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.

Towards a More Realistic In Vitro Meat: The Cross Talk between Adipose and Muscle Cells.

According to statistics and future predictions, meat consumption will increase in the coming years. Considering both the environmental impact of intensive livestock farming and the importance of protecting animal welfare, the necessity of finding alternative strategies to satisfy the growing meat demand is compelling. Biotechnologies are responding to this demand by developing new strategies for producing meat in vitro. The manufacturing of cultured meat has faced criticism concerning, above all, the practical issues of culturing together different cell types typical of meat that are partly responsible for meat's organoleptic characteristics. Indeed, the existence of a cross talk between adipose and muscle cells has critical effects on the outcome of the co-culture, leading to a general inhibition of myogenesis in favor of adipogenic differentiation. This review aims to clarify the main mechanisms and the key molecules involved in this cross talk and provide an overview of the most recent and successful meat culture 3D strategies for overcoming this challenge, focusing on the approaches based on farm-animal-derived cells.

Monitoring of Alarm Reactions of Red Deer (Cervus elaphus) in a Captive Population in Paneveggio Pale di San Martino Natural Park.

The study analyzes red deer responses to disturbances during the day and different exposures to tourists, to establish the more appropriate times to carry out activities inside the Paneveggio deer enclosure. The alarm reactions of red deer were observed after presenting different types of visual stimuli inside and outside the fence, in order to answer some questions: Which stimuli produce the strongest reactions from the animals? Do animals differently react to stimuli presented outside and inside the fence? On which days and times are the animals more sensitive to disturbances? Are there different reactions between the males and females? The results suggest that the red deer adversely react to the disturbance at different degrees of intensity in relation to day, sex, tourist and where the stimuli are presented. It was observed that during the days with the highest tourist presence, the animals were particularly alarmed; discomfort accumulation produced the highest number of alarm reactions on Monday. For these reasons, it would be opportune to manage the pasture on Tuesday, Wednesday and Thursday, scheduled at specific times of day, preferably far from the estimated presence of tourists.

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) [...].

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.

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.

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.

The Apelinergic System Immuno-Detection in the Abomasum and Duodenum of Sheep Grazing on Semi-Natural Pasture.

Apelin (APLN) is an adipokine mainly produced by adipose tissue and related to an individual's nutritional status as well as digestive apparatus functions. In this work, APLN and its receptor (APLNR) were investigated, by immunohistochemistry, in the abomasum and duodenum of 15 Comisana × Appenninica adult sheep reared in a semi-natural pasture. Organ samples were collected after maximum pasture flowering (M × F group) and after maximum pasture dryness (M × D group); the experimental group (E × p group) received a feed supplementation of 600 grams/day/head of barley and corn in addition to M × D group feeding. APLN and APLNR were identified in the lining epithelium and the fundic gland chief cells of the abomasum. APLNR was observed in the lining epithelium, in the crypts and the serotonin secreting cells of the duodenum. Similar reactivity was observed between the M × F and E × p groups, while the M × D group showed a lower intensity of immunostaining for both APLN and APLNR in all positive structures but the duodenal serotonin neuroendocrine cells. Hence, our findings show that the E × p group presents a picture quite overlapped with M × F and suggest that food supplementation has a maintaining effect on the apelinergic system expression in the investigated digestive tracts of the sheep.

Shape Analysis as an Additional Tool in Roe Deer (Capreolus capreolus) Management: A New Approach Based on the Relationship between Mandible Shape and Trophic Resources.

The analysis of body shape variability has always been a central element in biology. More recently, geometric morphometry has developed as a new field in shape analysis, with the aim to study body morphological variations and the identification of their causes. In wildlife management, geometric morphometry could be a useful tool to compare the anatomical structures of an organism and quantify its geometric information in order to relate them to environmental factors, thus identifying the causes and effects of the variation and acting management and/or conservation plans. The aim of our study is to evaluate the relationship between roe deer mandible shape and trophic resources available during autumn and winter. We applied a geometric morphometry approach consisting of a Relative Warp analysis of landmark data in 26 roe deer fawn mandibles. Each sample was assigned to an age category and to an environmental category based on the territory carrying capacity. The mandible shape of samples under 8 months of age is likely influenced by the availability of trophic resources. Our findings suggest that the mandible shape is a reliable instrument to assess resource availability. Geometric morphometry could thus represent an additional tool for roe deer management.

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.

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.

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.

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.

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.

The effects of feeding supplementation on the nutritional quality of milk and cheese from sheep grazing on dry pasture.

The effect of feeding supplementation on the nutritional characteristics of milk and cheese was studied in dairy sheep grazing on low mountain dry-grasslands during summer in typical sub-Mediterranean conditions of aridity. The control group (CG) of 25 sheep grazed on grass, while the experimental group (EG) of 25 sheep grazed on grass and received 600 g a day of a barley and corn mixture. Daily milk production showed a less pronounced decrease in EG than in CG (p ˂ 0.0368). After one month of supplementation, the concentrations of retinol and α-tocopherol in milk and cheese from EG were higher than CG (p < 0.05). Supplementary feeding had a positive effect on the fatty acid composition of the sheep milk. For the first time, positive effects on the volatile composition were found in EG cheese, displaying lower percentages of carboxylic acids associated with the pungent and rancid odour with respect to CG.

Apelin system detection in the reproductive apparatus of ewes grazing on semi-natural pasture.

Apelin (APLN) is an adipokine with pleiotropic effects involved in the regulation of metabolic, cardiovascular, immune, and electrolyte balance function. Recent studies demonstrated a pivotal role in the regulation of male and female reproduction. APLN and its receptor (APLNR) were found in the hypothalamic-pituitary-gonad axis tissues, regulating gonadotropin release and steroidogenesis. However, to date, there are no studies that describe APLN system in the reproductive apparatus of the sheep. The study was performed on 10 Comisana x Appenninica adult dry ewes reared in a semi-natural pasture. Organ samples were collected from five animals in the two pasture functional phases: after maximum pasture flowering (Group 1) and after maximum pasture dryness (Group 2). Experiments were devised to characterize the gene expression and protein localization of the APLN/APLNR system in ewe reproductive apparatus; in addition, the concentration of plasma APLN was evaluated during the trial. Through immunohistochemical analysis, a positive staining for APLN was observed in the large luteal cells, in the epithelial cell coat of the ampulla, in the uterus epithelial lining and in the uterine glands. APLNR was observed in the granulosa cells, in the large luteal cells, in the secreting cells of the ampulla, in the uterus epithelial lining and uterine glands. The transcripts for APLN and APLNR were evidenced in all organ tissues examined. The highest level of APLN mRNA was detected in the Group 2 ewes in the luteal phase of the ovarian cycle compared to Group 1 ewes in the anestrous one. The relative content of APLN transcript was respectively twofold higher in the ovary (P < 0.05) and uterus (P < 0.05) and threefold higher in the ampulla (P < 0.05) in the Group 2 vs Group 1. The same trend of APLN transcript was evaluated for APLNR mRNA in uterus (P < 0.05) and ovary (P < 0.05). No difference was evidenced between Group 1 and Group 2 for APLNR mRNA levels. The plasma APLN level was fairly constant during the trial period. In conclusion, the present data suggest that the apelinergic system is involved in the reproduction function of ewes, being differentially distributed and expressed in the organs of the reproductive apparatus of ewes; these variations could be related to the sexual cycle and to the cyclic activity of the reproductive apparatus.

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.

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.

Ontogenetic Pattern Changes of Nucleobindin-2/Nesfatin-1 in the Brain and Intestinal Bulb of the Short Lived African Turquoise Killifish.

Nesfatin-1 (Nesf-1) was identified as an anorexigenic and well conserved molecule in rodents and fish. While tissue distribution of NUCB2 (Nucleobindin 2)/Nesf-1 is discretely known in vertebrates, reports on ontogenetic expression are scarce. Here, we examine the age-related central and peripheral expression of NUCB2/Nesf-1 in the teleost African turquoise killifish Nothobranchiusfurzeri, a consolidated model organism for aging research. We focused our analysis on brain areas responsible for the regulation of food intake and the rostral intestinal bulb, which is analogous of the mammalian stomach. We hypothesize that in our model, the stomach equivalent structure is the main source of NUCB2 mRNA, displaying higher expression levels than those observed in the brain, mainly during aging. Remarkably, its expression significantly increased in the rostral intestinal bulb compared to the brain, which is likely due to the typical anorexia of aging. When analyzing the pattern of expression, we confirmed the distribution in diencephalic areas involved in food intake regulation at all age stages. Interestingly, in the rostral bulb, NUCB2 mRNA was localized in the lining epithelium of young and old animals, while Nesf-1 immunoreactive cells were distributed in the submucosae. Taken together, our results represent a useful basis for gaining deeper knowledge regarding the mechanisms that regulate food intake during vertebrate aging.