Mesenchymal stem cells (MSCs) have emerged as a promising alternative for treating neurological disorders due to their neuroprotective, neuroregenerative, immunomodulatory, anti-inflammatory, and anti-apoptotic properties. The use of allogeneic MSCs offers advantages such as the selection of specific cells and their immediate availability. This study aimed to evaluate the safety and feasibility of intrathecal transplantation of allogeneic equine bone marrow-derived MSCs (EqBM-MSCs) in healthy horses. Ten healthy horses (five–12 years old) were randomly divided into two groups. In the control group (n = 5), phosphate-buffered saline was administered intrathecally, while the MSCs group (n = 5) received 3 × 106 allogeneic EqBM-MSCs. Safety and feasibility were assessed through physical examinations monthly for one year, cerebrospinal fluid (CSF) analysis before and six days after transplantation, and magnetic resonance imaging (MRI) one-year post-transplantation. No significant differences were found between the groups in physical examinations or CSF analysis before and six days after transplantation. Furthermore, MRI showed no changes after one year. Matrix Metalloproteinase-2 (MMP-2) expression was detected in the CSF before and after transplantation but showed no significant differences. These findings suggest that intrathecal transplantation of allogeneic EqBM-MSCs is safe and viable, offering a potential therapeutic approach for neurological disorders in horses.

Cell-based therapy has emerged as a promising strategy for treating peripheral nervous system (PNS) injuries across different species. However, there is a scarcity in the literature regarding the transplantation of mesenchymal stem cells (MSCs) for treating PNS injuries in horses. This report aims to describe the therapeutic potential of equine MSC transplantation in two horses with chronic traumatic facial nerve paralysis. Both horses presented with lip ptosis and right deviation of the nostril and upper lip, being clinically diagnosed with left facial nerve paralysis. Due to the refractoriness to conventional anti-inflammatory treatments, cell-based therapy was chosen. One horse received an autologous transplant of equine bone marrow-derived MSCs (EqBM-MSCs) four months after the traumatic event, while the other underwent three transplants of allogeneic equine adipose tissue-derived MSCs (EqAT-MSCs) at 30-day intervals, starting two months after the injury. All transplants were performed at three different sites around the facial nerve, at the level of bifurcation of the buccal branch in both horses. Physical and neurological assessments revealed significant clinical recovery within three months for the first horse and four months for the second. These findings demonstrated that equine MSCs transplants have great therapeutic potential for chronic traumatic facial nerve paralysis in horses, highlighting the relevance of MSCs- based therapy for peripheral nerve injuries.

. This prospective, comparative, randomized, horizontal, and double-blind 31 clinical study investigated the clinical efficacy of leucocyte-poor platelet-rich plasma 32 (PRP, n=8) or allogeneic adipose-derived stem cells (ADSC, n=8) in dogs with bilateral 33 degenerative hip joint disease (DHJD). Sixteen dogs were treated with two intra-articular 34 injections of PRP or ADSCs, within a 30-day interval. The Canine Brief Pain Inventory 35 (CBPI), the Helsinki Chronic Pain Index (HCPI), and Visual Analogue Scales for pain 36 (VAS-pain) and locomotion (VAS-loc) were assessed by the dog owners. Analysis-of- 37 gait using a force plate, response to palpation (VAS-palp), and the descriptive numerical 38 scale for pain (DNS) were measured by a veterinarian. The assessments were performed 39 before (baseline), 30 and 60 days after the first treatment. Data were analyzed using the 40 unpaired-t test, paired Wilcoxon test, Fisher´s exact test, and Mann-Whitney and 41 Friedman tests (P < 0.05). Compared with baseline HCPI, CBPI, VAS-pain, and VAS- 42 palp scores reduced 41%, 52%, 51%, and 48% (P = 0.0001-0.03) at 60 days in the ADSC 43 group. In PRP-treated dogs, CBPI, VAS-loc, and DNS scores decreased by 43%, 43%, 44 and 33% at 60 days, respectively (P = 0.0003-0.011). Based on CBPI data, the rate of 45 success at 60 days was 75% and 25% in the ADSC and PRP groups (P = 0.13), 46 respectively. Both therapies were apparently safe and effective to reduce chronic pain in 47 dogs with bilateral DHJD during a 60-day period. However, a trend towards greater 48 improvement was provided by the ADSC treatment.

This prospective, comparative, randomized, horizontal, and double-blind clinical study investigated the clinical efficacy of leucocyte-poor platelet-rich plasma (PRP, n=8) or allogeneic adipose-derived stem cells (ADSC, n=8) in dogs with bilateral degenerative hip joint disease (DHJD). Sixteen dogs were treated with two intra-articular injections of PRP or ADSCs, within a 30-day interval. The Canine Brief Pain Inventory (CBPI), the Helsinki Chronic Pain Index (HCPI), and Visual Analogue Scales for pain (VAS-pain) and locomotion (VAS-loc) were assessed by the dog owners. Analysis-of-gait using a force plate, response to palpation (VAS-palp), and the descriptive numerical scale for pain (DNS) were measured by a veterinarian. The assessments were performed before (baseline), 30 and 60 days after the first treatment. Data were analyzed using the unpaired t test, paired Wilcoxon test, Fisher's exact test, and Mann-Whitney and Friedman tests (P<0.05). Compared with baseline HCPI, CBPI, VAS-pain, and VAS-palp scores reduced 41%, 52%, 51%, and 48% (P=0.0001-0.03) at 60 days in the ADSC group. In PRP-treated dogs, CBPI, VAS-loc, and DNS scores decreased by 43%, 43%, and 33% at 60 days, respectively (P=0.0003-0.011). Based on CBPI data, the rate of success at 60 days was 75% and 25% in the ADSC and PRP groups (P=0.13), respectively. Both therapies were apparently safe and effective to reduce chronic pain in dogs with bilateral DHJD during a 60-day period. However, a trend towards greater improvement was provided by the ADSC treatment.

Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.

Cell culture is an excellent alternative for the maintenance of cell lines and to obtain quality chromosome preparations of fishes. However, this methodology is still little employed, mainly because of the difficulty of standardization of cell cultures. In this study, we describe a methodology for the rapid acquisition of cell lineages and mitotic chromosomes for cytogenetic studies of fish species from muscle tissue cells. Our methodology is based on the use of a gelatin film, which provides better adhesion of a large number of cells and appropriate conditions for multiplication. The cells of Astyanax altiparanae, used as an experimental model, with fibroblast-like morphology, showed rapid cellular proliferation, resulting in a great number of cells. Chromosomal preparations of cultured cells showed the diploid number of the species, 2n = 50 chromosomes, in 80% of the cells examined, with chromosomes intact and distended. Cell populations were cryopreserved and after being recovered, these cells maintained their proliferative capacity. The development of this methodology represents an innovation for the fish cytogenetics area and it may bring a significant contribution to the conservation and study of several groups due to the difficulty of obtaining good-quality chromosome preparations.

Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis/necrosis rates, immunophenotypic profile (IP), chromosomal stability, clonicity, and differentiation potential of UCIM-MSCs cryopreserved with four different mediums (with FBS: M1, M3, M4 and without FBS: M2). After 3 months of cryopreservation, samples were thawed and analyzed. The potential of differentiation in the mesodermal lineages, clonicity, and the chromosomal stability were maintained after cryopreservation of UCIM-MSCs with medium containing FBS. Changes (P < 0.05) at IP for some markers were observed at cells cryopreserved with medium M1-M3. Only the UCIM-MSCs cryopreserved with the CM (M4) had similar viability post-thaw (P = 0.23) when compared with fresh cells. We proved the hypothesis that the medium of cryopreservation containing CM supports the cryopreservation of UCIM-MSCs, at the experimental conditions, being the medium that better maintains the biological characteristics observed at fresh cells. Thus, future studies of UCIM-MSCs secretome should be conducted to better understand the beneficial and protective effects of the CM during the freezing process.

ABSTRACT: Stem cells are undifferentiated cells with a high proliferation potential. These cells can be characterized by their in vivo ability to self-renew and to differentiate into specialized cell lines. The most used stem cell types, in both human and veterinary fields, are the mesenchymal stem cells (MSC) derived from bone marrow and adipose tissue. Nowadays, there is a great interest in using stem cells derived from fetal tissues, such as amniotic membrane (AM) and umbilical cord tissue (UCT), which can be obtained non-invasively at delivery time. Due to the scarcity of studies in bovine species, the aim of this study was to isolate, characterize, differentiate and cryopreserve MSC derived from the mesenchymal layer of amniotic membrane (AM), for the first time, and umbilical cord tissue (UCT) of dairy cow neonates after assisted delivery (AD) and from fetus at initial third of pregnancy (IT) obtained in slaughterhouse. Cells were isolated by enzymatic digestion of the tissue fragments with 0.1% collagenase solution. Six samples of AM and UCT at delivery time and six samples of AM and UCT at first trimester of pregnancy were subjected to morphology evaluation, imunophenotype characterization, in vitro osteogenic, adipogenic and chondrogenic differentiation and viability analysis after cryopreservation. All samples showed adherence to plastic and fibroblast-like morphology. Immunocytochemistry revealed expression of CD 44, NANOG and OCT-4 and lack of expression of MHC II in MSC from all samples. Flow cytometry demonstrated that cells from all samples expressed CD 44, did not or low expressed CD 34 (AM: IT-0.3%a, AD-3.4%b; UCT: 0.4%, 1.4%) and MHC II (AM: IT-1.05%a, AD-9.7%b; UCT: IT-0.7%a, AD-5.7%b). They were also capable of trilineage mesenchymal differentiation and showed 80% viability after cryopreservation. According to the results, bovine AM and UCT-derived cells, either obtained at delivery time or from slaughterhouse, are a painless and non-invasive source of MSC and can be used for stem cell banking.

To the best of our knowledge, this is the first study describing the proteome of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs) in a global and functional manner. The aim of this work was to analyze the proteome of previously characterized UCIM-MSCs to determine protein abundance and classify the identified proteins according to Gene Ontology (GO) terms. Protein classification analysis according to biological process, molecular function and cellular component was performed using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification System, which revealed enrichment for 42 biological processes, 23 molecular functions and 18 cellular components. Protein abundance was estimated according to the emPAI method (Exponential Modified Protein Abundance Index). The two most abundant proteins in the proteome of UCIM-MSCs were the cytoskeletal proteins actin and vimentin, which have important roles in cell stability and motility. Additionally, we identified 14 cell surface antigens. Three of them, CD44, CD90 and CD105, had been previously validated by flow cytometry. In the present study, we also identified important information about the biological properties of UCIM-MSCs such as differentiation potential, low immunogenicity (low MHC-II expression) and chromosomal stability, which reinforces their use for cell therapy. Together with the proteomic findings, this information allowed us to infer the functional relevance of several activities related to primary metabolic processes, protein synthesis, production of vesicle coats, vesicle-mediated transport and antioxidant activity. In addition, the identification of different cell surface markers may help establish an immunophenotypic panel suitable for the characterization of MSCs from equine fetal membranes.

The injection of mesenchymal stem cells (MSCs) directly into the bone of osteoporotic (OP) patients for rapid recovery has been studied worldwide. Scaffolds associated with MSCs are used to maintain and avoid cell loss after application. A unique heterologous fibrin sealant (HFS) derived from snake venom was evaluated for the cytotoxicity of its main components and as a three-dimensional biological scaffold for MSCs to repair a critical femur defect in osteoporotic rats.

The use of the conditioned medium (CM) for diseases treatment is based on its enrichment with biomolecules with therapeutic properties and themselves have a beneficial effect. Secretome of bovine endometrial mesenchymal progenitor/stem cells (eMSCs) using a proteomics approach is until now unknown. This work aimed to evaluate the secretome of bovine eMSCs-CM challenged or not with lipopolysaccharide (LPS). For this, eMSCs characterized were challenged (TG) or not (CG). The CM was collected 12h after stimulation and submitted to mass spectrometry analysis. The classification of identified proteins was done by PANTHER according to biological processes, molecular function, cellular component and protein class. 397 protein groups were identified in TG and 302 in CG. We observed positive enrichment for antibacterial response proteins, macrophage activation function, receptor-mediated endocytosis, hydrolase activity, inhibitory enzyme in TG, and for activity structural molecule and intermediate filament cytoskeleton in the CG. Our experimental model shows that eMSCs respond to LPS in the concentration used and can be used to study immune-inflammatory response, besides of the secretion of proteins mainly related to tissue remodeling, immune response and angiogenesis which is an interesting feature for use in cell therapy.

Staphylococcus aureus is a common pathogen that causes subclinical bovine mastitis due to several virulence factors. In this study, we analyzed S. aureus isolates collected from the milk of cows with subclinical mastitis that had 8 possible combinations of bap, icaA, and icaD genes, to determine their capacity to produce biofilm on biotic (bovine primary mammary epithelial cells and HeLa cells) and abiotic (polystyrene microplates) surfaces, and their ability to adhere to and invade these cells. We also characterized isolates for microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and agr genes, and for their susceptibility to cefquinome sulfate in the presence of biofilm. All isolates adhered to and invaded both cell types, but invasion indexes were higher in bovine primary mammary epithelial cells. Using tryptic soy broth + 1% glucose on abiotic surfaces, 5 out of 8 isolates were biofilm producers, but only the bap+icaA+icaD+ isolate was positive in Dulbecco's Modified Eagle's medium. The production of biofilm on biotic surfaces occurred only with this isolate and only on HeLa cells, because the invasion index for bovine primary mammary epithelial cells was too high, making it impossible to use these cells in this assay. Of the 5 biofilm producers in tryptic soy broth + 1% glucose, 4 presented with the bap/fnbA/clfA/clfB/eno/fib/ebpS combination, and all were protected from cefquinome sulfate. We found no predominance of any agr group. The high invasive potential of S. aureus made it impossible to observe biofilm in bovine primary mammary epithelial cells, and we concluded that cells with lower invasion rates, such as HeLa cells, were more appropriate for this assay.

BACKGROUND AND OBJECTIVES:Mesenchymal stromal cells (MSCs) have great therapeutic potential, particularly in the process of tissue repair and immunomodulation through the secretion of biomolecules. Thus, the aim of this study was to evaluate the hypothesis that intramuscular transplantation of allogeneic MSCs obtained from equine umbilical cord (UC-MSCs) is safe, demonstrating that this is a suitable source of stem cells for therapeutic use. METHODS AND RESULTS:For this, UC-MSCs were cultured, characterized and cryopreserved for future transplantation in six healthy mares. On day 0, transplantation of three million UC-MSCs diluted in Hank's Balanced Solution (HBSS) was performed on right and left sides of the rump muscle. As a control, HBSS injections were performed caudally in the same muscle. Muscle biopsies were obtained as a control 30 days before transplantation (D-30). The biopsies were collected again on day 2 (left side) and day 7 (right side) post transplantation and examined histologically. All procedures were preceded by ultrasound examination and blood sampling. Hematologic evaluation remained within normal limits and no differences were observed between time points (p>0.05). Ultrasound examination was suggestive of inflammation 48 hours after transplantation in both groups (control and treated). At histological evaluation it was found only discrete inflammation signals between D-30×D2 (p<0.05) in the treated group, without differences (p> 0.05) between the groups at different time points. CONCLUSIONS:Equine UC-MSCs under the experimental conditions did not promote severe inflammation that causes tissue damage or lead to its rejection by the host organism and therefore has a good potential for clinical use.

It is reported a case of necrotizing mastitis by Staphylococcus aureus in goat whose signs were right ceiling with alteration on coloration, tenderness, consistency and bloody discharge. Supportive treatment with parenteral fluid therapy and antibiotics were performed prior to surgery to remove the affected hemigland. Additionally, postoperative topical therapy with hydrogen peroxide, tincture of iodine 2% and copper sulfate was conducted at different times. The treatment applied and the emergency care of the animal in this case were effective for improvement of the clinical case and were extremely important to maintaining the animal´s life.

Uterine histological analysis by serial biopsies are an important tool not only for a better understanding and evaluation of physiological events involved in the process of uterine involution, but also for the identification and monitoring of pathological process that may compromise reproductive life. This study describes a temporal histological changes in endometrial tissues from multiparous Nelore cows in Southeast Brazil treated or not treated with Cloprostenol sodium 1 and 4 days postpartum (DPP), to determine if the treatment promotes histological endometrial involution. Cloprostenol sodium was administered on 1 and 4 DPP and endometrial biopsies were collected 1, 7, 14, 28, and 42 DPP using a Yomanns biopsy instrument. The presence or absence of myometrial and endometrial tissue and its integrity, number of endometrial glands (classification 0 to 3), edema, hemorrhage, vascular congestion and type of epithelial cell was assessed. The average number of leukocytes was recorded. There was observed difference between groups on the number of endometrial glands (P≤ 0.05) at one-time point. In addition, the degree of inflammation was reduced (P≤ 0.05) on time points. Cloprostenol sodium did not alter the histological appearance of uterine tissue during the postpartum period or reduce the period of uterine involution. Additionally, serial biopsies did not affect the fertility of the animals.