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The last 18 years have brought an increasing interest in the therapeutic use of perinatal derivatives (PnD). Preclinical studies used to assess the potential of PnD therapy include a broad range of study designs. The COST SPRINT Action (CA17116) aims to provide systematic and comprehensive reviews of preclinical studies for the understanding of the therapeutic potential and mechanisms of PnD in diseases and injuries that benefit from PnD therapy. Here we describe the publication search and data mining, extraction, and synthesis strategies employed to collect and prepare the published data selected for meta-analyses and reviews of the efficacy of PnD therapies for different diseases and injuries. A coordinated effort was made to prepare the data suitable to make statements for the treatment efficacy of the different types of PnD, routes, time points, and frequencies of administration, and the dosage based on clinically relevant effects resulting in clear increase, recovery or amelioration of the specific tissue or organ function. According to recently proposed guidelines, the harmonization of the nomenclature of PnD types will allow for the assessment of the most efficient treatments in various disease models. Experts within the COST SPRINT Action (CA17116), together with external collaborators, are doing the meta-analyses and reviews using the data prepared with the strategies presented here in the relevant disease or research fields. Our final aim is to provide standards to assess the safety and clinical benefit of PnD and to minimize redundancy in the use of animal models following the 3R principles for animal experimentation. [ABSTRACT FROM AUTHOR]

Context: Ovarian quiescence can be due to hormonal deficiency usually caused by apoptosis of granulosa cells responsible for oestrogen synthesis. Aim: This study evaluated the regenerative effect of platelet rich plasma (PRP) on bovine in vitro models to understand its effect on granulosa cells. Methods: Quiescent and healthy ovarian sections were cultured in the presence/absence of PRP for 72 h and, at different times (0, 24, 48 and 72 h), hematoxylin-eosin and immunohistochemical detection of Ki-67 were performed. Additionally, granulosa cells collected from healthy bovine ovaries were stressed with 100 ng/mL of lipopolysaccharide (LPS) in presence/absence of PRP and evaluated at 0, 4, 8 and 24 h for apoptosis by acridine orange and propidium iodide staining. Enzyme-linked immunosorbent assay tests were performed to evaluate oestrogen (E2) and anti-Müllerian hormone (AMH) concentrations on cultures of ovarian slices and granulosa cells. Key results: In slides of quiescent ovaries treated with PRP, a marked and widespread positivity to Ki-67 was expressed by 40–60% of the follicular wall cells at 48 h of culture. Levels of E2 and AMH were significantly higher compared to untreated quiescent samples reaching the levels of healthy control samples. PRP counteracted the LPS effect and apoptosis (at 24 h, there were 93.44 ± 3.51% live cells with LPS + PRP compared to 37 ± 1.32% with LPS) and significantly increased concentrations of E2 and AMH. Conclusions: PRP can stimulate granulosa cell proliferation and counteract inflammatory processes in vitro. Implications: This treatment could improve the reproductive ability of quiescent females. Ovarian quiescence in women and in animals can be due to hormonal deficiency caused by apoptosis of granulosa cells responsible for hormone synthesis. In an alternative to the classical approach with hormonal treatment, regenerative medicine by platelet rich plasma could be a new treatment available. In this study, an in vitro model represented by ovarian slices and granulosa cells treated by platelet rich plasma stimulated granulosa cell proliferation and increased hormone levels. This treatment could improve the reproductive ability of quiescent females. (a) Green apoptotic granulosa cells with membrane budding and red dead granulose cell. (b) Live granulosa cells with defined margins after platelet rich plasma treatment. [ABSTRACT FROM AUTHOR]

Persistent post-breeding induced endometritis (PPBIE) is considered a major cause of subfertility in mares. It consists of persistent or delayed uterine inflammation in susceptible mares. There are many options for the treatment of PPBIE, but in this study, a novel approach aimed at preventing the onset of PPBIE was investigated. Stallion semen was supplemented with extracellular vesicles derived from amniotic mesenchymal stromal cells (AMSC-EVs) at the time of insemination to prevent or limit the development of PPBIE. Before use in mares, a dose–response curve was produced to evaluate the effect of AMSC-EVs on spermatozoa, and an optimal concentration of 400 × 106 EVs with 10 × 106 spermatozoa/mL was identified. At this concentration, sperm mobility parameters were not negatively affected. Sixteen susceptible mares were enrolled and inseminated with semen (n = 8; control group) or with semen supplemented with EVs (n = 8; EV group). The supplementation of AMSC-EVs to semen resulted in a reduction in polymorphonuclear neutrophil (PMN) infiltration as well as intrauterine fluid accumulation (IUF; p < 0.05). There was a significant reduction in intrauterine cytokine levels (p < 0.05) for TNF-α and IL-6 and an increase in anti-inflammatory IL-10 in mares in the EV group, suggesting successful modulation of the post-insemination inflammatory response. This procedure may be useful for mares susceptible to PPBIE. [ABSTRACT FROM AUTHOR]

Abstract Background The identification of the adipocyte-derived obesity gene product, leptin (Ob), and subsequently its association with reproduction in rodents and humans led to speculations that leptin may be involved in the regulation of oocyte and preimplantation embryo development. In mice and pigs, in vitro leptin addition significantly increased meiotic resumption and promoted preimplantation embryo development in a dose-dependent manner. This study was conducted to determine whether leptin supplementation during in vitro maturation (IVM) to horse oocytes could have effects on their developmental capacity after fertilization by IntraCytoplasmic Sperm Injection (ICSI). Methods Compact and expanded-cumulus horse oocytes were matured in medium containing different concentrations (1, 10, 100, 1000 ng/ml) of recombinant human leptin and the effects on maturation, fertilization and embryo cleavage were evaluated. Furthermore, early developmental expression of Ob and leptin receptor (Ob-R) was investigated by immunocytochemical staining. Results In expanded-cumulus oocytes, the addition of leptin in IVM medium improved maturation (74% vs 44%, for 100 ng/ml leptin-treated and control groups, respectively; P < 0.05) and fertilization after ICSI (56% vs 23% for 10 ng/ml leptin-treated and control groups, respectively; P < 0.05). However, the developmental rate and quality of 8-cell stage embryos derived from leptin-treated oocytes (100 ng/ml) was significantly reduced, in contrast to previous data in other species where leptin increased embryo cleavage. Ob and Ob-R proteins were detected up to the 8-cell stage with cortical and cytoplasmic granule-like distribution pattern in each blastomere. Conclusion Leptin plays a cumulus cell-mediated role in the regulation of oocyte maturation in the mare. Species-specific differences may exist in oocyte sensitivity to leptin.

The current research was designed to evaluate if intra-ovarian administration of autologous platelet rich plasma (PRP) before superovulation could increase the number of follicles responsive to gonadotropin treatment in order to improve embryo recovery in donor cows. Eight Holstein-Friesian cows of proven fertility were employed. After estrous synchronization, at the 18th day of diestrous, the right ovary of each cow was left untreated and served as control while the left ovary was inoculated with 5 mL of PRP. Cows were left to spontaneously return to estrous, and nine days later, a standard superovulation was initiated for every cow. Seven days after artificial insemination (AI), putative embryos were collected by flushing the right and left uterine horns separately. All statistics were calculated by ANOVA. The mean number of follicles, evaluated by transrectal ultrasound scanning, did not statistically differ before PRP treatment between right (control) and left (treated) ovaries (9.18 ± 1.35 and 7.32 ± 1.67, p = 0.28, respectively) as well as at 48 h after PRP injection (7.67 ± 2.52 and 8.00 ± 2.00, p = 0.73, respectively). A statistical (p = 0.023) difference was found in the average number of follicles at the last gonadotropin injection between control and treated ovaries (11.33 ± 2.89 and 20.00 ± 9.17, respectively). The statistically different (p = 0.0037) number of grade 1-2 blastocysts harvested from the uterine horn ipsilateral to control ovaries in comparison to that collected from the treated ones (6.63 ± 2.92 and 14.75 ± 5.92, respectively) suggests that intra-ovarian injection of PRP before superovulation could exert beneficial effects both in latent follicle growth and in vivo embryo production. [ABSTRACT FROM AUTHOR]

In mammals, the sex of the embryo depends on the SRY gene. In the presence of at least one intact and functional copy of this genetic factor (XY embryo) undifferentiated gonads will develop as testicles that subsequently determine the male phenotype. When this factor is not present, i.e., in subjects with 2 X chromosomes, an alternative pathway induces the development of ovaries, hence a female phenotype. In this case study, we describe a female cattle affected by a disorder of sex development (DSD). The subject, despite having a chromosomal XY constitution, did not develop testicles but ovaries, although they were underdeveloped. Moreover, genetic analysis highlighted the presence of the SRY gene with a normal coding region in both blood- and tissue-derived DNA. A chimeric condition was excluded in blood by sexing more than 350 cells and by allele profile investigation of 18 microsatellite markers. Array CGH analysis showed the presence of a not yet described 99-kb duplication (BTA18), but its relationship with the phenotype remains to be demonstrated. Gonadal histology demonstrated paired ovaries: the left one containing a large corpus luteum and the right one showing an underdeveloped aspect and very few early follicles. To our knowledge, we describe the first case of XY (SRY+) DSD in cattle with a normal SRY gene coding sequence. [ABSTRACT FROM AUTHOR]

The effect of conditioned medium (CM) or microvesicles (MVs), secreted by multicellular spheroids of oviductal cells, and the involvement of some microRNAs (miRNAs) were investigated in canine oocyte maturation. To generate CM, spheroids were cultured for 3 days. MVs were obtained by ultracentrifugation of CM at 100,000 g and measured for size and concentration by NanoSight instrument. Cumulus-oocyte complexes (COCs) were matured at 38.5°C with 5% CO2 and 5% of O2 in synthetic oviductal fluid (SOF) in biphasic systems: for 24 h, with 5.0 μg/mL of LH and for other 48 h with 10% oestrous bitch serum. SOF was used as control (CTR) or supplemented with 10% CM or 25-50-75-100-150×106 MVs/mL labeled with PKH-26. Results show that multicellular aggregates secreted shedding vesicles. By fluorescence microscopy, the incorporation of labeled MVs was visible only at 72 h in oocyte cytoplasm. These MVs had a positive effect (P < 0.05) on maturation rate (MII) at the concentration of 75 and 100×106 MVs/ mL compared to CM and CTR (20.34% and 21.82% vs 9.09% and 8.66% respectively). The concentration of 150×106 MVs/mL provided only 9.26% of MII. The expression of three specific miRNAs (miR-30b, miR-375 and miR-503) was studied. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375. In conclusion, the oviductal MVs could be involved in cellular trafficking during oocyte maturation and their possible use in vitro could facilitate the exploitment of canine reproductive biotechnologies. [ABSTRACT FROM AUTHOR]

Background: It is known that a paracrine mechanism exists between mesenchymal stem cells and target cells. This process may involve microvesicles (MVs) as an integral component of cell-to-cell communication. Methods: In this context, this study aims to understand the efficacy of MVs in in-vitro endometrial stressed cells in view of potential healing in in-vivo studies. For this purpose, the presence and type of MVs secreted by amniotic mesenchymal stem cells (AMCs) were investigated and the response of endometrial cells to MVs was studied using a dose-response curve at different concentrations and times. Moreover, the ability of MVs to counteract the in vitro stress in endometrial cells induced by lipopolysaccharide was studied by measuring the rate of apoptosis and cell proliferation, the expression of some pro-inflammatory genes such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin 1β (IL-1β), and metalloproteinases (MMP) 1 and 13, and the release of some pro- or anti-inflammatory cytokines. Results: MVs secreted by the AMCs ranged in size from 100 to 200 nm. The incorporation of MVs was gradual over time and peaked at 72 h. MVs reduced the apoptosis rate, increased cell proliferation values, downregulated pro-inflammatory gene expression, and decreased the secretion of pro-inflammatory cytokines. Conclusion: Our data suggest that some microRNAs could contribute to counteracting in-vivo inflammation of endometrial tissue. [ABSTRACT FROM AUTHOR]

Background: Endometritis reduces fertility and is responsible for major economic losses in beef and dairy industries. The aim of this study was to evaluate an alternative therapy using platelet-rich plasma (PRP). PRP was tested in vivo, after bovine intrauterine administration, and in vitro on endometrial cells. Methods: Bovine endometrial cells were cultured until passage (P) 10 with 5 % or 10 % PRP. Effect of PRP on endometrial cell proliferation and on the expression of genes [prostaglandin-endoperoxide synthase 2 (COX2), tumor protein p53 (TP53), oestrogen receptors (ER-α and ER-β), progesterone receptor (PR) and c-Myc] involved in the regulation of oestrus cycle and fetal-maternal interaction were evaluated. Moreover, to evaluate the ability of PRP to counteract inflammation, 10 and 100 ng/ml of bacterial endotoxin lipopolysaccharide (LPS) were used to inflame endometrial cells in vitro for 1, 6, 12, 24 and 48 h. The expression of genes such as interleukin 1β (IL-1β), interleukin-8 (IL-8), inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), and the release of PGE-2, IL-1β and IL-8 were evaluated. Results: In vivo treatment with PRP increased the detection of PR. In vitro, 5 % PRP at passage 5 increased proliferation rate and induced a significant increase in the expression of all studied genes. Furthermore, the results revealed that 10 ng/ml of LPS is the most effective dose to obtain an inflammatory response, and that PRP treatment significantly down regulated the expression of pro-inflammatory genes. Conclusion: This study lays the foundations for the potential treatment of endometritis with PRP in vivo. [ABSTRACT FROM AUTHOR]

In human and swine, leptin (OB) has been identified in seminal plasma and leptin receptors (OB-R) on the cell surface of spermatozoa, indicating that spermatozoa are a target for OB. This hormone has also been detected in follicular fluid (FF) in women and mares, although its role requires further study. The aims of this study were to investigate the immunolocalisation and the expression of OB and OB-R in equine spermatozoa and to evaluate the involvement of OB in equine in vitro fertilisation (IVF). Since progesterone (P) andOBare both found in FF, the individual and combined effects of these two hormones were studied in equine IVF and compared with the results obtained from the use of FF for in vitro sperm preparation. For the first time, we were able to identify OB and OB-R mRNA and their corresponding proteins in equine spermatozoa. When spermatozoa were treated with OB, there was a decrease in the three motility parameters VSL, STR and LIN, commonly associated with hyperactivation, whilst the acrosome reaction rate increased (P,0.05). The fertilisation rate was 51% with FF, 46.15% with P, 43.64% with PþOB and 0% with OB alone. The percentage of eightcell stage embryos was 18.7% with FF, 17.1% with P and 16.7% with OBþP. OB alone did not permit oocyte fertilisation, indicating that, in the horse, OB is involved in capacitation and hyperactivation but not in sperm penetration. [ABSTRACT FROM AUTHOR]

Recent studies have revealed the presence of a mesenchymal stem cell (MSC) population in human and in gilt granulosa cells (GCs), thus increasing the interest in identifying the same population in the bovine species. We first isolated GCs by scraping from bovine preovulatory follicles and then tested several different media to define the ideal conditions to select granulosa-derived stem cells. Although expressing MSC-associated markers, none of the media tested proven to be efficient in selecting MSC-like cells that were able to differentiate into mesodermic or ectodermic lineages. We performed another experimental approach exposing cells to a chemical stress, such as lowering of pH, as a system to select a more plastic population. Following the treatment, granulosa-specific granulose markers [follicle-stimulating hormone receptor ( FSHR), follistatin (FST), and leukemia inhibitory factor receptor ( LIFR)] were lost in bovine GCs, whereas an increase in multi- ( CD29, CD44, CD73) and pluripotent ( Oct-4 and c-Myc) genes was noticed. The stress allowed up-regulation of tumor necrosis factor-α and interleukin-1β expression and the dedifferentiation of GCs, which was demonstrated by differentiation studies. Indeed, pH-treated cells were able to differentiate into the mesodermic and ectodermic lineages, thus suggesting that the chemical stress allows for the selection of cells that are more prone to adjust and respond to the environmental changes. [ABSTRACT FROM AUTHOR]

The aim of this work was to provide, for the first time, a protocol for isolation and characterization of stem cells from porcine amniotic membrane in view of their potential uses in regenerative medicine. From three samples of allanto-amnion recovered at delivery, the amniotic membrane was stripped from overlying allantois and digested with trypsin and collagenase to isolate epithelial (amniotic epithelial cells [AECs]) and mesenchymal cells, respectively. Proliferation, differentiation, and characterization studies by molecular biology and flow cytometry were performed. Histological examination revealed very few mesenchymal cells in the stromal layer, and a cellular yield of AECs of 10 × 106/gram of digested tissue was achieved. AECs readily attached to plastic culture dishes displaying typical cuboidal morphology and, although their proliferative capacity decreased to the fifth passage, AECs showed a mean doubling time of 24.77 ± 6 h and a mean frequency of one fibroblast colony-forming unit (CFU-F) for every 116.75 plated cells. AECs expressed mesenchymal stem cell (MSC) mRNA markers ( CD29, CD166, CD90, CD73, CD117) and pluripotent markers ( Nanog and Oct 4), whereas they were negative for CD34 and MHCII. Mesodermic, ectodermic, and endodermic differentiation was confirmed by staining and expression of specific markers. We conclude that porcine amniotic membrane can provide an attractive source of stem cells that may be a useful tool for biomedical research. [ABSTRACT FROM AUTHOR]

Background: A repeat breeder cow (RBC) can be defined as an animal that after 3 or more inseminations cannot get pregnant because of fertilization failure or early embryonic death. If no cause is identified precisely, inadequate uterine receptivity is responsible for implantation failures. Since a large number of identified molecular mediators, such as cytokines, growth factors and lipids have been postulated to be involved in early feto-maternal interaction, in this study a different approach to the treatment of RBC syndrome has been employed using a platelet concentrate (PC) that contains a significant amount of growth factors accumulated in its a-granules. Methods: Three explorative studies were performed. Initially, PC was supplemented in the in vitro embryo culture medium to study its effect on embryo-development. After the pilot study, 4 RBCs were treated with intrauterine administration of PC to evaluate proliferative potential of endometrium by immunohistochemical expression of the antigen Ki-67. Lastly, the effect of intrauterine administration of PC at 48 hrs after artificial insemination in RBCs was evaluated. Results: The in vitro results show that 5 % of PC and 5 % of fetal calf serum (FCS) increase the rate of blastocysts compared with the control containing 10 % FCS only (43.04 % vs 35.00 % respectively). The immunohistochemical study shows more proliferating nuclei in the treated uterine horn compared to the control one. After intrauterine insemination in RBCs, the percentage of pregnant cows in the control group was 33.33 % compared to 70 % of the treated animals. Conclusion: We suppose that when embryo descends in uterus could find a more appropriate environment for nesting and subsequent pregnancy. [ABSTRACT FROM AUTHOR]

Amniotic membrane-derived mesenchymal cells (AMCs) are considered suitable candidates for a variety of cell-based applications. In view of cell therapy application in uterine pathologies, we studied AMCs in comparison to cells isolated from the endometrium of mares at diestrus (EDCs) being the endometrium during diestrus and early pregnancy similar from a hormonal standpoint. In particular, we demonstrated that amnion tissue fragments (AM) shares the same transcriptional profile with endometrial tissue fragments (ED), expressing genes involved in early pregnancy (AbdB-like Hoxa genes), pre-implantantion conceptus development (Erα, PR, PGRMC1 and mPR) and their regulators (Wnt7a, Wnt4a). Soon after the isolation, only AMCs express Wnt4a and Wnt7a. Interestingly, the expression levels of prostaglandin-endoperoxide synthase 2 (PTGS2) were found greater in AM and AMCs than their endometrial counterparts thus confirming the role of AMCs as mediators of inflammation. The expression of nuclear progesterone receptor (PR), membrane-bound intracellular progesterone receptor component 1 (PGRMC1) and membrane-bound intracellular progesterone receptor (mPR), known to lead to improved endometrial receptivity, was maintained in AMCs over 5 passages in vitro when the media was supplemented with progesterone. To further explore the potential of AMCs in endometrial regeneration, their capacity to support resident cell proliferation was assessed by co-culturing them with EDCs in a transwell system or culturing in the presence of AMC-conditioned medium (AMC-CM). A significant increase in EDC proliferation rate exhibited the crucial role of soluble factors as mediators of stem cells action. The present investigation revealed that AMCs, as well as their derived conditioned media, have the potential to improve endometrial cell replenishment when low proliferation is associated to pregnancy failure. These findings make AMCs suitable candidates for the treatment of endometrosis in mares. [ABSTRACT FROM AUTHOR]

Introduction While amniotic mesenchymal cells have been isolated and characterized in different species, amniotic epithelial cells (AECs) have been found only in human and horse and are recently considered valid candidates in regenerative medicine. The aim of this work is to obtain and characterize, for the first time in the feline species, presumptive stem cells from the epithelial portion of the amnion (AECs) to be used for clinical applications. Methods In our study, we molecularly characterized and induced in vitro differentiation of feline AECs, obtained after enzymatic digestion of amnion. Results AECs displayed a polygonal morphology and the mean doubling time value was 1.94 ± 0.04 days demonstrating the high proliferating capacity of these cells. By RT-PCR, AECs expressed pluripotent (Oct4, Nanog) and some mesenchymal markers (CD166, CD44) suggesting that an epithelial-mesenchymal transition may occur in these cells that lack the hematopoietic marker CD34. Cells also showed the expression of embryonic marker SSEA-4, but not SSEA-3, as demonstrated by immunocytochemistry and flow cytometry. Moreover, the possibility to use feline AECs in cell therapies resides in their low immunogenicity, due to the absence of MHC-II antigen expression. After induction, AECs differentiated into the mesodermic and ectodermic lineages, demonstrating high plasticity. Conclusions In conclusion, feline AECs appear to be a readily obtainable, highly proliferative, multipotent and non-immunogenic cell line from a source that may represent a good model system for stem cell biology and be useful in allogenic cell-based therapies in order to treat tissue lesions, especially with loss of substance. [ABSTRACT FROM AUTHOR]

There is no published information about follicular-fluid leptin concentrations or the presence of leptin and leptin receptor in the equine ovary or oocyte. Three groups of mares - adult draft mares, draft fillies and adult Standardbred mares - were included in the study. Leptin and leptin receptor were detected in all immature oocytes by immunofluorescence with higher intensity in oocytes from draft mares compared with draft fillies and Standardbred mares. After in vitro maturation a higher proportion of oocytes reached metaphase II in draft mares than in draft fillies and Standardbred mares, and in all groups both leptin and leptin receptor became localised in the oocyte cortex but with higher immunopositivity in draft mares compared with draft fillies and Standardbred mares. These intensities were confirmed by the expression profiles of leptin and leptin receptor mRNA. Moreover, leptin was detected in ovarian blood vessels in all three types of animal and within the corpora lutea in adult mares. Serum and follicular-fluid concentrations of leptin were similar in draft and Standardbred mares but higher in draft mares than in draft fillies. This study supports the hypothesis that expression of leptin and leptin receptor mRNA and the rate of maturation can be related either to adiposity or to puberty. Leptin is an endocrine signal of nutritional status and body fat-mass that influences reproduction. We investigated if leptin serum and follicular fluid concentrations, in pre-pubertal animals and in horses, different for body adiposity, could have any influence on the ability of equine oocytes to undergo in vitro maturation. Our data show that maturation rate can be related either to adiposity or to puberty and that sport or obese horses represent a model for studying similar human conditions. [ABSTRACT FROM AUTHOR]

Stray cat colonies in urban and rural areas of Lombardy, northern Italy, were surveyed for seroprevalence of feline immunodeficiency virus (FIV) antibodies, feline leukaemia virus (FeLV) antigen and Toxoplasma gondii IgG. Of 316 cats tested, 6.6% were positive for FIV and 3.8% were positive for FeLV infection; 203 cats were tested for T gondii IgG antibodies and a prevalence of 30.5% was detected. Statistical analysis tested the influence of provenience, age, gender, health status and laboratory results on seroprevalence and found male gender and adult age were risk factors for FIV infection. FIV-infected cats were more likely to have a decreased red blood cell count than FIV seronegative cats. No predictors were significantly associated with FeLV and T gondii seropositivity. Colony cats in this study posed a limited risk for retrovirus infection to pet cats allowed outdoors, whereas toxoplasmosis exposure was comparable with the worldwide data. [ABSTRACT FROM PUBLISHER]

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 sizesieved 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 μm in diameter) and a small (<8 μm) 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 μM) in presence of high [Ca2+]o and 4) the CaSR antagonist NPS 2390 (10 μM 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. [ABSTRACT FROM AUTHOR]

The aim of this work was to give an evidence of the likely presence of interstitial cells in the canine lower urinary tract and to study their possible interactions with the musculature and the intramural innervation. Cryosections of normal canine bladder and urethra were immunofluorescently labelled with c-kit, a transmembrane, tyrosine kinase growth factor receptor, known to be expressed on the interstitial cells of Cajal (ICCs) of the gut. The relationship with antiactin positive smooth muscle cells and PGP9.5-positive intramural innervation was also investigated by confocal microscopy. Anti-c-kit labelling demonstrated a network of elongated and branched c-kit positive cells, which were located in interstitial spaces, oriented in parallel to the smooth muscle bundles that form the bladder muscular layer, irrespective of dog sex. Cells with a similar localization were also PAS- and NADPH-diaphorase-positive. A contact between c-kit immunofluorescent cells and intramural innervation was demonstrated, too. The roles of interstitial cells might include regulation of smooth muscle activity of the bladder detrusor, integrating neuronal signals during urine storage and voiding. [ABSTRACT FROM AUTHOR]

Simple Summary: Platelet rich plasma is a biological product obtained from blood and used for regenerative treatments of different pathologies. It is characterized by a high concentration of platelets (at least 3 times the physiological level) containing many growth factors with anti-inflammatory, bactericidal and regenerative properties. In human medicine, PRP is used in an autologous way, it means that the blood donor is also the recipient. In veterinary medicine, PRP is used to treat different diseases or lesions and in bovine species to treat mastitis. In this context, the opportunity to have PRP ready to use, stored from donor cows of the same farm where it will be used, would be very useful in treating this pathology immediately when it occurs. For this purpose, the present research aimed to detect parameters useful to identify the most suitable cows to be used as blood donors to obtain the highest yield of PRP (milliliters of PRP obtained with respect to milliliters of initial blood). Our results showed that blood collection from the mammary vein within three months of parturition, from nonpregnant cows at 5 years of age, but not the blood collection season, were associated with a high yield of PRP. Platelet rich plasma (PRP) has been shown to be beneficial in the treatment of bovine mastitis, with an action comparable to that of antibiotics. Autologous treatment is feasible in experimental conditions but is difficult to apply in field conditions, particularly in acute mastitis. The ideal scenario would be to have heterologous PRP stored on every farm so that it is readily available when needed. In this paper, we analysed data collected during bovine mastitis treatment with heterologous PRP produced by casual donor cows on several farms. We tried to identify parameters which might be useful to identify the most suitable cows to be used as blood donors, to obtain the highest yield of PRP. Variables considered for each animal were the age, the parity, the date of the last parturition, the season of blood collection, the site of blood collection (jugular or mammary vein) and the reproductive status e.g., pregnant or not pregnant. There were statistically significant differences for all the variables considered from the 135 blood cows, except for the blood collection season. The highest yield of PRP was associated with nonpregnancy blood collection within three months of parturition, parity 3 or 4, and blood collection from the mammary vein. [ABSTRACT FROM AUTHOR]

Abstract: Fusarium mycotoxins, such as trichothecenes and zearalenone, are common grain and foodstuffs contaminants. Some of these like deoxynivalenol (DON) can negatively impact pregnancy success in swine, but evidence for direct ovarian effects of DON, zearalenone, and its major metabolite, α-zearalenol (ZEA) is meager. To evaluate the effects of two mycotoxins, DON and ZEA on porcine granulosa cell(s) (GC) proliferation, steroidogenesis and gene expression, pig GC from small follicles (1–5mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing control (no mycotoxins) or mycotoxins (at various doses/combinations). Both DON and ZEA had biphasic effects on IGF-I-induced estradiol production, increasing estradiol production at smaller doses and inhibiting at larger doses. ZEA at 3000ng/mL (9.37μM) increased IGF-I-induced progesterone production and at 30ng/mL (0.0937μM) and 300ng/mL (0.937μM) were without effect, but these doses of ZEA increased FSH-induced progesterone production. ZEA at 3000ng/mL inhibited FSH plus IGF-I-induced CYP19A1 and CYP11A1 mRNA abundance. DON inhibited progesterone production at 100ng/mL (0.337μM) and 1000ng/mL (3.37μM) but at 10ng/mL (0.0337μM) was without effect. DON at 1000ng/mL (but not at 10ng/mL) completely inhibited FSH plus IGF-I-induced CYP19A1 and CYP11A1 mRNA abundance. The concomitant treatment of ZEA had little effect on the dose response to DON. DON increased IGF-I-induced cell numbers at 10 and 100ng/mL and inhibited cell numbers at 1000ng/mL, whereas ZEA had no effect on GC numbers. Only a combined treatment of DON and ZEA increased serum-induced cell proliferation. In conclusion, mycotoxins have direct dose-dependent effects on GC proliferation, steroidogenesis and gene expression. These direct ovarian effects could be one mechanism whereby contaminating Fusarium mycotoxins in feedstuffs could impact reproductive performance in swine. [Copyright &y& Elsevier]

Increasing evidence suggests a role for ghrelin in the control of articular inflammatory diseases like osteoarthritis (OA). In the present study we examined the ability of ghrelin to counteract LPS-induced necrosis and apoptosis of chondrocytes and the involvement of GH secretagogue receptor (GHS-R)1a in the protective action of ghrelin. The effects of ghrelin (10 −7 –10 −11  mol/L) on equine primary cultured chondrocytes viability and necrosis in basal conditions and under LPS treatment (100 ng/ml) were detected by using both acridine orange/propidium iodide staining and annexin-5/propidium iodide staining. The presence of GHS-R1a on chondrocytes was detected by Western Blot. The involvement of the GHS-R1a in the ghrelin effect against LPS-induced cytotoxicity was examined by pretreating chondrocytes with D-Lys3-GHRP-6, a specific GHS-R1a antagonist, and by using des-acyl ghrelin (DAG, 10 −7 and 10 −9  mol/L) which did not recognize the GHS-R 1a. Low ghrelin concentrations reduced chondrocyte viability whereas 10 −7  mol/L ghrelin protects against LPS-induced cellular damage. The protective effect of ghrelin depends on the interaction with the GHS-R1a since it is significantly reduced by D-Lys3-GHRP-6. The negative action of ghrelin involves caspase activation and could be due to an interaction with a GHS-R type different from the GHS-R1a recognized by both low ghrelin concentrations and DAG. DAG, in fact, induces a dose-dependent decrease in chondrocyte viability and exacerbates LPS-induced damage. These data indicate that ghrelin protects chondrocytes against LPS-induced damage via interaction with GHS-R1a and suggest the potential utility of local GHS-R1a agonist administration to treat articular inflammatory diseases such as OA. [ABSTRACT FROM AUTHOR]

Extrafetal tissues are a noncontroversial and inexhaustible source of mesenchymal stem cells that can be harvested noninvasively at low cost. In the veterinary field, as in man, stem cells derived from extrafetal tissues express plasticity, reduced immunogenicity, and have high anti-inflammatory potential making them promising candidates for treatment of many diseases. Umbilical cord mesenchymal cells have been isolated and characterized in different species and have recently been investigated as potential candidates in regenerative medicine. In this study, cells derived from bovine Wharton jelly (WJ) were isolated for the first time by enzymatic methods, frozen/thawed, cultivated for at least 10 passages, and characterized. Wharton jelly–derived cells readily attached to plastic culture dishes displaying typical fibroblast-like morphology and, although their proliferative capacity decreased to the seventh passage, these cells showed a mean doubling time of 34.55 ± 6.33 hours and a mean frequency of one colony-forming unit fibroblast like for every 221.68 plated cells. The results of molecular biology studies and flow cytometry analyses revealed that WJ-derived cells showed the typical antigen profile of mesenchymal stem cells and were positive for CD29, CD44, CD105, CD166, Oct-4, and c-Myc. They were negative for CD34 and CD14. Remarkably, WJ-derived cells showed differentiation ability. After culture in induced media, WJ-derived cells were able to differentiate into osteogenic, adipogenic, chondrogenic, and neurogenic lines as shown by positive staining and expression of specific markers. On polymerase chain reaction analysis, these cells were negative for MHC-II and positive for MHC-I , thus reinforcing the role of extrafetal tissue as an allogenic source for bovine cell–based therapies. These results provide evidence that bovine WJ-derived cells may have the potential to differentiate to repair damaged tissues and reinforce the importance of extrafetal tissues as stem cell sources in veterinary regenerative medicine. A more detailed evaluation of their immunologic properties is necessary to better understand their potential role in cellular therapy. [ABSTRACT FROM AUTHOR]

Abstract: The jelly-like gubernaculum testis (GT) is a hydrated structure consisting of a concentric sheath of dense connective tissue around a loose mesenchymal core, with two cords of skeletal muscle cells asymmetrically placed alongside. Expansion of the GT occurs during the transabdominal phase of testicular descent, linked to cell proliferation together with modifications of the hydric content of the organ. The aim of this study was to detect immunohistochemically the presence of aquaporins (AQPs), integral membrane proteins permitting passive transcellular water movement, in the canine GTs. Samples (n =15) were obtained from pregnancies of 9 medium sized bitches and dissected from healthy fetuses. Five fetuses were aged 35–45 days of gestation, 10 fetuses from 46 days of gestation to delivery, thus offering us the opportunity to study the progressive maturation of the gubernacula. The presence of AQP3, 4, 7, 8 and -9 was assessed in the muscular components of the GT, some of them (AQP3, AQP4, AQP7) with increasing intensity through the second half of pregnancy up to term. AQP1 was localized in the capillary and venous endothelia in the younger fetuses, also in the artery adventitia and in the nerve perineurium in progressively older fetuses. These data demonstrate the potential importance and contribution of AQP-mediated water flux in hydration and volume modification of the growing GT in a canine model. [Copyright &y& Elsevier]

Tendon injuries represent even today a challenge as repair may be exceedingly slow and incomplete. Regenerative medicine and stem cell technology have shown to be of great promise. Here, we will review the current knowledge on the mechanisms of the regenerative potential of mesenchymal stem cells (MSCs) obtained from different sources (bone marrow, fat, cord blood, placenta). More specifically, we will devote attention to the current use of MSCs that have been used experimentally and in limited numbers of clinical cases for the surgical treatment of subchondral-bone cysts, bone-fracture repair and cartilage repair. Based on the recently emerging role in regenerative mechanisms of soluble factors and of extracellular vesicles, we will discuss the potential of non-cellular therapies in horse tendon injuries. [ABSTRACT FROM AUTHOR]

Seminal plasma contains extracellular vesicles (EVs) that vehicle RNA, proteins, and other molecules able to influence the biological function of sperm. The aim of this study was to improve the fertilizing capacity of male gametes of low-fertility bulls using EVs isolated by ultracentrifugation from the seminal plasma of a bull of proven fertility. After dose-response curve, 10×106 sperm of low-fertility bulls were co-incubated for an hour with 400×106 EVs/ml. In addition, it has been verified that the incorporation of EVs, which takes place in the sperm midpiece, is maintained for 5 hours and even after cryopreservation. Subsequently, the spermatozoa of low-fertility bulls, with EVs incorporated, were used for the in vitro production of embryos. The rate of blastocyst at seventh day yield in vitro, with the use of sperm with EVs incorporated, increased by about twice the yield obtained with the same sperm in the absence of EVs: bulls having an average embryonic yield of 6.41±1.48%, 10.32±4.34% and 10.92±0.95% improved their yield to 21.21±1.99%, 22.17±6.09% and 19.99±5.78%, respectively (P<0.05). These encouraging results suggest that it might be possible to keep breeding bulls with poor fertility. Further studies will be needed to evaluate the in vivo fertility of sperm treated with EVs and understand how the content of EVs is involve in the sperm-vesicle interaction and in the improved sperm performance.

Reproductive diseases could lead to infertility and have implications for overall health, most importantly due to psychological, medical and socio-economic consequences for individuals and society. Furthermore, economical losses also occur in animal husbandry. In both human and veterinary medicine, hormonal and surgical treatments, as well as assisted reproductive technologies are used to cure reproductive disorders, however they do not improve fertility. With ovarian disorders being the main reproductive pathology in human and bovine, over the past 2 decades research has approached regenerative medicine in animal model to restore normal function. Ovarian pathologies are characterized by granulosa cell and oocyte apoptosis, follicular atresia, decrease in oocyte quality and embryonic development potential, oxidative stress and mitochondrial abnormalities, ultimately leading to a decrease in fertility. At current, application of mesenchymal stromal cells or derivatives thereof represents a valid strategy for regenerative purposes. Considering their paracrine/autocrine mode of actions that are able to regenerate injured tissues, trophic support, preventing apoptosis and fibrosis, promoting angiogenesis, stimulating the function and differentiation of endogenous stem cells and even reducing the immune response, are all important players in their future therapeutic success. Nevertheless, obtaining mesenchymal stromal cells (MSC) from adult tissues requires invasive procedures and implicates decreased cell proliferation and a reduced differentiation capacity with age. Alternatively, the use of embryonic stem cells as source of cellular therapeutic encountered several ethical concerns, as well as the risk of teratoma formation. Therefore, several studies have recently focussed on perinatal derivatives (PnD) that can be collected non-invasively and, most importantly, display similar characteristics in terms of regenerating-inducing properties, immune-modulating properties and hypo-immunogenicity. This review will provide an overview of the current knowledge and future perspectives of PnD application in the treatment of ovarian hypofunction., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lange-Consiglio, Capra, Herrera, Lang-Olip, Ponsaerts and Cremonesi.)

Platelet rich plasma (PRP) has been shown to be beneficial in the treatment of bovine mastitis, with an action comparable to that of antibiotics. Autologous treatment is feasible in experimental conditions but is difficult to apply in field conditions, particularly in acute mastitis. The ideal scenario would be to have heterologous PRP stored on every farm so that it is readily available when needed. In this paper, we analysed data collected during bovine mastitis treatment with heterologous PRP produced by casual donor cows on several farms. We tried to identify parameters which might be useful to identify the most suitable cows to be used as blood donors, to obtain the highest yield of PRP. Variables considered for each animal were the age, the parity, the date of the last parturition, the season of blood collection, the site of blood collection (jugular or mammary vein) and the reproductive status e.g., pregnant or not pregnant. There were statistically significant differences for all the variables considered from the 135 blood cows, except for the blood collection season. The highest yield of PRP was associated with nonpregnancy blood collection within three months of parturition, parity 3 or 4, and blood collection from the mammary vein.

Recent studies on cull cows have shown that ovarian abnormalities, particularly ovarian insufficiency, are the main cause of reproductive failure. The aim of this study was to treat bovine ovarian failure with intraovarian administration of autologous platelet rich plasma (PRP), which is rich in growth factors, chemokines, and cytokines that could stimulate follicular growth and steroidogenesis. Twelve cows with ovarian hypofunction were enrolled in the study and they were randomly allocated in control group (CTR) and treated group (six animal for group). In the treated group, only five animals received the PRP treatment because intraovarian administration was hindered in one by a rectovaginal fistula. Animals of control group were treated by intraovarian administration of physiological solution. In the 4 weeks after PRP injection, a mild to strong increase in progesterone (PRG) concentrations was detected in four of the five cows treated. Artificial insemination (AI) resulted in four pregnancies that are still ongoing (7th month). Intraovarian administration of PRP improved ovarian function after 2 months of treatment. This effect may be due to reduction of follicular atresia or to revitalization of dormant oocytes allowing restoration of fertility., (Copyright © 2020 Cremonesi, Bonfanti, Idda and Lange-Consiglio.)

There is increasing evidence to suggest that, in addition to their regenerative effect, mesenchymal stromal cells (MSCs), and their secretome have an anti-inflammatory and antimicrobial role in the innate immune response in conditions such as sepsis. However, there is no published information on the effect of MSCs in bovine mastitis. Mastitis often results in extensive tissue damage due to multi-microorganism co-infection. This study investigated the ability of amniotic-derived conditioned medium (CM), in vitro and in vivo , to counteract microbial action and restore healthy tissue capable of milk production. Following determination of a dose-response curve, 10,000 colony-forming units (CFU) of Staphylococcus aureus ( S. aureus ) were inoculated into bovine mammary epithelial cell culture with and without 10% CM (supplemented either at the time of bacteria inoculation or after 4 h). Acridine orange staining was used to assess cell viability/apoptosis. Additionally, an in vivo study was performed using 48 dairy cows with acute and chronic mastitis, treated with CM (treated group) or antibiotics (control group). In vitro results showed that CM can attenuate bacterial growth, as evaluated by the number of CFU. After 24 h of culture with S. aureus , 89.67% of mammary epithelial cells treated with CM were still alive, whereas all cells cultured without CM were dead. Rates of epithelial cell survival (60.67%) were similar when CM was added 4 h after bacteria inoculation. There was no difference in somatic cell count between cases of acute mastitis in the CM-treated or control group in the in vivo study. However, relapses in chronic mastitis were less common in the group receiving CM. Our results show that CM is able to mitigate bacterial growth in vitro and may be particularly useful in the treatment of chronic mastitis, aiding restoration of milk production in cows that would otherwise be removed from the production cycle., (Copyright © 2019 Lange-Consiglio, Gusmara, Manfredi, Idda, Soggiu, Greco, Bonizzi, Cremonesi and Zecconi.)

Cell-derived microvesicles (MVs) are a recently discovered mechanism of cell-to-cell communication. Our previous data show that MVs secreted by equine amniotic mesenchymal-derived cells (AMCs) are involved in downregulation of proinflammatory genes in lipopolysaccharide-stressed equine tendon and endometrial cells. The aim of the present study was to evaluate whether AMC-MVs contain selected microRNAs (miRNAs) involved in inflammation. Two pools of cells, derived from 3 amniotic membranes each, and their respective MVs were collected. Small RNAs were extracted and deep sequenced, followed by miRNA in silico detection. The analysis identified 1,285 miRNAs, which were quantified both in AMCs and MVs. Among these miRNAs, 401 were classified as Equus caballus miRNAs, 257 were predicted by homology with other species (cow, sheep, and goat), and 627 were novel candidate miRNAs. Moreover, 146 miRNAs differentially expressed (DE) in AMCs and MVs were identified, 36 of which were known and the remaining were novel. Among the known DE miRNAs, 17 showed higher expression in MVs. Three of these were validated by real time polymerase chain reaction: eca-miR-26, eca-miR-146a, and eca-miR-223. Gene ontology analysis of validated targets showed that the DE miRNAs in cells and MVs could be involved both in immune system regulation by modulating interleukin signaling and in the inflammatory process. In conclusion, this study suggests a significant role of AMCs in modulating immune response through cell-cell communication via MV-shuttling miRNAs.

Amniotic epithelial cells (AECs) spontaneously transform into amniotic mesenchymal cells (AMCs) in vitro during cell culture. Glycocalyx was analyzed to identify the glycan pattern in AECs, AMCs and epithelial-mesenchymal transdifferentiated cells (EMTCs). Pure cell cultures were derived using cloned AEC and AMC cell lines obtained by the dilution technique from amniotic membranes. Mesenchymal cells generated by differentiation of clonal epithelial cells were considered transdifferentiated. Immunocytoscreen, in vitro multipotent differentiation and molecular characterization of EMTCs were performed. In combination with saponification and sialidase digestion, a panel of 12 lectins was used to analyze the glycan pattern of AEC, AMC and EMTC glycocalyx. Cytokeratin cell markers were lost in EMTCs and typical mesenchymal markers, such as vimentin, appeared. These cells retained their differentiation potential. Lectin histochemistry revealed a cell-specific glycan profile. Galactose (Gal)β1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galβ1,3(±NeuNAcα2,6)GalNAc were highly expressed on the surface of all the amniotic cell cultures. AECs expressed asialoglycans with terminal GalNAc and GlcNAc. More highly mannosylated N-linked glycans and NeuNAcα2,3Galβ1,3GalNAc in O-linked glycans were expressed by EMTCs, but these cells had fewer glycans ending with fucose (Fuc), Gal, GlcNAc and GalNAc than AECs. GlcNAc- and GalNAc-terminating glycans were similarly expressed on the glycocalyx of the mesenchymal cell populations (EMTCs and AMCs). These results demonstrate for the first time that the spontaneous epithelial-mesenchymal transition (EMT) of equine amnion cells is characterized by cell surface glycan remodeling and that glycosylation changes result in a cell type-specific glycan profile. The glycopattern of equine amnion spontaneous EMTCs differs from EMT of tumoral cells., (© 2015 S. Karger AG, Basel.)