Attitude toward non-human animals and their welfare: do behaviorists differ from other veterinarians?

Veterinarians are an important source of information about animal care for owners. They provide general advice about topics important to an animal’s well-being, such as appropriate training, exercise and nutrition. Veterinary behaviorists, when dealing with undesired or abnormal behaviors, also perform an assessment of pet welfare and an evaluation of owners’ behavior and attitudes. It is likely that these assessments are affected by the attitude toward animals and toward animal welfare of the behaviorists. The aim of this study was to investigate whether veterinary behaviorists have a different attitude toward animals and animal welfare compared to other veterinarians. An online questionnaire, also including the 20-item Animal Attitude Scale, was completed by a convenience sample of 540 Italian veterinarians dealing with companion animals: 140 were behaviorists, 22 were starting to work in the field of behavioral medicine, and 378 were not involved in the field. Veterinary behaviorists showed a more positive attitude toward non-human animals and their welfare, which seems to be more related to the interest in behavioral medicine than to its practice. Moreover, behaviorists attached more importance to the psychological aspects of pet welfare that they perceived as less protected in their feline and canine patients. These findings suggest that being involved in one discipline or another of veterinary medicine do matter in the attitude toward non-human animals and their welfare

Plasmatic tryptophan/large neutral amino acids ratio in domestic dogs is affected by meal composition

Tryptophan (Trp) is involved in the synthesis of serotonin and melatonin and it competes with other large neutral amino acids (LNAAs) for its uptake into the brain [1]. The aim of this study was to assess the impact of three different diets on plasmatic Trp/LNAAs ratio. This study included five female Labrador Retrievers (2 spayed, 8.6 ± 3.8 years old) from the same bloodline, who were usually fed the same commercial dry food once a day. Each dog received three different diets for one single day each. Isocaloric and isonitrogenous diets, with a carbohydrates content of 47% and proteins content of 28% on dry matter basis, were provided in two meals, one in the morning and one after 12 hours. Dogs received the first diet (D1) and then they returned to their normal diet for 30 days. After that “washout” period, dogs were fed with the second diet (D2), and after 30 more days they received the third diet (D3). D1 was composed of a mix of puffed rice, minced meat and olive oil equally divided into the two meals. D2 was made up of two different meals. The morning meal was composed of puffed rice and olive oil, whereas the evening meal consisted of minced meat and olive oil. D3 consisted of two identical meals of the commercial dry food usually consumed by the sample dogs. Blood was collected right before the first meal (t0) and after 2, 4, 6, 8, 10 and 24 hours. Plasma samples were used for HPLC quantification of Trp and other LNAAs (isoleucine + leucine + phenylalanine + tyrosine + valine) using a method described in literature [2]. Their levels and ratios at t0 and after D1, D2 and D3 were compared using a mixed model for repeated measures (p<0.05). Trp concentrations showed no significant difference between D1, D2 and D3 samples at any sampling times. LNAAs levels were similar at t0 in the three experimental days, but they showed different trends depending on the composition of the meal provided. In particular, D2 led to a decrease in LNAAs levels and therefore to higher Trp/LNAAs ratios in the 6 hours period after the provision of carbohydrates. In detail, mean Trp/LNAAs ratio of D2 was statistically higher compared to both D1 and D3 at t2 (D1=0.224; D2=0.306; D3=0.217; p<0.001), t4 (D1=0.225; D2=0.327; D3=0.197; p<0.001), and t6 (D1=0.244; D2=0.303; D3=0.205; p<0.015). In addition, mean Trp/LNAAs ratio after D2 was higher than after D3 also at t8 (D2=0.280; D3=0.206; p<0.001) and t10 (D2=0.294; D3=0.224; p<0.001). The trend was different at t24, when Trp/LNAAs ratio was found to be significantly lower after being fed D2 compared to D1 (D1=0.210; D2=0.155; p=0.041). These results indicate that the diet affects Trp bioavailability. Therefore, it is worthwhile to investigate the effects of diet on Trp bioavailability at the brain level, serotonin and melatonin secretion and the real impact of Trp/LNAAs ratio on dog behaviour

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

Feeding Enrichment in a Captive Pack of European Wolves (Canis Lupus Lupus): Assessing the Effects on Welfare and on a Zoo's Recreational, Educational and Conservational Role

This study investigated the effects of two feeding enrichment programs on the behaviour of a captive pack of European wolves (Canis lupus lupus) and their correlation with both zoo visitors' interest towards the exhibit and their overall perception of the species. Behavioural data (exploration, stereotypies, social interactions, activity/inactivity rates) were collected on four male wolves during four two-week long phases: initial control, hidden food, novel object, final control. Three observation sessions were performed daily: before, during and after feeding. Number of visitors and their permanence in front of the exhibit were recorded. After watching the wolves, visitors were asked to fill out a brief questionnaire in order to investigate their perception of captive wolf welfare, as well as their attitude towards wolf conservation issues. Despite the high inter-individual variability in their behavioural response, all wolves seemed to benefit from feeding enrichment. With regard to visitors, interest in the exhibit increased when enrichment was provided. Visitors' perception of the level of welfare of wolves improved if they attended a feeding session, especially during the novel object phase. Visitors' attitude towards wolf conservation issues also improved during feeding sessions, regardless of enrichment provision

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

The correlation between play behavior, serum cortisol and neutrophil-to-lymphocyte ratio in welfare assessment of dairy calves within the first month of life

Welfare assessment of dairy calves is commonly conducted through the observation of their behavior and the analysis of physiological parameters. Despite the large number of studies on this topic, there is a lack of research on the possible correlation between the physiological parameters and behavior at basal level in dairy calves. For this reason, the present study aimed to investigate the possible correlations between serum cortisol, neutrophil-to-lymphocyte ratio (N/L) and locomotor play behavior in 21 Friesian dairy calves within their first month of life. Every week, an arena test after blood collection was performed. A significant positive correlation between serum cortisol levels and N/L (rrm = 0.381; 95% CI = 0.083, 0.617; p = 0.012) and a negative correlation between time spent running and N/L (rrm = −0.351; 95% CI = −0.575, −0.077; p = 0.012) were found. On the contrary, no significant correlation between time spent running and cortisol levels was detected. Cortisol levels and N/L were higher in the newborn and had a constant decrease until reaching a stable value after the 24th day of life. These findings suggest a better predictivity of N/L, compared to serum cortisol, on locomotor play behavior

Converging Role for REEP1/SPG31 in Oxidative Stress

Mutations in the receptor expression-enhancing protein 1 gene (REEP1) are associated with hereditary spastic paraplegia type 31 (SPG31), a neurological disorder characterized by lengthdependent degeneration of upper motor neuron axons. Mitochondrial dysfunctions have been observed in patients harboring pathogenic variants in REEP1, suggesting a key role of bioenergetics in disease-related manifestations. Nevertheless, the regulation of mitochondrial function in SPG31 remains unclear. To elucidate the pathophysiology underlying REEP1 deficiency, we analyzed in vitro the impact of two different mutations on mitochondrial metabolism. Together with mitochondrial morphology abnormalities, loss-of-REEP1 expression highlighted a reduced ATP production with increased susceptibility to oxidative stress. Furthermore, to translate these findings from in vitro to preclinical models, we knocked down REEP1 in zebrafish. Zebrafish larvae showed a significant defect in motor axon outgrowth leading to motor impairment, mitochondrial dysfunction, and reactive oxygen species accumulation. Protective antioxidant agents such as resveratrol rescued free radical overproduction and ameliorated the SPG31 phenotype both in vitro and in vivo. Together, our findings offer new opportunities to counteract neurodegeneration in SPG31

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

Lysosomal Proteomics Links Disturbances in Lipid Homeostasis and Sphingolipid Metabolism to CLN5 Disease

CLN5 disease (MIM: 256731) represents a rare late-infantile form of neuronal ceroid lipofuscinosis (NCL), caused by mutations in the CLN5 gene that encodes the CLN5 protein (CLN5p), whose physiological roles stay unanswered. No cure is currently available for CLN5 patients and the opportunities for therapies are lagging. The role of lysosomes in the neuro-pathophysiology of CLN5 disease represents an important topic since lysosomal proteins are directly involved in the primary mechanisms of neuronal injury occurring in various NCL forms. We developed and implemented a lysosome-focused, label-free quantitative proteomics approach, followed by functional validations in both CLN5-knockout neuronal-like cell lines and Cln5−/− mice, to unravel affected pathways and modifying factors involved in this disease scenario. Our results revealed a key role of CLN5p in lipid homeostasis and sphingolipid metabolism and highlighted mutual NCL biomarkers scored with high lysosomal confidence. A newly generated cln5 knockdown zebrafish model recapitulated most of the pathological features seen in NCL disease. To translate the findings from in-vitro and preclinical models to patients, we evaluated whether two FDA-approved drugs promoting autophagy via TFEB activation or inhibition of the glucosylceramide synthase could modulate in-vitro ROS and lipid overproduction, as well as alter the locomotor phenotype in zebrafish. In summary, our data advance the general understanding of disease mechanisms and modifying factors in CLN5 disease, which are recurring in other NCL forms, also stimulating new pharmacological treatments.Peer reviewe

Lysosomal Proteomics Links Disturbances in Lipid Homeostasis and Sphingolipid Metabolism to CLN5 Disease

CLN5 disease (MIM: 256731) represents a rare late-infantile form of neuronal ceroid lipofuscinosis (NCL), caused by mutations in the CLN5 gene that encodes the CLN5 protein (CLN5p), whose physiological roles stay unanswered. No cure is currently available for CLN5 patients and the opportunities for therapies are lagging. The role of lysosomes in the neuro-pathophysiology of CLN5 disease represents an important topic since lysosomal proteins are directly involved in the primary mechanisms of neuronal injury occurring in various NCL forms. We developed and implemented a lysosome-focused, label-free quantitative proteomics approach, followed by functional validations in both CLN5-knockout neuronal-like cell lines and Cln5−/− mice, to unravel affected pathways and modifying factors involved in this disease scenario. Our results revealed a key role of CLN5p in lipid homeostasis and sphingolipid metabolism and highlighted mutual NCL biomarkers scored with high lysosomal confidence. A newly generated cln5 knockdown zebrafish model recapitulated most of the pathological features seen in NCL disease. To translate the findings from in-vitro and preclinical models to patients, we evaluated whether two FDA-approved drugs promoting autophagy via TFEB activation or inhibition of the glucosylceramide synthase could modulate in-vitro ROS and lipid overproduction, as well as alter the locomotor phenotype in zebrafish. In summary, our data advance the general understanding of disease mechanisms and modifying factors in CLN5 disease, which are recurring in other NCL forms, also stimulating new pharmacological treatments