Platelet-rich plasma and ovarian quiescence: a bovine in vitro model for regeneration of the ovary.

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 72h and, at different times (0, 24, 48 and 72h), hematoxylin-eosin and immunohistochemical detection of Ki-67 were performed. Additionally, granulosa cells collected from healthy bovine ovaries were stressed with 100ng/mL of lipopolysaccharide (LPS) in presence/absence of PRP and evaluated at 0, 4, 8 and 24h 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 48h 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 24h, 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.

Amniotic Mesenchymal-Derived Extracellular Vesicles and Their Role in the Prevention of Persistent Post-Breeding Induced Endometritis.

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.

Insights into animal models for cell-based therapies in translational studies of lung diseases: Is the horse with naturally occurring asthma the right choice?

Human asthma is a widespread disease associated with chronic inflammation of the airways, leading to loss of quality of life, disability and death. Corticosteroid administration is the mainstream treatment for asthmatic patients. Corticosteroids reduce airway obstruction and improve quality of life, although symptoms persist despite treatment in many patients. Moreover, available therapies failed to reverse the lung pathology present in asthma. Animal models, mostly rats and mice, in which the disease is experimentally induced, have been studied to identify new therapeutic targets for human asthma. Alternative animal models could include horses in which naturally occurring asthma could represent an important step to test therapies, potentially designed around mouse studies, before being translated to human testing. Horses naturally suffer from asthma, which has striking parallels with human asthma. Severe equine asthma (SEA) is characterized by reversible bronchospasms and neutrophil accumulation in the lungs immunologically mediated mainly by Th2. Moreover, the pulmonary remodelling that occurs in SEA closely resembles that of human asthma, making the equine model unique for investigation of tissue repair and new therapies. Cell therapy, consisting on mesenchymal stromal cells (MSCs) and derivatives (conditioned medium and extracellular vesicles), could represent a novel therapeutic contribution for tissue regeneration. Cell therapy may prove advantageous over conventional therapy in that it may repair or regenerate the site of injury and reduce the reaction to allergens, rather than simply modulating the inflammatory process.

Oviductal microvesicles and their effect on in vitro maturation of canine oocytes.

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.

Effects of platelet-rich plasma in a model of bovine endometrial inflammation in vitro.

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-ß), 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 1ß (IL-1ß), interleukin-8 (IL-8), inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (COX2/PTGS2), and the release of PGE-2, IL-1ß 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.

Leptin and leptin receptor are detectable in equine spermatozoa but are not involved in in vitro fertilisation.

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) and OB are 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 P+OB and 0% with OB alone. The percentage of eight-cell stage embryos was 18.7% with FF, 17.1% with P and 16.7% with OB+P. OB alone did not permit oocyte fertilisation, indicating that, in the horse, OB is involved in capacitation and hyperactivation but not in sperm penetration.

Platelet concentrate in bovine reproduction: effects on in vitro embryo production and after intrauterine administration in repeat breeder cows.

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 α-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.

Cell Surface Glycan Changes in the Spontaneous Epithelial-Mesenchymal Transition of Equine Amniotic Multipotent Progenitor Cells.

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)ß1,4GlcNAc, Neu5Acα2,6Gal/GalNAc and N-acetyl neuraminic (sialic) acid (NeuNAc)α2,3Galß1,3(±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 NeuNAcα2,3Galß1,3GalNAc 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.

Investigating the efficacy of amnion-derived compared with bone marrow-derived mesenchymal stromal cells in equine tendon and ligament injuries.

BACKGROUND AIMS: This is the first study to compare the treatment of horse tendon and ligament injuries with the use of mesenchymal stromal cells (MSCs) obtained from two different sources: amniotic membrane (AMSCs) and bone marrow (BM-MSCs). The objective was to prove the ability of AMSCs to exert beneficial effects in vivo. METHODS: Five million allogeneic frozen-thawed AMSCs or autologous fresh BM-MSCs were injected intralesionally in horses belonging to group A (51 horses) and group B (44 horses). The interval lesion/implantation was of 6-15 days for the AMSCs and 16-35 days for the BM-MSCs. Healing was assessed clinically and ultrasonographically. Follow-up was monitored for 2 further years from return to full work. RESULTS: No significant adverse effects after MSCs treatment were seen in any of the horses studied, independent of the type of stromal cell implanted. All animals belonging to group A resumed their activities between 4-5 months after treatment, whereas animals of group B resumed their activities after 4-12 months. The rate of re-injury in horses treated with AMSCs is lower (4.00%) compared with the average observed when horses were treated with BM-MSCs (23.08%). CONCLUSIONS: The possibility to inject allogeneic AMSCs in real time, before any ultrasonographic change occurs within the injured tendon and ligament, together with the higher plasticity and proliferative capacity of these cells compared with BM-MSCs, represents the main features of interest for this novel approach for the treatment of equine tendon diseases. An obvious active proliferative healing in the area injected with AMSCs makes these cells more effective than BM-MSCs.

Follicular fluid leptin concentrations and expression of leptin and leptin receptor in the equine ovary and in vitro-matured oocyte with reference to pubertal development and breeds.

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.

Guidelines to Analyze Preclinical Studies Using Perinatal Derivatives.

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.

Equine bone marrow mesenchymal or amniotic epithelial stem cells as feeder in a model for the in vitro culture of bovine embryos.

Various studies have shown that the in vitro culture environment is one of the key determinants of the blastocyst output. In the present study we investigated the effects of co-culturing bovine embryos with equine bone marrow mesenchymal stem cells (BM-MSCs) or equine amniotic epithelial stem cells (AE-SCs) on in vitro blastocysts development. BM specimens were obtained aseptically from sternal aspirates of horses under local anaesthesia and the isolated cells were resuspended in Dulbecco Modified Earle's Medium supplemented with 10 ng/ml of basic fibroblast growth factor (bFGF). Amniotic membranes were obtained from fresh placentas and, to release the AE cells, amniotic fragments were incubated with 0.05% trypsin for 45 min. Separated AE cells were plated in standard culture medium containing 10 ng/ml epidermal growth factor (EGF). Seven hundred and five cumulus-oocyte complexes were used and, after IVM and IVF, cumulus-free presumptive zygotes were randomly transferred into one of three co-culture systems in which they were cultured up to day 7: (1) co-culture with cumulus cells (control); (2) co-culture with BM-MSCs; and (3) co-culture with AE-SCs. Statistical analyses were performed by ANOVA. Blastocyst developmental rates were significantly different (p < 0.001) between control, AE-SCs and BM-MSCs (respectively 35.45, 41.84 and 30.09%). In conclusion, the AE-SC monolayer create a more suitable microenvironment necessary for inducing local cell activation and proliferation of the growing embryos in comparison with BM-MSCs and cumulus cells. It can be suggested that these cells secrete biologically active substances, including signalling molecules and growth factors of epithelial nature, different to those of the BM cells of mesenchymal origin.

Application of Perinatal Derivatives in Ovarian Diseases.

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.

Endometrial and oviduct extra-cellular vescicles for in vitro equine sperm hyperactivation and oocyte fertilization.

Unlike humans and many other mammalian species, conventional in vitro fertilization (IVF) in equine species is not successful. To mimic in vitro equine spermatozoon-oviduct interaction as close as possible to that which occurs in vivo, extracellular vesicles (EVs) secreted by the female genital tract were used. Three female genital tracts were collected at slaughterhouse from mares in late estrus. Ipsilateral proximal and apical horn endometrial explants were digested with collagenase and trypsin and cells obtained were cultured on insert system to allow their polarization. Ipsilateral oviducts were squeezed out to obtain spheroids. To produce EVs, proximal and apical horn endometrial cells and oviductal spheroids were cultured for three days in serum free medium. To trace interaction between spermatozoa and EVs by fluorescence microscopy, EVs were differently labeled. Pooled samples of ejaculated spermatozoa from three stallions were incubated in capacitating medium (CM) for 6 h and to induce hyperactivation for other 6 h in CM supplemented with different kind of EVs alone or in combination. A control was performed in absence of EVs. Sperm were assessed for motility by CASA system, EV incorporation by confocal microscopy and acrosomal reaction (AR) by staining with FITC-PNA/PI. In vitro fertilization was performed, and presumed zygotes were subjected to chromatin configuration. The results show that incorporation of EVs of the proximal horn does not take place, while apical horn EVs are incorporated in the head of the spermatozoon in 4 h. The EVs of oviductal spheroids are incorporated in the middle tract in 1 h. The rate of AR with EVs of the apical horn and oviductal spheroids were respectively 50.25% and 57.14%. When these EVs were added in combination, the rate of AR was 71.42%. In the control, the rate of AR was of 15%. After in vitro fertilization, 44% of oocytes showed male and female pronuclei, whereas no fertilization is obtained in the control. In conclusion, EVs from apical horn and oviduct could be involved in cell trafficking during equine semen hyperactivation, and their possible use in vitro could facilitate the development of equine reproductive biotechnologies.

Extracellular vesicles from seminal plasma to improve fertilizing capacity of bulls.

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.

Physiological Parameters to Identify Suitable Blood Donor Cows for Preparation of Platelet Rich Plasma.

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.

Platelet Rich Plasma for Regenerative Medicine Treatment of Bovine Ovarian Hypofunction.

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.

Improvement of Embryo Recovery in Holstein Cows Treated by Intra-Ovarian Platelet Rich Plasma before Superovulation.

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 hours 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.

Case Report: Use of Amniotic Microvesicles for Regenerative Medicine Treatment of a Mare With Chronic Endometritis.

Chronic endometritis is an inflammation in the inner layer of uterine mucosa, with or without an infectious process, which affects the animal's fertility but not its general health. A variety of treatments has been adopted over the years but to date, no effective cures have been able to renew the injured tissue. Since the defects in the fetal-maternal communication are caused by degenerative changes due to chronic endometrial inflammation, our working hypothesis was a new approach to this disease by the regenerative medicine using amniotic derived microvesicles (MVs) for their anti-inflammatory and regenerative effects. The MVs are responsible for horizontal transfer of genetic materials, including microRNA (miRNAs) that are involved in paracrine communication between origin cells and target cells. Thus, intrauterine MV infusion may be beneficial in degenerative chronic endometritis and in the fetal-maternal talk. The selected mare was an 11-year-old Friesian, with a history of failed pregnancies despite numerous insemination attempts. Punctual and evident heats characterized the reproductive history, but no insemination attempts had been made for many years. The first (failed) insemination was when the mare was 9-years-old. In the next two reproductive seasons, other attempts were made at regular intervals but none was successful. After a final insemination attempt using a stallion of proven fertility, the collection of an 8-day old embryo suggested that the mare was affected by implantation failure related to endometritis. The mare was treated with two cycles of intrauterine administration of amniotic-derived MVs. The success of the intrauterine administration of MVs was demonstrated by an improvement in the classification of endometritis and in a successful artificial insemination (AI) with implantation of an embryo, as detected at day 14 and with a pregnancy that is still ongoing. Probably, MVs were able to restore the injured endometrium and re-establish the proper communication for a successful embryo implantation.

Amniotic microvesicles impact hatching and pregnancy percentages of in vitro bovine embryos and blastocyst microRNA expression versus in vivo controls.

Embryo development and implantation are dynamic processes, responsive to external signals, and can potentially be influenced by many environmental factors. The aims of this study were to evaluate the effects of a culture medium supplemented with amniotic-derived microvesicles (MVs) on in vitro embryo hatching after cryopreservation, and pregnancy rate following embryo transfer. In addition, miRNA profiling of blastocysts produced in vitro, with or without (control; CTR) amniotic MV supplementation, was also evaluated using blastocysts produced in vivo. In vitro embryos were cultured with and without amniotic MV supplementation. In vivo blastocysts were obtained from superovulated cows. Samples for RNA isolation were obtained from three pools of 10 embryos each (in vivo, in vitro-CTR and in vitro + MVs). Our results show that the hatching percentage of cryopreserved in vitro + MVs embryos is higher (P < 0.05) than in vitro-CTR embryos and the pregnancy rate with fresh and cryopreserved in vitro + MVs embryos is higher than in vitro-CTR embryos. In addition, the analysis of differently expressed (DE) microRNAs showed that embryos produced in vivo are clearly different from those produced in vitro. Moreover, in vitro-CTR and in vitro + MVs embryos differ significantly for expression of two miRNAs that were found in higher concentrations in in vitro-CTR embryos. Interestingly, these two miRNAs were also reported in degenerated bovine embryos compared to good quality blastocysts. In conclusion, MV addition during in vitro production of embryos seems to counteract the adverse effect of in vitro culture and partially modulate the expression of specific miRNAs involved in successful embryo implantation.