An Update on Applications of Cattle Mesenchymal Stromal Cells

Attention on mesenchymal stromal cells (MSCs) research has increased in the last decade mainly due to the promising results about their plasticity, self-renewal, differentiation potential, immune modulatory and anti-inflammatory properties that have made stem cell therapy more clinically attractive. Furthermore, MSCs can be easily isolated and expanded to be used for autologous or allogenic therapy following the administration of either freshly isolated or previously cryopreserved cells. The scientific literature on the use of stromal cells in the treatment of several animal health conditions is currently available. Although MSCs are not as widely used for clinical treatments in cows as for companion and sport animals, they have the potential to be employed to improve productivity in the cattle industry. This review provides an update on state-of-the-art applications of bovine MSCs to clinical treatments and reproductive biotechnologies

Equine Bone Marrow and Adipose Tissue Mesenchymal Stem Cells: Cytofluorimetric Characterization, In Vitro Differentiation, and Clinical Application

The aim of the present work was to isolate, cultivate, differentiate, and conduct cellular characterization of mesenchymal stem cells (MSCs) derived from equine adipose tissue (eAT) and bone marrow (eBM). Isolated and characterized cells were used in racehorses suffering from a superficial flexor tendon injury. Equine adipose tissue collection was performed at the base of the horse tail, whereas eBM was aspirated from iliac crest. Mononuclear cell fraction was isolated and cultured. In vitro differentiation and molecular characterization at P3 of culture were performed. No statistically significant differences in the number of cell doublings were found among different culture passages (P > .05). Doubling time was greater for eBM than eAT (3.2 1.5 vs. 1.3 0.7; P < .05). Positive von Kossa and Alizarin Red staining confirmed osteogenesis. Alcian Blue and Oil Red O staining illustrated chondrogenesis and adipogenesis, respectively. Isolated cells resulted positive for CD90, CD44, and CD105, whereas negative for hematopoietic markers, CD14, CD45, and CD34. Using isolated cells for injured tendon therapy, no adverse reactions were observed, and all inoculated horses returned to race competitions. In vitro results revealed the immunophenotypic characterization of isolated cells similar to that observed in human MSCs from the same sources; furthermore, in the present study, their clinical use proves the safety of eBM-derived and eAT-derived MSCs and a successful outcome for the treated animals that returned to their previous level of sport activity

Ultrastructural characteristics and immune profile of equine MSCs from fetal adnexa

Both in human and equine species, mesenchymal stem cells (MSCs) from amniotic membrane (AM) and Wharton\ue2\u80\u99s jelly (WJ), may be particularly useful for immediate use or in later stages of life, after cryopreservation in cell bank. The aim of this study was to compare equine AM- and WJ-MSCs in vitro features that may be relevant for their clinical employment. MSCs were more easily isolated from WJ, even if MSCs derived from AM exhibited more rapid proliferation (P< 0.05). Osteogenic and chondrogenic differentiation were more prominent in MSCs derived from WJ. This is also suggested by the lower adhesion of AM cells, demonstrated by the greater volume of spheroids after hanging drop culture (P< 0.05). Data obtained by PCR confirmed the immunosuppressive function of AM and WJ-MSCs and the presence of active genes specific for anti-inflammatory and angiogenic factors (IL-6, IL 8, IL-\uce\ub21). For the first time, by means of transmission electron microscopy (TEM), we ascertained that equine WJ-MSCs constitutively contain a very impressive number of large vesicular structures, scattered throughout the cytoplasm. Moreover, an abundant extracellular fibrillar matrix was located in the intercellular spaces among WJ-MSCs. Data recorded in this study reveal that MSCs from different fetal tissues have different characteristics that may drive their therapeutic use. These finding could be noteworthy for horses as well as for other mammalian species, including humans

Effects of Two Different Cooling Devices for Testicles Transport on Stallion Epididymal Sperm Quality

This study evaluates the effects of two cooling devices and temperature for testicles storage on epididymal sperm quality after 24 hours; different levels of seminal plasma (0% and 10%) were evaluated on sperm after recovering. Testicles from six stallions were recovered immediately after castration (2) or at the slaughterhouse (4); of the same animal, one testicle was placed in Equitainer (+8\ub0C), the other in a styrofoam box with ice (+3\ub0C). After 24 hours, the temperature of parenchyma was measured, and testicles and epididymal were weighted. Sperm were flushed from the cauda epididymides with Kenney extender, total sperm number recorded and motility and viability evaluated immediately after flushing (T0) with or without 10% SP (G1 Eq 0%, G2 Eq 10%, G3 Ice 0%, G4 Ice 10%). Motility and viability were evaluated after 24 hours and 48 hours of storage at +4\ub0C. Temperature of the parenchyma was lower in testicles stored in ice compared to Equitainer (3.2 \ub1 0.6\ub0C and 8.6 \ub1 2.5\ub0C, respectively; P < .05). Motility and viability at T0 were similar (P > .05) in G1 and G3, whereas addition of SP after recovery significantly improved motility only in samples stored in Equitainer (G2). Viability was higher (P < .05) in G2 than in G4. At T24 and T48, no differences (P > .05) in sperm quality were found between storage methods or samples with or without SP. In conclusion, equine testicles can be safely stored either at lower (+3\ub0C) or higher (+8\ub0C) temperature than +5\ub0C. This can be useful, especially when testicles are shipped in a hot climate, where devices cannot guarantee optimal refrigeration conditions

Macroscopic characteristics of the umbilical cord in Standardbred, Thoroughbred and Warmblood horses

The umbilical cord (UC), the connection between mother and fetus via the umbilical vessels, carries nutrients and oxygenated blood to the fetus through the umbilical vein and removes deoxygenated blood and waste products via the umbilical arteries. It is designed to protect blood flow to the fetus during pregnancy. In equine medicine, only a few studies have described the UC, and most of these involved Thoroughbreds. The present study describes and compares the macroscopic features of the equine umbilical cord in three different breeds and in relation to the foal's gender. In addition, a possible correlation between UC features and maternal and perinatal factors is investigated. One hundred and twenty four healthy mares with normal pregnancies were enrolled in the study and were divided into three groups according to their breed: 70 Standardbreds (STB), 38 Thoroughbreds (THB) and 16 Warmbloods (WAB). The following data were recorded: mare's age and parity, gestation length, placental weight, presence of fetal membrane alterations, UC length and number of coils in the amniotic and allantoic portions, and the Umbilical Coiling Index (UCI), which is the ratio between total coils and total UC length. The UCI has not been investigated previously in veterinary medicine. Furthermore, immediately after foaling, APGAR score, foal's weight and sex were recorded. All the STB and WAB were housed in Italy and the THB were housed in New Zealand. Mares\ue2\u80\u99 mean age was higher in WAB than in THB and STB; the latter had a significantly shorter gestation length. The foal's weight was positively correlated with placental weight in all breeds; and in STB, foal weight was positively related to parity and gestation length. Mean total UC length was comparable to previous reports in THB, STB and WAB. The lengths of the two UC portions were statistically different between STB and THB, where the amniotic portion was longer than the allantoic one. In each breed, total UC length was correlated with total number of coils (THB and STB = 5 \uc2\ub1 1; WAB = 6 \uc2\ub1 1), the UC amniotic length was positively correlated with the number of amniotic coils and the allantoic length was positively correlated with the number of allantoic coils. The UCI values were 0.09 in STB and THB and 0.1 in WAB. This study provides reference values for UCI that could be included in the gross placental evaluation if its clinical importance were demonstrated

Variation in foraging activity influences area-restricted search behaviour by bottlenose dolphins

Open Access via the Royal Society Agreement Beatrice Offshore Wind Ltd COWRIE Department of Energy & Climate Change, Scottish Government Fundación la Caixa (becas Posgrado, 2015) Marine Mammal Monitoring Programme (MMMP) Marine Scotland Science Moray Offshore Wind Farm (East) Ltd NatureScot Funding This project was made possible through the integration of O.F.B.'s PhD into a broader NatureScot and joint industry funded Marine Mammal Monitoring Programme (MMMP) that supports statutory monitoring of the Moray Firth SAC and offshore windfarm construction. We thank NatureScot, Marine Scotland Science, Beatrice Offshore Wind Ltd, Moray Offshore Wind Farm (East) Ltd, Department of Energy & Climate Change, Scottish Government, Oil & Gas UK and COWRIE for contributing funds or equipment to the MMMP. O.F.B. was funded through a studentship from the Fundación ‘la Caixa’ (Becas Posgrado, 2015). I.M.G., B.J.C. and P.M.T. were core funded by the University of Aberdeen but with salary support for the period of this study though contract to MMMP. V.I.M. and S.M.P. were funded through the MMMP. R.X.C. was core funded by Leibniz Institute for Zoo and Wildlife Research (IZW). Acknowledgements The authors gratefully acknowledge the support of the Maxwell High Performance Computing Cluster, funded by the University of Aberdeen, during the development of DOLPHIN-SPOT. We would also like to thank Claudia Aparicio Estaella for her help during the validation of the automatic detector. We acknowledge Bill Ruck, Moray First Marine and colleagues from the University of Aberdeen for assistance with the data collection and anonymous reviewers for comments that helped improve the manuscript.Peer reviewedPublisher PD

Sex-sorted canine sperm cryopreservation: Limits and procedural considerations

The aim of this study was to define a protocol to store dog sperm before and after sorting to obtain an insemination dose sufficient to allow the conception by artificial insemination. Experiment 1 and 2 were performed to evaluate the more appropriate extender for preserving at room temperature dog sperm before and after sorting. Four extenders were tested: (1) Tris-fructose-citrate (TFC), (2) Tris-glucose-citrate (TGC), (3) modified Tyrode\u2019s albumin lactate pyruvate medium (mTALP), and (4) third fraction of the ejaculate (after centrifugation at 5000 g for 10 minutes; III FRAC). Experiment 3 and 4 were performed to evaluate the ability of dog semen to withstand sex sorting and freezing/thawing. Modified Tyrode\u2019s albumin lactate pyruvate medium was the best extender for canine sperm storage at room temperature (20 C\u201325 C) before (total motility: TFC, 8.3 1.7; TGC, 50.0 11.5; mTALP, 70.0 0.1; III FRAC, 25.0 1 0.4; P < 0.05) and after sorting (total motility: TFC, 7.3 1.5; TGC, 10.3 1.5; mTALP, 33.3 6.7; III FRAC, 8.7 5.8; P < 0.05), even if at 24- hour sorted sperm quality was impaired in all extenders tested herein. Sperm quality decreased after sorting (total motility: control, 92.5 0.9; sorted, 52.9 6.0; P < 0.05) and, especially, after freezing/thawing (total motility: frozen control, 25.7 4.1; frozen sorted, 2.4 1.2; P < 0.05). In conclusion, mTALP is an appropriate medium for canine sperm storage before and soon after sorting (hours), but a long storage period of sexed sperm at room temperature is not adequate. Cryopreservation greatly impaired sperm quality, and further studies are needed to optimize the freezing protocol for sexed dog sperm

Heterologous Wharton's Jelly Derived Mesenchymal Stem Cells Application on a Large Chronic Skin Wound in a 6-Month-Old Filly

A complex feedback of growth factors, secreted by a variety of cell types, is responsible for the mediation of skin healing. Despite the recent advances in wound healing management, this fails up to 50% and skin wounds can still be considered one of the main causes of morbidity, both in human and veterinary medicine. Regenerative medicine, involving mesenchymal stromal cells (MSCs), is nowadays a promising solution for skin wound healing. Indeed, MSCs are involved in the modulation of the inflammatory local response and cell replacing, by a paracrine mode of action. Local application of equine umbilical cord Wharton's jelly MSCs (WJMSCS) was carried out in a 6-months-old filly with a non-healing skin wound. Heterologous WJMSCs were applied four times using a carboxymethylcellulose (CMC) gel, produced dissolving CMC in autologous plasma. At first application the mean wound area was 7.28 ± 0.2 cm2. Four days after the last application of WJMSCs, the mean wound area was 1.90 ± 0.03 cm2, and the wound regression rate was +74%. No local or systemic side effects were registered after WJMSCs application and no evident exuberant scar was observed after wound healing. At discharge, the mean wound area was 0.38 ± 0.01 cm2 and the total regression rate was +80%. Five days later, the wound was completely healed. In the present clinical case report, the use of WJMSCs led to promising clinical results, paving the way for possible future applications in the treatment of chronic wounds in horses