Search

Fast and effective anesthesia is the key for refining many invasive procedures in fish and gaining reliable data. For fish as for all vertebrates, it is also required by European law to reduce pain, suffering, and distress to the unavoidable minimum in husbandry and experiments. The most often used substance to induce anesthesia in zebrafish is tricaine (MS-222). When properly prepared and dosed, tricaine causes rapid loss of mobility, balance and reaction to touch. These signs are interpreted as a stage of deep anesthesia although its effects on the central nervous system have not convincingly been shown. Therefore, it might be possible that tricaine first acts only on the periphery, resulting in a paralyzed instead of an anesthetized fish. This has severe implications for animals undergoing procedures. To investigate the effects of tricaine on the central nervous system, we used zebrafish larvae [Tg( elavl3 :H2B-GCaMP6s)] at 4 days post fertilization (dpf), expressing a calcium indicator (GCaMP6s) in all neurons, that allows monitoring and quantifying the neuronal activity. After treating larvae with 168 mg/L tricaine, a rapid loss of neuronal activity in the forebrain was observed in confocal microscopy. In contrast, only mild effects were seen in the midbrain and hindbrain. In conclusion, the different larval brain areas showed differences in the sensitivity to tricaine treatment. The effects on the central nervous system are indicative of tricaine's anesthetic function and are consistent with behavioral observations of inactivity and unresponsiveness to touch., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ohnesorge, Wilzopolski, Steinfath, Lewejohann, Banneke and Heinl.)

Tricaine is a common anesthetic used in the long-distance transport of live fish. Recently, its negative impact on human health has aroused extensive concern. Thus, rapid and reliable techniques for tricaine residue analysis are essential to ensuring the quality of aquatic products. Herein, a specific anti-tricaine monoclonal antibody (Mab) was prepared. Then, a sensitive and robust ratiometric fluorescence ELISA (RF-ELISA) was constructed for detecting tricaine based on two MnO 2 nanoflake-mediated (MnO 2 NFs) fluorogenic reactions. In the RF-ELISA protocol, MnO 2 NFs with oxidase-like activity can trigger the formation of fluorescent 2,3-diaminophenazine (oxOPD) with an emissive peak at 570 nm from non-fluorescent o -phenylenediamine (OPD), while ascorbic acid (AA) can decompose MnO 2 NFs to lose their oxidase-mimicking activity, which is accompanied by the oxidation of AA into dehydroascorbic acid (DHAA). The subsequent reaction between the generated DHAA and OPD will result in the production of 3-(1,2-dihydroxy ethyl)furo[3,4- b ]quinoxalin-1(3H)-on (DFQ), which has a potent emission peak at 445 nm. By virtue of the alkaline phosphatase (ALP) labeled on the antibody, which can catalyze the production of AA from ascorbic acid 2-phosphate (AAP), the concentration of tricaine can be linked to the variation of the RF signal (F 445 /F 570 ) via a competitive immunoreaction. After optimization, RF-ELISA displayed a detection limit (LOD) of 0.28 ng/mL toward tricaine (in buffer solution), which was 376-fold lower than that of the traditional colorimetric ELISA. For practical application, the LODs of RF-ELISA for tricaine detection in shrimp and tilapia samples were determined to be 2.8 and 5.6 ng/g, respectively. Recoveries for spiked shrimp and tilapia samples, as well as the validation data from LC-MS/MS, showed that RF-ELISA exhibited good accuracy, precision, and reliability. This RF-ELISA protocol opened up new ways for tricaine and other-target analyses in food safety detection.

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.

Tricaine, or MS-222, is the most commonly used chemical anesthetic in zebrafish research. It is thought to act via blocking voltage-gated sodium channels, though its mechanism of action, particularly at the neuronal level, is not yet fully understood. Here, we first characterized the effects of tricaine on both body balance and touch responses in freely swimming animals, before determining its effect on the neural activity underlying the optokinetic response at the level of motion perception, sensorimotor signaling and the generation of behavior in immobilized animals. We found that the standard dose for larvae (168 mg/L) induced loss of righting reflex within 30 seconds, which then recovered within 3 minutes. Optokinetic behavior recovered within 15 minutes. Calcium imaging showed that tricaine interferes with optokinetic behavior by interruption of the signals between the pretectum and hindbrain. The motion sensitivity indices of identified sensory neurons were unchanged in larvae exposed to tricaine, though fewer such neurons were detected, leaving a small population of active sensory neurons. We then compared tricaine with gradual cooling, a potential non-chemical alternative method of anesthesia. While neuronal tuning appeared to be affected in a similar manner during gradual cooling, gradual cooling induced a surge in calcium levels in both the pretectum and hindbrain. This calcium surge, alongside a drop in heartrate, is potentially associated with harmful changes in physiology and suggests that tricaine is a better anesthetic agent than gradual cooling for zebrafish laboratory research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Leyden, Brüggemann, Debinski, Simacek, Dehmelt and Arrenberg.)

Zebrafish are often euthanized by overdose of anaesthesia. However, fish may have aversion towards some anaesthetics, and protocol efficacy varies between species. Using wild type adult Danio rerio , we assessed time to loss of opercular beat, righting, and startle reflexes during induction of anaesthetic overdose by either tricaine (0.5 g/L or 1 g/L), benzocaine (1 g/L), 2-phenoxyethanol (3 mL/L), clove oil (0.1%), isoeugenol (540 mg/L), lidocaine hydrochloride (1 g/L), or etomidate (50 mg/L). Initial screening demonstrated that benzocaine and buffered lidocaine hydrochloride achieved the fastest loss of reflexes. The rapid induction times were confirmed when retesting using larger batches of fish. The fastest induction was obtained with 1 g/L lidocaine hydrochloride buffered with 2 g/L NaHCO 3 , in which all adult zebrafish lost reflexes in less than 2 min. Next, we monitored signs of distress during benzocaine or buffered lidocaine hydrochloride overdose induction. The results indicated that buffered lidocaine hydrochloride caused significantly less aversive behaviors than benzocaine. Finally, we tested several buffers to refine the lidocaine hydrochloride immersion. The most efficient buffer for euthanasia induction using 1g/L lidocaine hydrochloride was 2 g/L NaHCO 3 with 50 mL/L 96% ethanol, inducing immobility in less than 10 s and with only 2% of adult zebrafish displaying aversive behaviors during treatment.

Tricaine methanesulfonate is one of most commonly used anesthetics in fish during blood sampling, artificial propagation and long-distance transportation. In this study, an accurate method for the quantitative determination of tricaine in fish samples by a stable isotope dilution assay coupled with high-performance liquid chromatography-triple quadrupole mass spectrometry was developed. Tricaine-D 5 was synthesized and used as an isotopically labeled internal standard for the determination of tricaine. The analytical performance of the method was validated for tricaine determination in marine fish and freshwater fish. The determination of tricaine was linear in the range of 2.0-200.0 μg L -1 . The limit of detection and limit of quantitation for fish muscle tissues were 1.0 and 4.0 μg kg -1 , respectively. Good recoveries were obtained in the range of 92.08-97.50%. The inter- and intra-assay relative standard deviations (RSD values) were investigated, and the values were 0.39-3.01 and 0.85-2.77%, respectively. The values of CCα and CCβ were 10.21-10.43 and 10.42-10.87 μg kg -1 , respectively. The clearance of MS-222 from grass carp was further studied using our method. The results demonstrate that MS-222 could be well absorbed and rapidly eliminated after bath administration., (© 2019 John Wiley & Sons, Ltd.)

Long-term in vivo imaging in adult zebrafish (i.e., 1-24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1-24 h.

Cytochrome P450 1A (CYP1A) expression in fish is used as a biomarker of exposure to organic contaminants, such PAHs, PCBs and dioxins, in the aquatic environment. South American guppy fish Jenynsia multidentata were exposed to the prototypical aryl hydrocarbon receptor (AHR) agonist beta-naphthoflavone (BNF; 1μM) and the fins were biopsied to characterize different aspects of CYP1A induction. RTq-PCR was used to quantify CYP1A mRNA levels in fish tissues. CYP1A induction in the gill, liver and anal fin (gonopodium) occurred within the first hour of waterborne exposure to BNF and persisted throughout 2, 4, 8, 24, 48 and 96h compared to controls (DMSO vehicle; p<0.05). The organ-specific temporal pattern of induction was marked by mRNA levels consistently augment as duration of exposure increases and tend to a sustained induction from 24h to 96h for gill and liver (∼15-fold and ∼50-fold over control, respectively). In gonopodium, there was a maximum CYP1A mRNA level at 4h (∼34-fold over control). Basal CYP1A mRNA levels and its induction following BNF exposure were not affected by administration of a chemical anesthetic (fish immersion in 100mgl(-1) MS-222 for 2-5min) in the gill, liver, gonopodium, dorsal or tail fin (p<0.05). In an ex vivo assay, in which small pieces of biopsied fins were exposed to BNF for 4h, high CYP1A induction was observed in the tail and gonopodium (∼49-fold and ∼69-fold, respectively) but not in the dorsal fin compared to controls. To our knowledge, this is the first study to show that a 1h waterborne exposure to an AHR agonist is sufficient to cause CYP1A induction in fish organs and fins. The present study added new information to the field regarding the use of MS-222 as an anesthetic on fish and the analysis of biopsied fins as an alternative non-lethalex vivo assay for evaluating the CYP1A biomarker in fish. This observation could be useful for planning fish toxicological bioassays and biomonitoring studies on the aquatic environments in South America., (Copyright © 2014. Published by Elsevier Inc.)