The African turquoise killifish Nothobranchius furzeri has emerged as a valuable vertebrate model for ageing research. Despite its increasing use in laboratory studies, systematic welfare monitoring approaches in N. furzeri is still emerging, and only a limited number of structured health-assessment frameworks have been proposed. Here, we present a welfare scoring system designed for daily monitoring of N. furzeri across its entire lifespan under laboratory conditions. Building on established zebrafish welfare frameworks and adapting them to the biological and ethological characteristics of N. furzeri, we identified a set of morphometric, behavioural and clinical parameters, including body condition score, external appearance, food intake, respiratory pattern, swimming activity, tank occupancy and clinical signs. Each parameter was assigned a numerical score ranging from 0 to 3, reflecting increasing degrees of welfare impairment. We further defined standardized intervention criteria, including humane endpoints, to support decision-making in cases of compromised welfare. Importantly, we established five distinct age stages to enable age-specific welfare assessment in two widely used wild-type strains, GRZ/GRZ-AD and MZM04-10. This scoring system provides a practical, reproducible, and easily implementable tool that can be seamlessly integrated into routine husbandry procedures. Its application is expected to promote standardized welfare assessment, improve experimental reproducibility, and enhance animal welfare standards in research using N. furzeri.

The use of olive by-products in livestock farming is a valuable resource, given their high levels of bioactive compounds with antioxidant and health-promoting properties. This preliminary study adopted an integrated approach to evaluate the influence of dietary Olea europaea L. polyphenols on animal welfare, physiological stress response, intestinal morphofunctional traits, and meat quality in Neroametà finishing pigs, a novel Casertana × Large White genetic line (Neroametà). Thirty pigs reared under extensive farming conditions were randomly allocated to two groups (n = 15): a control group fed a standard diet (C) and a treatment group (OL) supplemented with 300 mg/head/day of olive polyphenolic extract for 90 days. The study focused on the systemic correlation between host health and product quality. Meat composition, rheological properties, meat antioxidant activity, stress parameters, and fatty acid profiles of the longissimus lumborum and psoas major muscles were analyzed. Results showed that the OL diet significantly modulated the HPA axis, as evidenced by a marked reduction in plasma ACTH and cortisol levels, alongside improved antioxidant status. These physiological changes were positively associated with a trophic effect on the intestinal mucosa, characterized by increased villus height and a more favorable villus/crypt ratio. Regarding meat quality, the OL group exhibited superior oxidative stability, optimized pH decline, and an improved intramuscular fatty acid profile (increased MUFA and n-3 PUFA, reduced SFA). Despite the pilot scale of 30 animals, these findings provide a solid foundation for characterizing the Neroametà breed. In conclusion, Olea europaea L. polyphenols act as a multi-level modulator, enhancing physiological resilience and meat quality, offering a sustainable strategy for high-quality pork production in line with circular economy and One Health principles.

Fish welfare is increasingly gaining attention in both aquaculture and research, aligning with the "One Welfare" principles that connect animal welfare, human wellbeing, and environment. Here we evaluate whether short-term feed withdrawal, a common practice used in research and aquaculture prior to handling, transportation, and sedation determines a stress response in the adult zebrafish (Danio rerio), through the assessment of key biomarkers across different biological matrices. Adult zebrafish were assigned either to a control group, fed with standard diet twice daily, or to a group subjected to 96 h of complete food deprivation. Cortisol levels were measured in fins, while advanced oxidation protein products (AOPP) and malondialdehyde (MDA) levels were analyzed in muscle tissue. Cellular and oxidative stress markers were assessed through the immunohistochemical localization of Heat Shock Protein-70 (HSP70) and 8-hydroxy-2'-deoxyguanosine (8-OHdG). The levels of cortisol did not have significant differences by treatment, sex, or their interaction. AOPP levels exhibited a significant interaction between treatment and sex, with a 10 % increase in fasted males compared to control females, while MDA levels were unchanged. Consistently, immunohistochemical analyses revealed no significant differences between groups in HSP70 and 8-OHdG positivity. These findings suggest that short-term feed withdrawal does not significantly affect key physiological stress markers in zebrafish. Finally, we validated the use of the caudal fin as a matrix for measuring individual cortisol levels in zebrafish, providing an alternative to terminal whole-body analyses as well as to group-level water-borne cortisol.

In aquaculture and in laboratory settings, the development of sustainable and functional feeds is crucial in order to promote fish welfare, growth, and reproductive performance. Among natural dietary supplements, spirulina (Arthrospira platensis), a blue-green microalga rich in proteins, essential fatty acids, vitamins, and antioxidant molecules, represents a promising bioactive ingredient capable of influencing both physiological and behavioral traits. A 32-week longitudinal study was conducted on adult zebrafish (Danio rerio) to evaluate the effects of spirulina supplementation (5%, 25%, 50%, 75%, and 100%) compared to a standard diet. Parameters related to survival, growth, reproductive fitness, and reproductive behavior were assessed in vivo and supported by a morphometric analysis of the gonads. Supplementation with 5% improved survival rate and the Body Condition Index, while 25% supplementation increased survival, enhanced reproductive behavior and spawning success (140% egg production vs. control), and supported optimal gonadal development and gamete maturation timing. Higher percentages of spirulina (50-100%) seem to cause nutritional imbalance, impairing health and reproductive fitness. This study demonstrates that moderate spirulina supplementation (5-25%) supports health, reproductive physiology, and behavior in zebrafish in a dose-dependent manner. These results highlight the potential of spirulina as a functional supplement for precision nutrition approaches, with implications for fish welfare, reproductive performance, and aquaculture sustainability.

Recent studies have highlighted the potential of plant-based and agro-industrial by-products as valuable sources of bioactive compounds for animal feed formulation. This study aims to evaluate the impact of dietary supplementation with Olea europaea L. leaf extract on the fatty acid composition of pig muscle. Thirty commercial hybrid pigs (Large White × (Landrace × Duroc)), with an initial body weight of 169 ± 7.90 kg and an average age of 10 ± 1 months, were randomly assigned to two experimental groups (n = 15 per group): one fed a standard diet containing Sulla (C) and the other receiving the same diet enriched with olive leaf extract (OL). Over 90 days, the OL group was supplemented with 300 mg/head/day of olive leaf extract, primarily containing oleuropein diglucoside, luteolin-7-glucoside, and verbascoside. The phenolic content, antioxidant activity, bioactive compounds, and fatty acid profiles of both meats and diets were analyzed. Results showed that OL supplementation did not significantly affect the chemical composition of the meat or pig performance, but it did influence the lipid profile. Specifically, OL supplementation led to a significant reduction in saturated fatty acids (SFAs) and an increase in oleic acid, thus enhancing the proportion of monounsaturated fatty acids (MUFAs). Moreover, the n-6/n-3 PUFA ratio in the meat was significantly reduced, suggesting potential improvements in the nutritional and functional quality of pig meat, enhancing the nutritional and functional quality of the meat by improving its fatty acid profile.

Background: The setup of experimental protocols able to preserve the anatomical integrity also in terms of organ microarchitecture is mandatory to ensure result translatability. Also, the maintenance of structural integrity perfectly aligns with the refinement implementation aiming to reduce procedure severity, a key issue in animal studies deemed compulsory from both ethical and legal standpoints. Here we report a detailed description of all peri-operative and post-operative care and clinical evaluation in a surgical rat model to test the efficacy of a catheter functionalized by a peptide coating with antimicrobial and antibiofilm properties, whose efficacy was previously tested in vitro. Methods: We used male and female adult Fischer 344 rats (tot n = 44, n = 22 each sex), which were divided into four experimental groups. Each animal underwent refined surgery for the implantation of a functionalized or standard catheter, depending on the group, and was observed for 7 and 14 days. The surgical refinement strategy was based on the placement of the catheter into the bladder lumen rather than in the urethra. Still in the refinement perspective, ultrasound examination of the bladder was conducted to confirm the in situ position of the medical device at an intermediate time point, 4 or 10 days post-surgery depending on the group, while, at the same time, but also at days 0, 7, or 14 post-surgery, an ultrasound-guided cystocentesis was performed to collect sterile urine. The imaging approach was used in place of metabolic cages to minimize distress to the animals and to ensure reliable and unbiased scientific outcomes. Hematological and biochemical parameters were monitored along the preclinical trial; namely, blood sampling was performed at the beginning (day 0) and at the end of the trial (day 7 or 14 post-surgery depending on the group). Results: Clinical scores and biochemical analyses of all animals did not reveal signs of distress or disease. At the endpoints, histological analyses of urinary bladder displayed a well-preserved anatomical structure of the organ without significant signs of inflammatory infiltration into the urothelium. Conclusions: Our model represents a refined strategy to achieve the scientific goals required by the preclinical setting related to catheter-associated urinary tract infections. In particular, it ensures the preservation of bladder morphology and urothelium microarchitecture, maintaining an adequate level of animal health and welfare while monitoring the onset of urinary tract infections through the sterile collection of urine in long-lasting experiments.

The orexinergic system is anatomically and functionally conserved in almost all vertebrates, and the role in healthy ageing and age-associated diseases has been studied in mammals. Here, we review the main findings on the age-related regulation of orexinergic system in mammals, including human patients and highlights how the fish Nothobranchius furzeri serves as an exceptional model to spearhead research and unravel the intricate mechanisms underlying orexinergic regulation during ageing. The ageing brain of this teleost is characterized by the presence of neurodegenerative processes similar to those associated with human pathologies rather than those of healthy ageing. We present an in-depth summary and discussion on the groundbreaking advances in understanding the neuroanatomical organization of the orexinergic system, its pivotal role in mammalian and fish models, and its profound involvement in healthy ageing and age-associated diseases.

BackgroundShort- or mid-term fasting, full or partial, triggers metabolic response known to have in turn health effects in an organism. At central level, the metabolic stimulus triggered by fasting is known to be perceived firstly by hypothalamic neurons.In the field of neuroscience, ribosomal protein S6 (S6) phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signalling activation or as a marker for neuronal activity. The aim of this study is addressed to evaluate whether the phosphorylation of S6 occurs in the central neurons of zebrafish exposed to four (short-term) and seven (mid-term) days of complete fasting.MethodsGroup-housed adult zebrafish were exposed to four and seven days of complete food withdrawal. At the end of the experimental period, Western blotting analyses were carried out to measure the expression levels of the phosphorylated S6 (pS6) by comparing the two experimental conditions versus the control group. The same antibody was then used to identify the distribution pattern of pS6 immunoreactive neurons in the whole brain and in the taste buds.ResultsWe did not observe increased pS6 levels expression in the brain of animals exposed to short-term fasting compared to the control, whereas the expression increased in brain homogenates of animals exposed to mid-term fasting. pS6 immunoreactivity was reported in some hypothalamic neurons, as well as in the dorsal area of telencephalon and preoptic area, a neurosecretory region homolog to the mammalian paraventricular nucleus. Remarkably, we observed pS6 immunostaining in the sensory cells of taste buds lining the oral epithelium.ConclusionsTaken together, our data show that in zebrafish, differently from other fish species, seven days of fasting triggers neuronal activity. Furthermore, the immunostaining on sensory cells of taste buds suggests that metabolic changes may modulate also peripheral sensory cells. This event may have valuable implications when using zebrafish to design metabolic studies involving fasting as well as practical consequences on the animal welfare, in particularly stressful conditions, such as transportation.

Undisturbed home cage recording of mouse activity and behavior has received increasing attention in recent years. In parallel, several technologies have been developed in a bid to automate data collection and interpretation. Thanks to these expanding technologies, massive datasets can be recorded and saved in the long term, providing a wealth of information concerning animal wellbeing, clinical status, baseline activity, and subsequent deviations in case of experimental interventions. Such large datasets can also serve as a long-term reservoir of scientific data that can be reanalyzed and repurposed upon need. In this review, we present how the impact of Big Data deriving from home cage monitoring (HCM) data acquisition, particularly through Digital Ventilated Cages (DVCs), can support the application of the 3Rs by enhancing Refinement, Reduction, and even Replacement of research in animals.

Vincenzo Mazza (1793-1859) is a neglected figure in the history of Italian veterinary medicine. His collection represents the first nucleus of the future Anatomical Museum of the Neapolitan Veterinary School. After the Napoleonic wars, in which he took part as a military veterinarian, he attended the School of Milano, where he learnt to prepare the anatomical specimens from Jean Louis Leroy and Giambattista Volpi. In 1816 he graduated in Medicine and Philosophy in Pisa, where he learnt Andrea Vacc & agrave; Berlinghieri's innovative surgical techniques and established a private veterinary school. In 1821, he was appointed Professor in Theoretical and Practical Clinic at the Veterinary School in Napoli, where he also directed the adjoining hospital. Here, he privately and independently produced his anatomical preparations for educational purposes and tried in vain to create a museum within the School modelled on the one in Milano and other major European schools. Unfortunately, after 1848, he fell victim to Borbonic political repression and was forced to leave teaching. Most of his specimens are still preserved in excellent condition in the "Museo di Anatomia Veterinaria" of the Federico II Napoli University.

Smell and taste are extensively studied in fish species as essential for finding food and selecting mates while avoiding toxic substances and predators. Depending on the evolutionary position and adaptation, a discrete variation in the morphology of these sense organs has been reported in numerous teleost species. Here, for the first time, we approach the phenotypic characterization of the olfactory epithelium and taste buds in the African turquoise killifish (Nothobranchius furzeri), a model organism known for its short lifespan and use in ageing research. Our observations indicate that the olfactory epithelium of N. furzeri is organized as a simple patch, lacking the complex folding into a rosette, with an average size of approximately 600 µm in length, 300 µm in width, and 70 µm in thickness. Three main cytotypes, including olfactory receptor neurons (CalbindinD28K), supporting cells (β-tubulin IV), and basal cells (Ki67), were identified across the epithelium. Further, we determined the taste buds’ distribution and quantification between anterior (skin, lips, oral cavity) and posterior (gills, pharynx, oesophagus) systems. We identified the key cytotypes by using immunohistochemical markers, i.e. CalbindinD28K, doublecortin, and neuropeptide Y (NPY) for gustatory receptor cells, glial fibrillary acidic protein (GFAP) for supporting cells, and Ki67, a marker of cellular proliferation for basal cells. Altogether, these results indicate that N. furzeri is a microsmatic species with unique taste and olfactory features and possesses a well-developed posterior taste system compared to the anterior. This study provides fundamental insights into the chemosensory biology of N. furzeri, facilitating future investigations into nutrient-sensing mechanisms and their roles in development, survival, and ageing.

This study evaluated the effect of pig diet supplementation with an Olea europaea L. extract on the muscle fatty acid composition. Ten commercial hybrid pigs (Large White x (Landrace x Duroc)) were randomly assigned to two experimental treatments and fed either a standard diet (C) or diet with olive leaves extract (OL). For 90 days, the OL group received the olive leaf extract (300 mg/head/day), which mainly comprises oleuropein diglucoside, luteolin-7-glucoside, and verbascoside. The phenolic content, antioxidant activity, bioactive molecules’ characterization, and the fatty acids profile of meat and diet were performed. The results indicated that while the OL supplementation did not significantly alter the overall chemical composition of the meat or the pigs' performance, it did modify the lipid profile of the meat. The OL diet results in a significant reduction in SFA and an increase in oleic acid in meat, which correspondingly elevates the total MUFA content. Dietary treatment reduced the sum of n-6 PUFA in the meat of the OL group, and resulted in a significantly lower n-6/n-3 PUFA ratio. Therefore, these findings suggest that incorporating olive leaf extract into the pig diet enhances the nutritional and functional quality of the meat by improving its fatty acid profile.

The mechanism underlying the transition from the pre-symptomatic to the symptomatic state is a crucial aspect of epileptogenesis. SYN2 is a member of a multigene family of synaptic vesicle phosphoproteins playing a fundamental role in controlling neurotransmitter release. Human SYN2 gene mutations are associated with epilepsy and autism spectrum disorder. Mice knocked out for synapsin II (SynII KO) are prone to epileptic seizures that appear after 2 months of age. However, the involvement of the endocannabinoid system, known to regulate seizure development and propagation, in the modulation of the excitatory/inhibitory balance in the epileptic hippocampal network of SynII KO mice has not been explored. In this study, we investigated the impact of endocannabinoids on glutamatergic and GABAergic synapses at hippocampal dentate gyrus granule cells in young pre-symptomatic (1–2 months old) and adult symptomatic (5–8 months old) SynII KO mice. We observed an increase in endocannabinoid-mediated depolarization-induced suppression of excitation in young SynII KO mice, compared to age-matched wild-type controls. In contrast, the endocannabinoid-mediated depolarization-induced suppression of inhibition remained unchanged in SynII KO mice at both ages. This selective alteration of excitatory synaptic transmission was accompanied by changes in hippocampal endocannabinoid levels and cannabinoid receptor type 1 distribution among glutamatergic and GABAergic synaptic terminals contacting the granule cells of the dentate gyrus. Finally, inhibition of type-1 cannabinoid receptors in young pre-symptomatic SynII KO mice induced seizures during a tail suspension test. Our results suggest that endocannabinoids contribute to maintaining network stability in a genetic mouse model of human epilepsy.

In this study, we have investigated the immunolocalization of NGF (Nerve Growth Factor) and BDNF (Brain-Derived Neurotrophic Factor) in the pancreas of two species of marine mammals: Tursiops truncatus (common bottlenose dolphin), belonging to the order of the Artiodactyla, and Otaria flavescens (South American sea lion), belonging to the order of the Carnivora. Our results demonstrated a significant presence of NGF and BDNF in the pancreas of both species with a wide distribution pattern observed in the exocrine and endocrine components. We identified some differences that can be attributed to the different feeding habits of the two species, which possess a different morphological organization of the digestive system. Altogether, these preliminary observations open new perspectives on the function of neurotrophins and the adaptive mechanisms of marine mammals in the aquatic environment, suggesting potential parallels between the physiology of marine and terrestrial mammals.

Tissue-engineered skin substitutes are promising tools to cover large and deep skin defects. However, the lack of a synergic and fast regeneration of the vascular network, nerves, and skin appendages limits complete skin healing and impairs functional recovery. It has been highlighted that an ideal skin substitute should mimic the structure of the native tissue to enhance clinical effectiveness. Here, we produced a pre-vascularized dermis (PVD) comprised of fibroblasts embedded in their own extracellular matrix (ECM) and a capillary-like network. Upon implantation in a mouse full-thickness skin defect model, we observed a very early innervation of the graft in 2 weeks. In addition, mouse capillaries and complete epithelialization were detectable as early as 1 week after implantation and, skin appendages developed in 2 weeks. These anatomical features underlie the interaction with the skin nerves, thus providing a further cue for reinnervation guidance. Further, the graft displays mechanical properties, collagen density, and assembly features very similar to the host tissue. Taken together our data show that the pre-existing ECM components of the PVD, physiologically organized and assembled similarly to the native tissue, support a rapid regeneration of dermal tissue. Therefore, our results suggest a promising potential for PVD in skin regeneration.

Morphological mouse phenotyping plays a pivotal role in the translational setting and even more in the area of auditory research, where mouse is a central model organism due to the evolutionary genetic relationship and morpho-functional analogies with the human auditory system. However, some results obtained in murine models cannot be translated to humans due to the inadequate description of experimental conditions underlying poor reproducibility. We approach the characterization of the aging process of the mouse cochlea in animals up to 18 months of age belonging to two of the most used outbred (CD1) and inbred (C57BL/6N) strains. Striving to reduce any environmental variable we performed our study compliantly to the ARRIVE guidelines. We integrated instrumental data (auditory brainstem response test), with morphological analyses to correlate functional discrepancies to morphological changes and track the differences in the evolution of sensorineural hearing loss in the two strains. We featured the localization of Gipc3, Myosin VIIa, and TMC1 in hair cells of the Corti organ as well as NF 200 and the density of type I neuron in the spiral ganglion. We outlined age-related hearing loss (ARHL) in both strains, and a clear drop in the selected marker localization. However, in CD1 we detected a different trend allowing the identification of potential strain-specific mechanisms, namely an increase in myosin VIIa in 6 months aging mice in comparison to 2 months old animals. Our findings represent an asset to investigate the strain-dependent physiological trigger of ARHL providing new insights in the translational area.

Here, we evaluated the effect of dietary supplementation with an Olea europaea L. extract on the animal welfare and milk quality of dairy cows. Thirty Italian Holstein–Friesian dairy cows in the mid-lactation phase (90 to 210 days) were blocked into experimental groups based on parity class (namely, primiparous (P) (n = 10), secondiparous (S) (n = 10) and pluriparous (PL) (n = 10)) and received, for 60 days, Phenofeed Dry® at 500 mg/cow/day. Milk and blood samples were collected before the start of the treatment (T0), subsequently every 15 days (T1–T4) and at 45 days after the end of treatment (T5). In the serum, glucose and triglycerides, stress, the thyroid, lactation and sex hormones were measured; in the milk, lysozyme content as well as the fatty acid profile were assessed. In the whole animal, the enriched feed helped to maintain hormonal parameters in the physiological range while producing hypoglycemic (T4 vs. T0, for P and PL p < 0.001) and hypolipidemic effects (T4 vs. T0, for P p < 0.001 and for PL p < 0.01). At the milk level, it resulted in a reduction in total fat (T5 vs. T0, for P, S and PL p < 0.001) and in the saturated fatty acids (SFAs)/monounsaturated fatty acids (MUFAs) ratio paralleled by an increase in polyunsaturated fatty acids (PUFAs) (T5 vs. T0, for P, S and PL p < 0.001), protein content (lysozyme (T4 vs. T0, for P and PL p < 0.001)) and lactose (T5 vs. T0, for P, S and PL p < 0.001). Thus, the inclusion of natural bioactive molecules such as O. europaea L. polyphenols in the dairy cow diet may help to improve animal welfare and milk quality.

This study aimed to examine the impact of different surface properties of poly(lactic-co-glycolic) acid (PLGA) nanoparticles (P NPs) and PLGA-Poloxamer nanoparticles (PP NPs) on their in vivo biodistribution. For this purpose, NPs were formulated via nanoprecipitation and loaded with diphenylhexatriene (DPH), a fluorescent dye. The obtained NPs underwent comprehensive characterization, encompassing their morphology, technological attributes, DPH release rate, and thermodynamic properties. The produced NPs were then administered to wild-type mice via intraperitoneal injection, and, at scheduled time intervals, the animals were euthanized. Blood samples, as well as the liver, lungs, and kidneys, were extracted for histological examination and biodistribution analysis. The findings of this investigation revealed that the presence of poloxamers led to smaller NP sizes and induced partial crystallinity in the NPs. The biodistribution and histological results from in vivo experiments evidenced that both, P and PP NPs, exhibited comparable concentrations in the bloodstream, while P NPs could not be detected in the other organs examined. Conversely, PP NPs were primarily sequestered by the lungs and, to a lesser extent, by the kidneys. Future research endeavors will focus on investigating the behavior of drug-loaded NPs in pathological animal models.

Tissue engineered skin substitutes are promising tools to cover large and deep skin defects. However, the lack of a synergic and fast regeneration of the vascular network, nerves and skin appendages limits the complete skin healing and impairs functional recovery. It has been highlighted that to enhance clinical effectiveness, the structure of an ideal skin substitute should mimic that of normal skin. Here, we used a well-established bioengineering approach to produce a pre vascularized dermis (PVD) comprised of fibroblasts embedded in their own extra cellular matrix (ECM) in addition to a functional capillary-like network. Upon implantation in a mouse full-thickness skin defect model, surprisingly we observed a very early innervation of the graft just 2 weeks after implantation. Nerve recruitment from the host tissue was sustained by mouse capillary networks detectable as early as 1 week after PVD implantation. In addition, we found complete epithelialization and skin appendage development. These anatomical features underlie the interaction with the skin nerves, thus providing a further cue for reinnervation guidance. Further, from the structural side the graft displays mechanical properties, collagen density and assembly features very similar to those of the host tissue. Taken together our results show that the pre-existing ECM components of the PVD, physiologically organized and assembled similarly to the native tissue, support a rapid regeneration of dermal tissue, suggesting a promising potential for PVD in skin regeneration.