Zebrafish Feed Intake: A Systematic Review for Standardizing Feeding Management in Laboratory Conditions.

Zebrafish are one of the most used animal models in biological research and a cost-effective alternative to rodents. Despite this, nutritional requirements and standardized feeding protocols have not yet been established for this species. This is important to avoid nutritional effects on experimental outcomes, and especially when zebrafish models are used in preclinical studies, as many diseases have nutritional confounding factors. A key aspect of zebrafish nutrition is related to feed intake, the amount of feed ingested by each fish daily. With the goal of standardizing feeding protocols among the zebrafish community, this paper systematically reviews the available data from 73 studies on zebrafish feed intake, feeding regimes (levels), and diet composition. Great variability was observed regarding diet composition, especially regarding crude protein (mean 44.98 ± 9.87%) and lipid content (9.91 ± 5.40%). Interestingly, the gross energy levels of the zebrafish diets were similar across the reviewed studies (20.39 ± 2.10 kilojoules/g of feed). In most of the reviewed papers, fish received a predetermined quantity of feed (feed supplied). The authors fed the fish according to the voluntary intake and then calculated feed intake (FI) in only 17 papers. From a quantitative point of view, FI was higher than when a fixed quantity (pre-defined) of feed was supplied. Also, the literature showed that many biotic and abiotic factors may affect zebrafish FI. Finally, based on the FI data gathered from the literature, a new feeding protocol is proposed. In summary, a daily feeding rate of 9-10% of body weight is proposed for larvae, whereas these values are equal to 6-8% for juveniles and 5% for adults when a dry feed with a proper protein and energy content is used.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression.

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

A Review on the Bioactivity of Cannabinoids on Zebrafish Models: Emphasis on Neurodevelopment.

For centuries, the cannabis plant has been used as a source of food, fiber, and medicine. Recently, scientific interest in cannabis has increased considerably, as its bioactive compounds have shown promising potential in the treatment of numerous musculoskeletal and neurological diseases in humans. However, the mechanisms that underlie its possible effects on neurodevelopment and nervous-system functioning remain poorly understood and need to be further investigated. Although the bulk of research on cannabis and cannabinoids is based on in vitro or rodent models, the zebrafish has now emerged as a powerful in vivo model for drug-screening studies and translational research. We here review the available literature on the use of cannabis/cannabinoids in zebrafish, and particularly in zebrafish models of neurological disorders. A critical analysis suggests that zebrafish could serve as an experimental tool for testing the bioactivity of cannabinoids, and they could thus provide important insights into the safety and efficacy of different cannabis-extract-based products. The review showed that zebrafish exhibit similar behaviors to rodents following cannabinoid exposure. The authors stress the importance of analyzing the full spectrum of naturally occurring cannabinoids, rather than just the main ones, THC and CBD, and they offer some pointers on performing behavioral analysis in zebrafish.

Trehalose Treatment in Zebrafish Model of Lafora Disease.

Mutations in the EPM2A gene encoding laforin cause Lafora disease (LD), a progressive myoclonic epilepsy characterized by drug-resistant seizures and progressive neurological impairment. To date, rodents are the only available models for studying LD; however, their use for drug screening is limited by regulatory restrictions and high breeding costs. To investigate the role of laforin loss of function in early neurodevelopment, and to screen for possible new compounds for treating the disorder, we developed a zebrafish model of LD. Our results showed the epm2a-/- zebrafish to be a faithful model of LD, exhibiting the main disease features, namely motor impairment and neuronal hyperexcitability with spontaneous seizures. The model also showed increased inflammatory response and apoptotic death, as well as an altered autophagy pathway that occurs early in development and likely contributes to the disease progression. Early administration of trehalose was found to be effective for rescuing motor impairment and neuronal hyperexcitability associated with seizures. Our study adds a new tool for investigating LD and might help to identify new treatment opportunities.

Milk composition, fatty acids profile and fat globule size of bottlenose dolphin (Tursiops truncatus, Montagu 1821) milk at early lactation.

Research data on milk composition in cetaceans are scattered and fragmentary. This paper analyses the gross and mineral composition, the fatty acids profile and the fat globule size of bottlenose dolphin (Tursiops truncatus) colostrum and milk at early lactation. The milk samplings were carried out on three lactating female of bottlenose dolphins at the 1st, 4-5 and 20- 42 day post partum. High percentages of dry matter (51.88%), fat (26.08%) and protein (13.83%) were found in the colostrum on the first day, while there was a tendency for these components to decrease in the milk. The average diameter the milk fat globule (7.07 µm) of this species was assessed for the first time. The milk was rich in unsaturated fatty acids, which were more than twice compared to the saturated fatty acids (unsaturated / saturated ratio = 2.6). The main fatty acids of dolphin milk were C16: 0, C16:1 n-7, C18: 1n-9, C20: 1 n-11 C20: 5 and C22:6n-3. Several FA tended to increase from colostrum to milk: C12: 0 doubled, C20: 1 n-7 and C22:1 n-11 tripled, C20: 1 n-9 quintupled in later milk samples. Differently, other FA tended to decrease: C18: 3 n-3 was approximately 4 times lower and C24: 0 and C24: 1 halved in milk compared to colostrum. In conclusion, this paper contributes to the knowledge of the nutritional value of milk from bottlenose dolphins maintained under human care. Higher content of nutritional components such as fat and protein was found in the first day colostrum compared to milk. Moreover, the findings of this study highlight abundance of unsaturated fatty acids and large sized milk fat globules in dolphin milk.

Dietary Supplementation with a Blend of Hydrolyzable and Condensed Tannins Ameliorates Diet-Induced Intestinal Inflammation in Zebrafish (Danio rerio).

The current study evaluated the effects of hydrolyzable and condensed tannins from chestnut and quebracho wood, respectively (TSP, Silvafeed®), on zebrafish with intestinal inflammation induced by a plant-based diet (basal diet). Four experimental diets were prepared as follows: the basal diet + 0 TSP, the basal diet + TSP at 0.9 g/kg of feed, the basal diet + TSP at 1.7 g/kg of feed, and the basal diet + TSP at 3.4 g/kg of feed. Eighty-four zebrafish (Danio rerio) were fed for 12 days with the experimental diets. In zebrafish fed the basal diet, intestine integrity appeared to be altered, with damaged intestinal villi, high immunoexpression of tumor necrosis factor-α (TNFα) and cyclooxygenase 2 (COX2), and high expression of the cox2, interleukin 1 (il-1b), interleukin 8 (cxcl8-l1), and tnfα genes. The tannin treatment partially restored intestinal morphology and downregulated the expression of cytokines. The best activity was detected with 1.7 and 3.4 g/kg of feed. In the guts of all groups, Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes were the most represented phyla. The most represented genera were Plesiomonas and Sphingomonas, belonging to the Proteobacteria phylum; Cetobacterium, belonging to the Fusobacteria phylum; and Lactobacillus, belonging to the Firmicutes phylum. No significant differences were detected among groups, except for a slight decrease in the Fusobacteria phylum and slight increases in the Shewanella and Bacteroides genera with TSP. In conclusion, these results suggest that tannins can improve the zebrafish intestinal inflammation caused by a terrestrial-plant-based diet in a dose-dependent manner.

In Vivo Evaluation of Cannabis sativa Full Extract on Zebrafish Larvae Development, Locomotion Behavior and Gene Expression.

Historically, humans have been using Cannabis sativa for both recreational and medical purposes. Nowadays, cannabis-based products have gained scientific interest due to their beneficial effects on several syndromes and illnesses. The biological activity of cannabinoids is essentially due to the interaction with the endocannabinoid system, and zebrafish (Danio rerio) is a very well-known and powerful in vivo model for studying such specific interactions. The aim of the study was to investigate the effects of different doses of a Cannabis sativa whole extract [dissolved in dimethyl sulfoxide (DMSO)] on zebrafish eggs' hatchability, embryo post-hatching survival, larvae locomotion behavior and mRNA gene expression. The results showed the absence of toxicity, and no significant differences were observed between treatments for both embryo hatching and survival rate. In addition, larvae exposed to the cannabis extract at the highest dose [containing 1.73 nM and 22.3 nM of ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively] showed an increased locomotion compared to the control and DMSO treated groups. Moreover, qRT-PCR analysis showed that the highest dosage of cannabis induced an over-expression of cnr1 and cnr2 cannabinoid receptors. In conclusion, the exposition of zebrafish larvae to the whole extract of Cannabis sativa showed no negative effects on embryo development and survival and enhanced the larvae's locomotor performances. These findings may open up possible Cannabis sativa applications in human pharmacology as well as in other animal sectors.

Efficient Neuroprotective Rescue of Sacsin-Related Disease Phenotypes in Zebrafish.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a multisystem hereditary ataxia associated with mutations in SACS, which encodes sacsin, a protein of still only partially understood function. Although mouse models of ARSACS mimic largely the disease progression seen in humans, their use in the validation of effective therapies has not yet been proposed. Recently, the teleost Danio rerio has attracted increasing attention as a vertebrate model that allows rapid and economical screening, of candidate molecules, and thus combines the advantages of whole-organism phenotypic assays and in vitro high-throughput screening assays. Through CRISPR/Cas9-based mutagenesis, we generated and characterized a zebrafish sacs-null mutant line that replicates the main features of ARSACS. The sacs-null fish showed motor impairment, hindbrain atrophy, mitochondrial dysfunction, and reactive oxygen species accumulation. As proof of principle for using these mutant fish in high-throughput screening studies, we showed that both acetyl-DL-leucine and tauroursodeoxycholic acid improved locomotor and biochemical phenotypes in sacs-/- larvae treated with these neuroprotective agents, by mediating significant rescue of the molecular functions altered by sacsin loss. Taken together, the evidence here reported shows the zebrafish to be a valuable model organism for the identification of novel molecular mechanisms and for efficient and rapid in vivo optimization and screening of potential therapeutic compounds. These findings may pave the way for new interventions targeting the earliest phases of Purkinje cell degeneration in ARSACS.

ß-Glucans as Dietary Supplement to Improve Locomotion and Mitochondrial Respiration in a Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular childhood disorder that causes progressive muscle weakness and degeneration. A lack of dystrophin in DMD leads to inflammatory response, autophagic dysregulation, and oxidative stress in skeletal muscle fibers that play a key role in the progression of the pathology. ß-glucans can modulate immune function by modifying the phagocytic activity of immunocompetent cells, notably macrophages. Mitochondrial function is also involved in an important mechanism of the innate and adaptive immune responses, owing to high need for energy of immune cells. In the present study, the effects of 1,3-1,6 ß-glucans on five-day-old non-dystrophic and dystrophic (sapje) zebrafish larvae were investigated. The effects of the sonication of ß-glucans and the dechorionation of embryos were also evaluated. The results showed that the incidence of dystrophic phenotypes was reduced when dystrophic embryos were exposed to 2 and 4 mg L-1 of 1,3-1,6 ß-glucans. Moreover, when the dystrophic larvae underwent 8 mg L-1 treatment, an improvement of the locomotor performances and mitochondrial respiration were observed. In conclusion, the observed results demonstrated that 1,3-1,6 ß-glucans improve locomotor performances and mitochondrial function in dystrophic zebrafish. Therefore, for ameliorating their life quality, 1,3-1,6 ß-glucans look like a promising diet supplement for DMD patients, even though further investigations are required.

Nutraceutical Screening in a Zebrafish Model of Muscular Dystrophy: Gingerol as a Possible Food Aid.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an inherited neuromuscular disorder that causes loss of muscle mass and motor skills. In the era of genomic medicine, there is still no known cure for DMD. In clinical practice, there is a growing awareness of the possible importance of nutrition in neuromuscular diseases. This is mostly the result of patients' or caregivers' empirical reports of how active substances derived from food have led to improved muscle strength and, thus, better quality of life. In this report, we investigate several nutraceutical principles in the sapje strain of zebrafish, a validated model of DMD, in order to identify possible natural products that, if supplemented in the diet, might improve the quality of life of DMD patients. Gingerol, a constituent of fresh ginger, statistically increased the locomotion of mutant larvae and upregulated the expression of heme oxygenase 1, a target gene for therapy aimed at improving dystrophic symptoms. Although three other compounds showed a partial positive effect on locomotor and muscle structure phenotypes, our nutraceutical screening study lent preliminary support to the efficacy and safety only of gingerol. Gingerol could easily be proposed as a dietary supplement in DMD.

Donkey Feeding During Maintenance, Pregnancy, and Lactation: Effects on Body Weight, Milk Production, and Foal Growth.

We evaluated the daily intake in donkeys during maintenance, late pregnancy, and early lactation. The growth curves of the foals in the first eight weeks of life and the milk production in lactating jennies were also investigated. Donkeys were separated into two groups: seven pregnant jennies (group 1: pregnant/lactating) and seven nonpregnant, nonlactating jennies (group 2). The groups were fed two different diets each. The feeding period for group 1 covered eight weeks before parturition and eight weeks postpartum. Group 2 was managed during the same time period (16 weeks). Diet 1 consisted of ad libitum hay, which was fed to group 1 during late pregnancy and to group 2 from weeks 1-8. Diet 2 consisted of the same ad libitum hay supplemented with 2 kg/head/day of concentrate, which was fed to group 1 during lactation and to group 2 from weeks 9-16. The daily dry matter intake (diet 1) was 2.56 kg/100 kg of body weight (BW) for nonpregnant jennies and 31% lower in the pregnant donkeys. In the lactating jennies, the total dry matter intake was similar to the nonpregnant group fed diet 2. The nonpregnant jennies lost 1.9% of their initial BW when fed diet 1, whereas they gained 8.4% of their initial BW when fed with diet 2. A weight loss was also found in the lactating donkeys. The foals more than doubled in their birth weight within two months.

Social Preference Tests in Zebrafish: A Systematic Review.

The use of animal models in biology research continues to be necessary for the development of new technologies and medicines, and therefore crucial for enhancing human and animal health. In this context, the need to ensure the compliance of research with the principles Replacement, Reduction and Refinement (the 3 Rs), which underpin the ethical and human approach to husbandry and experimental design, has become a central issue. The zebrafish (Danio rerio) is becoming a widely used model in the field of behavioral neuroscience. In particular, studying zebrafish social preference, by observing how an individual fish interacts with conspecifics, may offer insights into several neuropsychiatric and neurodevelopmental disorders. The main aim of this review is to summarize principal factors affecting zebrafish behavior during social preference tests. We identified three categories of social research using zebrafish: studies carried out in untreated wild-type zebrafish, in pharmacologically treated wild-type zebrafish, and in genetically engineered fish. We suggest guidelines for standardizing social preference testing in the zebrafish model. The main advances gleaned from zebrafish social behavior testing are discussed, together with the relevance of this method to scientific research, including the study of behavioral disorders in humans. The authors stress the importance of adopting an ethical approach that considers the welfare of animals involved in experimental procedures. Ensuring a high standard of animal welfare is not only good for the animals, but also enhances the quality of our science.

Short communication: Technological and seasonal variations of vitamin D and other nutritional components in donkey milk.

Vitamin D is an essential nutrient that plays a crucial role in calcium homeostasis and bone metabolism and also acts as a hormone. Although several studies on the content of vitamin D in bovine milk have been conducted, little information is available regarding donkey milk. In the context of the nutritional assessment of donkey milk, the aim of this study was to assess the vitamin D content in donkey milk and its chemical profile, with particular reference to seasonal and technological modifications after pasteurization. The study was conducted on a dairy farm that produces donkey milk for human consumption located in central Italy. At sampling time, an aliquot of total bulk milk production was sampled before and after pasteurization (63°C for 30 min without homogenization) with a total of 20 raw and 20 pasteurized milk samples. The samples were collected for 10 mo, every 15 d, from May to February 2017. All the samples were analyzed for the chemical composition and vitamin D2 and D3 content by HPLC after saponification. The donkey milk analyzed showed a higher average vitamin D content (raw milk: vitamin D2 = 1.68, vitamin D3 = 0.60 µg/100 mL; pasteurized milk: vitamin D2 = 1.38, vitamin D3 = 0.30 µg/100 mL) than reported for bovine and human milk. The results of the effect of pasteurization on milk did not highlight significant differences in the total content of vitamin D. However, vitamin D3 has a poor thermal stability, which led to a significant reduction in content in pasteurized milk compared with raw milk. The total vitamin D content of donkey milk did not show significant variations between seasons; however, a higher concentration of vitamin D3 was found in spring and summer. In conclusion, raw and pasteurized donkey milk showed a high content of vitamin D, which could be useful in meeting the deficiencies of this vitamin in humans. Further investigations are needed to improve the vitamin D content in donkey milk by increasing its endogenous synthesis or its transfer in milk and to clarify other variability factors.