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.

Presence, Tissue Localization, and Gene Expression of the Adiponectin Receptor 1 in Testis and Accessory Glands of Male Rams during the Non-Breeding Season.

Adiponectin (ADIPOQ) is a member adipocytokines, and its actions are supported by two receptors, ADIPOQ receptor 1 and -2, respectively (ADIPOR1 and -R2). Our study was performed to evaluate the ADIPOR1 presence and location and its gene expression in reproductive tissues of the male ram, during its non-breading season. The different portions of the male ram reproductive system (testis, epididymis, seminal vesicle, ampoule vas deferens, bulb-urethral gland) were collected in a slaughterhouse. Immunohistochemistry showed ADIPOR1 positive signals in the cytoplasm of all the glandular epithelial cells, with a location near the nucleus; in the testes, the positive reaction was evidenced in the cytoplasm in the basal portion of the germinal epithelial cells. The immune reaction intensity was highest (p < 0.001) in the prostate and seminal vesicles glands than that of other parts of the ram reproductive tract. RT-qPCR detected the ADIPOR1 transcript in the testes, epididymis, vas deferens, bulbourethral glands, seminal vesicles, and prostate; the expression levels were high (p < 0.01) in the prostate and low (p < 0.01) in the testis, epididymis, and bulbourethral glands. The present results evidenced the possible ADIPOQ/ADIPOR1 system's role in regulating the testicular activity of male rams during the non-breading season.

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.

Metabolic milieu and localization of ovarian leptin and receptor in queens under different reproduction phases.

Metabolic profile and leptin presence and expression of receptor in the ovary of 8 queens were analyzed. All queens were assessed for nutritional state and underwent ovariectomy in accordance with Italian law for the control of stray cats and dogs. Three queens were at early gestation, three prepubescent and two non-pregnant. All animals appeared healthy but underweight. Haemogram and biochemical profile did not show to be of prognostic value. On microscopy, granulosa cells of secondary follicles tested positive to leptin in pregnant queens, while testing negative in non-pregnant ones. Muscular layer of vessels of the ovary in non-pregnant queens tested positive to receptor, but negative in those found pregnant. Results suggest that leptin is produced and receptor expressed in the different tissues of the ovary in underweight queens. Additionally, the expression of receptor did not necessarily imply local leptin production and clearly displayed to vary according to the reproduction phase. Taken altogether, these findings allow to conclude that the expression of receptor can be interpreted as the pillar bridging ovary tissues as target sites of circulating leptin produced elsewhere. Arguably, ovary removal may affect hormonal cross-talk at the systemic level, likely affecting different systemic functions, including the nutritional status.

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.

Effects of Obesity on Adiponectin System Skin Expression in Dogs: A Comparative Study.

Obesity is an important health issue in dogs since it influences a plethora of associated pathologies, including dermatological disorders. Considering the scarcity of information in pets, this work aimed to evaluate the localization and expression of adiponectin (ADIPOQ) and its two receptors (ADIPOR1 and ADIPOR2) in the skin of 10 obese dogs, compared with serum ADIPOQ level. Through immunohistochemistry, ADIPOQ and ADIPOR2 were observed in the adipose tissue, sweat and sebaceous glands, endothelium, and some connective cells. Both receptors were observed in the epidermis and the hair follicles, other than in the sweat and sebaceous glands. Real-time PCR evidenced that the ADIPOQ and ADIPOR2 transcripts were expressed 5.4-fold (p < 0.01) and 2.3-fold less (p < 0.01), respectively, in obese than in normal weight dogs, while ADIPOR1 expression did not change. Obese dogs showed lower serum ADIPOQ levels than the normal weight group. Accordingly, ADIPOQ and ADIPOR2 expression in the skin appear negatively correlated with obesity in the same way as the serum ADIPOQ level. These findings evidence that ADIPOQ system changes in the skin of obese dogs and suggest that the ADIPOQ effect on the skin is at least in part regulated by the reduced expression of ADIPOR2.

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.

Leptin System in Obese Dog Skin: A Pilot Study.

Obesity predisposes to several health problems including skin diseases. However, information on the relationship between obesity and skin disorders in pets is very scarce. Leptin (LEP) is mainly produced by adipose tissue and has a prominent role in skin biology. This study evaluated the LEP system in the skin of obese dogs compared to normal-weight animals. The investigation was carried out on 10 obese (Obese group) and 10 normal-weight (Normal-weight group) dogs through Real-time PCR and immunohistochemistry. Cells of skin associated immune system were also evaluated. No differences were evidenced between the two groups as well as skin inflammation. LEP differences were no significant, while LEPR transcript appeared 10-fold higher in obesedogs than in normal-weight ones. Immunostaining for both molecules was observed in several skin structures such as the epidermis, hair follicles, and glands. No differences appeared in the skin associated immune system composition. This study is a preliminary report showing that LEP system changes in obese dog skin. The increased LEPR expression observed in the obese group suggests that the receptor plays a modulating role in the system control. However, the exact role of LEPin the skin under obesity conditions needs further elucidation.

Seasonal Expression of NGF and Its Cognate Receptors in the Ovaries of Grey Squirrels (Sciurus carolinensis).

The grey squirrel is an invasive alien species that seriously threatens the conservation of the native red squirrel species. With the aim of characterizing the reproductive physiology of this species due to its great reproductive success, the function of the ovarian nerve growth factor (NGF) system was analyzed in a grey squirrel population living in central Italy. During the breeding and nonbreeding seasons, the ovarian presence, distribution, and gene expression of NGF, neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR), as well as NGF plasma concentrations, were evaluated in female grey squirrels. NGF was found in the luteal cells and in the thecal and granulosa cells of follicles, while NTRK1 and NGFR were only observed in follicular thecal and granulosa cells. NGF and NGFR transcripts were almost two-fold greater during the breeding season, while no seasonal differences were observed in NTRK1 gene expression. During the breeding season, NGFR was more expressed than NTRK1. Moreover, no changes were observed in NGF plasma levels during the reproductive cycle. The NGF system seems to be involved in regulating the ovarian cycle mainly via local modulation of NGF/NGFR, thus playing a role in the reproductive physiology of this grey squirrel population.

Shedding light on cashmere goat hair follicle biology: from morphology analyses to transcriptomic landascape.

BACKGROUND: Cashmere goat is known for its precious undercoat. Being photoperiod-dictated, cashmere growth has been studied focusing mainly on hair follicle cycle phases (anagen, catagen and telogen). An accurate molecular knowledge of the goat hair follicle cycle, disentangling gene expression changes during phases and recognizing timing boundaries, could be useful to improve cashmere goat management and ultimately cashmere production. RESULTS: To better describe goat's hair follicle transcriptome we applied RNA-sequencing to isolated hair follicles from five Italian cashmere goats, during the anagen and catagen phase, identifying total of 214 differentially expressed genes (DEGs): 97 were up-regulated while 117 were down-regulated in catagen with respect to anagen. Gene Ontology and pathway analysis were performed. We detected 144 significant pathways spanning from estrogen, pluripotency of stem cells, thermogenesis and fatty acid metabolism that were strongly expressed during the hair follicle phases analysed. Finally, we validated promising DEGs by RT-qPCR in the same set of samples as well as in hair follicles and entire skin biopsies of another cashmere goats cohort accounting for early anagen, anagen, early catagen, and catagen phases. CONCLUSIONS: As in the isolated hair follicles, some target genes were homogenously modulated during the four hair follicle phases. Ceruloplasmin (CP) and Keratin 4 (K4), confirmed their clear cut expression between growing and resting phase. In fact, K4 was almost absent in catagen phases while CP was barely expressed in anagen phases. In particular, the strong expression of K4 in early anagen makes it an eligible marker to track the beginning of a new hair cycle, and therefore defining the optimum time for cashmere harvesting.

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.

Reproductive Traits of an Invasive Alien Population of Grey Squirrel (Sciurus carolinensis) in Central Italy.

The reproductive cycle of an invasive alien Italian grey squirrel population was studied to understand its adaptation and limit its spread, in order to conserve the autochthonous red squirrel. Female and male genital traits were evaluated throughout the reproductive cycle, including the ovary, uterus, testicle, epididymis, seminiferous tubule morphometry, and germinative epithelium histology. Moreover, individual female fecundity was determined by counting uterine scars. Ovary width and uterus weight, length, and width reached their highest values in the luteal and pregnancy phases. On conducting a histological evaluation of the testicular germinal epithelium, four morphotypes related to the different reproductive phases of the male squirrels were identified: immature, pubertal, spermatogenesis, and regressive. Testicle and epididymis weights and seminiferous tubule diameters reached their largest values during spermatogenesis. Uterine scar analysis showed that 69% of the females had given birth to one or two litters, while 31% had no uterine scars. Litters were larger in the first breeding period than in the second; annual fecundity was 4.52 ± 1.88 uterine scars/female. Umbrian grey squirrels have adapted to their non-native range, showing two annual mating periods at times similar to those in their native range, and high reproductive success.

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.

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.

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze-drying rabbit (Oryctolagus cuniculus) spermatozoa.

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.

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.

PDGFA in Cashmere Goat: A Motivation for the Hair Follicle Stem Cells to Activate.

The cashmere hair follicle (HF) perpetually goes through cycles of growth, involution and rest. The photoperiod is the main factor in the control of seasonal coat change in cashmere goats while stem cells play a crucial role in the HF growth. Several factors, including Platelet-Derived Growth Factor A (PDGFA), Bone Morphogenetic Protein 2 (BMP2) and Lim-Homeobox gene 2 (LHX2) are implicated in HF morphogenesis and cycle. In this work, the mentioned molecules were investigated to evaluate their role in follicular cycle activation. The study was performed on skin samples collected at different periods of HF cycle and the molecular expression of PDGFA, BMP2 and LHX2 was evaluated by Real-Time PCR (qPCR) at each time point. Since PDGFA showed the most variation, the goat PDGFA gene was sequenced and the protein localization was investigated by immunohistochemistry together with PDGF receptor α (PDGFRα). PDGFA immunostaining was observed in the basal layer of the HF outer root sheath and the immunoreaction appeared stronger in the regressive HFs compared to those in the anagen phase according to qPCR analysis. PDGFRα was observed in the HF epithelium, proving the effect of PDGFA on the follicular structure. The data obtained suggest that PDGFA and BMP2 are both implicated in HF cycle in goat. In particular, PDGFA secreted by the HF is involved in the anagen activation.

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.

Apelin System in Mammary Gland of Sheep Reared in Semi-Natural Pastures of the Central Apennines.

Sheep are the most bred species in the Central Italy Apennine using the natural pastures as a trophic resource and grazing activity is fundamental to maintain the grassland biodiversity: this goal can be reached only ensuring an economical sustainability to the farmers. This study aimed to investigate the apelin/apelin receptor system in ovine mammary gland and to evaluate the differences induced by food supplementation, in order to shed light on this system function. A flock of 15 Comisana x Appenninica adult dry ewes were free to graze from June until pasture maximum flowering (MxF). From this period to pasture maximum dryness (MxD), in addition to grazing, the experimental group (Exp) was supplemented with 600 g/day/head of cereals. Apelin and apelin receptor were assessed by Real-Time PCR and immunohistochemistry on the mammary glands of subjects pertaining to MxF, MxD and Exp groups. They were detected in alveolar and ductal epithelial cells. The pasture maximum flowering group showed significant differences in apelin expression compared with experimental and MxD groups. Apelin receptor expression significantly differed among the three groups. The reduced apelin receptor expression and immunoreactivity levels during parenchyma involution enables us to hypothesize that apelin receptor plays a modulating role in the system control.