Influence of Transportation on Stress Response and Cellular Oxidative Stress Markers in Juvenile Meagre (Argyrosomus regius).

In aquaculture, the transportation of live fish is a crucial but stress-inducing practice, necessitating a thorough understanding of its impact on fish welfare. This study aimed to assess the physiological stress response of meagre (Argyrosomus regius) juveniles during a 24 h commercial transport by quantifying muscle cortisol levels using a specific radioimmunoassay. Additionally, an immunohistochemical approach was used to detect and localize the cellular distribution of oxidative-stress-related biomarkers within various tissues and organs. The results demonstrated a significant increase in muscle cortisol levels following the loading procedure, remaining elevated above basal levels throughout the 24 h transport period. This effect may be attributed to either insufficient time for recovery from the loading stress or prolonged transportation-related stress. Immunostaining for all the antibodies we examined was observed in multiple tissues and organs, but we found no notable variations among the various transport phases. In conclusion, the observed stress response appears to be mainly linked to loading stress and the transport process itself, emphasizing the importance of implementing appropriate operational procedures to safeguard fish well-being during transport. Nonetheless, the unaltered distribution of oxidative stress markers between the control and transported groups suggests that the experienced stress might be within tolerable limits.

Productive Results, Oxidative Stress and Contaminant Markers in European Sea Bass: Conventional vs. Organic Feeding.

In the present study European sea bass (Dicentrarchus labrax) subjected to two different diets (organic vs. conventional) were evaluated in terms of growing performances, oxidative stress, and contaminant markers. Growing performances were evaluated using biometric measures and condition factor (K), whereas insulin-like growth factor (IGF-I and IGF-II) levels were assessed trough Real-Time PCR analysis. For oxidative stress, immunohistochemical staining for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 4-hydroxy-2-nonenal (HNE) was performed, whereas total glutathione (GSH) in blood serum was determined by an enzymatic method adapted. Cytochrome P4501A (CYP1A) and melanomacrophage centers (MMCs) were evaluated as contaminant markers trough immunohistochemical and histochemical approaches, respectively. The growing performances showed a positive trend in both groups but a greater productivity in conventional fed fish compared to the organic ones. A significant higher expression of MMCs was observed in organic vs. conventional diet fed fish. Fillet analysis showed a higher MUFA content and a lower PUFAs n-6 content in organically fed sea bass indicating that diets with a content in fatty acids closer to that of wild fish will definitely affect the fatty acid profile of the fish flesh. On the other hand, the diet composition did not seem to affect neither the oxidative stress parameters (GSH, 8-OHdG, HNE) nor the CYP1A expression.

Safety assessment of antibiotic administration by magnetic nanoparticles in in vitro zebrafish liver and intestine cultures.

Different in vitro models have been suggested to replace in vivo studies. In vitro studies are of great interest and give the opportunity to analyze cellular responses in a closed system with stable experimental conditions and to avoid direct animal exposure and distress during the experiments. These methods are useful to test drugs and chemicals toxicity in order to better understand their environmental impact. In the present study, fish organ cultures have been used to test different oxytetracycline exposure methods, including oxide nanoparticles (IONPs), using zebrafish as experimental model. Results showed that oxytetracycline accumulation at the end of the experiment (24 h) in the exposed organs did not show any significant difference in the analyzed samples and was not dependent on the exposure way (free or IONPs-bound oxytetracycline). However, as regards molecular analysis, the different exposure ways tested in this study showed some differences in the expression of genes involved in stress response. The present data did not completely agree with a previous in vivo study performed in zebrafish using IONPs, underlying that replacement of in vivo models with in vitro studies cannot always represent the complexity of interactions typical of a biological system.

Self-assembly of chlorin-e6 on γ-Fe2O3 nanoparticles: Application for larvicidal activity against Aedes aegypti.

Aedes aegypti mosquitos are widespread vectors of several diseases and their control is of primary importance for biological and environmental reasons, and novel safe insecticides are highly desirable. An eco-friendly photosensitizing magnetic nanocarrier with larvicidal effects on Aedes aegypti was proposed. The innovative core-shell hybrid nanomaterial was synthesized by combining peculiar magnetic nanoparticles (called Surface Active Maghemite Nanoparticles - SAMNs, the core) and chlorin-e6 as photosensitizer (constituting the shell) via self-assembly in water. The hybrid nanomaterial (SAMN@chlorin) was extensively characterized and tested for the photocidal activity on larvae of Aedes aegypti. The SAMN@chlorin core-shell nanohybrid did not present any toxic effect in the dark, but, upon light exposure, showed a higher photocidal activity than free chlorin-e6. Moreover, the eco-toxicity of SAMN@chlorin was determined in adults and neonates of Daphnia magna, where delayed toxicity was observed only after prolonged (≥4 h) exposure to intense light, on the green alga Pseudokirchneriella subcapitata and on the duckweed Lemna minor on which no adverse effects were observed. The high colloidal stability, the physico-chemical robustness and the magnetic drivability of the core-shell SAMN@chlorin nanohybrid, accompanied by the high photocidal activity on Aedes aegypti larvae and reduced environmental concerns, can be proposed as a safe alternative to conventional insecticides.

Mapping of the Early Intrauterine Morphogenesis in the Alpaca (Vicugna pacos): External Features and Development of the Cephalic Vesicle in Comparison with the Progressive Carnegie Scale.

In this study, we characterized the morphological aspects of the early development of the head of the alpaca (Vicugna pacos) and identified the main structures of the central nervous system during the first trimester of pregnancy. The topography and the cytoarchitecture of the fetal brain regions were described by histological analysis of the brain sections. We performed this analysis on alpaca embryos and fetuses presumably aged 20, 30, 45, and 90 days. For the description of the external body structures we considered the shape of the head, the development of the optic primordium, the dorsal curvature of the body, the limb buds, the umbilical cord and relative vessels, and the thickness and transparency of the skin. The prosencephalic, mesencephalic, and the rhomboencephalic vesicles were described by analyzing sagittal sections of the head. The present article provides the first progressive morphological and anatomical description of alpaca brain during early development. A detailed study represents an important basis to further understand the phases of prenatal development in this species, since information about alpaca embryology in incomplete and reproductive failure is a relevant factor. These data are important also for interspecies comparisons and application of reproductive biotechnologies. Anat Rec, 302:1226-1237, 2019. © 2018 Wiley Periodicals, Inc.

Vascularization of the trachea in the bottlenose dolphin: comparison with bovine and evidence for evolutionary adaptations to diving.

The rigid structure of the mammalian trachea is functional to maintain constant patency and airflow during breathing, but no gas exchange takes place through its walls. The structure of the organ in dolphins shows increased rigidity of the tracheal cartilaginous rings and the presence of vascular lacunae in the submucosa. However, no actual comparison was ever made between the size and capacity of the vascular lacunae of the dolphin trachea and the potentially homologous structures of terrestrial mammals. In the present study, the extension of the lacunae has been compared between the bottlenose dolphin and the bovine, a closely related terrestrial Cetartiodactyla. Our results indicate that the extension of the blood spaces in the submucosa of dolphins is over 12 times larger than in the corresponding structure of the bovines. Furthermore, a microscopic analysis revealed the presence of valve-like structures in the walls of the cetacean lacunae. The huge difference in size suggests that the lacunae are not merely a product of individual physiological plasticity, but may constitute a true adaptive evolutionary character, functional to life in the aquatic environment. The presence of valve-like structures may be related to the regulation of blood flow, and curtail excessive compression under baric stress at depth.

Aging and Veterinary Care of Cats, Dogs, and Horses through the Records of Three University Veterinary Hospitals.

The present article examines over 63,000 medical records belonging to the Veterinary Hospitals of the Universities of Bologna, Torino, and Padova, all in Northern Italy, and relative to dogs (approximately 50,000), cats (approximately 12,000), and companion horses (slightly less than 1,000). The animals of the three species were divided into age classes and categorized per sex into males, females, and neutered individuals. The mean age at visit and the effects of age classes and category (analyzed via ANOVA) are presented and discussed. The data indicate that many animals are presented to the hospitals either in the early phases of their life (presumably for vaccination and, in cats and dogs, gonadectomy) or in the advanced age (over 10 years in dogs, over 15 years in cats, and over 17 years in horses). The records of very old individuals of the three species are also reported. On the whole, the results suggest that a growing population of mature to old domestic carnivores or companion horses reaches ages that were considered exceptional only a few years ago. The data also testify an evolution in the animal-owner relationship and a renewed respect for the value of life in companion domestic mammals.

The Brain of the Giraffe (Giraffa Camelopardalis): Surface Configuration, Encephalization Quotient, and Analysis of the Existing Literature.

The anatomy of the giraffe (Giraffa camelopardalis Linnaeus, 1758) has been poorly studied, except for the circulatory system. In particular, only a handful of studies have concerned the brain of this species since the first description in 1839. Accordingly, only a very few articles discussing encephalization mentioned the giraffe or used it in their calculations. In this article, we performed a thorough examination of the literature including old and grey, regarding the central nervous system of the giraffe. Furthermore, we examined the brain of 3 giraffes, and calculated the encephalization quotient (EQ) of the species, based on our own data and the values found in the literature. We also revised the pre-existing literature and re-mapped the main sulci based on current comparative interpretation and anatomical nomenclature. Our results were compared to those of other selected significant mammals. The mean brain weight was of 719.9 ± 12.5 g. Our data indicate that the EQ of the giraffe is 0.64 and matches that of the typical ungulate, despite having the largest brain among terrestrial Cetartiodactyla. This emphasizes that the giraffe is a highly specialized mammal, within the limitations of its clad. Anat Rec, 300:1502-1511, 2017. © 2017 Wiley Periodicals, Inc.

The Brain of the Domestic Bos taurus: Weight, Encephalization and Cerebellar Quotients, and Comparison with Other Domestic and Wild Cetartiodactyla.

The domestic bovine Bos taurus is raised worldwide for meat and milk production, or even for field work. However the functional anatomy of its central nervous system has received limited attention and most of the reported data in textbooks and reviews are derived from single specimens or relatively old literature. Here we report information on the brain of Bos taurus obtained by sampling 158 individuals, 150 of which at local abattoirs and 8 in the dissecting room, these latter subsequently formalin-fixed. Using body weight and fresh brain weight we calculated the Encephalization Quotient (EQ), and Cerebellar Quotient (CQ). Formalin-fixed brains sampled in the necropsy room were used to calculate the absolute and relative weight of the major components of the brain. The data that we obtained indicate that the domestic bovine Bos taurus possesses a large, convoluted brain, with a slightly lower weight than expected for an animal of its mass. Comparisons with other terrestrial and marine members of the order Cetartiodactyla suggested close similarity with other species with the same feeding adaptations, and with representative baleen whales. On the other hand differences with fish-hunting toothed whales suggest separate evolutionary pathways in brain evolution. Comparison with the other large domestic herbivore Equus caballus (belonging to the order Perissodactyla) indicates that Bos taurus underwent heavier selection of bodily traits, which is also possibly reflected in a comparatively lower EQ than in the horse. The data analyzed suggest that the brain of domestic bovine is potentially interesting for comparative neuroscience studies and may represents an alternative model to investigate neurodegeneration processes.

The claustrum of the bottlenose dolphin Tursiops truncatus (Montagu 1821).

The mammalian claustrum is involved in processing sensory information from the environment. The claustrum is reciprocally connected to the visual cortex and these projections, at least in carnivores, display a clear retinotopic distribution. The visual cortex of dolphins occupies a position strikingly different from that of land mammals. Whether the reshaping of the functional areas of the cortex of cetaceans involves also modifications of the claustral projections remains hitherto unanswered. The present topographic and immunohistochemical study is based on the brains of eight bottlenose dolphins and a wide array of antisera against: calcium-binding proteins (CBPs) parvalbumin (PV), calretinin (CR), and calbindin (CB); somatostatin (SOM); neuropeptide Y (NPY); and the potential claustral marker Gng2. Our observations confirmed the general topography of the mammalian claustrum also in the bottlenose dolphin, although (a) the reduction of the piriform lobe modifies the ventral relationships of the claustrum with the cortex, and (b) the rotation of the telencephalon along the transverse axis, accompanied by the reduction of the antero-posterior length of the brain, apparently moves the claustrum more rostrally. We observed a strong presence of CR-immunoreactive (-ir) neurons and fibers, a diffuse but weak expression of CB-ir elements and virtually no PV immunostaining. This latter finding agrees with studies that report that PV-ir elements are rare in the visual cortex of the same species. NPY- and somatostatin-containing neurons were evident, while the potential claustral markers Gng2 was not identified in the sections, but no explanation for its absence is currently available. Although no data are available on the projections to and from the claustrum in cetaceans, our results suggest that its neurochemical organization is compatible with the presence of noteworthy cortical inputs and outputs and a persistent role in the general processing of the relative information.

The brain of the horse: weight and cephalization quotients.

The horse is a common domestic animal whose anatomy has been studied since the XVI century. However, a modern neuroanatomy of this species does not exist and most of the data utilized in textbooks and reviews derive from single specimens or relatively old literature. Here, we report information on the brain of Equus caballus obtained by sampling 131 horses, including brain weight (as a whole and subdivided into its constituents), encephalization quotient (EQ), and cerebellar quotient (CQ), and comparisons with what is known about other relevant species. The mean weight of the fresh brains in our experimental series was 598.63 g (SEM ± 7.65), with a mean body weight of 514.12 kg (SEM ± 15.42). The EQ was 0.78 and the CQ was 0.841. The data we obtained indicate that the horse possesses a large, convoluted brain, with a weight similar to that of other hoofed species of like mass. However, the shape of the brain, the noteworthy folding of the neocortex, and the peculiar longitudinal distribution of the gyri suggest an evolutionary specificity at least partially separate from that of the Cetartiodactyla (even-toed mammals and cetaceans) with whom Perissodactyla (odd-toed mammals) are often grouped.

Immunofluorescence evidence of melanotrophs in the pituitary of four odontocete species. An immunohistochemical study and a critical review of the literature.

Cetaceans share peculiar features of their pituitary glands, with a complete separation of pars distalis and pars nervosa by a dural septum and the absence of an intermediate lobe and cleft. In most mammals the pars intermedia is the main source of circulating α-melanocyte stimulating hormone (α-MSH), derived from a large precursor called proopiomelanocortin (POMC), which also generates adrenocorticotropic hormone (ACTH) in the adenohypophysis. The lack of an intermediate lobe in cetaceans led us to investigate whether their glands are able to produce α-MSH, and if this hormone is secreted by a distinct population of melanotrophs or by corticotrophs in the pars distalis. Immunofluorescence evidences seem to support the first assumption, with ACTH-immunoreactive (-ir) elements rarely overlapping with α-MSH-ir ones. The discovery of a population of true melanotrophs in the hypophysis of some odontocetes underscores the need for further research on the melanocortin system of cetaceans.

Estradiol effects on intracellular Ca(2+) homeostasis in bovine brain-derived endothelial cells.

Estrogens diversely affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Additional to the trophic effects of estrogens promoting cell growth and differentiation, recent experimental evidence highlights their involvement in the regulation of intracellular Ca(2+) homeostasis. The effects of estrogens on excitable cells are well documented. However, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. We have focused our attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. Estradiol (E(2)) effects on intracellular Ca(2+) homeostasis were tested by varying the exposure time to the hormone (8, 24, 48 h). Calcium measurements were performed with genetically encoded Ca(2+) probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca(2+) homeostasis (cytosol, endoplasmic reticulum, mitochondria). Mitochondrial Ca(2+) uptake significantly decreased after 48-h exposure to E(2), whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E(2) on mitochondrial Ca(2+) handling was blocked by ICI 182,780, a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor-mediated. To evaluate whether the decrease of Ca(2+) uptake affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester; no significant changes were seen between cells treated with E(2) and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E(2), suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process.

Evidence of melatonin secretion in cetaceans: plasma concentration and extrapineal HIOMT-like presence in the bottlenose dolphin Tursiops truncatus.

The pineal gland is generally believed to be absent in cetaceans, although few and subsequently unconfirmed reports described the organ in some species. The recent description of a complete and photographed pineal body in a bottlenose dolphin (Tursiops truncatus) prompted us to examine a series of 29 brains of the same species, but no gland was found. We then decided to investigate if the main product of the gland, melatonin, was nevertheless produced and present in the plasma of this species. We collected plasma and serum samples from a series of captive bottlenose dolphins for a period of 7 months spanning from winter to summer and we determined the indoleamine concentration by radio-immunoassay (RIA). The results demonstrated for the first time a quantitative assessment of melatonin production in the blood of a cetacean. Melatonin levels were comparable to those of terrestrial mammals (5.15-27.74 pg/ml daylight concentration), with indications of both seasonal and daily variation although the presence of a circadian rhythm remains uncertain. Immunohistochemical analyses using as a marker hydroxyindole-O-methyl-transferase (HIOMT, the key enzyme involved in the biosynthesis of the hormone), suggested extrapineal melatonin production by the retina, the Harderian gland and the gut. The enzyme was unequivocally localized in all the three tissues, and, specifically, ganglion cells in the retina showed a very strong HIOMT-immunoreactivity. Our results suggest that further research might reveal unexplored aspects of melatonin production in cetaceans and deserves special attention and further efforts.

Primary cultures of astrocytes from fetal bovine brain.

We describe here a method to obtain primary cell cultures from the cerebral cortex and the hypothalamus of bovine fetuses. We report how tissue origin, developmental stages, and culture medium conditions influence cell differentiation and the prevalence of glial cells vs. neurons. We compare explants from early, middle, and late stages of development and two different fetal calf serum concentrations (1 and 10%) to identify the best conditions to obtain and grow viable astrocytes in culture. In addition, we describe how to cryopreserve and obtain viable cortical astrocytes from frozen fetal bovine brain samples.

Primary cell cultures from fetal bovine hypothalamus and cerebral cortex: a reliable model to study P450Arom and alpha and beta estrogen receptors in vitro.

Estrogens synthesized by neural P450 aromatase (P450Arom) are implicated in many aspects of mammalian brain development and particularly in sexual differentiation of the central nervous system (CNS). This study analyzes the usefulness of an in vitro model based on bovine primary cell cultures from the hypothalamus and frontal cortex to investigate the role of P450Arom and estrogen receptors (ERs) in the development of fetal neural structures. The mRNA expression of P450Arom, ERalpha and ERbeta was detected using RT-PCR analysis in both hypothalamic and cortical primary cell cultures. P450Arom was identified and localized by immunocytochemistry in both neurons and astrocytes. Our results indicate that, within our experimental settings, astrocytes do not express ERalpha. The experimental model that we propose may represent a standardized dynamic model to study cellular and molecular mechanisms involved in the complex process of brain sexual differentiation.

Ontogenesis of brain aromatase P450 expression in the bovine hypothalamus.

Aromatase P450 (P450(AROM)), converting testosterone (T) into estradiol (E), plays an important role in sexual differentiation of neural structures in the developing mammalian brain. The aim of the present study was to characterize the qualitative and quantitative profile of P450(AROM) mRNA expression in the bovine hypothalamus (the region of the central nervous system in which the enzyme is mainly localized) using RT-PCR and quantitative real-time RT-PCR analysis, respectively. P450(AROM) expression was examined in the developing hypothalamus in a series of experimental groups investigated at 10 weeks interval one from the other. Our data indicate that in the bovine fetal hypothalamus P450(AROM) expression peaks at the second quarter of gestation. The presence of neural cells containing P450(AROM) in the bovine fetal hypothalamus was confirmed by immunohistochemistry, and localized in the medial preoptic area. We conclude that second quarter of the gestation is the developmental stage which represents a critical period for hypothalamic differentiation in bovine ontogenesis, an important difference with the rat and mouse, short gestation species in which P450(AROM) activity peaks around delivery.

Antidiuretic hormone release in the bovine: values at rest, and evidence for sex differences under stressful conditions including severe hemorrhage.

OBJECTIVES: This study investigates antidiuretic hormone (ADH) plasma concentration in bulls and heifers at rest, after transportation stress, trauma and massive hyper-acute hemorrhage at slaughter, with special attention to differences between sexes. We also quantified ADH concentration in the ADH-producing hypothalamic area. SETTING AND DESIGN: The study was carried out on animals at the abattoir, exploiting the routinely procedure of slaughtering to obtain the experimental stressful conditions. The control group was represented by a group of animals matched for breed, age and gender. RESULTS: ADH plasma values suggest that transportation stress has little or no effect in heifers in contrast to bulls. In both sexes ADH release increases after open head trauma due to stunning prior to slaughter. Hemorrhagic hyper-acute stress caused by bleeding induces a rapid hundredfold rise of ADH plasma concentration that becomes diergic (physiologically different between sexes), with males showing far higher values. Females show a higher ADH concentration in the hypothalamus. MAIN FINDING: This study points out a difference in ADH output between bulls and heifers under conditions of severe hemorrhagic stress, as produced by severing of the neck vessels at slaughtering. CONCLUSION: Extreme stress unmasks endocrine differences in bovine ADH release that may be relevant only during specific circumstances, such as fighting.

Primary cultures from fetal bovine brain.

This study describes a method of obtaining primary cultures from the cerebral cortex and the hypothalamus of bovine fetuses. We describe here the influence of tissue origin, developmental stage and culture medium conditions on cell differentiation and prevalence of neurons vs glial cells. To identify optimal conditions for obtaining and growing viable neurons and astrocytes in culture, we tested early, middle and late stages of development. Explants from cortex, early stages (week 10 of pregnancy out of 36) and low fetal calf serum concentration (1%) yielded maximum amounts of neurons. Fresh and thawed tissues gave comparable results.

Human Doppel and prion protein share common membrane microdomains and internalization pathways.

Doppel is the first identified homologue of the prion protein (PrPc) implicated in prion disease. Doppel is considered an N-truncated form of PrPc, and shares with PrPc several structural and biochemical features. When over expressed in the brain of some PrP knockout animals, it provokes cerebellar ataxia. As this phenotype is rescued by reintroducing the PrP gene, it has been suggested that Doppel and PrPc have antagonistic functions and may compete for a common ligand. However, a direct interaction between the two proteins has recently been observed. To investigate whether the neuronal environment is suitable for such possibility, human Doppel and PrPc were expressed separately, or together, in neuroblastoma cells, and then studied by biochemical and immunomicroscopic tools, as well as in intact cells expressing fluorescent fusion constructs. The results demonstrate that Doppel and PrPc co-patch extensively at the plasma membrane, and get internalized together after ganglioside cross-linking by cholera toxin or addition of an antibody against only one of the proteins. These processes no longer occur if the integrity of rafts is disrupted. We also show that, whereas each protein expressed alone occupies Triton X-100-insoluble membrane microdomains, co-transfected Doppel and PrPc redistribute together into a less ordered lipidic environment. All these features are consistent with interactions occurring between Doppel and PrPc in our neuronal cell model.