Zebrafish Embryos and Larvae as Alternative Animal Models for Toxicity Testing

Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.

Free Feeding of CpG-Oligodeoxynucleotide Particles Prophylactically Attenuates Allergic Airway Inflammation and Hyperresponsiveness in Mice

CpG-oligodeoxynucleotides (CpG-ODNs) constitute an attractive alternative for asthma treatment. However, very little evidence is available from studies on the oral administration of CpG-ODNs in animals. Previously, we developed acid-resistant particles (named ODNcap) as an oral delivery device for ODNs. Here, we showed that free feeding of an ODNcap-containing feed prophylactically attenuates allergic airway inflammation, hyperresponsiveness, and goblet cell hyperplasia in an ovalbumin-induced asthma model. Using transcriptomics-driven approaches, we demonstrated that injury of pulmonary vein cardiomyocytes accompanies allergen inhalation challenge, but is inhibited by ODNcap feeding. We also showed the participation of an airway antimicrobial peptide (Reg3γ) and fecal microbiota in the ODNcap-mediated effects. Collectively, our findings suggest that daily oral ingestion of ODNcap may provide preventive effects on allergic bronchopulmonary insults via regulation of mechanisms involved in the gut-lung connection.

Optimal Standardized Ileal Digestible Total Sulfur Amino Acids to Lysine REQUIREMENTS Are Increased in Nursery Pigs Raised under Antibiotic-Free Feeding Regime

This study aimed to investigate the effect of increasing the standardized ileal digestible (SID) total sulfur amino acid to lysine (TSAA:Lys) on the growth performance of nursery pigs raised with or without antibiotics (AGP) and to determine the optimal SID TSAA:Lys in nursery pigs raised without AGP. In Exp. 1, 924 nursery pigs (7.9 ± 1.3 kg), blocked by initial BW and sex, were randomly allotted to one of six treatments, with seven pens per treatment and twenty-two pigs per pen. The treatments were arranged in a 2 × 3 factorial design, with two AGP levels (0 or 50 mg/kg Carbodox) and three levels of SID TSAA:Lys (51.0, 58.5 or 66.0%). In Exp. 2, 990 weaned piglets (5.1 ± 0.9 kg), blocked by initial BW and sex, were randomly allotted to one of five dietary treatments (SID TSAA:Lys at 51, 58, 65, 72 or 79%) in the absence of AGP, with nine pens per treatment and twenty-two pigs per pen. Competing heteroskedastic models including broken-line linear (BLL), broken-line quadratic (BLQ), and quadratic polynomial (QP) were fitted for the growth performance data to estimate the optimal TSAA:Lys. In Exp. 1, AGP supplementation increased (p < 0.05) ADG and ADFI during the 21 d period. Increasing SID TSAA:Lys in the diets with AGP did not affect growth performance; however, increasing SID TSAA:Lys in the diets without AGP resulted in a linear increase (p < 0.05) in ADG and G:F. In Exp. 2, the best-fitting models for ADG and G:F from d 0 to 21 post-weaning were BLL, which yielded the optimal SID TSAA:Lys of 62% and 72%, respectively. The best-fitting models for ADG and G:F from d 21 to 42 post-weaning were BLL, which yielded the optimal SID TSAA:Lys of 59% and 58%, respectively. In conclusion, SID TSAA to Lys requirements under an antibiotic-free feeding regime during the first 21 d post-weaning were 62% and 72% in terms of ADG and G:F, respectively, whereas an SID TSAA:Lys of approximately 58% was required to maximize ADG and G:F for the late nursery phase.

Bioaccumulation And Biotransformation of BDE-47 Using Zebrafish Eleutheroembryos (Danio Rerio)

Polybrominated diphenyl ethers (PBDEs) industrially used as flame retardants are nowadays considered emerging pollutants as they are endocrine disrupting chemicals (EDCs), persistent in the environment, bioaccumulative and in addition, its hydroxylated (OH-BDEs) and methoxylated (MeO-BDEs) metabolites have similar ecotoxic properties. The aim of this work was to develop an analytical method to be applied in the study of the bioconcentration and biotransformation of BDE-47 due to its bioavailability, toxicity and high persistence and abundance in environmental samples, including humans. So, a dependable ultrasonic extraction process followed to dispersive SPE clean-up step and GC-MS-μECD detection has worked out for the determination of BDE-47 and its main biotransformation products (MeO-BDEs and OH-BDEs), considering the polarity difference. In addition, an alternative method to bioconcentration official guideline OECD 305, developed previously with zebrafish (Danio rerio) eleutheroembryos (i.e., hatched but not yet free feeding embryos) is used, reducing dramatically the animal suffering but also time and reagents. Bioconcentration factors (BCF) were calculated using first order one-compartment toxicokinetic model. The profiles found show rapid absorption in the first hours of larval development and great bioaccumulative with capacity, finding bioconcentration factors (BCF) of 6631 and 44210 at nominal concentrations of 10 and 1 μg·L-1 (< 1% LC50), respectively. Metabolization studies show increasing concentrations of the metabolites BDE-28, 2'-OH-BDE-28 and 5-MeO-BDE-47 throughout the exposure time. The results obtained show the feasibility of the method for bioaccumulation and opens the possibility of metabolic studies with zebrafish eleutheroembryos, which is a very underdeveloped field without official testing or regulation.

Hippocampal Circuits for the Hunger-Dependent Control of Feeding Behaviour

Background: Feeding behavior is a complex motivated behavior that requires organisms to integrate features of the environment, such as food availability and value, and internal states, such as hunger, in deliberating over the decision to eat. The hippocampus - a brain region classically thought to support spatial cognition and episodic memory - is increasingly recognised to contribute to such decision-making processes. This function makes the hippocampus a likely candidate in supporting the higher-order decisions that underpin motivated behaviors such as feeding. However, the role of the hippocampus during free-feeding behavior has not been examined. Methods and Results: To address this question, we used in vivo calcium imaging during feeding behavior in mice to monitor the neural activity of the ventral subiculum (vS) - one of the main output structures of the ventral hippocampus. In a free-feeding task, we found that the vS encoded the investigative approach phase of feeding behavior and that activity during this period correlated with the probability of transitioning from food investigation to consumption. Calcium imaging during an operant task confirmed the specific encoding of preparatory behaviour preceding food consumption. Furthermore, the sensitivity of vS to the hunger state could be mapped to vS neurons projecting to the nucleus accumbens (vS-NAc). Ghrelin - a hormone signalling the hunger state - altered synaptic transmission specifically in vS-NAc neurons, and molecular knockdown of the ghrelin receptor was required for the hunger sensitivity of vS-NAc. Consequently, both reducing ghrelin signalling in vS-NAc neurons through molecular knockdown and artificially elevating vS-NAc activity through optogenetics were sufficient to shift the feeding strategy of animals, effectively curtailing overall food consumption. Conclusion: In summary, these results provide evidence for a hippocampal circuit that integrates hunger state signals to regulate the decision to eat.

Characteristics of Salmonella From Chinese Native Chicken Breeds Fed on Conventional or Antibiotic-Free Diets

Salmonella is a common food-borne Gram-negative pathogen with multiple serotypes. Pullorum disease, caused by Salmonella Pullorum, seriously threatens the poultry industry. Many previous studies were focused on the epidemiological characteristics of Salmonella infections in conventional antibiotic use poultry. However, little is known about Salmonella infections in chicken flocks fed on antibiotic-free diets. Herein, we investigated and compared Salmonella infections in three Chinese native breeders fed on antibiotic-free diets, including the Luhua, Langya, and Qingjiaoma chickens, and one conventional breeder, the Bairi chicken, via analyzing 360 dead embryos in 2019. The results showed that the main Salmonella serotypes detected in a total of 155 isolates were S. Pullorum (82.6%) and S. Enteritidis (17.4%). Coinfection with two serotypes of Salmonella was specifically found in Bairi chicken. The sequence type (ST) in S. Pullorum was ST92 (n = 96) and ST2151 (n = 32), whereas only ST11 (n = 27) was found in S. Enteritidis. The Salmonella isolates from three breeder flocks fed on antibiotic-free diets exhibited phenotypic heterogeneity with a great variety of drug resistance spectrum. Most of the isolates among three chicken breeds Luhua (64.9%, 50/77), Langya (60%, 12/20) and Qingjiaoma (58.3%, 7/12) fed on antibiotic-free diets were resistant to only one antibiotic (erythromycin), whereas the rate of resistance to one antibiotic in conventional Bairi chicken isolates was only 4.3% (2/46). The multidrug-resistance rate in Salmonella isolates from layer flocks fed on antibiotic-free diets (20.2%, 22/109) was significantly (P &lt; 0.0001) lower than that from chickens fed on conventional diets (93.5%, 43/46). However, high rate of resistance to erythromycin (97.4%~100%) and streptomycin (26%~41.7%) were also found among three breeder flocks fed on antibiotic-free diets, indicating resistance to these antibiotics likely spread before antibiotic-free feeding in poultry farms. The findings of this study supplement the epidemiological data of salmonellosis and provide an example of the characteristics of Salmonella in the chicken flocks without direct antibiotic selective pressure.

Activation of Basolateral Amygdala to Nucleus Accumbens Projection Neurons Attenuates Chronic Corticosterone-Induced Behavioral Deficits in Male Mice

The basolateral amygdala (BLA) is critical for reward behaviors via a projection to the nucleus accumbens (NAc). Specifically, BLA-NAc projections are involved in reinforcement learning, reward-seeking, sustained instrumental responding, and risk behaviors. However, it remains unclear whether chronic stress interacts with BLA-NAc projection neurons to result in maladaptive behaviors. Here we take a chemogenetic, projection-specific approach to clarify how NAc-projecting BLA neurons affect avoidance, reward, and feeding behaviors in male mice. Then, we examine whether chemogenetic activation of NAc-projecting BLA neurons attenuates the maladaptive effects of chronic corticosterone (CORT) administration on these behaviors. CORT mimics the behavioral and neural effects of chronic stress exposure. We found a nuanced role of BLA-NAc neurons in mediating reward behaviors. Surprisingly, activation of BLA-NAc projections rescues CORT-induced deficits in the novelty suppressed feeding, a behavior typically associated with avoidance. Activation of BLA-NAc neurons also increases instrumental reward-seeking without affecting free-feeding in chronic CORT mice. Taken together, these data suggest that NAc-projecting BLA neurons are involved in chronic CORT-induced maladaptive reward and motivation behaviors.

Hibernation Period in Some Land Snail Species (Gastropoda: Helicidae): Epiphragmal Structure and Hypometabolic Behaviors

In this study, the hypometabolism behavior, epiphragm formation process, and the mineral composition of the epiphragmal structure of the Helix pomacella, Eobania vermiculata, Helix melanostoma, and Helix asemnis land snails were investigated during the hibernation period. 100 snails selected among mature individuals, 25 from each species, were collected from nature for the research. The snails were placed in four 50x50x5 cm pans containing 20-cm-deep humus soil. Snails were fed with lettuce by free feeding until the hibernation period. Hypometabolism behavior of snails that had hibernated between November and December 2018, and January and February 2019 were examined as entering the hibernation stage, hibernation stage, and leaving the hibernation stage. The ionic concentration (Calcium, Iron, and Phosphorus) of the epiphragm layer formed during the hibernation process was analyzed, its formation duration, and its degradation at the end of the hibernation period were examined. In the study, it was determined that the weight before hibernation reduced by 18-22% in all examined species (H. pomacella, E. vermiculata, H. melanostoma, and H. asemnis). The highest values in terms of epiphragm formation duration (19±0.30 days), weight (0.41±0.26 g), and thickness (0.310±0.22 mm) were determined in H. asemnis. In terms of the ionic concentration, the highest Calcium level was determined in H. asemnis (29.96±0.12 mg/g) while the highest Phosphorus level (4.02±0.20 mg/g) was determined in E. vermiculata, and the highest Iron content was found in H. pomacella (19.50±0.33 mg/g). The degradation duration of the epiphragm layer at the end of hibernation was determined the longest in E. vermiculata (7 days) whereas the shortest in H. pomacella (3 days). As a result of the present research, it was seen that the epiphragm layer is important for snails in terms of a sustainable life cycle and that the ecological tolerances of snails depend on the epiphragmal structure.