Introduction
The white leg shrimp, Penaeus vannamei is the worldwide leader of aquaculture production for crustaceans, with over 7 million tonnes farmed in 2022 alone (FAO, 2024). Production at these volumes can lead to poor welfare standards, especially if done in remote or inaccessible places. Welfare standards include the best practices for manipulation and before euthanasia, especially in farms with large volumes of production.
However, for shrimps, the effect/response to anaesthetics is vaguely known (Rotllant et al., 2023). A newly developed definition should tackle EFSA’s triangulation principle, correlating the organisms’ responses at different levels of organisation. Farm standard practices involve using slurry ice as an anaesthetic before dispatch, although its effect remains unknown. In this work, we conducted a three-part experiment to characterize behavioural, metabolic, and neurophysiological responses to ‘cold’ seawater (~10°C) as an anaesthetic to determine if this practice reduces this shrimp species’ neural and physiological activities.
Materials and methods
Juveniles of P. vannamei of 21.1±1.3 mm carapace length (CL) and 6.3±0.5 g wet weight (ww) were delivered to the aquaria facilities of the Autonomous University of México (UNAM), Sisal, and left for two weeks at 24°C and 34UPS. Animals were fed commercial pellet food daily and maintained under a 12L:12D period. The working temperature was 10°C, defined as the thermal condition in which animals were rendered unresponsive in less than 10 min and recovered their normal position in under 20 min.
The first set of experiments on the effects of temperature focused on the behavioural responses of shrimp, particularly aiming to find the time when animals remain at the bottom of the tank lying on their side, and after were unresponsive when stimulated by touching the legs. This last marked the desired response and after which water was changed to let the animals recover (at 24°C). Animals were sampled at the end of the anaesthesia stage (n=7), or the end of the recovery (n=7) to measure metabolic and immunologic stress markers in haemolymph, muscle and midgut gland.
The second set of experiments focused on the effects of temperature on the metabolic rate (MR) of P. vannamei. The oxygen consumption (VO2) was measured using a continuous flow respirometer of a closed chamber connected to a recirculating seawater system. Animals were placed in the respirometry chamber 12h before the experimental measurements, with a shrimp-less chamber used as a baseline. Once 120 min of baseline was recorded, the cooled seawater was added to the system, allowing the temperature inside the respiratory chambers to drop to ~10°C after 17 min. After 10 min of exposure, the system was refilled with seawater at 24°C. Metabolic rate was measured after, during, and before the seawater temperature change.
Lastly, neurophysiological activity and heart rate were recorded. Electrodes were implanted flanking the supraesophageal ganglion to measure electroencephalogram (EEG), visual evoked responses (VERs), and flanking the pericardial zone to measure heart rate (HR). Once a baseline was measured, water was changed to add the anaesthetic (cold water, ~10.5°C) and measures continued for 5 min, before water was changed again, to measure the parameters of recovery.
Analysis of variance (ANOVA) was used to evaluate if there were differences in each experimental process; analyses were carried out using R version 4.4.2.
Results
Exposing animals to cold water induced an almost immediate equilibrium loss, after several body spasms, and, unresponsiveness after 1.3±0.3 min. The recovery happened after 11.0±5.9 min in renewed seawater. The haemolymph lactate levels remained unaltered after anaesthesia (initial, 0.060±0.053 mg/mL vs unresponsive, 0.141±0.062 mg/mL). Interestingly, the lactate levels were significantly increased after recovery (0.422±0.025 mg/mL, p < 0.0008) when compared to the original levels. The opposite is seen for cholesterol in the midgut gland, where it decreased after the recovery (initial, 0.815±0.023 mg/mL vs. recovery, 0.668±0.016 mg/mL, p<0.05). The MR of animals exposed to cold seawater, was 20% lower than the MR before the cold exposure. After recovery, the MR remained 3% higher than before exposure to cold water.
During the first minute of exposure to cold sweater (~10.5°C) VERs were lost, and remained undetected for the 5 min. Once the water was changed, animals recovered VERs within the first minute. Similarly, the HR had a sharp drop (ca.50%) in the first minute of exposure, dropping to 35% of the basal response (168±17 bpm vs 59±11 bpm, baseline vs cold seawater). Interestingly, the recovery of the HR took about 2 min, but it remained 16% lower than during the baseline (168±17 bpm vs 141±31 bpm).
Discussion and conclusions
During experimental trials, the loss of equilibrium was expected (called ‘stage II’) and the following stage (III), both characteristics previously described as inactivity of the cephalothoracic appendages and lack of response when stimulated (De Souza Valente C., 2022). Exposure to ‘cold’ (~10.5°C) seawater had animals losing their balance almost instantly, although they performed some vigorous escape response before it. During the respirometry analysis some animals lost their balance, displaying vigorous movements since the drop in temperature happened progressively (17 min). In such a condition, the physiological stress was revealed, showing mobilization of reserves and increased lactate.
On a neurophysiological level, the loss of the VERs could indicate the desired anaesthetic stage. The sharp drop in the HR reinforces the idea that cold water could be and effective anaesthetic. It is necessary to point out that the temperature chosen is around this species’ minimum critical temperature (CTmin). This could explain the increase in lactate (and metabolic rate) during recovery. Acute exposure to near CTmin temperature could instantly achieve the desired anaesthesia stage, since some sensorial pathways are shut down. Yet, other organizational levels could be irreversibly damaged and only be seen after a late recovery of P. vannamei. The effect of cold water studied on the different levels of organization evaluated show that shrimps were quickly anesthetized, however their recovery was as well quick. Hence, we suggest that the use of slurry ice in farms is followed by a quick slaughter process.
Acknowledgments
This work was co-funded by the European Union’s Horizon Europe Project 101136346 EUPAHW, CEREBAL project INTER23001 (CSIC - AECID) and by the Spanish government through the “Severo Ochoa Center of Excellence” accreditation (CEX2019-000928-S).
Bibliography
FAO. (2024) The State of World Fisheries and Aquaculture 2024. Blue Transformation in Action.
De Souza Valente, C. (2022) . Anaesthesia of decapod crustaceans. Veterinary and Animal Science, vol. 16, p. 100252.
Rotllant, G., et al. (2023). "Methods to Induce Analgesia and Anesthesia in Crustaceans: A Supportive Decision Tool." Biology 12(3): 387.