in duration.

Figure 7

Heart rate measures at various temperatures while increasing from 10 °C to 84 °C in crayfish conditioned at 10 °C for 24 h. The ECG traces are shown for crayfish after 24 h of being maintained at 10 °C prior to the heating and during the heating at a rate of 46 °C/min. The heart rhythm stopped at 80 °C. The arrow shown in E represents artifacts from re-zeroing the impedance detector while recording. The sequence of events is in alphabetical order. Each trace is 10 s in duration.

Figure 8

Heart rate measures at 11 °C and boiling water at 98 °C. The crayfish was conditioned to 10 °C for 24 h. (A) A representative ECG trace of a crayfish placed in a container at 10 °C after 24 h. (B) The crayfish was moved from one container to another with water at 11 °C to determine if the ECG trace was maintained with movement. (C) The impedance began recording immediately when placing the crayfish into the boiling water. Each trace is 20 s in duration.

Figure 9

Impedance measures of a single crayfish. (A) Impedance measure of the heart rate in the live crayfish for 1 min at 20 °C. (B) A 30 s impedance measure of the heart rate of the crayfish after being euthanized with CO₂. (C) Impedance measure of the dead crayfish placed in boiling water in the first min for 1 min and (D) after 1 min. (E) After 2 min in boiling water the impedance traces flatlined. The arrows shown represent artifacts from re-zeroing the impedance detector while recording. The sequence of events is in alphabetical order.

Figure 10

The activity of the MRO at various temperatures. (A1) Representative trace of the extracellular 10 s activity of the MRO segmental nerve in saline at 20 °C. (A2) Representative trace of the extracellular 10 s activity of the MRO segmental nerve at 44 °C. (A3) Representative trace of the extracellular 10 s activity at 20 °C after being at 44 °C for 1 min. (B) The mean number of spikes for the 10 s activity for each MRO preparation at each temperature. The heating to 44 °C had a significant effect in reducing the neural activity (N = 6, p < 0.05 non-parametric sign test). Each line represents individual preparations and some of the symbols overlap.

Figure 11

The excitatory junction potentials (EJPs) recorded from the opener muscle at various temperatures. (A1) Representative trace of the EJPs recorded with an intracellular electrode from the distal muscle fibers in opener muscle of a crayfish walking leg at 20 °C. The responses show a marked facilitation that occurs throughout the stimulation train delivered at 40 Hz for 25 stimuli. (A2) The EJPs are not able to be observed at 44 °C. (A3) Upon returning the preparation to 20 °C, the EJPs are still absent. The vertical bars represent the 25 stimuli pulses. (B) The mean 25th EJP amplitude (mV) for each opener muscle preparation at each temperature is shown (N = 6, significant difference between 20 °C and 44 °C, p < 0.05 paired t-test).