Resistin in endocrine pancreas of sheep: Presence and expression related to different diets.

Resistin (RETN), a recently discovered adipokine, is a cysteine-rich and secretory protein produced by adipocytes. RETN has been detected in several tissues, including human and laboratory animals' pancreas, wherein impairs glucose tolerance and insulin (INS) action and causes INS resistance. This study aims to evaluate the presence and expression of RETN in the pancreas of 15 adult female sheep reared on Apennine pastures, which show a decrease in their nutritional value due to the drought stress linked to the increasing summer aridity. The sheep were divided into 3 groups according to the diet they were subjected to: maximum pasture flowering (MxF) group, maximum pasture dryness (MxD) group, and experimental (Exp) group which received a feed supplementation in addition to the MxD group feeding. Immunohistochemistry and immunofluorescence were performed on formalin-fixed and paraffin-embedded sections of the pancreas to detect the RETN presence and to evaluate the co-localization of RETN with both glucagon (GCG)- and INS-producing cells. In addition, the expression of the three molecules was evaluated also in relation to different diets. RETN was observed only in the endocrine pancreas, showing a wide distribution throughout the pancreatic islets with few negative cells and the RETN producing cells colocalized with both α cells and ß cells. No differences in distribution and immunostaining intensity of RETN, GCG and INS were observed among the three groups. Quantitative PCR showed the expression of RETN, GCG and INS in all tested samples. No significant differences were observed for RETN and GCG among all three groups of sheep. Instead, a high statistically significant expression of INS was detected in the MxF group with respect to the Exp and MxD groups. These results highlight the localization of RETN in GCG- and INS-secreting cells involved in glucose homeostasis suggesting a modulatory role for RETN. Furthermore, the RETN expression is not influenced by food supplementation and thus is not affected by diet.

Central and Peripheral NPY Age-Related Regulation: A Comparative Analysis in Fish Translational Models.

NPY is among the most abundant neuropeptides in vertebrate brain and is primarily involved in the regulation of food intake. The NPY system is also associated with the aging process showing beneficial effects on neuronal survival via autophagy modulation. Here, we explore the age-related regulation of NPY in the brain and foregut of the shortest- and longest-lived fish species, Nothobranchius furzeri and Danio rerio, respectively. These two research models, despite some similarities, display profound biological differences making them attractive vertebrates to elucidate the mechanisms underlying the regulation of neuropeptide synthesis and function. It is noteworthy that in both fish species only Npya has been identified, while in the other teleosts two classes of NPY (Npya and Npyb) have been annotated. Our findings document that in both species: (i) NPY is centrally regulated; (ii) NPY levels increase in the brain during aging; (iii) NPY is localized in the enteroendocrine cells as well as in the myenteric plexus and drastically decreases in old animals. According to our data, the age-related regulation in the gut resembles that described in other vertebrate species while the increased levels in the brain offer the unique possibility to explore the role of NPY in model organisms to develop future experimental and translatable approaches.

Anatomical templates for tissue (re)generation and beyond.

Induced pluripotent stem cells (iPSCs) represent a valuable alternative to stem cells in regenerative medicine overcoming their ethical limitations, like embryo disruption. Takahashi and Yamanaka in 2006 reprogrammed, for the first time, mouse fibroblasts into iPSCs through the retroviral delivery of four reprogramming factors: Oct3/4, Sox2, c-Myc, and Klf4. Since then, several studies started reporting the derivation of iPSC lines from animals other than rodents for translational and veterinary medicine. Here, we review the potential of using these cells for further intriguing applications, such as "cellular agriculture." iPSCs, indeed, can be a source of in vitro, skeletal muscle tissue, namely "cultured meat," a product that improves animal welfare and encourages the consumption of healthier meat along with environmental preservation. Also, we report the potential of using iPSCs, obtained from endangered species, for therapeutic treatments for captive animals and for assisted reproductive technologies as well. This review offers a unique opportunity to explore the whole spectrum of iPSC applications from regenerative translational and veterinary medicine to the production of artificial meat and the preservation of currently endangered species.

Mutation studies in ascidians: a review.

Historically, mutations have had a significant impact on the study of developmental processes and phenotypic evolution. Lesions in DNA are created by artificial methods or detected by natural genetic variation. Random mutations are then ascribed to genetic change by direct sequencing or positional cloning. Tunicate species of the ascidian genus Ciona represent nearly fully realized model systems in which gene function can be investigated in depth. Additionally, tunicates are valuable organisms for the study of naturally occurring mutations due to the capability to exploit genetic variation down to the molecular level. Here, we summarize the available information about how mutations are studied in ascidians with examples of insights that have resulted from these applications. We also describe notions and methodologies that might be useful for the implementation of easy and tight procedures for mutations studies in Ciona.

TigarB causes mitochondrial dysfunction and neuronal loss in PINK1 deficiency.

OBJECTIVE: Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink(-/-) ) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency. METHODS: The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function, and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation. RESULTS: PINK1 deficiency results in loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink(-/-) larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalization of mitochondrial function, with resulting rescue of dopaminergic neurons in pink(-/-) larvae. There was also marked microglial activation in pink(-/-) larvae, but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis. INTERPRETATION: Pink1(-/-) zebrafish are the first vertebrate model of PINK1 deficiency with loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD.

Immunolocalization of Two Neurotrophins, NGF and BDNF, in the Pancreas of the South American Sea Lion Otaria flavescens and Bottlenose Dolphin Tursiops truncatus.

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.

Fasting duration impacts ribosome protein 6 phosphorylation in zebrafish brain: New insights in aquatic organisms' welfare.

BACKGROUND: Short- 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. METHODS: Group-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. RESULTS: We 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. CONCLUSIONS: Taken 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.

Zebrafish bcl2l is a survival factor in thyroid development.

Regulated cell death, defined in morphological terms as apoptosis, is crucial for organ morphogenesis. While differentiation of the thyroid gland has been extensively studied, nothing is yet known about the survival mechanisms involved in the development of this endocrine gland. Using the zebrafish model system, we aim to understand whether genes belonging to the Bcl-2 family that control apoptosis are implicated in regulation of cell survival during thyroid development. Evidence of strong Bcl-2 gene expression in mouse thyroid precursors prompted us to investigate the functions played by its zebrafish homologs during thyroid development. We show that the bcl2-like (bcl2l) gene is expressed in the zebrafish thyroid primordium. Morpholino-mediated knockdown and mutant analyses revealed that bcl2l is crucial for thyroid cell survival and that this function is tightly modulated by the transcription factors pax2a, nk2.1a and hhex. Also, the bcl2l gene appears to control a caspase-3-dependent apoptotic mechanism during thyroid development. Thyroid precursor cells require an actively maintained survival mechanism to properly proceed through development. The bcl2l gene operates in the inhibition of cell death under direct regulation of a thyroid specific set of transcription factors. This is the first demonstration of an active mechanism to ensure survival of the thyroid primordium during morphogenesis.

Immunohistochemical and Physiological Research on Farm Animals.

This Special Issue "Immunohistochemical and Physiological Research on Farm Animals" is dedicated to the application of immunohistochemical and physiological studies carried out on farm animals, including traditional (pig, cow, buffalo, horse, sheep, goat, rabbit, turkey and trout) and emerging farm species (yak, sea bass and zebrafish) [...].

Leptin system is not affected by different diets in the abomasum of the sheep reared in semi-natural pastures of the Central Apennines.

The growing summer drought stress is affecting the nutritional value of pastures, no longer sufficient to support the nutritional status of sheep in extensive rearing. Adipokines affect organ and tissue functionality can be useful to evaluate animal welfare and prompt an improvement in the management of the grazing animals. Leptin (Lep) is an adipokine mainly produced by adipose tissue that regulates food intake by an anorexigenic action. Lep has also been detected in the human and rat gastrointestinal tract, where it regulates the rate of gastric emptying. In this study, Lep system was evaluated in the abomasum of 15 adult sheep reared on Apennine pastures and subjected to different diets. Until the maximum pasture flowering (MxF group), the sheep fed on fresh forage; from that moment until the maximum pasture dryness (MxD group), the experimental group (Exp group) received a feed supplementation in addition to MxD group feeding. The Lep system was investigated in the abomasum samples by immunohistochemistry (IHC) and RT-qPCR. Double-label localisation of Lep and leptin receptor (LepR) with neuroendocrine hormones was conducted to distinguish the gland cell types. The analysis performed revealed the presence of Lep and LepR in the chief and neuroendocrine cells of the fundic glands of the abomasum. RT-qPCR evidenced the transcript for Lep and LepR also identifying the long isoform (LepRb). No significant differences were observed among the three groups of sheep subjected to different diets. The abundant immunostaining observed in the fundic glands suggests that the Lep intervenes in the regulation of abomasum in sheep with a similar pattern to monogastric species while long term food supplementation seems do not influence the local function of the Lep system. A better understanding of the gastrointestinal system can contribute to improving sheep management and optimising the sustainability of livestock production.

A Morphological and Ultrastructural Study of the Anterior Digestive Tract of Adult Nile Tilapia Oreochromis niloticus.

Among the most-used fish species in aquaculture is the Nile tilapia, due to its rapid growth rate and its adaptation to a wide range of farming conditions. A careful description of the morphology of the digestive tract, particularly the esophagus and stomach, allows a better understanding of the relationship between structure and function. Combining scanning and light microscopy we highlighted the presence of five different zones in the stomach (1: esophagus-gastric lumen passage; 2: descending glandular portion; 3: fundic portion; 4: ascending glandular portion; 5: gastric-pyloric transition portion). Histochemical investigation showed a secretion of carboxylates mucopolysaccharides along the esophagus and sulphated complex carbohydrates in the stomach. These results suggest that mucins play a protective role of the epithelial lining, which is essential for a correct digestive process. Finally, the characterization of the main cellular structures may be inspiring for more advanced studies aiming to decipher the role of specific molecules, such as neuropeptides, involved in the physiological digestive process.

Immunolocalization of Nesfatin-1 in the Gastrointestinal Tract of the Common Bottlenose Dolphin Tursiops truncatus.

First identified as an anorexigenic peptide, in the last decades, several studies have suggested that Nesfatin-1 (Nesf-1) is a pleiotropic hormone implicated in numerous regulatory processes in peripheral organs and tissues. In vertebrates, Nesf-1 is indeed expressed in the central nervous system and peripheral organs. In this study, we characterized the pattern of Nesf-1 distribution within the digestive tract of the common bottlenose dolphin (Tursiops truncatus), composed of three gastric chambers and an intestine without a clear subdivision in the small and large intestine, also lacking a caecum. Our results indicated that Nesf-1 is widely distributed in cells of the mucosal epithelium of the gastric chambers. Most of the immunoreactivity was observed in the second chamber, compared to the first and third chambers. Immunopositivity was also found in nerve fibers and neurons, scattered or/and clustered in ganglion structures along all the examined gastrointestinal tracts. These observations add new data on the highly conserved role of Nesf-1 in the mammalian digestive system.

Effects of Feed Supplementation on Nesfatin-1, Insulin, Glucagon, Leptin, T3, Cortisol, and BCS in Milking Ewes Grazing on Semi-Natural Pastures.

This study aimed to investigate the effects of feed supplementation on body condition score (BCS) and different metabolic hormones concentration in lactating sheep reared in Italian Central Apennine pastures during the grazing summer period. In this study, 24 Comisana x Appenninica pluriparous ewes from June until August were divided into two homogenous groups: the control group (UNS) was free to graze, while the other group (SUP), in addition to grazing, was supplemented with 600 g/day/head of cereals. At the start of the supplementation and at an interval of 9-10 days until the end of experimentation, BCS evaluation and blood withdrawal to assay nesfatin-1, insulin, glucagon, leptin, triiodothyronine and cortisol levels were performed. Univariable analysis showed no remarkable differences between the groups, while multivariable analysis suggested that the UNS group was characterized by a lower BCS and greater nesfatin-1 than the SUP group. These findings can be considered in relation to milk production, which shows a clear better persistence in the SUP group. Our results indicate that nutritional supplementation has protected ewes from the usual lowering of the body state linked to lactation and provides a good maintenance of milk production, determining also a better overall body and metabolic state of the animals, which is important at the beginning of the sexual season.

Distinct Pattern of NPY in Gastro-Entero-Pancreatic System of Goat Kids Fed with a New Standardized Red Orange and Lemon Extract (RLE).

The use of natural compounds as feed additive is also increasing in farm animals, thanks to the beneficial effect on both animals and consumers health. Here, we questioned whether natural extracts, such as red orange and lemon extract (RLE) rich in flavanones, anthocyanins, and other polyphenols, used as feed additives could display an effect on the neuropeptide Y (NPY) in the gastro-entero-pancreatic tract of goat kids. NPY is one of the most abundant neuropeptides in mammals, known for its orexigenic role although it is involved in many central and peripheral functions. We carried out immunohistochemical analyses on samples of abomasum, duodenum and pancreas collected from two experimental groups: one fed with standard diet and one with standard diet + RLE. For the first time we document NPY distribution in the abomasum, duodenum and pancreas of goats and observe the highest number of NPY positive cells in neuroendocrine cells of duodenum. Remarkably, upon RLE feed supplementation, NPY immunoreactive cells increased significantly in abomasal epithelium and pancreatic islets but not in duodenum, likely due to pH variation of abomasum and duodenum. Our observations represent a baseline for future studies on the interaction between neuropeptides and polyphenols, used as feed additive.

Oregano Feed Supplementation Affects Glycoconjugates Production in Swine Gut.

This study evaluated the effects of adding oregano aqueous extract (OAE) to the diet of pig slaughtered at finisher stage. Study was performed to identify glycoconjugates and evaluate the oxidative stress levels in the duodenum and colon intestinal tracts. Glycohistochemistry was performed by staining with Periodic acid-Schiff (PAS), Alcian blue (AB) pH 2.5, AB-PAS, AB pH 1, AB pH 0.5, low iron diamine, and high iron diamine. Serial sections were pre-treated with sialidase V before staining with AB pH 2.5 (Sial-AB) preceded or not by saponification. To study oxidative stress, an immunohistochemical analysis was applied to investigate the presence of the oxidative stress target molecule Bcl-2 Associate X protein (BAX). Findings show that oregano aqueous extract supplementation improves the production of the secretion glycoconjugates involved in direct and indirect defense, thus enhancing the protection of the pig intestinal mucosa. Moreover, the reduced BAX protein immunostaining observed in both duodenum and colon of swine of the oregano-supplemented group respect to that observed in the control group suggests an enhanced antioxidant action by oregano adding. Findings could be useful for other studies aiming to reduce antibiotic use and prevent antimicrobial resistance.

Influence of Different Feed Physical Forms on Mandibular Gland in Growing Pigs.

A study was performed on the mandibular gland obtained from growing pigs enrolled in a wide research project aiming to test the effects of different feed physical forms on animal health, production and welfare. We used 48 pigs fed for four weeks with different dietary treatments based on different grinding intensities and compactions of the same diet, namely coarsely ground meal (CM), finely ground pelleted (FP) and coarsely ground pelleted (CP) diets. Samples were analyzed by conventional histochemistry to identify the glycohistochemical profile and by immunohistochemistry to localize aquaporin 5, apelin and apelin receptor. Statistical elaborations were performed using the Stats R-package, version 3.5.3. Pig mandibular gland adenomere increased both the quantity and acidity of produced glycoconjugates from CM to FP and CP diets. This probably calls forth higher watery saliva, thus promoting a better feed softening facilitating the amalgamation of the bolus. Mandibular gland increased aquaporin 5 positivity in the CP diet, supporting the hypothesis of an augmented demand for water. Based on apelin/receptor localization, it was hypothesized that in pig mandibular gland the apelinergic system likely performs an endocrine control on the demilunes activity and a paracrine control on ducts, facilitating the production of a more fluid saliva.

A Novel Method for Increasing the Numerousness of Biometrical Parameters Useful for Wildlife Management: Roe Deer Mandible as Bone Model.

Study of dimensions (biometry) and shapes (geometric morphometry) of bone structures in ungulates is of extreme importance in wildlife population management. Unlike classical biometry, which involves the use of a caliper for measurements, geometric morphometry acquires, through software, a series of reference points (landmarks) from digital photos, providing a series of linear measures. A method to convert values obtained from the GeoGebra software into biometric measures is described. We took photos of 25 mandibles of adult roe deer and at the same time measured mandible length and teeth row length using a caliper. After image processing using GeoGebra, we calculated the conversion factor as the mean ratio between measures taken using GeoGebra and the caliper. The series of measurements, taken with two different methods (direct measurement using the caliper and conversion from GeoGebra output), showed a good degree of agreement. We used the conversion factor to obtain, from the GeoGebra database, four additional parameters of 50 mandibles. The analysis of variance showed that one parameter was significantly different between sexes (p = 0.04), demonstrating the usefulness of the measurement conversion. The conversion factor is helpful to improve classical biometric databases to better clarify the relationship between environment and wildlife status.

Immuno- and glyco-histochemistry as a tool to evaluate the oregano supplemented feed effects in pig gut.

Among oregano properties, its antioxidant and antibacterial effects are particularly interesting. Oregano is also able to induce a higher glycoconjugate production in gut, creating a physical barrier against microorganisms. This study evaluated the effects of adding an aqueous extract of oregano (OAE) to the diet of two homogenous groups of pigs during the finisher phase. The diets were as follows: control commercial diet (CTR group) and CTR diet supplemented (2 g/kg) with OAE (O group). Samples of ileum and caecum from the two groups were examined by conventional histochemistry to analyze complex carbohydrates and by immunohistochemistry to detect Bcl-2 Associate X protein (BAX), an indicator of oxidative stress. Glyco-histochemistry showed significant differences between the two groups. Immunohistochemistry revealed a lower presence of BAX in O group. The OAE supplementation improved the production of glycoconjugates, able to enhance in pig the protection of intestinal mucosa by means of direct and indirect defense actions. The reduced BAX immunostaining observed in O group may be an indicator of enhanced antioxidant action promoted by oregano. The results of this study can be used in further research to identify ways to improve endogenous defence ability, with the aim of reducing antibiotic use and preventing antimicrobial resistance.

Epithelial expression of the hormone leptin by bovine skin.

Leptin (Lep) stimulates keratinocytes to proliferate, intervenes in the wound healing and participates to hair follicle morphogenesis and cycle. While it is secreted by skin structures including epidermis and hair follicles, intradermal adipose tissue also seems to have a role in Lep secretion and accordingly in the control of hair follicle growth in mice and humans. Lep was investigated in the skin of humans and laboratory animals but there are not data regarding bovine species. The aim of this work was to study the expression of Lep and its receptor (LepR) in the skin of bovine and, at the same time, to investigate the presence and extension of intradermal adipose tissue. A morphological evaluation of the skin was performed while the presence and localization of Lep and LepR were analyzed by RT-PCR and immunohistochemistry. A high and thick dermis without adipocytes was observed. Hair follicles and sebaceous and sweat glands were located in the proximal part of the skin while a thick layer of connective tissue, lacking adipose cells, separated these structures by subcutis. RT-PCR evidenced the transcripts for both molecules. By immunohistochemistry, Lep and LepR were observed in the epidermis and hair follicles. Based on the absence of intradermal adipose tissue and the presence of both Lep and LepR in the epidermis and in the hair follicle epithelium, it can be posited that in bovine skin Lep participates to the control of epidermis growth and hair follicle cycle through a paracrine and autocrine mechanisms.

Immunohistochemical identification of resistin in the uterus of ewes subjected to different diets: Preliminary results.

Resistin is a polypeptide hormone of the adipokine-family, primarily, but not exclusively, produced by the adipose tissue. Recent studies suggested that resistin may affect the male and female reproductive activity. The study aim was to immunohistochemically evaluate the presence and distribution of resistin in the ovine uterus. Uterine samples were collected from two groups of ewes at the end of an experimental trial during which the animals of the first group (CTRL) were fed only by grazing while those of the second one (EXP) were supplemented with barley and corn. Using a monoclonal antibody against resistin, tested by Western Blot, the immunopositive reaction was identified in the cytoplasm of epithelial lining cells and uterine glands. The endogenous production of resistin seemed to be affected by different diet, as evidenced by staining differences between the CTRL and EXP groups. Our findings support the existence of a peripheral resistin system in the sheep uterus. It is possible that this system is involved in the functionality of the uterus, which is also affected by the animal's nutritional status.