A Multi-Site Assessment of Anesthetic Overdose, Hypothermic Shock, and Electrical Stunning as Methods of Euthanasia for Zebrafish (Danio rerio) Embryos and Larvae.

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.

From Mating to Milk Access: A Review of Reproductive Vocal Communication in Mice.

Vocalisations play a central role in rodent communication, especially in reproduction related behaviours. In adult mice (Mus musculus) the emission of ultrasonic vocalisations (USVs) has been observed in courtship and mating behaviour, especially by males. These have been found to have distinctive individual signatures that influence female choice of mating partner. The most recent findings show that vocal communication also has a role in parental cooperation, in that female mice communicate with male partners in ultrasonic frequencies to induce paternal behaviour. Infant vocalisations form the other important part of reproductive vocal communication. Although born deaf, neonatal mice are capable of producing vocalisations since birth. As an altricial species, successful mother-infant communication is essential for survival, and these vocalisations are important modulators of maternal behaviour. Three main types of infant vocalisations have been identified and characterised. Most research has addressed pure USVs, related to stressful situations (e.g., cold, isolation, handling, presence of unfamiliar males or predators), which usually elicit maternal search and retrieval. In addition, broad-band spectrum signals, emitted post-partum during cleaning of foetal membranes, inhibit biting and injury by adults and "wriggling calls," emitted during suckling, release maternal behaviour (such as licking). Several variables have been identified to modulate vocalisations in mice, including individual characteristics such as strain/genotype, age, sex, and experimental factors such as pharmacological compounds and social context. In recent years, there has been a big increase in the knowledge about the characteristics of vocal communication in rodents due to recent technological advances as well as a growing interest from the neuroscience community. Vocalisation analysis has become an essential tool for phenotyping and evaluating emotional states. In this review, we will (i) provide a comprehensive summary of the current knowledge on mouse reproductive vocal communication and (ii) discuss the most recent findings in order to provide a broad overview on this topic.

Screening of Anaesthetics in Adult Zebrafish (Danio rerio) for the Induction of Euthanasia by Overdose.

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 NaHCO3, 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 NaHCO3 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.

Tissue macrophages and interferon-gamma signalling control blood-stage Plasmodium chabaudi infections derived from mosquito-transmitted parasites.

Natural infection with Plasmodium parasites, the causative agents of malaria, occurs via mosquito vectors. However, most of our knowledge of the immune response to the blood stages of Plasmodium is from infections initiated by injection of serially blood-passaged infected red blood cells, resulting in an incomplete life cycle in the mammalian host. Vector transmission of the rodent malaria parasite, Plasmodium chabaudi chabaudi AS has been shown to give rise to a more attenuated blood-stage infection in C57Bl/6J mice, when compared to infections initiated with serially blood-passaged P. chabaudi-infected red blood cells. In mouse models, the host immune response induced by parasites derived from natural mosquito transmission is likely to more closely resemble the immune responses to Plasmodium infections in humans. It is therefore important to determine how the host response differs between the two types of infections. As the spleen is considered to be a major contributor to the protective host response to P. chabaudi, we carried out a comparative transcriptomic analysis of the splenic response to recently mosquito-transmitted and serially blood-passaged parasites in C57Bl/6J mice. The attenuated infection arising from recently mosquito-transmitted parasites is characterised by an earlier and stronger myeloid- and IFNγ-related response. Analyses of spleen lysates from the two infections similarly showed stronger or earlier inflammatory cytokine and chemokine production in the recently mosquito-transmitted blood-stage infections. Furthermore, tissue macrophages, including red pulp macrophages, and IFNγ-signalling in myeloid cells, are required for the early control of P. chabaudi recently mosquito-transmitted parasites, thus contributing to the attenuation of mosquito-transmitted infections. The molecules responsible for this early activation response to recently-transmitted blood-stage parasites in mice would be important to identify, as they may help to elucidate the nature of the initial interactions between blood-stage parasites and the host immune system in naturally transmitted malaria.

Generation and Surgical Analysis of Genetic Mouse Models to Study NF-κB-Driven Pathogenesis of Diffuse Large B Cell Lymphoma.

Enforced activation of NF-κB signaling can be achieved by constitutive NF-κB-inducing kinases, IKK2 and NIK, or via lymphoma-associated mutants of MYD88, CARD11, and CD79B. In order to model Diffuse Large B Cell Lymphoma (DLBCL) in mice, conditional alleles for these proteins are combined with alleles targeting Cre recombinase expression in mature B cells. However, unopposed NF-κB signaling promotes plasmablast differentiation, and as a consequence the model system must be complemented with further mutations that block differentiation, such as Prdm1/BLIMP1 inactivation or overexpression of BCL6. Here, we describe the currently available tools for DLBCL models in mice and their relative advantages and drawbacks. Furthermore, we describe methods to monitor lymphomagenesis, using ultrasound tomography of the spleen, and the technique of partial splenectomy surgery with recovery. These powerful techniques allow paired comparison of individual lymphoma cases before and after interventions, including therapies, and to study the evolution of lymphoma over time. NF-κB activation also promotes widespread nodal involvement with lymphoma and we describe the post-mortem dissection of major nodal groups.

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems.

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa have the potential to impair animal welfare and research. The fish are typically analyzed post mortem to detect microbes. The use of sentinels is a suggested way to improve the sensitivity of the surveillance and to reduce the number of animals to sample. The setting of a pre-filtration sentinel tank out of a recirculating system is described. The technique is developed to prevent water pollution and to represent the fish population by a careful selection of age, gender, and strains. In order to use the minimum number of animals, techniques to screen the environment are also detailed. Polymerase Chain Reaction (PCR) on surface sump swabs is used to significantly improve the detection of some prevalent and pathogenic mycobacterial species such as Mycobacterium fortuitum, Mycobacterium haemophilum, and Mycobacterium chelonae. Another environmental method consists of processing the sludge at the bottom of a holding tank or sump to look for P. tomentosa eggs. This is a cheap and fast technique that can be applied in quarantine where a breeding device is submerged into the holding tank of imported animals. Finally, PCR is applied to the sludge sample and A. hydrophila is detected at the sump's bottom and surface. Generally, these environmental screening techniques applied to these specific pathogens have led to an increased sensitivity compared to the testing of pre-filtration sentinels.