This paper describes serum and milk oxidative profiles during early postpartum (10th–16th day) of dairy cows, after three different treatments: b-carotene and a-tocopherol (Dalmavital, Fatro, Italy) (VAE Group); L-carnitine, d, L-acetylmethionine, cyanocobalamin (Adiuxan, Ceva Vetem, Italy) (LCAR Group) and Dalmavital + Adiuxan (VAE/LCAR Group). Antioxidant administrations resulted in a general reduction in ROS concentrations which decreased more after a single drug administration (VAE and LCAR Groups) vitamin A and E serum levels increased in the three treated groups compared to control one (p < 0.05). Milk lipoperoxide content was statistically lower in treated cows than in control ones, whereas, as to vitamins, only vitamin E increased in all the treated groups compared to control. Taken together, the results of this study confirm the efficacy of antioxidant administration in the early postpartum of dairy cows and suggest that an excess in antioxidant administration may result, paradoxically, in an increase in ROS generation.

This study investigates the mechanisms of action by which a GnRH analogue may modulate the contractility of the bovine ovarian follicular wall. The in vitro evaluation of the spontaneous basal contractility of bovine preovulatory and cystic follicles was performed, followed by testing the effects of lecirelin, a GnRH analogue, on their basal contractility. Strips of tissue in isolated organ bath were employed.In addition, to better investigate the mechanism of action of lecirelin, the study of the effects of cumulative doses of nifedipine (a calcium channel blocker), phentolamine (an α-adrenoceptor antagonist) and reserpine (an inhibitor of the vesicular up-take of catecholamines) alone and, at the highest doses employed, associated to lecirelin, was set up.The results demonstrate that in basal conditions and after the addition of lecirelin, the strips from preovulatory follicles contract significantly more than strips from cysts.Furthermore, among the patterns of contractility evoked by the three drugs employed, the one induced by nifedipine was the only one unaffected by the addition of lecirelin.The data obtained provide the hypothesis that one of the main mechanisms of action of GnRH, could involve calcium channels.

This in vitro study investigated the modulatory effect of rifaximin on bovine uterus contractility, in both phases of the oestrous cycle, with and without the steroid hormones that are predominant in the respective phases: oestrogen in the follicular phase and progesterone in the luteal phase. The procedures were conducted in an isolated organ bath by using rifaximin alone (104 M) and in association with the steroid hormone antagonists (105 M) tamoxifen (oestrogen antagonist) in the follicular phase and mifepristone (progesterone antagonist) in the luteal phase. The results indicated that rifaximin can stimulate uterine contractility. Indeed, the administration of rifaximin in the presence of tamoxifen or mifepristone increased the tonic activity of the uterus in both phases of the cycle. This result is clinically significant because rifaximin might also enable, in vivo, a simultaneous increase in uterine cleaning and the antimicrobial action against bacteria during the first 14 days postpartum, during the development of acute metritis.

Thyroid activity affects the functionality of the reproductive axis and thyroid dysfunction has been associated with ovarian hyperstimulation syndrome and polycystic ovarian syndrome, in human medicine. This study investigates serum17- < beta> estradiol, progesterone, thyrotropic and thyroid hormone levels, in cyclic dairy cows on heat (Group H) and in dairy cows with ovarian follicular cysts (Group FC). Both 17- < beta> estradiol and progesterone serum concentrations were statistically higher in cystic than in cyclic cows (estradiol: 8.51 ± 1.91 vs 6.32 ± 1 pg/mL) (progesterone: 0.49 ± 0.17 vs 0.13 ± 0.03 ng/mL), whereas TSH and fT4 serum concentrations were statistically lower in cows with cystic ovarian follicles (COF), compared to cyclic ones (TSH: 2.48 ± 1.31 vs 3.56 ± 1.03 ng/mL) (fT4: 5,86 ± 1.69 vs 8.63 ± 1.08). fT3 serum levels were similar, in both cystic and cyclic subjects (2.94 ± 0.65 vs 3.02 ± 0.9, respectively). Standing on these results, a look to the functioning of thyrotropic axis should be given in dairy cows with COF, just like it is routinely performed in human gynaecology, in case of ovarian diseases. If severe hypothyroidism should be found, a hormone replacement therapy could be attempted in cystic cows refractory to “ordinary” therapies.

Introduction: D-cloprostenol is a synthetic PGF2a, commonly used in bovine reproduction, that increases myometral contractility. However, little is known regarding its contractile behaviour and how it depends on the reproductive state and uterine topography (regions and muscular layers). Hypothesis and objectives: These aspects would affect the action of d-cloprostenol on the uterus. Therefore, we hypothesize a possible use of this drug at the time of artificial insemination, to improve conception rates and, in the post-partum, in order to accelerate uterine involution in dairy cattle. The purpose of the present study was to investigates the modulatory effect of d-cloprostenol on contractility of the bovine uterine region (horn and corpus) and their muscle layers (circular and longitudinal), in follicular and luteal phases. To our knowledge, motility effects of d-cloprostenol on different regions from healthy bovine uterus have not been described up to now. Materials and methods: Uterine specimens were collected from uterine body and horn of cattle in the follicular (n ¼ 28) and luteal phase (n ¼ 32) of the estrous cycle at slaughter. Two strips were prepared from each regions corresponding to the circular and the longitudinal muscular layers, respectively. Samples were cultured in an organ bath, exposed to synthetic prostaglandin (1 mM d-cloprostenol) and their contractile activity was monitored for 10 min. The functionality of the strip throughout the experiment was tested by a dose of carbachol (105 M). Results: The mean basal amplitude of contractions was higher in the follicular compared to the luteal phase in uterine horn samples, but not in muscles collected from the uterine body. The amplitude of contractions increased after d-cloprostenol administration in all tissues with a greater increase in samples from cattle in the follicular phase. The frequency of contractions increased after d-cloprostenol administration in longitudinal but not in circular fibrees. Conclusion: The contractile responses to d-cloprostenol in both horn and corpus were strongest in the circular muscles but weak in the longitudinal muscles.

Kisspeptin (Kp) and Kiss-1 receptor (Kiss-1R) expressions have been reported to be in the placenta, and a possible involvement of the Kiss-1R/Kps system in regulating trophoblast invasion and proliferation has been hypothesized. The aim of the present study was to investigate whether Kiss-1R activation by kisspeptin-10 (Kp-10) could modulate in vitro proliferation and progesterone (P4) secretion of bovine primary placental cell lines isolated from cotyledons of fetuses in the first trimester of pregnancy. The involvement of Kiss-1R in the cell responses observed was also analyzed. Uteri from cows at the first trimester of pregnancy were obtained from local abattoirs. Fetal cotyledon fragments were digested with collagenase in low glucose Dulbecco's Modified Eagle's Medium and cell lines were isolated. After being characterized for epithelial polygonal morphology, the presence of binucleate cells, male gender, and the expression of cytokeratin and zona occludens 2, cell lines were cultured in a low glucose Dulbecco's Modified Eagle's Medium–based expansion medium in the presence of 0.01, 0.1, 1, and 10 μM Kp-10. Control cells were cultured in the absence of Kp-10. Cell population doubling time was evaluated for each culture passage (P) from P1 to P10. Cells were tested for Kiss-1R mRNA expression analysis by real-time reverse transcription–polymerase chain reaction, and culture media were analyzed for P4 concentration by radioimmunoassay. Kisspeptin-10 modulated in vitro proliferation of epithelial cell lines isolated from cotyledons recovered from bovine fetuses in the first trimester of pregnancy. Inhibitory (line A) or stimulatory (line B) effects of Kp-10 on cell proliferation were found in different cell lines and observed cell responses were found to be related to Kiss-1R mRNA levels. Inhibition of cell proliferation matched with not significant variation of Kiss-1R expression, whereas stimulation of cell proliferation was found to be related to Kiss-1R upregulation. In both cell lines, no effect of Kp-10 on P4 secretion was found at any tested concentration. These results lead to the conclusion that the Kiss-1R/Kps system is involved in the regulation of cell proliferation of bovine placental cotyledon cell lines isolated at the first trimester of pregnancy but, at this gestational stage, it may not be involved in modulating placental P4 secretion.

Kisspeptin (Kp) and Kiss-1 receptor (Kiss-1R) expressions have been reported to be in the placenta, and a possible involvement of the Kiss-1R/Kps system in regulating trophoblast invasion and proliferation has been hypothesized. The aim of the present study was to investigate whether Kiss-1R activation by kisspeptin-10 (Kp-10) could modulate in vitro proliferation and progesterone (P4) secretion of bovine primary placental cell lines isolated from cotyledons of fetuses in the first trimester of pregnancy. The involvement of Kiss-1R in the cell responses observed was also analyzed. Uteri from cows at the first trimester of pregnancy were obtained from local abattoirs. Fetal cotyledon fragments were digested with collagenase in low glucose Dulbecco's Modified Eagle's Medium and cell lines were isolated. After being characterized for epithelial polygonal morphology, the presence of binucleate cells, male gender, and the expression of cytokeratin and zona occludens 2, cell lines were cultured in a low glucose Dulbecco's Modified Eagle's Medium-based expansion medium in the presence of 0.01, 0.1, 1, and 10 μM Kp-10. Control cells were cultured in the absence of Kp-10. Cell population doubling time was evaluated for each culture passage (P) from P1 to P10. Cells were tested for Kiss-1R mRNA expression analysis by real-time reverse transcription-polymerase chain reaction, and culture media were analyzed for P4 concentration by radioimmunoassay. Kisspeptin-10 modulated in vitro proliferation of epithelial cell lines isolated from cotyledons recovered from bovine fetuses in the first trimester of pregnancy. Inhibitory (line A) or stimulatory (line B) effects of Kp-10 on cell proliferation were found in different cell lines and observed cell responses were found to be related to Kiss-1R mRNA levels. Inhibition of cell proliferation matched with not significant variation of Kiss-1R expression, whereas stimulation of cell proliferation was found to be related to Kiss-1R upregulation. In both cell lines, no effect of Kp-10 on P4 secretion was found at any tested concentration. These results lead to the conclusion that the Kiss-1R/Kps system is involved in the regulation of cell proliferation of bovine placental cotyledon cell lines isolated at the first trimester of pregnancy but, at this gestational stage, it may not be involved in modulating placental P4 secretion.

Bovine follicular cysts are an ovarian disorder of dairy cows associated with abnormal estrous behaviour and infertility. The treatment of choice is intramuscular administration of a GnRH analogue, which acts by triggering pituitary release of LH. However, the presence of GnRH and GnRH receptors on spinal cord and ovary in some species, and the kind of innervation of the ovary, let us hypothesize that GnRH and its analogues may also act when administered by epidural route, as happens for other drugs. Therefore the aim of this study was to compare the effects of epidural vs intramuscular administration of lecirelin (a GnRH analogue) on FC regression, estrus detection and pregnancy outcomes.The study was conducted on 220 Friesian cows affected by follicular cysts, divided among 4 groups: Group Lepid and Group Lim received, respectively 50μg of lecirelin in the epidural space and intramuscular; Group Cepid and Group Cim were used as control groups. In Group Lepid, estrus induction and pregnancy rates were significantly higher than in Group Lim.The results of this study show that the epidural administration of lecirelin promoted the remission of follicular cysts and an improvement of reproductive parameters compared to intramuscular administration.Thus, an alternative therapeutical approach is available for FC treatment, in order to obtain an easier restoration of the ovarian activity, especially in those cases refractory to classical therapeutic approaches.

In dairy cows, the intensity of metabolic activity, associated with the negative energy balance (NEBAL), is responsible for an increased production of reactive oxygen species (ROS) and, subsequently, for the development of the condition of oxidative stress, which may overwhelm the antioxidant potential of the bovine maternal organism, making it prone to the development of many puerperal dysfunctions, as well as to an alteration of colostrum and milk quality. Given these premises, the aims of this study are to evaluate serum and milk concentrations of ROS and lipoperoxides, vitamins A and E, on the 10th, 12th, 14th and 16th day postpartum of dairy cows, a particularly critical period during which the NEBAL reaches its nadir, and to compare the trends of these parameters in two different bovine breeds. The study was performed in pluriparous Italian Friesian and Brown dairy cows. On the 10th day postpartum, all cows underwent a clinical examination to exclude the presence of alterations; furthermore, on the same day, a milk sample was collected from each cow, in order to perform the somatic cell count (SCC; (CE) N. 853/2004) and to establish which of them had an SCC ≤400 000/ml or >400 000/ml. In this study, among the 110 cows that were initially selected, the evaluation of these parameters allowed the inclusion of 80 animals, which were divided into four groups of 20 subjects each: Group F and F1: Italian Friesian healthy cows, with SCC ≤400 000/ml and >400 000/ml, respectively; Group B and B1: Italian Brown healthy cows, with SCC ≤400 000/ml and >400 000/ml, respectively. On the 10th, 12th, 14th and 16th day postpartum, peripheral blood and milk samples were collected. The results obtained show that in group B1 there were higher concentrations of ROS and milk antioxidants compared with Friesian group cows. This datum let us suppose that even in the presence of higher ROS concentrations the antioxidant status found in group B1 seems to be able to counteract the oxidative damage, which is more likely to develop in these cows.

Complex cytokine networks play an important role in a wide range of pregnancy-related processes. During physiological pregnancy, the balance of T helper lymphocytes Th1 and Th2 is strongly shifted toward Th2, which has a protective role in the feto–maternal interaction. The aim of the present study is to establish the serum concentrations, ranges, and trends of anti-inflammatory interleukin (IL)-4 and IL-10, and inflammatory IL-12A, during three phases of gestation in the bitch (20–30, 31–40, and 41–57 days of gestation). Our results indicate that, in early gestation, IL-4 and -10 serum concentrations are elevated and might depend on progesterone that could act as a potent inhibitor of Th1 responses inducing, conversely, the production of Th2-type cytokines (i.e., IL-10 and IL-4). On the other hand, between 30 and 40 days of gestation, the concentrations of the antiinflammatory ILs decrease probably because of high concentrations of prolactin, which is endowed with immunostimulatory properties on different immune cell types. In the third phase of gestation (41–57 days), an increase in IL-10 occurs, which might depend on high levels of 17b-estradiol that, during pregnancy, interfere with the ability of dendritic cells to stimulate T lymphocytes, acting as an anti-inflammatory factor. Conversely, the low and persistent concentrations of inflammatory IL-12A, throughout pregnancy, compared with anti-inflammatory ILs, might depend on the characteristic cytokine products of Thl and Th2 cells that are known to be mutually inhibitory. The monitoring of immunological status via the levels of cytokines during pregnancy in the bitch, could represent a diagnostic tool to predict and/or prevent pregnancy abnormality, as demonstrated in women

Background: The present study investigates the effects of high external calcium concentration ([Ca2+]o) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level. Methodology/Principal Findings: A large (.8 mm in diameter) and a small (,8 mm) cell line were cultured in medium containing: 1) low [Ca2+]o (0.37 mM); 2) high [Ca2+]o (2.87 mM); 3) NPS R-467 (3 mM) in presence of high [Ca2+]o and 4) the CaSR antagonist NPS 2390 (10 mM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca2+]o. Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca2+]o was not effective in this cell line. In small cells, both higher [Ca2+]o and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca2+]o and/or NPS R-467 reduced doubling time values. Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level. Conclusions/Significance: In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes.

This paper reviews puerperal metritis in the cow, particularly the complex and multi-factorial pathogenesis characterized by an altered cross-talk among infectious agents, endocrine and immune systems. Uterine infections impair fertility and is one of the main causes of economic losses in dairy production. The early postpartum is a period characterized by an increased exposition to infectious agents and the disruption of the metabolic homeostasis, leading to endocrine and immunologic disorders. Dysregulation of uterine defence mechanisms results in the development of metritis. Because there is a complex interaction between infectious, endocrine and immune factors during metritis, there is need to use safer and cheaper drugs which are able to strengthen the anti-infective actions of the routine therapies.

LH/hCG receptor has been found in extragonadal tissues in human and animals. The myometrium presents such receptors but their functional role is still not clear. Aim of our study was to test the activity of human chorionic gonadotropin (hCG) on bovine uterine contractility. Uterine strips from cows both during follicular and luteal phases were mounted in an organ bath and then exposed to increased doses of hCG. The amplitude of the myometrium contractions were significantly decreased in the follicular and luteal phase but the frequency was not affected. These findings prove a relaxing effect of hCG in the bovine uterus, as already shown in the sow and human, and its possible functional role in modulating uterine contractility.

Contractions of ovarian tunica albuginea, the teleostean cystovary wall layer containing smooth muscle fibres, facilitate oocytes and fluids movements within the ovary, oocytes ovulation and spawning. Fish isotocin, the homologue hormone of mammalian oxytocin, plays a significant role in ovulation, oviduct contraction and spawning. In the present study, ovarian wall spontaneous contraction, as well as isotocin in vitro effect on tunica albuginea contractility, was analysed in female seabream in different reproductive conditions: vitellogenesis, regressing (post-spawning) and extensive atresia. Tunica albuginea spontaneous contractility was recorded using ovary wall strips mounted in an organ bath containing modified Ringer's solution. The strips were then exposed to cumulative doses of isotocin (6, 30, 60 μg/ml). Female seabream in regressing condition exhibited the highest level of tunica albuginea spontaneous contraction amplitude compared with the other two groups. Only fish in vitellogenesis state showed a significant increase in contraction amplitude after isotocin administration at the dose of 30 μg/ml. The same group exhibited also a significant isotocin dose-dependent decrease in the contractile frequency. These results confirm the involvement of isotocin in stimulating tunica albuginea contractile activity during the oestrogen-regulated phase of vitellogenesis, whereas the absence of significant effects of isotocin on ovarian contractility in fish at the regressing state might be ascribed to the occurrence of a contractile activity autonomously regulated by the internal pacemaker system. The absence of exposed isotocin receptors could explain the lack of effects of the isotocin administration in seabream showed extensive atresia of the follicular cells.

The L-Arginine-Nitric Oxide Synthase-Nitric Oxide (L-Arg-NOS-NO) system exerts a pivotal role in the maintenance of uterine quiescence during pregnancy, whereas Homocysteine (Hcy) promotes uterine contractility. The aim of this study was to test the in vitro effects of L-Arg on spontaneous and Hcy-induced contractions of uteri excised from pregnant bitches. 104 strips cut from pregnant uteri were mounted in an organ bath. 40 out of 104 strips (16 from mid-gestation uteri and 24 from close to term uteri, respectively) were exposed to cumulative doses of L-Arg; 40 strips (16 from mid-gestation-uteri and 24 from close to term-uteri, respectively) were exposed to N-nitro-L-arginine methyl ester (L-NAME), a NOS antagonist; the remaining 24 strips (from close-to-term uteri) were first exposed to a single dose of Hcy and then to increasing doses of L-Arg. L-Arg showed no effects on spontaneous contractility both in mid-gestation- and close to term-uterine strips, whereas it promoted a relaxant effect on Hcy-induced contractility. On the contrary, L-NAME increased amplitude of contraction both in mid-gestation and close to term strips. These findings suggest that the L-Arg-NO system is present in the uterus of pregnant bitches and that Hcy is able to modulate its actions. Further investigation of this system may provide the basis of future obstetrical therapies in bitches.

Bovine follicular cysts (FCs) are a common cause of economic loss in modern dairy herds. Their aetiopathogenesis is not completely understood, even though an inadequate hypothalamic release of GnRH at the time of ovulation is considered to be their main cause. Much evidence, however, suggests a role for adrenergic innervation in ovarian functions, such as follicular development, steroid hormone secretion, and follicular contractility, the latter being an event important for ovulation. Moreover, in humans, polycystic ovary syndrome, a disease very similar to bovine follicular cysts, is characterised by increased density of adrenergic nerves. Given these premises, the aim of our study was to evaluate the effectiveness and mode of action of a novel strategy for the treatment of bovine follicular cysts. In the in vivo experiment, 170 Friesian cows diagnosed with follicular cysts were assigned to four groups (groups A, B, C, and D) to assess the effects of epidural administration of a β-adrenergic antagonist (carazolol) alone or in combination with a GnRH analogue (lecirelin). The four groups underwent the following treatments: Group A was administered lecirelin in combination with carazolol; Group B was administered carazolol; Group C was administered lecirelin; and Group D was administered only normal saline solution. In the in vitro experiment, strips of the walls of cystic follicles recovered post-mortem were suspended in an organ bath, connected to an isometric force transducer and exposed to increasing doses of epinephrine or to the same treatment after exposure to carazolol for 15 min (n = 10). The amplitude and frequency of the contractile activity were recorded. None of the control cows was observed in oestrus or was submitted to AI. The combination of lecirelin and carazolol induced a significant increase in the number of cows in oestrus (88%) compared to lecirelin alone or to carazolol alone (P < 0.05 and P < 0.01, respectively). The combination of lecirelin and carazolol and lecirelin alone were significantly more efficacious than carazolol alone (P < 0.01 and P < 0.05, respectively). In the in vitro experiment, epinephrine increased the amplitude of the contractions of the strips in a dose-dependent manner. This response was significantly enhanced in strips pre-treated with carazolol. The treatments had no effect on the frequency of contractions. In conclusion, our work demonstrates that the epidural administration of a GnRH analogue and a β-adrenergic blocker is an effective means of treating cows with cystic ovarian disease. Moreover, it confirms, from a clinical point of view, that alterations of the adrenergic system and of the contractility of the follicular wall can be considered aetiopathogenic factors involved in the development of FCs. The results of this study lay the basis for a new therapeutic approach to FCs.

Human mastocytosis are heterogeneous group of neoplastic diseases characterized by a different degree of uncontrolled mast cell (MC) proliferation and activation. Interestingly, human mastocytosis share several biological and clinical features with canine mast cell disorders, so called canine mast cell tumors (CMCTs). These CMCTs are the most common spontaneous cutaneous tumors found in dogs representing a valid model to study neoplastic mast cell disorders. It has been discovered that the pathological activation of c-Kit receptor (c-KitR), expressed by MCs, has been involved in the pathogenesis of neoplastic MC disorders. In this review we have focused on human mastocytosis in terms of: (i) epidemiology and classification; (ii) pathogenesis at molecular levels; (iii) clinical presentation. In addition, we have summarized animal models useful to study neoplastic MC disorders including CMCTs and murine transgenic models. Finally, we have revised therapeutic approaches mostly common in human and canine MCTs and novel tyrosine kinase inhibitors approved for CMCTs and recently translated in human clinical trials.

Background. Experimental evidence suggests a relationship between the vasodilatory effect of hCG and the NOS system in the testis. The influence of hCG administration on testicular vascular NOS gene expression has not been fully investigated. OBJECTIVE: This study aimed to evaluate the presence of the nitric oxide syntheses gene in ram testicular arteries and the influence of hCG administration on its expression. MATERIALS AND METHODS: Both testicular arteries of sixteen rams were extracted before and after i.v. administration of 5000 IU of hCG or placebo. The expression of the iNOS gene was investigated by real time PCR. Data were analyzed by means of Wilcoxon and Mann-Whitney tests. A p value of < 0.05 was considered statistically significant. RESULTS: PCR revealed the presence of iNOS mRNA in all basal samples but the expression of the iNOS gene was significantly reduced in all arteries obtained 24 h after the administration of either hCG or placebo. A significant reduction in the expression of iNOS gene was observed in the testicular arteries extracted after 24 h in both treated and placebo groups. On the other hand hCG stimulation did not significantly influence iNOS expression following its administration compared to a placebo. CONCLUSION: Ram testicular arteries express the iNOS gene but hCG stimulation did not significantly influence iNOS expression. A significant reduction in the expression of this gene was observed in the testicular arteries extracted after 24 h in both treated and placebo groups, suggesting that iNOS expression on the testicular artery could be influenced by the spermatic vessel ligation of the controlateral testis.

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the &gt;8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P &lt; 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the &lt;8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P &lt; 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P &lt; 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P &lt; 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes.

The calcium sensing receptor (CaSR) plays a key role in cells involved in calcium (Ca2+) homeostasis by directly sensing changes in extracellular Ca2+ concentration ([Ca2+]o), and external Ca2+ is a potent mediator of cell proliferation. The present study investigated the effects of high [Ca2+]o and of the CaSR agonist NPS R-467 on growth and proliferation of equine size-sieved umbilical cord matrix mesenchymal stem cells (UCM-MSC). The involvement of CaSR on observed cell response was analysed at the mRNA and protein level. Two subpopulations of UCM-MSC, isolated using multi-dishes with transwell inserts of 8-μm pores and expressing MSC markers (CD105, CD44, CD29; Corradetti et al. 2010 Reprod. Fertil. Dev. 22, 347–348), were analysed. Cells were cultured in medium containing: (A) low [Ca2+]o (0.37 mM), (B) high [Ca2+]o (2.87 mM), (C) NPS R-467 (3 μm) in the presence of high [Ca2+]o, and (D) the CaSR antagonist NPS 2390 (10 μm for 30′) followed by NPS R-467 in the presence of high [Ca2+]o. Growth and proliferation rates were compared among treatments (Student’s t-test). The CaSR expression and subcellular localization were investigated by real-time quantitative RT-PCR, immunofluorescence, and confocal microscopy. In the &gt;8-μm cell line, the addition of NPS R-467, in the presence of [Ca2+]o, significantly increased cell growth after day 7 of culture (C v. A and B; P &lt; 0.001). Increasing [Ca2+]o was not effective in this cell line (B v. A; not significant). In the &lt;8-μm cell line, NPS R-467 increased cell growth, even at a lower extent (C v. A; P &lt; 0.05), as observed on day 9 of culture. In this cell line, an increased proliferation rate was observed upon [Ca2+]o increase (B v. A; P &lt; 0.05). In both cell lines, preincubation with NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, a stimulatory effect of additional calcium and NPS R-467 on cell proliferation, in terms of reduced DT values, was observed. In the 2 cell lines, CaSR expression was down-regulated in the presence of high calcium and in NPS R-467-treated cells compared with controls (B and C v. A cells; P &lt; 0.001). Treatment with high calcium or NPS R-467 reduced CaSR labelling in the cytosol and increased it at the cortical level. We found that CaSR is expressed at mRNA and protein levels in equine UCM-MSC, and it is functionally active because the selective CaSR agonist NPS R-467 induced a stimulatory effect on cell growth and proliferation, which was reversed by the CaSR antagonist NPS 2390. The different responses to treatments between the 2 UCM-MSC subpopulations suggest that CaSR could be differentially activated in these cell lines. The calcimimetic NPS R-467 might be useful as an adjunctive component of media for UCM-MSC culture to obtain enough cells for down-stream purposes.

Leptin has been proven to be crucial in the ovulatory process. Aims of this study are to assess the expression of leptin receptor (Ob-R) in the ovaries of queens at estrus and to evaluate the capability of leptin in modulating ovarian contractility in vitro. Right ovaries underwent immunoblot analysis. Left ovaries were mounted in an organ bath under physiological condition and exposed to murine leptin (10(-6)M). Immunoblot analysis showed that the queen ovary expresses leptin receptor at estrus. Leptin at the dose of 10(-6)M significantly reduced the contractile activity of the ovary. The presence of ovarian Ob-R and leptin inhibitory effects on ovarian contractility suggest leptin implication in the modulation of ovarian activity, as well as in ovulatory disorders.

The receptor of the Thyroid Stimulating Hormone (TSHr) and thyroglobin (TGB), are two proteic factors necessary for the synthesis of hormones, in the thyrocite. In mammals, many immuno-histochemical reports indicate the presence of the TSHr in extra-thyroidal tissues, but not in the ovary. Triiodothyronine (T(3)) and thyroxine (T(4)) have been widely shown to affect ovarian functions and the synthesis of progesterone (P(4)). The aim of this study was to determine if by immunohistochemistry techniques TSHr and TGB could be found in the bovine corpora haemorragica, lutea and albicantia. A primary rabbit polyclonal antibody against human TSHr and a primary rabbit polyclonal antibody against human TGB were employed. Furthermore, the accuracy of bovine thyroid to the antibodies used in this study was tested. A positivity reaction for the anti-TSHr serum in the large luteal cells and immunostaining of both small and large luteal cells with the anti-TGB serum occurred only in mature corpora lutea. No immunostaining was detected in stromal cells, blood and lymphatic vessels and in corpora haemorragica and albicantia. Bovine thyroid tissue showed immunostaining to both the antibodies employed. These data suggest that the luteal cells of mature corpora lutea may be involved in the synthesis of thyroid hormones, which may modulate P(4) synthesis, acting in an autocrine and paracrine way.

This in vitro study investigates the modulatory effect of three antibiotics (amoxicillin, enrofloxacin, and rifaximin) on contractility of the bovine uterine tissue in follicular and luteal phases. The effects of these antibiotics at three single doses (106, 105, and 104 M) on their basal contractility were evaluated in isolated organ bath. The functionality of the strip throughout the experiment was evaluated by a dose of carbachol (105 M); the obtained effect had to be repeatable (difference of 20%) that is comparable to that induced by the previous administration of the same substance. The results demonstrate the different modulatory activities of these antibiotics on uterine contractility in follicular and luteal phases. The effects induced by amoxicillin and enrofloxacin are opposite: the first relaxes and the second increases the uterine contractility in both cycle phases. Instead, the activity of rifaximin varies depending on the phase of estrous cycle: it increases in the follicular phase and relaxes in the luteal phase. The obtained data provide the hypothesis of possible implications of these drugs in the pharmacologic modulation of uterine contractions. Their action at this level, associated with their specific antimicrobial effects, could suggest using these antibiotics for the treatment of diseases related to postpartum or infections that may occur in pregnant cattle, by virtue of their effects on myometrial contractility too.

During physiological pregnancy, all tissues and, mostly, placenta and foetus require high amounts of oxygen. Reactive oxygen species (ROS), generated both by mother and foetus, are implicated in foetal growth because they promote replication, differentiation and maturation of cells and organs. Nevertheless, ROS excess, if not properly counterbalanced, may lead to an alteration in cell constituents, with harmful effects both on mother and foetus.ROS exert a biphasic effect because adequate ROS concentration is essential for embryo development, implant, foetal defence against uterine infections, steroidogenesis, pregnancy maintainance and partum. On the other hand, an uncontrolled ROS generation, beyond physiological antioxidant defences, may lead to embryo resorption, placental degeneration with subsequent alteration in maternal-foetal exchanges, delay in foetal growth, pregnancy interruption, stillbirths. This review investigates the mechanisms underlying ROS generation and effects, throughout physiological and pathological pregnancy in sheep, with a look to antioxidants and their importance in such a critical phase of the reproductive cycle of the sheep.

Free radicals are highly reactive oxidizing agents containing one or more unpaired electrons. Both in human and veterinary neonathology, it is generally accepted that oxidative stress functions as an important catalysator of neonatal disease. Soon after birth, many sudden physiological and environmental conditions make the newborn vulnerable for the negative effects of oxidative stress, which potentially can impair neonatal vitality. As a clinician, it is important to have in depth knowledge about factors affecting maternal/neonatal oxidative status and the cascades of events that enrol when the neonate is subjected to oxidative stress. This report aims at providing clinicians with an up-to-date review about oxidative stress in neonates across animal species. It will be emphasized which handlings and treatments that are applied during neonatal care or resuscitation can actually impose oxidative stress upon the neonate. Views and opinions about maternal and/or neonatal antioxydative therapy will be shared.

Any peritoneal inflammatory process consequent to infections or surgical injuries may induce abdominal adhesion formation. Peritoneal adhesions are connective laciniae that develop among abdomino-pelvic organs that limit physiologic visceral motion. Consequently, fertility may be impaired, and intestinal obstruction and pelvic pain may develop, mainly in subjects that had undergone gynaecological surgery. This review illustrates the pathogenic steps of adhesiogenesis and the therapeutic scenario that evolved over the years to tackle the threat of peritoneal adhesions, both in domestic animals and in women.

Reactive oxygen species (ROS) are highly reactive oxidizing agents. Cells, under aerobic conditions, have a defence system against ROS, and in normal circumstances, there is an appropriate balance between pro-oxidants and antioxidants. When an overproduction of ROS develops or the body fails to eliminate ROS in excess, oxidative stress arises, during which ROS accumulate and damage cells and tissues. Besides their noxious effects, accumulating data have shown that controlled and adequate ROS concentrations exert physiologic functions. Different studies have confirmed the presence of ROS and the transcripts of the various antioxidant enzymes in the female reproductive tract. When ROS production overwhelms antioxidant defences, oxidative stress occurs, which may deeply threaten the anatomical and functional integrity of the genital tract. This review addresses the main physiological and pathological roles exerted by ROS and their scavenging systems in several processes involved in the main physiological functions of the female reproductive tract of both women and domestic animals. Particularly, the involvement of the oxidant system in several reproductive processes is investigated, such as follicular development, ovarian steroidogenesis, ovulation, corpus luteum formation and function, luteolysis, germ cell function, maintenance of pregnancy and beginning of parturition.

Angiogenesis and signaling through the RASlRAF/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERKcascade have been reported to play important roles in the development of hepatocellular carcinoma (HCC). Sorafenib (Nexavar), a novel bi-aryl urea SAY 43-9006, is an orally administered multikinase inhibitor with activity against RASIRAF kinases multikinase inhibitor with activity against RAP kinases and several receptor tyrosine kinases, including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (pDGFR), FLT3, Ret, and c-Kit. It is involved in angiogenic pathway and cell proliferation. Sorafenib has demonstrated potent anti-tumor activity in in vitro studies, preclinical xenograft models of different tumor types and human clinical trials. This review summarizes the history of sorafenib from its discovery by the medicinal chemistry approach through to clinical development and ongoing trials on the combination between sorafenib and trans-arterial chemoembolization (TACE) in HCC patients.

Leptin is a Ob gene product secreted mainly by adipose tissue. Several reports showed leptin production by other tissue including the ovary. The action of leptin is mediated upon binding to its receptor widely expressed in reproductive tissues in different species. In fact, there are growing evidences that leptin plays an important role in the modulation of reproductive functions. Therefore, the aim of this study was to evaluate in the queen, the expression of leptin receptor during the functional ovarian cycle and pregnancy. We found that the ovaries of the queen express leptin receptor in all the examined phases. The highest leptin receptor expression was found in the luteal phase (pseudopregnancy, pregnancy) compared to other phases of the cycle (anestrus, proestrus, estrus). The variations in the expression of leptin receptor suggest a likely implication of leptin in the modulation of ovarian activity, in the examined species.

This manuscript investigates, by Color Doppler ultrasonography, the basal hemodynamic features of the left cranial epigastric vein (milk vein) of 210 Holstein–Friesian cows, throughout lactation. Blood velocity, diameter, blood flow of the milk vein (before and after milking) and milk production detected in cows administered oxytocin were compared with the same parameters found in untreated cows. Blood velocity tended to decrease throughout lactation as did milk production, whereas both diameters and blood flows tended to increase towards the end of the study. All the four parameters showed higher values in the treated cows than in the untreated ones. This study provides a detailed picture of the hemodynamic features of the milk vein of the dairy cows throughout lactation and suggest that oxytocin may exert a vasodilatory effect, in vivo, at least on the cranial epigastric vein of the dairy cow.

The Gonadotropin-Releasing Hormone (GnRH) exists in two isoforms, GnRH-I and GnRH-II, in most vertebrates, including humans. Both of these isoforms and their respective receptors have been found in many healthy and pathologic extra nervous system tissues, such as cells found in cancers of the reproductive systems and, in particular, in breast cancer. GnRH analogues are used as therapeutic agents in the case of sex-hormone-dependent tumours. Besides acting as suppressors of steroidogenesis, GnRH analogues seem to interfere with mitogenic signal transduction pathways, thus behaving as negative regulators of tumour growth and progression. GnRH analogues counteract the proliferating effects of both epidermal growth factor (EGF) and insulin like growth factor (IGF-I); additionally, it affects the mitogen-activated protein kinase (MAPK) cascade and modulates the activity of the urokinase-type plasminogen activator (uPA)/plasminogen activator inhibitory (PAI) system, which is involved in the process of metastasis. In addition, GnRH analogues decrease the expression of many growth factors involved in the development of human uterine myomas (as well as endometriotic tissue), such as the vascular endothelial growth factor (VEGF), which is deeply implied in the angiogenesis of many benign and malignant tumours, including breast cancer. Angiogenesis is one of the primary processes leading to the progression and metastasis of breast cancer cells, and a key therapeutic goal in the fight against tumours is the blocking of new vessel sprouts. Given these premises, this review provides an update on the background of anti-neoplastic properties of GnRH analogues.

In infants delivered by cesarian section, the immune system could be influenced by anesthetic drugs that may cross the placenta and reach the fetal circulation. Factors that determine placental transfer of anesthetics and their distribution into and ultimate removal from fetal tissue include physiochemical properties of the compound; the anatomic features of the maternal circulation, placenta, and fetus; and the hemodynamic and pharmacokinetic events that occur within them. Because the anesthetic agents can interfere with many neonatal function, the aim of this manuscript is to review the interference of regional or general anesthesia on neonatal immune response

Background. Energetic substrates and hormonal phase are important for uterine contractions. Etomoxir a muscle carnitine palmitoyltransferase-1 inhibitor, able to diverge uterine metabolic pathways towards glycolysis, facilitates glucose utilisation. The aim of this study was to evaluate its effect on uterine contractility in different hormonal situations. Methods. Uterine samples were collected from 60 cows during follicular phase, luteal phase and pregnancy. The cows were slaughtered at a local abattoir. Longitudinal strips were mounted vertically in a 30-ml organ bath connected to an isometric force transducer. Contractions were recorded with an ink-writing polygraph. After the equilibration period, etomoxir was added to the organ bath at different concentrations. The amplitude and frequency of contractions were registered before and after addition of etomoxir. Results. In 17 strips from pregnant cows, etomoxir increased the amplitude (p < 0.05) of contractions but not the frequency in comparison with basal conditions. In 15 strips from cows in the luteal phase, etomoxir increased the amplitude (p < 0.05) and frequency of contractions (p < 0.05 at 5 μM and p < 0.01 at 8 and 10 μM). In 18 strips from cows in the follicular phase, etomoxir increased the frequency of contractions but not the amplitude (p < 0.01 at 5 μM and p < 0.05 at 8 and 10 μM). Conclusions. This study confirms the importance of glucose for uterine contractility and, moreover, it underlines different patterns of contraction with regard to the hormonal status.