The effect of urethane and MS-222 anesthesia on the electric organ discharge of the weakly electric fish Apteronotus leptorhynchus.

Urethane and MS-222 are agents widely employed for general anesthesia, yet, besides inducing a state of unconsciousness, little is known about their neurophysiological effects. To investigate these effects, we developed an in vivo assay using the electric organ discharge (EOD) of the weakly electric fish Apteronotus leptorhynchus as a proxy for the neural output of the pacemaker nucleus. The oscillatory neural activity of this brainstem nucleus drives the fish's EOD in a one-to-one fashion. Anesthesia induced by urethane or MS-222 resulted in pronounced decreases of the EOD frequency, which lasted for up to 3 h. In addition, each of the two agents caused a manifold increase in the generation of transient modulations of the EOD known as chirps. The reduction in EOD frequency can be explained by the modulatory effect of urethane on neurotransmission, and by the blocking of voltage-gated sodium channels by MS-222, both within the circuitry controlling the neural oscillations of the pacemaker nucleus. The present study demonstrates a marked effect of urethane and MS-222 on neural activity within the central nervous system and on the associated animal's behavior. This calls for caution when conducting neurophysiological experiments under general anesthesia and interpreting their results.

Effects of transportation on physiological indices and metabolomics of the large yellow croaker Larimichthys crocea.

The aim of this study was to investigate the survival rate, biochemical indices, and metabolome changes of the large yellow croaker after 48 h of live transportation. Two hundred and forty large yellow croakers (body weight: 23.4 ± 5.3 g, total length: 12.2 ± 0.7 cm) were used in this experiment. The transport buckets were filled with fresh seawater and the parameters of the water were a temperature of 16 ± 0.5 °C and a dissolved oxygen content of 6.0-7.2 mg/L. Large yellow crokers were first divided to 0, 10, 20, and 30 mg/L MS-222 groups to observe the 12 h survival rate. The survival rate of 10 mg/L MS-222 group (T1) was the 95%, highest of all, and was further analyzed. The results of liver biochemical indices indicated inhibition of gluconeogenesis and pentose phosphate pathway metabolism. In addition, metabolomics analysis identified significantly differentially expressed metabolites between T1 group and 0 mg/L MS-222 control (C) groups. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) results revealed that the pathways of amino acid metabolism, especially the lysine, aspartate, and homoserine in the liver were significantly affected. In conclusion, the combination of metabolomics and liver biochemical assays provided a characterization of the response mechanism of L. crocea exposed to live transportation.

Malformations and mortality in zebrafish early stages associated with elevated caspase activity after 24 h exposure to MS-222.

Tricaine methanesulfonate (MS-222) is a commonly used anaesthetic agent for immobilization of aquatic species. However, delayed development and malformations have been observed in 24 hpf (hours post-fertilization) zebrafish embryos after long-term immobilization. Still, no comprehensive study has been described regarding zebrafish exposure to MS-222 during the first hours of development, which are one of the most sensitive life stages to toxicants. Therefore, this research aimed to assess the toxicity of a 24 h exposure to MS-222 on zebrafish embryonic development. Based on the MS-222 LC50, early blastula stage embryos (~2 hpf) were exposed to 0, 12.5, 25 and 50 mg L-1 for 24 h and then allowed to develop up to 144 hpf. The chromatographic analysis showed that this anaesthetic agent bioaccumulates in 26 hpf zebrafish larvae in a concentration-dependent manner. In addition, increased mortalities and skeletal abnormalities were observed at 144 hpf, namely in the highest tested concentration. Yet, no craniofacial anomalies were observed either by alcian blue or calcein staining methods. Independently of the tested concentration, decreased speed and distance travelled were perceived in 144 hpf larvae. At the biochemical level, decreased in vivo reactive oxygen species (ROS) generation and apoptosis was observed. Additionally, catalase activity was increased at 26 hpf while results of mRNA expression showed a decreased gclc transcript content at the same time-point. Overall, data obtained highlight the toxicological risk of MS-222 and support ROS-mediated cell death signalling changes through the elevation of catalase activity as an adaptative or protective response.

Differential effects of aquatic anaesthetics on the pharmacokinetics of antibiotics: Examples using florfenicol in Nile tilapia (Oreochromis niloticus).

Anaesthetics are commonly applied in pharmacokinetic (PK) studies to assure smooth handling of experimental procedures or to promote animal welfare. However, the influence of anaesthetics on the PK of co-administered drug is generally unknown but assumes ignorable. The goal of the study was to investigate the effect of tricaine methanesulfonate (MS-222), 2-phenoxyethanol (2-PE) and eugenol (EUG) on the PK of florfenicol (FF) in Nile tilapia. Twenty-eight fish were repeatedly exposed to 90 ppm EUG, 300 ppm MS-222 or 900 ppm 2-PE before FF oral administration (15 mg/kg) and each successive blood sampling. The serum concentration-time profiles were analysed by a 2-compartmental model, and the generated parameters in the control (without anaesthetic) and anaesthetic groups were statistically compared. The results demonstrated that the serum concentrations of each anaesthetic were similar at every FF sampling times (70 µg/ml for MS-222; 277 µg/ml for 2-PE; and 61 µg/ml for EUG). In comparison with the control group, the repeated use of MS-222 did not result in a statistical difference in most of the PK parameters. In contrast, the elimination half-lives of the 2-PE and EUG groups were significantly longer whereas the absorption and distribution half-lives of the 2-PE group were significantly shorter than the control, resulting in altered optimal dosages in the simulation modelling. Whether or not the numbers and extent of PK parameters change mitigate subsequent estimations of other PK-derived secondary values such as dosing regimen and withdrawal time remains to be elucidated, but the auxiliary use of anaesthetics in PK studies should not assume uninfluential.

Concentration effects of three common fish anesthetics on Pacific hagfish (Eptatretus stoutii).

The efficacy of three common fish anesthetics (clove oil, 2-phenoxyethanol, and tricaine methanesulfonate) was evaluated in the Pacific hagfish (Eptatretus stoutii). The overarching aim of our study was to identify the best anesthetic and concentration for the purposes of routine laboratory use of Pacific hagfish (i.e., short and consistent induction and recovery times and minimized stress and safety risk to hagfish). The objectives of our study were fourfold: (1) identify anesthetic stages of Pacific hagfish using clove oil anesthesia; (2) establish standardized anesthesia preparation procedures; (3) determine the optimal anesthetic and concentration for safely achieving stage V anesthesia; and (4) investigate the effects of repeatedly exposing Pacific hagfish to anesthesia. Experimental concentrations, ranging from 50 to 400 mg/L, of each anesthetic were tested on at least three Pacific hagfish individuals. We found the following: (1) Pacific hagfish exhibited similar stages of anesthesia to those described for bony fishes; (2) sufficient mixing of clove oil with seawater had a considerable effect on the consistency and timing of anesthetic induction; (3) concentration and anesthetic significantly impacted induction and recovery timing, whereas body mass had no impact on anesthetic trends; and (4) repeatedly exposing Pacific hagfish to optimal concentrations of clove oil or MS-222 had no effect on induction or recovery timing, whereas exposure number significantly impacted induction timing when using 2-PE. Due to consistent induction and recovery times, low risk of accidental overdose, and high safety margins for both handler and hagfish, we recommend 175 mg/L of clove oil as the ideal anesthetic and concentration for the routine laboratory use of Pacific hagfish.

Quantifying the Effects of Two Local Anesthetics on the Crayfish Stretch Receptor Organ: An Integrated Neurophysiology Lab.

The crayfish stretch receptor organ (SRO) preparation represents a robust experimental model for undergraduate laboratory experiences. For example, this preparation may be included as part of a course-based undergraduate research experience (CURE), where students work independently to plan and carry out their own experiments. In the current paper, we provide an example of how local anesthetics may be used to manipulate the SRO preparation and to perform quantitative analyses of SRO action potential firing rates. Local anesthetics provide interesting tools for manipulating physiological responses within the nervous system. A variety of inexpensive anesthetics are available for student use and each of these is expected to inhibit neurophysiological responses. While specific anesthetics exhibit subtle differences in chemical organization, they are generally understood to block voltage gated sodium channels. In the current study, we investigated the effects of two local anesthetics, MS-222 and procaine, on the action potential firing rate from the crayfish SRO. Using quantitative analyses of SRO action potential generation, we determined that each anesthetic has unique inhibitory effects on action potential firing rate that may be explained by their neuropharmacological properties. This manipulation may thus be utilized as an interesting experimental tool in undergraduate teaching laboratories. Local anesthetics applied to crayfish SRO preparations can thus be used to deepen student understanding of local anesthetics, exercise quantitative analyses, and provide experimental tools for independent experimental design.

Transcriptomic and cortisol analysis reveals differences in stress alleviation by different methods of anesthesia in Crucian carp (Carassius auratus).

Stress response has negative effect on fish in aquaculture and research, which can be alleviated with anesthetic. To determine the optimal anesthetic, we investigated the physiological response of crucian carp (Carassius auratus) treated with three different anti-stress treatments: MS-222, eugenol and percussive stunning. Stress responses were evaluated by analyzing serum cortisol level and gene expression in blood. We determined the optimal concentrations of MS-222 (100 mg L-1) and eugenol (20 mg L-1) by dose selection. We found that the control group had significantly higher cortisol levels (172.78 ±â€¯19.95 ng mL-1) compared to the MS-222 treated group (46.85 ±â€¯3.22 ng mL-1), the eugenol treated group (72.78 ±â€¯9.07 ng mL-1), and the stunning treatment group (82.78 ±â€¯8.16 ng mL-1). Transcriptome analysis revealed 1572 differentially expressed genes (DEGs), including 155 DEGs related to the stress response, mainly involved in oxidative-stress response, heat shock proteins, and cold shock domain-containing protein. The heat shock protein genes were the primary DEGs in response to stress. RT-qPCR analysis confirmed differential expression of Hsps. We analyzed the function of the DEGs, which were enriched in genes involved in cellular response to stress and antigen processing and presentation. Combining the results from biochemical, transcriptome, and gene expression analysis, our data suggest that eugenol is more effective than MS-222 and percussive stunning in alleviating stress in crucian carp.

Anesthetic MS-222 eliminates nerve and muscle activity in frogs used for physiology teaching laboratories.

Frogs are routinely used in physiology teaching laboratories to demonstrate important physiological processes. There have been recent directives that promote the use of the anesthetic MS-222 (tricaine methanesulfonate), rather than lowering body temperature with a cold water bath to prepare reptiles and amphibians for physiological experiments or euthanasia. Indeed, the most recent edition of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals proclaims that chilling in water is not an appropriate method and advocates for the usage of MS-222 or other anesthetics. However, prominent researchers have responded to this position by highlighting evidence that cooling ectothermic vertebrates is, in fact, an effective and appropriate method. Furthermore, MS-222 is a known voltage-gated Na+ channel blocker, and this anesthetic's impact on the physiology of excitable tissues suggests that its use might be incompatible with experiments on nerve and muscle tissues. In the present study, I examined the effects of MS-222 at a concentration of 1.5 g/l on nerve, skeletal muscle, and cardiac muscle physiology of frogs. I found that immersion of frogs in this anesthetic blocked basic nerve and muscle physiology, making the frogs unsuitable for laboratory experiments. Applying MS-222 directly to the sciatic nerve dramatically blocked normal excitation-contraction coupling in skeletal muscle preparations, and direct application to the heart caused the organs to stop contracting. Based on these results, I conclude that MS-222 at the concentration studied may be incompatible with physiological preparations that rely on electrically excitable tissues for their normal function. Physiology educators who must use MS-222 with frogs should empirically determine an appropriate dosage and recovery time before using the anesthetic in the teaching laboratory.

The effects of commonly used anaesthetics on colour measurements across body regions in the poeciliid fish, Girardinus metallicus.

The effects of common anaesthetics on the hue, saturation and brightness measurements of the poeciliid fish Girardinus metallicus were investigated in two experiments. For both experiments the coloration of four body regions was measured from digital images of the same males obtained under three conditions: (1) control (in a water-filled chamber); (2) anaesthetised with MS-222; and (3) anaesthetised with eugenol (clove oil). In experiment 1 anaesthetised fish were photographed out of water. In experiment 2 all photographs were taken in a water-filled chamber. Anaesthetics altered coloration in both experiments. In the more methodologically consistent experiment 2 we found significantly different hue, increased saturation and decreased brightness in anaesthetic v. control conditions, consistent with darkening caused by the anaesthetics. The body regions differed in coloration consistent with countershading but did not differentially change in response to anaesthesia. These findings suggest that photographing fish in a water-filled chamber without anaesthetic is preferable for obtaining digital images for colour analysis and that multiple body regions of fish should be measured when assessing coloration patterns meaningful in behavioural contexts, to account for the gradients caused by countershading. We are encouraged that some researchers employ such methods already and caution against using anaesthetics except when absolutely necessary for immobilisation.

IMMERSION IN TRICAINE METHANESULFONATE (MS-222) IS NOT SUFFICIENT FOR EUTHANASIA OF SMOKEY JUNGLE FROGS (LEPTODACTYLUS PENTADACTYLUS).

Although tricaine methanesulfonate (MS-222) immersion has historically been standard of care for fish and anuran euthanasia, recent research has proven it insufficient for euthanasia of goldfish. To assess appropriateness for humane euthanasia of anurans, this study evaluated the efficacy of MS-222 in Smokey Jungle Frogs (Leptodactylus pentadactylus). Eighteen frogs (21-33 g) were exposed to one of three MS-222 concentrations via partial immersion: 2.5 g/L for 90 min (M2.5/90), 5 g/L for 60 min (M5/60), or 10 g/L for 60 min (M10/60). Physiologic parameters and times to loss of spontaneous movement, righting reflex, and noxious stimulus response were recorded. Following exposure, frogs were rinsed with dechlorinated water, and time to cessation of heart beat was recorded. Survival in M2.5/90, M5/60, and M10/60 was one of six, zero of six, and zero of six, respectively. In M2.5/90, three of six frogs had continued purposeful, spontaneous movement throughout exposure. In M5/60 and M10/60, median (range) time to initial loss of movement was 14.3 (5.5-30.0) and 7.6 (4.8-19.7) min, respectively. Twelve of 18 frogs among all groups demonstrated a median (range) of two (one to six) episodes of regained consciousness with purposeful, spontaneous movement following loss of noxious stimulus response. Median (range) time to heart beat cessation in M2.5/90, M5/60, and M10/60 was 150 (135-210), 157.5 (60-225), and 90 (75-210) min, respectively. Although death was achieved in 17 of 18 frogs, given the repeated events of regained consciousness, MS-222 immersion when used at concentrations ≤10 g/L did not result in rapid and distress-free death and is not sufficient for humane euthanasia in this species.

ENDOSCOPIC REMOVAL OF A FOREIGN BODY IN A MEXICAN AXOLOTL (AMBYSTOMA MEXICANUM) WITH THE USE OF MS222-INDUCED IMMOBILIZATION.

This communication briefly describes the use of tricaine methanesulfonate (MS222) to induce chemical restraint/general anesthesia of a Mexican axolotl (Ambystoma mexicanum) for the endoscopic retrieval of a gastric foreign body. There is very little published scientific literature concerning the anesthesia of Mexican axolotls. The anesthesia used in this case was an immersion bath of tricaine methanesulfonate where the concentration of tricaine methanesulfonate was gradually increased to 500 mg/L (ppm) over a 15-min period. A loss of righting reflex was observed within 3 min of attaining the final concentration of the anesthetic bath. The first voluntary movements following the transfer to a freshwater bath occurred within 7 min. The recovery was uneventful. Tricaine methanesulfonate in this case proved to be an effective anesthetic agent for a short, minimally invasive procedure.

[Determination of 5 kinds of fish anesthetics residues in fish by ultra-high performance liquid chromatography-tandem mass spectrometry].

OBJECTIVE: A method for the simultaneous determination of 5 kinds of fish anesthetics residues in fish has been developed by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS). Eugenol, methyl-eugenol, methyl-isoeugenol, acetyl-isoeugenol and tricaine methanesulfonate(MS-222) were concerned. METHODS: After homogenization fish samples were extracted by acetonitrile-water(80↿0, V/V), purified by Oasis PRiME HLB solid-phase extraction column. Then after centrifuged and concentrated, the samples were separated by Waters ACQUITY UPLC BEH Phenyl column(2. 1 mm×100 mm, 1. 7 µm). The detection was confirmed and quantified by mass spectrum of triple quadrupole in the multiple reaction monitoring(MRM) mode. RESULTS: The calibration curves showed good linearity in each range with correlation coefficients greater than 0. 995. Three levels spiked recovery experiments were carried out using blank fish mud extraction as substrate, the recoveries ranged from 72. 6% to 106. 0%, the relative standard deviations(RSDs) ranged from 2. 2% to 20. 1%(n=6). The qualitative limits of detections(S/N>3) were 0. 14-0. 30 µg/kg and the quantitative limits(S/N>10) were 0. 5-1. 0 µg/kg. CONSLUSION: The method is simple and easy to operate, with less organic reagent, high sensitivity and good stability. The isomers of methyl eugenol and methyl isoeugenol were successfully separated. It is suitable for the detection of 5 kinds of fish anesthetics in fish.

Effects of repeated anaesthesia on gill and general health of Atlantic salmon, Salmo salar.

Fish are the second most widely utilized vertebrate group used for scientific procedures in the United Kingdom, but the development and application of 3Rs (the principles of replacement, reduction, and refinement) in aquaculture disease research lags behind methodologies in place for mammalian studies. With a need for individual monitoring and non-lethal sampling, the effect of repeat anaesthesia on experimental fish needs to be better understood. This study analyses the effect of repeat anaesthesia with MS-222, metomidate and AQUI-S upon the gill and general health of post-smolt Atlantic salmon Salmo salar. A single, lethal dose of anaesthetic was compared with seven anaesthetizing time points over 28 days, terminating in a lethal dose. No anaesthetic showed significant differences in accumulation in the muscle tissue, or changes in plasma glucose after repeated or single dosing. Fish repeatedly anaesthetized with MS-222 or AQUI-S exhibited upregulation of osmoregulatory genes in the gill and AQUI-S-treated individuals showed, histologically, epithelial lifting from the lamellae capillary irrespective of whether they had a single or repeated dose history. No significant changes were seen in inflammatory or stress genes in the head kidney of fish repeatedly anaesthetized with AQUI-S or metomidate, however MS-222 treatment resulted in upregulation of tnfα3. Repeated anaesthesia with MS-222 and metomidate gave a significant decrease and increase in peripheral blood neutrophils, respectively. This study concludes that no increase in cumulative stress or inflammation is induced by the repeated anaesthetization of S. salar with any of the tested anaesthetics, however gill osmotic regulation and blood parameters may be affected.

Cold block of in vitro eyeblink reflexes: evidence supporting the use of hypothermia as an anesthetic in pond turtles.

Use of hypothermia as a means of anesthesia for amphibians and reptiles is prohibited by agencies that establish veterinary guidelines. This has recently been called into question by members of the scientific community based on reviews of published literature. Using pond turtles (Trachemys scripta elegans), hypothermia as a method for anesthesia to precede euthanasia by decapitation was assessed. Turtles were subjected to hypothermia using a cooling followed by freezing protocol. Body temperature measurements ranged between -1 and -2°C while core body temperature was -1°C. Ice crystal formation was never observed. A protective reflex to noxious stimuli, the eyeblink response, was recorded from in vitro brainstem preparations subjected to cold. At 5-6°C, reflex responses were suppressed, demonstrating minimal synaptic transmission in brain circuits above temperatures used for hypothermia induction. These and previous data indicate that a re-evaluation of the use of hypothermia as an anesthetic in amphibians and reptiles is warranted.

MEASURING INTRAOCULAR PRESSURE IN WHITE'S TREE FROGS (LITORIA CAERULEA) BY REBOUND TONOMETRY: COMPARING DEVICE, TIME OF DAY, AND MANUAL VERSUS CHEMICAL RESTRAINT METHODS.

Ocular diseases reported in frogs include uveitis and glaucoma, which are associated with changes in intraocular pressure (IOP). The objectives of this study were to characterize the normal IOP for White's tree frogs ( Litoria caerulea ) using two types of rebound tonometers, and to assess whether time of day or method of restraint affected IOP. Eighteen conscious, unrestrained, ophthalmologically normal frogs were used to measure IOP using TonoVet® and TonoLab® tonometers, at three time points during the day. In a subset of 12 frogs, IOP was measured while under manual restraint using the TonoVet. Anesthesia was induced in 9 frogs using two different concentrations of MS-222 (0.5 g/L and 2 g/L) in order to evaluate for changes in IOP with the TonoVet. Mean (± SD) IOP values for the TonoLab (16.8 ± 3.9 mm Hg) were significantly higher than TonoVet values (14.7 ± 1.6 mm Hg; P < 0.01). TonoVet IOP values did not significantly change with time of day. TonoLab values were significantly lower in the evening (1600-1800; 14.5 ± 3.1 mm Hg), compared with morning and midday measurements (0800-1000 and 1200-1400; 18.0 ± 3.8 mm Hg; P < 0.01). Manually restrained frogs had significantly lower IOP (13.4 ± 1.5 mm Hg) compared with unrestrained frogs (15.3 ± 1.2 mm Hg; P < 0.01). Chemical restraint did not cause significant changes in IOP. Intraocular pressure can be measured with both types of rebound tonometers in White's tree frogs, but time of day and manual restraint can affect IOP values.

Salinity effects on plasma ion levels, cortisol, and osmolality in Chinook salmon following lethal sampling.

Studies on hydromineral balance in fishes frequently employ measurements of electrolytes following euthanasia. We tested the effects of fresh- or salt-water euthanasia baths of tricaine mesylate (MS-222) on plasma magnesium (Mg(2+)) and sodium (Na(+)) ions, cortisol and osmolality in fish exposed to saltwater challenges, and the ion and steroid hormone fluctuations over time following euthanasia in juvenile spring Chinook salmon (Oncorhynchus tshawytscha). Salinity of the euthanasia bath affected plasma Mg(2+) and Na(+) concentrations as well as osmolality, with higher concentrations in fish euthanized in saltwater. Time spent in the bath positively affected plasma Mg(2+) and osmolality, negatively affected cortisol, and had no effect on Na(+) concentrations. The difference of temporal trends in plasma Mg(2+) and Na(+) suggests that Mg(2+) may be more sensitive to physiological changes and responds more rapidly than Na(+). When electrolytes and cortisol are measured as endpoints after euthanasia, care needs to be taken relative to time after death and the salinity of the euthanasia bath.

TRICAINE METHANESULFONATE (MS-222) SEDATION AND ANESTHESIA IN THE PURPLE-SPINED SEA URCHIN (ARBACIA PUNCTULATA).

The purple-spined sea urchin ( Arbacia punctulata ) is commonly found in shallow waters of the western Atlantic Ocean from the New England area of the United States to the Caribbean. Sea urchins play a major role in ocean ecology, echinoculture, and biomedical research. Additionally, sea urchins are commonly displayed in public aquaria. Baseline parameters were developed in unanesthetized urchins for righting reflex (time to regain oral recumbency) and spine response time to tactile stimulus. Tricaine methanesulfonate (MS-222) was used to sedate and anesthetize purple-spined sea urchins and assess sedation and anesthetic parameters, including adhesion to and release from a vertical surface, times to loss of response to tactile stimulus and recovery of righting reflex, and qualitative observations of induction of spawning and position of spines and pseudopodia. Sedation and anesthetic parameters were evaluated in 11 individuals in three circumstances: unaltered aquarium water for baseline behaviors, 0.4 g/L MS-222, and 0.8 g/L MS-222. Induction was defined as the release from a vertical surface with the loss of righting reflex, sedation as loss of righting reflex with retained tactile spine response, anesthesia as loss of righting reflex and loss of tactile spine response, and recovery as voluntary return to oral recumbency. MS-222 proved to be an effective sedative and anesthetic for the purple-spined sea urchin at 0.4 and 0.8 g/L, respectively. Sodium bicarbonate used to buffer MS-222 had no measurable sedative effects when used alone. Anesthesia was quickly reversed with transfer of each individual to anesthesia-free seawater, and no anesthetic-related mortality occurred. The parameters assessed in this study provide a baseline for sea urchin anesthesia and may provide helpful comparisons to similar species and populations that are in need of anesthesia for surgical procedures or research.

Prolonged in vivo imaging of Xenopus laevis.

BACKGROUND: While live imaging of embryonic development over long periods of time is a well established method for embryos of the frog Xenopus laevis, once development has progressed to the swimming stages, continuous live imaging becomes more challenging because the tadpoles must be immobilized. Current imaging techniques for these advanced stages generally require bringing the tadpoles in and out of anesthesia for short imaging sessions at selected time points, severely limiting the resolution of the data. RESULTS: Here we demonstrate that creating a constant flow of diluted tricaine methanesulfonate (MS-222) over a tadpole greatly improves their survival under anesthesia. Based on this result, we describe a new method for imaging stage 48 to 65 X. laevis, by circulating the anesthetic using a peristaltic pump. This supports the animal during continuous live imaging sessions for at least 48 hr. The addition of a stable optical window allows for high quality imaging through the anesthetic solution. CONCLUSIONS: This automated imaging system provides for the first time a method for continuous observations of developmental and regenerative processes in advanced stages of Xenopus over 2 days. Developmental Dynamics 243:1011-1019, 2014. © 2014 Wiley Periodicals, Inc.

Evaluation of the Effectiveness of Eugenol and MS-222 as Anesthetics in Zebrafish in Repeated Exposures and Post-Anesthesia Behaviour.

The increasing use of the zebrafish (Danio rerio) in scientific experiments has made it necessary to implement anesthesia protocols guaranteeing minimum pain and suffering for these animals and ensuring the reliability of the results obtained from their research. Therefore, we aimed to compare the effectiveness of two anesthetics, eugenol and MS-222, in consecutive administrations and evaluate the zebrafish behaviour after repeated anesthesia. Thus, several zebrafish were anaesthetized with eugenol, MS-222, and buffered MS-222 three times repeatedly with a 24-h interval between each exposure. The induction and recovery periods were also timed. Their swimming frequency was determined after each exposure to assess their behaviour after the anesthesia. Anesthesia induction was quicker with eugenol compared to MS-222. However, eugenol presented longer recovery times, which were prolonged after each exposure. Also, the swimming frequency was reduced after each anesthesia with eugenol. The buffered version of MS-222 was more efficacious than the non-buffered one. Both versions of MS-222 did not affect the swimming frequency. Based on these findings, we recommend the utilization of MS-222 buffered rather than eugenol when repeated, brief-duration anesthesia is necessitated for a study.

Analysis of protein expression in zebrafish during gonad differentiation by targeted proteomics.

The molecular mechanisms governing sex determination and differentiation in the zebrafish (Danio rerio) are not fully understood. To gain more insights into the function of specific genes in these complex processes, the expression of multiple candidates needs to be assessed, preferably on the protein level. Here, we developed a targeted proteomics method based on selected reaction monitoring (SRM) to study the candidate sex-related proteins in zebrafish which were selected based on a global proteomics analysis of adult gonads and representational difference analysis of male and female DNA, as well as on published information on zebrafish and other vertebrates. We employed the developed SRM protocols to acquire time-resolved protein expression profiles during the gonad differentiation period in vas::EGFP transgenic zebrafish. Evidence on protein expression was obtained for the first time for several candidate genes previously studied only on the mRNA level or suggested by bioinformatic predictions. Tuba1b (tubulin alpha 1b), initially included in the study as one of the potential housekeeping proteins, was found to be preferentially expressed in the adult testis with nearly absent expression in the ovary. The revealed changes in protein expression patterns associated with gonad differentiation suggest that several of the examined proteins, especially Ilf2 and Ilf3 (interleukin enhancer-binding factors 2 and 3), Raldh3 (retinaldehyde dehydrogenase type 3), Zgc:195027 (low density lipoprotein-related receptor protein 3) and Sept5a (septin 5a), may play a specific role in the sexual differentiation in zebrafish.