Search Results (19)

Chemical euthanasia protocols are routinely applied across zebrafish developmental stages, but the effects of formulation, time exposure, and developmental stage remain unclear and could introduce variability in transcriptional results and interfere with interlaboratory reproducibility of molecular endpoints. Wild-type AB zebrafish were exposed at three developmental windows (6 hpf, gastrulation; 30 hpf, pharyngula; 100 hpf, pre-feeding larva) to three operational euthanasia formulations (clove oil 0.1% v/v, tricaine/MS-222 1 g/L, or lidocaine 1 g/L), for 3, 10, or 25 min. Expression of ten genes covering immediate-early activation, cellular stress, metabolism, neuroendocrine signalling, apoptosis, and inflammation was quantified by qPCR. Expression responses were analysed using a three-way factorial framework including the developmental phase, formulation, and exposure time. The developmental phase explained substantial developmental context in several genes. The Phase × Treatment interaction was significant after global FDR correction for nine of ten genes, whereas treatment main effects did not provide a stable cross-stage ranking of formulations. Descriptively, gene expression varied across formulations and stages, with several Phase III markers showing positive shifts, but no operational formulation showed universally consistent effects after multiple-testing correction, while effect-size magnitudes should be interpreted cautiously. Acute transcriptional responses to operational chemical euthanasia formulations in zebrafish embryos and larvae were context-dependent. Developmental stage, rather than a universal formulation effect, emerged as the most consistent modulator. Because euthanasia efficacy and non-recovery were not experimental endpoints, these transcriptional data do not identify a best formulation for each developmental phase. These results support stage-aware reporting of euthanasia conditions. Full article

Fish are continuously exposed to stress factors throughout their life cycle, making the use of anaesthetics essential for a wide range of experimental procedures. Currently, the most commonly used and FDA approved anaesthetic for fish research is Tricaine Methanesulfonate (MS-222). However, its use has been associated with several undesirable effects, including hypoxemia, hypercapnia and hypoglycaemia, as well as environmental concerns due to its release through aquaculture effluents. These limitations highlight the need for alternative anaesthetic strategies. Natural compounds such as clove oil, menthol and thymol have been investigated as potential alternatives, demonstrating effective anaesthetic properties. However, their low aqueous solubility, represents a significant challenge, which may be overcome through nanoencapsulation. This approach can enhance solubility, enable controlled release, and reduce the effective dose required. Accordingly, the present study aims to provide an overview of the recent advances in nanoparticle-based encapsulation strategies for anaesthetic delivery in fish, with a focus on their efficacy, safety and environmental impact. Some studies have demonstrated the benefits of nanoencapsulation. In adult zebrafish (Danio rerio), lower concentrations of benzocaine were required when encapsulated in chitosan-PLGA nanoparticles, while lidocaine-loaded lipid NPs reduced bradycardia. In Nile Tilapia (Oreochromis niloticus), clove oil encapsulated in lipid-based nanocapsules enabled effective anaesthesia and prolonged release of the active compound eugenol. Similarly, mucoadhesive zein NPs, reduced the effective concentration of Eugenol by up to 50%. Monoterpenes such as menthol and thymol also show promise for zebrafish anaesthesia, demonstrating efficacy at 50 mg/L. These findings suggest that nanoparticle-based delivery systems can improve the efficacy and safety of fish anaesthetics while reducing required doses and potential environmental impact. Future research should focus on optimizing nanoparticle-anaesthetic systems by combining natural compounds with biocompatible and biodegradable nanocarriers (e.g., zein, chitosan or PLGA) to achieve controlled release, targeted delivery and minimization of side effects. Full article

The black shinner Pseudopungtungia nigra is an endangered freshwater fish endemic to Korea, and standardized anesthetic protocols are needed for conservation-related hatchery handling. This study evaluated the effects of water temperature and MS-222 concentration on anesthetic induction and recovery responses in hatchery-reared juvenile P. nigra of approximately 3 cm total length. Juveniles were exposed to four MS-222 concentrations, 80, 100, 150, and 200 mg L⁻¹, at three water temperatures, 21, 24, and 27 °C. Induction time, recovery time, and recovery success within 600 s were assessed using behavioral endpoints. The 80 mg L⁻¹ treatment induced anesthesia within 600 s only at 27 °C, whereas fish exposed at 21 and 24 °C failed to reach the defined anesthetic stage within 600 s; therefore, this treatment was treated as a low-concentration induction-failure condition. In the main 3 × 3 factorial analysis using 100, 150, and 200 mg L⁻¹, induction time decreased significantly with increasing MS-222 concentration and water temperature, with significant effects of temperature, concentration, and their interaction. In contrast, recovery time increased with increasing MS-222 concentration, indicating a clear trade-off between rapid induction and recovery stability. Although 200 mg L⁻¹ produced the shortest induction times, it also resulted in the longest recovery times and delayed recovery at 24 and 27 °C. The 100 mg L⁻¹ treatment showed stable recovery but required prolonged induction, especially at lower temperatures. Overall, 150 mg L⁻¹ provided the most balanced behavioral response by substantially reducing induction time compared with 100 mg L⁻¹ while avoiding the greater recovery burden observed at 200 mg L⁻¹. These findings suggest that 150 mg L⁻¹ MS-222 is a practical concentration for routine short-term handling of hatchery-reared juvenile P. nigra under the tested temperature and handling conditions. However, this recommendation should be interpreted as a behavioral handling guideline because physiological stress responses and long-term post-anesthetic outcomes were not evaluated. Full article

During production, fish are exposed to multiple environmental, physiological, and physical stressors, which compromise development, productivity, and welfare and urge the implementation of effective and safe stress-mitigating strategies, particularly during early developmental stages. Larval zebrafish (Danio rerio) constitute a powerful model for studying acute stress responses due to the numerous advantages they offer, such as developmental transparency, a conserved hypothalamic–pituitary–interrenal (HPI) axis, and suitability for high-throughput screening. This review examines the potential of natural monoterpenes as stress-reducing compounds and compares their performance with conventional synthetic anesthetics. Evidence from vortex-flow stress paradigms, behavioral profiling and biochemical assays shows that acute stress in zebrafish larvae triggers metabolic disruption, behavioral hyperactivity and enzyme imbalance, with cortisol responses depending on stimulus intensity. Monoterpenes such as thymol and menthol consistently reduce stress-induced hyperactivity, support redox homeostasis and display favorable safety profiles at low doses and short exposures. Nevertheless, as research into these substances is still recent, evidence of any potential adverse effects is still limited. Although individual monoterpenes may act on different subsets of molecular targets, their multimodal mechanisms, including gamma-aminobutyric acid (GABA)ergic enhancement, voltage-gated ion channel and transient receptor potential (TRP) modulation, suggest broader and potentially safer actions compared to single-target anesthetics as tricaine methane sulfonate (MS-222). Collectively, these findings suggest that monoterpenes offer promising natural alternatives for stress mitigation in aquaculture and the refinement of research procedures involving early life stages. Full article

Background/Objectives: MS-222 is a commonly used anesthetic for fish. Research on the anesthetic mechanism of MS-222 is scarce, especially in largemouth bass. Therefore, this study investigated the tissue-specific transcriptomic and metabolomic effects of MS-222 anesthesia on largemouth bass (Micropterus salmoides). Methods: Experimental groups exposed to 40 mg/L MS-222 for 12 h were compared with untreated controls, and then transcriptomic and metabolomic analyses were performed on gill and liver samples. Results: Gill tissues exhibited 3252 differentially expressed genes (DEGs; 2309 upregulated and 943 downregulated) enriched in cardiac muscle contraction, cytoskeletal regulation, glycolysis, and toll-like receptor pathways for anesthetic adaptation. In contrast, liver tissues showed fewer DEGs (1140; 654 upregulated and 486 downregulated) primarily linked to metabolic network reorganization such as endoplasmic reticulum protein processing, PPAR signaling, and ribosome biogenesis. Metabolomic profiling demonstrated inverse patterns, with 173 differential metabolites in gills versus 297 in liver samples. Methyl nicotinate and N-acetyl-L-phenylalanine were the most significantly upregulated in the gill and liver samples. Metabolic pathway enrichment analysis revealed that MS-222-induced differential metabolites in the gill and liver of largemouth bass were predominantly associated with pathways involved in amino acid, fatty acid, phenylalanine, and nucleotide metabolism. Conclusions: These findings reveal that MS-222 anesthesia triggers organ-specific physiological adaptations through the differential regulation of metabolic and immune pathways, which provide multi-omics insights into the mechanistic basis of anesthetic responses in fish, highlighting distinct tissue strategies for managing chemical stress. Full article

Anisotremus scapularis is a commercially important species in Peru, where its cultivation and management require technological strategies to minimize stress during aquaculture and fishing practices. Fish handling and transport can induce adverse physiological responses, making anesthetic use a key tool to mitigate these effects and enhance animal welfare. However, information on optimal doses, safety margins, and induction and recovery times of anesthetics in this species remains limited. This study evaluated the effects of eugenol and tricaine (MS-222) on the sedation of A. scapularis juveniles. A total of 450 individuals (9–10 cm total length) were exposed to different concentrations of eugenol and MS-222 (20, 40, 60, 80, and 100 mg/L) via immersion. Induction and recovery times were recorded, determining that the lowest concentrations allowing an optimal induction time (<6 min) and adequate recovery (<16 min) were 20–60 mg/L for eugenol and 80 mg/L for MS-222. These results provide essential information to optimize sedation protocols for A. scapularis juveniles, promoting more efficient handling with minimal impact on fish welfare in production and fishery management systems. Full article

The biological early warning system with fish behavior as the detection index is an efficient and rapid early warning technology for the ecological damage caused by water pollutants. However, the attempt to apply the scototaxis (dark preference) behavior of fish to biological early warning is still relatively lacking. In this study, we delved into the dark and light preferences of the rare minnows (Gobiocypris rarus), employing three distinct tank configurations. Additionally, we systematically examined the modulating effects of environmental illumination, nutritional status, and the number of test subjects on this behavior, aiming to establish optimal experimental parameters for its observation. Furthermore, cadmium ions [Cd²⁺], tricaine methanesulfonate [MS222], and p-chloroaniline were employed as representative heavy metal ions, neuroactive agents, and organic toxicants, respectively, to test the impact of chemicals on scototaxis in gradient concentrations. The results demonstrated that the rare minnow exhibited a clear scototaxis (dark preference), and this behavior was not affected by the nutritional status of the test fish, the illumination, or the number of subjects. While the dark chamber was consistently the preferred location of rare minnows during the chemical exposure tests, the degree of scototaxis by the rare minnow significantly decreased at Cd²⁺ ≥ 3 mg/L, MS222 ≥ 11 mg/L, and p-chloroaniline ≥ 29 mg/L, suggesting a potential disruption of their innate behavioral patterns by these chemicals. These findings underscore the sensitivity of rare minnows to water pollutants. Therefore, the scototaxis behavior of rare minnows can be a potential and useful behavioral indicator for biological early warning, which can be used for early biological warning of sudden water pollution caused by chemicals such as Cd²⁺, MS222, and p-chloroaniline. Full article

Anesthesia serves as an effective method to mitigate the stress response in aquatic animals during aquaculture and product transportation. In this study, we assessed the anesthetic efficacy of clove oil, tricaine methane-sulfonate (MS-222), ethanol, and magnesium chloride by anesthesia duration, recovery time, 24-hour survival rate, and the behavior of mud crabs (Scylla paramamosain). Additionally, the optimal anesthetic concentration for varying body weights of mud crabs was also investigated. The results revealed that clove oil emerged as the optimal anesthetic for mud crabs, with a 24-hour survival rate surpassing those observed in MS-222 and magnesium chloride treatments. Ethanol caused amputation and hyperactivity in mud crabs. Regression analyses between the optimal anesthetic concentration of clove oil and the weight categories of 0.03–27.50 g and 27.50–399.73 g for mud crabs yielded the following equations: y = 0.0036 x³ − 0.1629 x² + 1.7314 x + 4.085 (R² = 0.7115) and y = 0.0437 x + 2.9461 (R² = 0.9549). Clove oil exhibited no significant impact on serum cortisol, glucose, lactate content, aspartate aminotransferase (AST), alanine aminotransferase (ALT) activities, or superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) levels in mud crabs across different treatment groups. Anesthesia induced by clove oil in mud crabs resulted in an increase in inhibitory neurotransmitters such as glycine. However, the recovery from anesthesia was associated with elevated levels of the excitatory neurotransmitters L-aspartic acid and glutamate. In conclusion, clove oil proves to be a safe and optimal anesthetic agent for mud crabs, exerting no physiological stress on the species. Full article

This study was conducted to determine the optimal doses and minimum effective concentrations (MECs) of tricaine methanesulfonate (MS-222) in marketable-size Asian seabass reared at two temperatures (22 and 28 °C). Serum biochemical parameters, pharmacokinetics, and tissue distributions of MS-222 following immersion at the determined optimal doses were also evaluated in order to delineate possible mechanisms dictating the temperature difference. The definition of optimal dose is set as the dose when fish attain stage III anesthesia within 5 min, sustain this stage for 3 min, and re-attain equilibrium within 5 min. The MEC is the fish serum MS-222 concentration when stage III anesthesia is reached. The results showed that water temperature exerted no or minimal impact on the designated parameters. The optimal doses at 22 and 28 °C were 140 and 150 µg/mL, while the MECs were 70.48 and 78.27 µg/mL, respectively. Fish exposed to the optimal doses of MS-222 had significantly elevated blood concentrations of lactate, glucose, calcium, magnesium, and sodium, while the blood pH was significantly decreased. The fish eliminated MS-222 faster at 28 °C than at 22 °C, with serum half-lives of 18.43 and 37.01 h, respectively. Tissue-specific distribution patterns were evident. Irrespective of water temperature, MS-222 peaked at 5 min for the brain and gill but peaked slightly later at 10–20 min for the liver and kidney. Most tissues exhibit a gradual decline of drug concentration except for the gill, which was maintained at a steady level. Muscle is the least perfused tissue with the lowest drug concentration throughout the 90 min period. This study provided physiological and pharmacokinetic evidence contributing to a better understanding of the actions of MS-222 in Asian seabass at different temperatures. Full article

Giant Pacific octopuses (Enteroctopus dofleini) (GPOs) are commonly housed in zoos or aquaria, and sedation, anesthesia, and/or euthanasia may be indicated for a variety of reasons. Despite this need, evidence-based data on best practices is limited and focuses on smaller or more tropical species. The objectives of this study were to survey the aquatic community regarding the husbandry and veterinary care of GPOs, with a specific focus on anesthetic and euthanasia protocols. A two-part web-based survey was distributed to four aquatic and/or veterinary email listservs. Individuals from fifty-two institutions participated in phase one. Results documented that 40 (78 percent) participating institutions currently house GPOs, with most housing one and nine institutions housing two to three GPOs. The median (range) habitat volume is 5405 (1893–16,465) L, and 78 percent of systems are closed. Of the institutions surveyed, 23 have anesthetized or sedated a GPO for nonterminal procedures, including wound care, biopsies, and hemolymph collection. Reported methods of sedation or anesthesia include magnesium chloride, ethanol, isoflurane, tricaine methanesulfonate (MS-222), magnesium sulfate, benzocaine, and dexmedetomidine. Drugs or methods used for euthanasia include magnesium chloride, ethanol, mechanical decerebration, pentobarbital, isoflurane, MS-222, magnesium sulfate, benzocaine, potassium chloride, dexmedetomidine, and freezing. Reported observed side effects include ineffectiveness or inadequate sedation, inking, prolonged drug effects, and behavior changes. Survey data have the potential to guide the husbandry and veterinary care of GPOs and build the framework for future prospective studies on GPO sedation and anesthesia. Full article

CHARGE syndrome typically results from mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7). CHD7 is involved in regulating neural crest development, which gives rise to tissues of the skull/face and the autonomic nervous system (ANS). Individuals with CHARGE syndrome are frequently born with anomalies requiring multiple surgeries and often experience adverse events post-anesthesia, including oxygen desaturations, decreased respiratory rates, and heart rate abnormalities. Central congenital hypoventilation syndrome (CCHS) affects ANS components that regulate breathing. Its hallmark feature is hypoventilation during sleep, clinically resembling observations in anesthetized CHARGE patients. Loss of PHOX2B (paired-like homeobox 2b) underlies CCHS. Employing a chd7-null zebrafish model, we investigated physiologic responses to anesthesia and compared these to loss of phox2b. Heart rates were lower in chd7 mutants compared to the wild-type. Exposure to tricaine, a zebrafish anesthetic/muscle relaxant, revealed that chd7 mutants took longer to become anesthetized, with higher respiratory rates during recovery. chd7 mutant larvae demonstrated unique phox2ba expression patterns. The knockdown of phox2ba reduced larval heart rates similar to chd7 mutants. chd7 mutant fish are a valuable preclinical model to investigate anesthesia in CHARGE syndrome and reveal a novel functional link between CHARGE syndrome and CCHS. Full article

This study aimed to evaluate the effect of different transport densities on water deterioration, physiological response, nutrients, and fresh quality of sea bass (Lateolabrax maculatus) at 30 mg/L tricaine methanesulphonate (MS-222) before and after simulated live transport. The results indicated that the addition of MS-222 could effectively decrease mortality compared with the control (CK) sample during the simulated live transport. The concentration of dissolved oxygen was lower and the total ammonia nitrogen was higher in the high transport density samples than those of low transport density samples after 72 h in transport. The level of blood cortisol (COR), glucose (GLU), lactic acid (LD), aspartate aminotransferase (AST), alanine aminotransferase (ALT) for the sea bass were significantly higher compared with the CK sample (p < 0.05) during the simulated live transport and after 12 h of recovery. These results indicated that the sea bass presented a strong stress response in high-density transport. The glycogen, fat, and protein of the sea bass were degraded to supply the energy for the body in the process of surviving the transportation, resulting in the decreased nutrient content in the muscle, which recovered to the initial level (CK) after 12 h. The increase in flavor substance content, such as free amino acids, nucleotides, organic acids, and minerals, enhanced the special flavor of the muscle during the simulated live transport. This study demonstrates that the addition of MS-222 at 30 mg/L to the transport water is an effective method for live fish to realize low mortality and physiological response during high-density and long-distance transport. Full article

An international survey was conducted regarding species used for research, methods of euthanasia, health monitoring, and biosecurity in fish laboratories. A total of 145 facilities from 23 countries contributed. Collectively, over 80 different species (or groups of species) were reported to be used for research, of which zebrafish (Danio rerio) was the most common by far. About half of the participating laboratories used multiple species. Anesthetic overdose was the preferred method for euthanasia for adult, fry (capable of independent feeding), and larval (not capable of independent feeding) fish. For all developmental stages, the most popular anesthetic compound was tricaine (MS-222), a substance associated with distress and aversion in several species. Moreover, around half of the respondents did not perform a completion method to ensure death. One-quarter of the responding facilities did not have a health monitoring system in place. While most respondents had some form of quarantine process for imported fish, only a small fraction reported quarantine routines that ensure reliable biological barriers. Furthermore, less than one in five screened fish for pathogens while in quarantine. In sum, there was little consensus amongst facilities in how to perform biosecurity measures. Regarding euthanasia, health monitoring, and biosecurity processes, there is a need for updated and universal guidelines and for many laboratories to adjust their practices. Full article

In this research, we designed a rapid tricolour immunochromatographic test strip with double test lines (TS-DTL) and two-colour AuNP probes, which realised the simultaneous detection of tricaine mesylate (TMS) and malachite green (MG). Through a distinct tricolour system (red T₁ line, blue T₂ line and purple C line), a visual identification of TMS (0.2 μg/mL) and MG (0.5 μg/mL) was quickly achieved on site, which improved the accuracy of naked eye observations. The LODs of TMS in aquaculture water, fish and shrimp were 11.0, 29.6 and 61.4 ng/mL, respectively. MG LODs were 47.0 ng/mL (aquaculture water), 82.8 ng/mL (fish) and 152.4 ng/mL (shrimp). The LOD of MG was close to the similar TS methods. However, visual detection of TMS could meet the requirements of the residue limit (1 μg/mL) of TMS in the USA, and the quantitative detection of TMS was over 16 times lower than the USA standard. The developed platform was rapid (~20 min, HPLC~3 h) and accurate, which was verified using a traditional HPLC method. The recovery rates ranged from 82.2% to 108.6% in three types of real samples, indicating a potential application in on-site fast screening or multiple detection for TMS and MG residues in aquatic products. Full article

Euthanasia in zebrafish (Danio rerio) younger than 5 days post fertilization (dpf) is poorly described in the literature, and standardized protocols are lacking, most likely because larvae not capable of independent feeding are often not protected under national legislations. We assessed the euthanasia efficacy in laboratories in different countries of a one hour anesthetic overdose immersion with buffered lidocaine hydrochloride (1 g/L, with or without 50 mL/L of ethanol), buffered tricaine (1 g/L), clove oil (0.1%), benzocaine (1 g/L), or 2-phenoxyethanol (3 mL/L), as well as the efficacy of hypothermic shock (one hour immersion) and electrical stunning (for one minute), on zebrafish at <12 h post fertilization (hpf), 24 hpf, and 4 dpf. Based on the survival/recovery rates 24 h after treatment, the most effective methods were clove oil, lidocaine with ethanol, and electrical stunning. For 4 dpf larvae, signs of aversion during treatment demonstrated that all anesthetics, except lidocaine, induced aversive behavior. Therefore, the most suited euthanasic treatment was lidocaine hydrochloride 1 g/L, buffered with 2 g/L of sodium bicarbonate and mixed with 50 mL/L of ethanol, which euthanized both embryos and larvae in an efficient and stress-free manner. Electrical stunning also euthanized embryos and larvae efficiently and without signs of aversion; this method needs further assessment in other laboratories to draw firm conclusions. Full article