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, for which the use of sedatives or anaesthetics is recommended. An ideal anaesthetic should be affordable and readily available in most places, with known working concentrations (Rotllant et al., 2023). Yet, for this group of animals, the effect/response to anaesthetic is vaguely known (De Souza Valente, 2022). A newly developed definition should tackle EFSA’s the triangulation principle, correlating the organisms’ responses in different levels of organisation. In this work, we conducted a three-part experiment in order to characterize behavioural, metabolic and neurophysiologic responses to oregano oil as an anaesthetic.
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) in 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 concentration of oregano oil was established once animals were rendered unresponsive in less than 10 min, and were able to recover their normal position under 20 min. The first set of experiments focused on evaluating the effects of oregano oil (150 µL/L) on shrimp’s behavioural responses. It aimed to find out when animals remained on the bottom of the tank, lying on their sides, and 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. Animals were also sampled at the end of the anaesthesia (n=8) and recovery (n=9) to measure metabolic and immunological stress markers in haemolymph, muscle and digestive gland. The second set of experiments focused on the effects of oregano oil (150 µL/L) on the metabolic rate (RMR) 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 oregano oil was introduced into the chambers via de flow-through seawater system, allowing for a complete water change after 17 min. After 10 min of exposure, the system was refilled with aerated seawater, with constant change, and measures continued for 4 h. Metabolic rate was measured after, during, and before the seawater temperature change. Lastly, we recorded neurophysiological and heart rate parameters. 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 oregano oil (150 µL/L) and measures continued for 6 min, before water was changed again, to measure during recovery phase.
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
The oregano oil induced an equilibrium loss after 6.1±0.7 min, a complete unresponsiveness after 10.0±1.1 min, and recovered after 15.3±5.7 min. Before losing balance, animals experience multiple spasms, after which they lost equilibrium. After the animals were unresponsive the lactate levels were increased by 10-fold (0.8±0.2 mg/mL), and after the recovery period they were still increased by 20-fold (1.7±0.3 mg/mL), when compared with basal levels (0.1±0.1 mg/mL). The RMR was 10% higher than observed in shrimp exposed to control seawater or ethanol (oregano’s solvent). Yet, the recovery of the metabolic rate at the end of the experiment showed no difference. The exposure with oregano oil did not affect the VERs, since the beta waves amplitude during light stimulation was always distinguishable from dark or control periods (>10 ratio of magnitude), during and after the anaesthesia. For the HR, a slight decrease was observed from control levels during the exposure to oregano oil (a 17% drop). The HR returned to control levels after water change (~180 BPM).
Discussion and conclusions
As a starting point when doing trials with anaesthetics, the loss of equilibrium was expected, and noted as stage II, yet no other considerations (as what it took to be achieved) were taken into account. The following stage (III) of the anaesthetic involved the total inactivity of cephalothoracic appendices and lack of response when they were stimulated, while pleopod movement continued; cessation of pleopod movements resulted in no recovery (shrimp died). The exposure with oregano oil had animals lose their balance, but not before they performed an escape attempt from the aquaria. Similarly, during the respirometry analysis, after some time of avoidant response (or escape), animals lost their balance. The escape response could explain the higher metabolic rate found after the oregano oil was applied, and the loss of equilibrium could be a result of fatigue. This fatigue hypothesis could be reinforced by the unaffected VERs, since the loss of balance or inactivity does not involve any frontal ganglion dysfunction or failure on the primary sensory pathway. Interestingly, the mild drop in the HR is not consistent with a rise on the metabolic rate, but it is worth mentioning that the movement of these shrimps was restricted for electrodes placement and that could impact the oregano oil uptake. Overall, our results considering behavioural, physiological and neurological approach might indicate that oregano oil has not an anaesthetic effect on juvenile shrimps, although the animals have lost equilibrium after exposure. Therefore, without further results to ensure replicability and robustness, we will not suggest the use of oregano oil for shrimp aquaculture.
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.