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Apelin, a member of the adipokine family, is a novel endogenous peptide which regulates the male reproduction system of men and male laboratory animals by interacting with a specific receptor. Recent studies have highlighted that apelin may play a role in the regulation of reproduction by reducing testerone production and inhibiting LH secretion. To the best of our knowledge, there is no available data on the presence of the apelin and its receptor in canine testes. Therefore, the aim of this study was to reveal its presence and evaluate its distribution in the canine testes using immunohistochemical and RT-PCR techniques. For this purpose, five fertile and healthy male dogs were subjected to elective orchiectomy. The immunohistochemical reaction revealed the presence of apelin and its receptor in the canine testes. Apelin was localized in spermatids and spermatozoa with a positive signal in the “acrosomal bodies”. As regards the apelin receptor, a positive immunoreaction was detected in the cytoplasm of the cells localized near to the basal membrane of the seminiferous tubules and in the cytoplasm of Leydig cells. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all of the samples under study. A 35 kDa band confirmed apelin receptor protein expression in all of the samples analysed. In conclusion, the paracrine and endocrine role of apelin and its cognate receptor on male reproduction reported in humans and laboratory animals, could also be hypothesized in dogs.

The objectives of this study were to evaluate in horse testes the expression of kisspeptin (KiSS) and GnRH1 neuropeptides and their cognate receptors, KiSS1R and GnRH1R, as well as their action on testosterone, GnRH1, prostaglandin F2α (PGF2α), and PGE2 synthesis and cyclooxygenase 1 (COX1) and COX2 activity by Leydig cells in vitro. Testes were obtained from 9 sexually mature horses by surgical castration. Immunohistochemistry, evidenced the presence of KiSS, KiSS1R, GnRH, and GnRH1R in Leydig cells, whereas germinal and Sertoli cells were positive only for GnRH1. Transcripts for both neuropeptides and their cognate receptors were revealed in isolated Leydig cells by RT-PCR. Isolated and purified Leydig cells were in vitro cultured with agonists and antagonists of KiSS (KiSS-10 and KiSS-234, respectively) and GnRH1 (buserelin and antide, respectively). KiSS-10 and buserelin increased (P 0.01) COX1 activity and testosterone and PGF2α basal secretion, while decreased (P 0.01) that of PGE2. KiSS-10 and buserelin did not affect COX2 activity. GnRH1 basal production was increased (P 0.01) by KiSS-10, but not by buserelin. Antide counteracted the KiSS and GnRH1 effects, whereas KiSS-234 influence only those of KiSS. Summarizing, the KiSS/GnRH1 system is present in horse Leydig cells and modulates their endocrine activity. In particular, the endocrine effects of KiSS are mediated by GnRH1, so suggesting that hypothalamic-like interaction between KiSS and GnRH1 occurs also in Leydig cells.

Apelin, a member of the adipokine family, is a new endogenous peptide and it’s involved, by interacting with a specific receptor, in the control of human and laboratory animals’male reproduction. As far as we know, no data is available about the presence of the apelinergic system in dog testicles. Therefore the aim of this study was to show, for the first time, its presence and distribution in the canine testis by immunoistochemistry and molecular biology studies. For this purpose, five fertile and healthy male dogs were used and subjected to elective orchiectomy. The immunohistochemical reaction evidenced the presence of apelin and its receptor in the canine testis. In particular, apelin appeared localized in spermatids and spermatozoa with a positive signal in the “acrosomal bodies”. Regarding to the apelin receptor, a positive immunoreaction was evidenced in the cytoplasm of the cells localized in the basal portion of the seminiferous tubules, likely Sertoli’s cells, and in the cytoplasm of Leydig cells. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all the samples examined. In conclusion our result allows us to hypothesize that the role of the paracrine and endocrine apelinergic system reported in laboratory animals can also be found in the dog.

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.

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 &lt, 0.01) and 2.3-fold less (p &lt, 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.

In addition to the well-known central modulatory role of orexins, we recently demonstrated a peripheral involvement in swine granulosa cells for orexin A and in adipose tissue for orexin B (OXB). The aim of present research was to verify immunolocalization of OXB and its potential role in modulating the main features of swine granulosa cells. In particular, we explored the effects on granulosa cell proliferation (through the incorporation of bromodeoxyuridine), cell metabolic activity (as indirect evaluation by the assessment of ATP), steroidogenic activity (by immunoenzymatic examination) and redox status (evaluating the production of superoxide anion by means of the WST test, production of nitric oxide through the use of the Griess test and the non-enzymatic reducing power by FRAP test). Our data point out that OXB does not modify granulosa cell growth, steroidogenesis and superoxide anion generation. On the contrary, the peptide stimulates (p &lt, 0.05) nitric oxide output and non-enzymatic reducing power. Since new vessel growth is crucial for ovarian follicle development, a further aim of this study was to explore the expression of prepro-orexin and the effects of OXB on swine aortic endothelial cells. We found that the peptide is ineffective in modulating cell growth, while it inhibits redox status parameters. In addition, we demonstrated a stimulatory effect on angiogenesis evaluated in fibrin gel angiogenesis assay. Taken together, OXB appears to be potentially involved in the modulation of redox status in granulosa and endothelial cells and we could argue an involvement of the peptide in the follicular angiogenic events.

The aim of this study was to evaluate the effect of dietary polyphenols on the expression of the effectors involved in inflammation and apoptosis in rabbit ovary. New Zealand White female rabbits were fed a basal control diet (CTR), or the same diet supplemented with a polyphenolic concentrate (POL, 282.4 mg/kg) obtained from olive mill waste waters. The follicle counts and the relative mRNA (RT-qPCR) and protein (immunohistochemistry) expression of the effectors involved in inflammation (cyclooxygenase-2, interleukin-1beta, tumor necrosis factor-alpha, TNFA) and apoptosis (BCL2-associated X protein, BAX), detected in the ovaries of both groups, were examined. The POL diet increased the primary and total follicles number. Cyclooxygenase-2 gene expression was higher (p &lt, 0.05) in the POL group than in the CTR group, whereas BAX was lower (p &lt, 0.05) in POL than CTR. Immunohistochemistry revealed the presence of all the proteins examined, with weaker (p &lt, 0.05) COX2 and BAX signals in POL. No differences between the CTR and POL groups were observed for IL1B and TNFA gene and protein expression. These preliminary findings show that dietary polyphenols modulate inflammatory and apoptotic activities in rabbit ovary, regulating cyclooxygenase-2 and BAX expression, thus suggesting a functional involvement of these dietary compounds in mammalian reproduction.

Simple Summary Invasive alien species pose a significant threat to biodiversity, as once they have adapted to their new environment, they cause the reduction and even extinction of native species. In this framework, the American grey squirrel (Sciurus carolinensis) poses a serious threat to the European red species squirrel (Sciurus vulgaris), especially in the Umbria region of Italy. In fact, an invasive grey squirrel population has adapted well to the Umbrian territory, showing high reproductive success. In addition to its role in the development of the vertebrate nervous system, nerve growth factor (NGF) has recently been found to play an important role in reproduction. In order to investigate the reproductive physiology of female grey squirrels, the ovarian presence, distribution, and gene expression of NGF and its cognate receptors were evaluated during both breeding and nonbreeding seasons. The presence and gene expression of this system at the ovarian level, mainly during the breeding season, confirm the possible involvement of NGF and its receptors in the gonadal activity of this invasive grey squirrel population. Abstract 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.

Simple Summary The wildlife expansion in the Italian Apennines caused a general development in hunting activities, together with the necessity of using biometry (size analysis) and geometric morphometry (shape analysis) as methods for monitoring the status of wildlife populations. Thus, in the last decades, study of the sizes and shapes of structures in wildlife populations has been extensively investigated. Biometric surveys and analysis of the resulting cranial and body data are now crucial in management decisions and new possibilities of improving datasets should be considered. Thus, we attempted to identify a conversion factor between shape and size analysis methods, using the mandible of adult roe deer as a bone model. The availability of this conversion factor enhances the numerousness of parameters into the classical biometric database, by means of the conversion of shape measures into size measures. Therefore, the relationship among biometric parameters, animal and environmental features can be better studied. The obtained data can be very useful to assess both wildlife population status and its management. Abstract 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.

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.

Apelin is a potent inotropic agent causing endothelium-mediated vasodilation and is involved in vessel formation by interacting with a specific receptor. Its cardiovascular profile suggests a role in the regulation of gestational hemodynamic changes. The expression of apelin and its receptor has been reported in some portions of the reproductive tract of different mammalian species. As far as we know, there are no reports describing the expression of apelin and apelin receptor in bitch’s placenta. Therefore, the aim of this study was to investigate, for the first time, the presence and distribution of apelin and apelin receptor in bitch placenta by molecular biology and immunohistochemical techniques. Sixteen adult female half-breed bitches were used. The animals were divided into two groups based on the stage of pregnancy: group 1 (mid-gestation n = 8) and group 2 (end gestation n = 8). These bitches were subjected to ovariohysterectomy (group1) or non-conservative caesarean section (group 2). The immunohistochemical technique revealed the presence of positive immune reaction for apelin and apelin receptor in all the samples examined at 30 days and at the end of pregnancy. In particular, apelin and apelin receptor staining was evident in the cytoplasms of cytotrophoblasts and in epithelial cells of the maternal portion. Even if not included into the structure of the placenta, the uterine glands also exhibited a positive immune reaction for apelin and apelin receptor. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all the placenta samples examined. On the basis of our results it was also possible to hypothesize a potential role of apelin in the control of local placenta blood flow during pregnancy development in bitches.

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&ndash, 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.

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.

Trace element status and metabolic milieu are sometimes overlooked in common veterinary clinical practice across animal species. The evaluation of requirements of trace elements, in fact, may be useful to prevent the perturbation of tissue-specific metabolic impair. In particular, essential trace elements in the diet play key roles within sub-cellular metabolic patterns with macro effects at the systemic level, like blood cell stability and semen quality. This effect was studied in breeding stallions, in which semen quality and haemogram are important for reproduction. A case-control feeding trial involved 40 stallions (age: 8–21 years; body weight, BW: 510–531 kg) of one stud centre, allotted to two experimental groups (n = 20 control, CON vs. n = 20 supplemented, SUPPL100), following a matched-pairs approach based on age. Supplemented stallions (SUPPL100) received a mixed mineral and vitamin supplement of Zn/Se and α-tocopherol (α-TOH) (100 g/day stallion) to compound feed, fed as control diet to horses of the control group (CON). Horses resulted deficient in circulating α-TOH and Zn at the start, though clinically healthy. After supplementation, different plasmatic levels of α-TOH, Zn and Se were found between groups. Circulating basophils (BASO) and mean cell haemoglobin concentration (MCHC) were affected by the dietary treatment (p 13 years marginal circulating levels of α-TOH (p = 0.04). Ameliorating the micromineral status showed to improve the haemogram of stallions in view of circulating levels of Cu. Semen quality appeared to be strongly dependent on animal effects.

In the present study, we evaluated the dynamic changes of intra-ovarian blood flow, by real-time colour-coded and pulsed Doppler ultrasonography, as well as the immunopresence of prostaglandin F2α (PGF2α) receptor (FP) and peripheral plasma progesterone concentrations in pseudopregnant rabbit after PGF2α treatments at either early- (4 days) and mid-luteal (9 days) stages. During the pre-treatment observation interval of one hour, the ovarian blood flows showed a fluctuating pattern. Independently of luteal stage, PGF2α administration caused a fourfold decline in the blood flow within 40 min that was followed 50 min later by a reactive hyperaemia that lasted several hours, while the resistive index showed an opposite trend. Twenty-four hour later, the blood flow was one half that measured before PGF2α injection. At day 4 of pseudopregnancy, PGF2α did not affect peripheral plasma progesterone concentrations, but at day 9, it caused functional luteolysis as progesterone levels declined 6 hr later to reach basal values after 24 hr. The changes in the ovarian blood flows of pseudopregnant rabbits receiving PGF2α were accompanied by simultaneous changes in the resistance index. This biphasic response in the blood flow and vascular resistances likely reflects reactive hyperaemia following vasoconstriction. By immunohistochemistry, strong positive immune reaction for FP was detected in the cytoplasm of endothelial cells of ovarian arteries, veins and capillaries. In conclusion, these results suggest that PGF2α could acutely regulate the ovarian blood flow of pseudopregnant rabbits, even if there is no evidence of a blood flow reduction anticipating luteolysis.

Several functions are acknowledged for saliva secretion in different animal species following prehension and mastication of feed. Most of such are linked to the specific role of lubrication and softening of the bolus to allow taste perception and easy swallowing. Moreover, enzymatic components are produced in the saliva, some of which are destined to contribute to the digestion of different nutrients (to various extents according to animal species) and to exert antimicrobial activity (lysozyme). In addition, the buffering power and the virtuous recycle of water, electrolytes, and other metabolites are of particular importance for proper digestion and for nutrition–related aspects. Moreover, salivation appears to be involved in a number of other functions. Recent studies on salivary production and roles point to salivary glands as target organs of neuroendocrine regulation in response to many external stimuli coming from the outer world, for which feed still represents the chief external stimulus. Various animal species establish an adaptive strategy when coming into contact with different feeding stuffs and/or dietary substances by modifying both the composition and amount of saliva produced. In the light of recent updates, this review provides a focus on the functional roles of saliva secretions, showing the broad involvement of salivary response in several mechanisms beyond the digestive function and influencing feed selection.

This study investigated the location of hyaluronic acid (HA)- and chondroitin sulphate (CS)-coated gold nanoparticles in rabbit bladder and evaluated gene expression of CD44, RHAMM and ICAM-1 receptors involved in HA and CS transport into the cell. Gold nanoparticles were synthesised by reduction of gold salts with HA or CS to form HA-AuNPs and CS-AuNPs. Bladder samples were incubated with CS-AuNPs and HA-AuNPs or without glycosaminoglycans. Transmission electron microscopy, optic microscopy and scanning electron microscopy were used to determine the location of the synthesised AuNPs. Real-time PCR was used to analyse expression of urothelial cell receptors CD44, RHAMM, ICAM-1, after ex vivo administration of CS-AuNPs and HA-AuNPs. We showed that HA-AuNPs and CS-AuNPs were located in the cytoplasm and tight junctions of urothelial umbrella cells; this appearance was absent in untreated bladders. There were no significant differences in gene expression levels for CD44, RHAMM and ICAM-1 receptors in treated versus control bladder tissues. In conclusion, we clearly showed the presence of exogenous GAGs in the bladder surface and the tight junctions between umbrella cells, which is important in the regeneration pathway of the urothelium. The GAGs-AuNPs offer a promising approach to understanding the biophysical properties and imaging of urothelial tissue.

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.

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 (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.