Equine Tissue Research Articles

Bioactive glass ceramic nanoparticles-coated poly(l-lactic acid) scaffold improved osteogenic differentiation of adipose stem cells in equine.

Horses with big bone fractures have low chance to live mainly due to the lake of a proper treatment strategy. We believe that further attempts in equine bone tissue engineering will probably be required to meet all the needs for the lesion therapies. Therefore in this study we aimed to investigate the osteogenic differentiation capacity of equine adipose-derived stem cells (e-ASCs) on nano-bioactive glass (nBGs) coated poly(l-lactic acid) (PLLA) nanofibers scaffold (nBG-PLLA). Using electrospinning technique, PLLA scaffold was prepared successfully and coated with nBGs. Fabricated nanofibers were characterized by MTT, SEM, and FTIR analyses, and then osteogenic differentiation potential of isolated e-ASCs was investigated by the most key osteogenic markers, namely Alizarin red-S, ALP, calcium content and bone related (RUNX2, Collagen I, Osteonectin, and ALP) gene markers. Our results indicated that nBGs was successfully coated on PLLA scaffold and this scaffold had no negative (p>0.05) effect on cell growth rate as indicated by MTT assay. Moreover, e-ASCs that differentiated on nBGs-PLLA scaffold showed a higher (p<0.05) ALP activity, more (p<0.05) calcium content, and higher (p<0.05) expression of bone-related genes than that on uncoated PLLA scaffold and TCPS. According to the results, a combination of bioceramics and biopolymeric nanofibers hold valuable promising potentials to use for bone tissue engineering application and regenerative medicine.

The Analysis of Phenylbutazone and Its Active Metabolite, Oxyphenbutazone, in Equine Tissues (Muscle, Kidney, and Liver), Urine, and Serum by LC-MS/MS.

This study reports the use of two validated LC with tandem MS (MS/MS) methods to study the residue depletion profile of phenylbutazone (PBZ) and its metabolite oxyphenbutazone (OXPBZ) from equine serum, urine, and muscle, kidney, and liver tissues. One LC-MS/MS method, with an LOQ of 1.0 ng/mL for PBZ and 2.0 ng/mL for OXPBZ, was used for the analysis of the two drugs in the biological fluids (equine urine and serum); the other LC-MS/MS method, with an LOQ of 0.5 ng/g for PBZ and OXPBZ, was used for the analysis of the drugs in the equine tissue samples. PBZ was administered intravenously to two horses dosed with 8.8 mg/kg PBZ once daily for 4 days and sacrificed humanely at a slaughter plant 7 days after the last drug administration. Urine, serum, and kidney, liver, and muscle tissues were collected from the two horses and shipped on ice to the laboratory and stored at -20°C until analysis. The concentrations of PBZ and OXPBZ residues in the biological fluid and tissue samples collected at slaughter were measured with the two validated LC-MS/MS methods using deuterated internal standards. The results demonstrate that the validated methods are fit for studying the depletion kinetics of PBZ residues in equine tissues and biological fluids.

A finite element model to study the effect of tissue anisotropy on ex vivo arterial shear wave elastography measurements

Shear wave elastography (SWE) is an ultrasound (US) diagnostic method for measuring the stiffness of soft tissues based on generated shear waves (SWs). SWE has been applied to bulk tissues, but in arteries it is still under investigation. Previously performed studies in arteries or arterial phantoms demonstrated the potential of SWE to measure arterial wall stiffness—a relevant marker in prediction of cardiovascular diseases. This study is focused on numerical modelling of SWs in ex vivo equine aortic tissue, yet based on experimental SWE measurements with the tissue dynamically loaded while rotating the US probe to investigate the sensitivity of SWE to the anisotropic structure. A good match with experimental shear wave group speed results was obtained. SWs were sensitive to the orthotropy and nonlinearity of the material. The model also allowed to study the nature of the SWs by performing 2D FFT-based and analytical phase analyses. A good match between numerical group velocities derived using the time-of-flight algorithm and derived from the dispersion curves was found in the cross-sectional and axial arterial views. The complexity of solving analytical equations for nonlinear orthotropic stressed plates was discussed.

Use of contrast media in computed tomography and magnetic resonance imaging in horses: Techniques, adverse events and opportunities.

The use of contrast media in computed tomography (CT) and magnetic resonance imaging (MRI) is increasing in horses. These contrast-enhanced imaging techniques provide improved tissue delineation and evaluation, thereby expanding diagnostic capabilities. While generally considered safe, not all contrast media exhibit the same safety profiles. The safety of contrast media use and descriptions of adverse events occurring in horses are sparsely reported. This review summarises the reported evidence of contrast media use and adverse events that occur in horses, with added contribution from other veterinary species and studies in man for comparison. This comprehensive data set empowers equine clinicians to develop use and monitoring strategies when working with contrast media. Finally, it summarises the current state-of-the-art and highlights the potential applications of contrast-enhanced CT and MRI for assessment of diseased or injured equine tissues, as well as (patho)physiological processes.

Insulin signaling in various equine tissues under basal conditions and acute stimulation by intravenously injected insulin

The aim of the study was to analyze key proteins of the equine insulin signaling cascade and their extent of phosphorylation in biopsies from muscle tissue (MT), liver tissue (LT), and nuchal AT, subcutaneous AT, and retroperitoneal adipose tissues. This was investigated under unstimulated (B1) and intravenously insulin stimulated (B2) conditions, which were achieved by injection of insulin (0.1 IU/kg bodyweight) and glucose (150 mg/kg bodyweight). Twelve warmblood horses aged 15 ± 6.8 yr (yr), weighing 559 ± 79 kg, and with a mean body condition score of 4.7 ± 1.5 were included in the study. Key proteins of the insulin signaling cascade were semiquantitatively determined using Western blotting. Furthermore, modulation of the cascade was assessed. The basal expression of the proteins was only slightly influenced during the experimental period. Insulin induced a high extent of phosphorylation of insulin receptor in LT (P < 0.01) but not in MT. Protein kinase B and mechanistic target of rapamycin expressed a higher extent of phosphorylation in all tissues in B2 biopsies. Adenosine monophosphate protein kinase, as a component related to insulin signaling, expressed enhanced phosphorylation in MT (P < 0.05) and adipose tissues (nuchal AT P < 0.05; SCAT P < 0.01; retroperitoneal adipose tissue P < 0.05), but not in LT at B2. Tissue-specific variations in the acute response of insulin signaling to intravenously injected insulin were observed. In conclusion, insulin sensitivity in healthy horses is based on a complex concerted action of different tissues by their variations in the molecular response to insulin.

Effects of FSH addition to an enriched medium containing insulin and EGF after long-term culture on functionality of equine ovarian biopsy tissue

The effect of FSH supplementation on an enriched cultured medium containing insulin (10 ng/mL) and EGF (50 ng/mL) was investigated on in vitro culture of equine ovarian biopsy tissue. Ovarian tissue fragments were collected from mares (n = 10) and distributed in the following treatments: noncultured control, cultured control, and cultured + FSH. Both treated groups were cultured for 7 or 15 days. The end points evaluated were: follicular morphology, estradiol levels in the culture medium, fluorescence intensity for TUNEL, EGFR and Ki-67 detection, and gene expression of GDF-9, BMP-15, and Cyclin-D2 in the ovarian tissue. After seven days of culture, medium supplemented with FSH had a similar (P > 0.05) percentage of morphologically normal follicles compared to the noncultured control group. Estradiol levels increased (P < 0.05) from Day 7 to Day 15 of culture for both treated groups. No difference (P > 0.05) was observed for TUNEL and EGFR intensity between the noncultured control group and the treated groups after 15 days of culture. Ki-67 intensity did not differ (P > 0.05) between treated groups after 15 days of culture, but decreased (P < 0.05) when compared with the noncultured control group. Similar (P > 0.05) mRNA expression for GDF-9, BMP-15, and Cyclin-D2 was observed among all treatments after 15 days of culture. In conclusion, an enriched medium supplemented or not with FSH was able to maintain the functionality of equine ovarian biopsy tissue after a long-term in vitro culture.

Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo

Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Sensitivity and repeatability were established, and specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old fully differentiated (mature), newly seeded (immature) primary equine respiratory epithelial cell (ERECs), and equine dermal cell cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 14days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with equine herpesvirus 1 (EHV-1). The qPCR assay detected EHV-5 at stable concentrations throughout 14days in inoculated mature EREC and equine dermal cell cultures (peaking at 202 and 5861 viral genomes per 106 cellular β actin, respectively). EHV-5 copies detected in the immature EREC cultures increased over 14days and reached levels greater than 10,000 viral genomes per 106 cellular β actin. Moreover, EHV-5 was detected in the lymphocytes of 76% of horses and in the nasal swabs of 84% of horses experimentally infected with EHV-1 pre-inoculation with EHV-1. Post-inoculation with EHV-1, EHV-5 was detected in lymphocytes of 52% of horses while EHV-5 levels in nasal swabs were not significantly different from pre-inoculation levels. In conclusion, qPCR was a reliable technique to investigate viral load in in vivo and in vitro samples, and EHV-5 replication in equine epithelial cells may be influenced by cellular stages of differentiation.

Equine ovarian tissue viability after cryopreservation and in vitro culture

Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmentation. Vitrification with EG had the highest rate of morphologically normal preantral follicles, while DMSO had the lowest (76.1 ± 6.1% and 40.9 ± 14.8%, respectively; P < 0.05). In slow-freezing, despite that DMSO had the highest percentage of morphologically normal follicles (77.7 ± 5.8%), no difference among the CPAs was observed. Fluorescence intensity of EGFR and Ki-67 was greater when vitrification with EG was used. Regardless of the cryopreservation treatment, DMSO had the highest (P < 0.05) Bax/Bcl-2 ratio; however, DNA fragmentation was similar (P > 0.05) among treatments after thawing. After in vitro culture, the percentage of normal follicles was similar (P > 0.05) between slow-freezing and vitrification methods; however, vitrification had greater (P < 0.05) stromal cell density than slow-freezing. In summary, equine ovarian tissue was successfully cryopreserved, increasing the viability of the cells in the ovarian tissue after thawing when using DMSO and EG for slow-freezing and vitrification methods, respectively. Therefore, these results are relevant for fertility preservation programs.

Isolation and characterization of equine native MSC populations

BackgroundIn contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell populations in that species. Moreover, a comparison between perivascular CD146+ and CD34+ cell populations has not been performed in any species.MethodsImmunohistochemistry was used to identify adventitial cells (CD34+) and pericytes (CD146+) and to determine their localization in relation to MSCs in equine tissues. Isolation of CD34+ (CD34+/CD146–/CD144–/CD45–) and CD146+ (CD146+/CD34–/CD144–/CD45–) cell fractions from equine adipose tissue was achieved by fluorescence-activated cell sorting. The isolated cell fractions were cultured and analyzed for the expression of MSC markers, using qPCR and flow cytometry, and for the ability to undergo trilineage differentiation. Angiogenic properties were analyzed in vivo using a chorioallantoic membrane (CAM) assay.ResultsBoth CD34+ and CD146+ cells displayed typical MSC features, namely growth in uncoated tissue culture dishes, clonal growth when seeded at low density, expression of typical MSC markers, and multipotency shown by the capacity for trilineage differentiation. Of note, CD146+ cells were distinctly angiogenic compared with CD34+ and non-sorted cells (conventional MSCs), demonstrated by the induction of blood vessels in a CAM assay, expression of elevated levels of VEGFA and ANGPT1, and association with vascular networks in cocultures with endothelial cells, indicating that CD146+ cells maintain a pericyte phenotype in culture.ConclusionThis study reports for the first time the successful isolation and culture of CD146+ and CD34+ cell populations from equine tissues. Characterization of these cells evidenced their distinct properties and MSC-like phenotype, and identified CD146+ cells as distinctly angiogenic, which may provide a novel source for enhanced regenerative therapies.

Panfungal Polymerase Chain Reaction for Identification of Fungal Pathogens in Formalin-Fixed Animal Tissues

Identification of fungal organisms often poses a problem for pathologists because the histomorphology of some fungal organisms is not specific, fresh tissues may not be available, and isolation and identification in culture may take a long time. The purpose of this study was to validate the use of panfungal polymerase chain reaction (PCR) to identify fungal organisms from formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed paraffin-embedded curls were tested from 128 blocks containing canine, feline, equine, and bovine tissues with cutaneous, nasal, pulmonary, and systemic fungal infections, identified by the presence of fungi in histologic sections. Quantitative scoring of histologic sections identified rare (11.9%), occasional (17.5%), moderate (17.5%), or abundant (53.1%) fungal organisms. DNA was isolated from FFPE tissues and PCR was performed targeting the internal transcribed spacer 2 (ITS-2) region, a segment of noncoding DNA found in all eukaryotes. Polymerase chain reaction products were sequenced and identified at ≥97% identity match using the Basic Local Alignment Search Tool and the NCBI database of ITS sequences. Of the 128 blocks, 117 (91.4%) yielded PCR products and high-quality sequences were derived from 89 (69.5%). Sequence and histologic identifications matched in 79 blocks (61.7%). This assay was capable of providing genus- and species-level identification when histopathology could not and, thus, is a beneficial complementary tool for diagnosis of fungal diseases.

Comprehensive characteristics of microRNA expression profile of equine sarcoids

Equine sarcoids are the most common neoplasms occurring in horses. Despite frequent occurrence, they are still not well described at the molecular level. Thus, in the present study, we performed a comprehensive comparative analysis of sarcoid miRNAome profile to identify aberrantly expressed microRNAs, along with their structural variants, potentially useful as biomarkers and, in a wider perspective, broaden the knowledge about this tumor and underlying mechanisms. To this end, we conducted next generation sequencing and as a result we identified both known and potentially novel miRNAs. Differential expression analysis revealed the existence of almost one hundred miRNAs being over- or underexpressed in sarcoids in comparison to healthy tissue (p-adj<0.05), of which many are known for their involvement in processes crucial for neoplastic transformation. Among upregulated miRNAs there were those associated with decreased cell adhesion abilities as well as engaged in global protein production, while downregulation of some miRs i.a. increased cell expansion abilities. Moreover, we identified altered expression levels of miRNA variants (isomiRs) between the investigated tissues. Further analysis revealed that 5′ isomiRs comprise different seed sequences leading to target gene switching followed by activation of different biological pathways. Our results are the first which revealed the complexity of microRNA profiles in equine sarcoids and skin tissue, along with the dynamism of their growing in importance concomitants, namely isomiRs. They also showed miRNA molecules and biological pathways important from the sarcoid oncogenesis point of view.

Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C.

The method of transportation and the conditions imposed on the ovarian tissue are pivotal aspects for the success of ovarian tissue cryopreservation (OTC). The aim of this study was to evaluate the effect of the size of the ovarian tissue (e.g. whole ovary, biopsy size and transplant size) during different times of storage (0, 6, 12 and 24 h) on the structural integrity of equine ovarian tissue transported at 4°C. Eighteen pairs of ovaries from young mares (<10 years old) were harvested in a slaughterhouse and processed to simulate the fragment sizes (biopsy and transplant size groups) or kept intact (whole ovary group) and stored at 4°C for up to 24 h in α-MEM-enriched solution. The effect of the size of the ovarian tissue was observed on the morphology of preantral follicles, stromal cell density, DNA fragmentation and mitochondrial membrane potential. The results showed that (i) biopsy size fragments had more morphologically normal preantral follicles after 24 h of storage at 4°C; (ii) mitochondrial membrane potential was the lowest during each storage time when the whole ovary was used; (iii) DNA fragmentation rate in the ovarian cells of all sizes of fragments increased as storage was prolonged and (iv) transplant size fragments had increased stromal cell density during storage at cool temperature. In conclusion, the biopsy size fragment was the best to preserve follicle morphology for long storage (24 h); however, transportation/storage should be prior determined according to the distance (time of transportation) between patient and reproduction centers/clinics.

Xenografting of isolated equine (Equus caballus) testis cells results in de novo morphogenesis of seminiferous tubules but not spermatogenesis.

The study of spermatogenesis in the horse is challenging because of the absence of an invitro system that is capable of reproducing efficient spermatogenesis and because of the difficulties and costs associated with performing well-controlled studies invivo. In an attempt to develop novel methods for the study of equine spermatogenesis, we tested whether cells from enzymatically digested pre-pubertal equine testicular tissue were capable of de novo tissue formation and spermatogenesis following xenografting under the back skin of immunocompromised mice. Testes were obtained from normal pre-pubertal colts and dissociated into cell suspensions using trypsin/collagenase digestion. Resulting cell pellets, consisting of both somatic and germ cells, were injected into fascial pockets under the back skin of immunocompromised, castrated mice and maintained for between 1 and 14months. Mice were killed and grafts were recovered and analyzed. As has been reported for testis cell suspensions from pigs, mice, cattle, and sheep, de novo formation of equine testicular tissue was observed, as evidenced by the presence of seminiferous tubules and an interstitial compartment. There was an increased likelihood of de novo testicular formation as grafting period increased. Using indirect immunofluorescence, we confirmed the presence of spermatogonia in de novo formed seminiferous tubules. However, we found no evidence of meiotic or haploid cells. These results indicate that dissociated pre-pubertal equine testis cells are capable of reorganizing into the highly specialized endocrine and spermatogenic compartments of the testis following ectopic xenografting. However, in spite of the presence of spermatogonia within the seminiferous tubules, spermatogenesis does not occur. Although this technique does allow access to the cells within the seminiferous tubule and interstitial compartments of the equine testis prior to reaggregation, the absence of spermatogenesis will limit its use as a method for the study of testicular function in the horse.

Immunohistochemical characterisation of immune cells in fixed equine endometrial tissue: a diagnostic relevant method

Immunohistochemical characterisation of immune cells in fixed equine endometrial tissue: a diagnostic relevant method

Segmental diversity of phosphate transport along the intestinal axis in horses.

For horses, distinct differences in intestinal phosphate transport have been postulated to account for the unique features of hind gut fermentation compared to other monogastric animals and ruminants. So far published data on mechanisms and underlying transport proteins involved in intestinal phosphate transport in the horse are still missing. Therefore we investigated intestinal phosphate transport in horses at both functional and molecular levels. Segmental diversity of intestinal phosphate transport along the intestinal axis was documented using the Ussing chamber technique. A transcellular phosphate secretion in the jejunum was confirmed. Furthermore, 2 sodium-dependent phosphate cotransporters, NaPiIIb and PiT1, were first detected in the equine intestine at mRNA level with PiT1 being expressed in both the small and large intestine, and NaPiIIb being solely expressed in the large intestine. In the colon, unidirectional net flux rates of phosphate were significantly greater compared to flux rates in other segments ( < 0.005) suggesting the colon as a major site for phosphate absorption in horses. Phosphate transport in the colon was mainly transcellular and mediated by a sodium-gradient as documented by Ussing chamber experiments and uptake of phosphate into colonic brush border membrane vesicles. In summary, the present study demonstrated mechanisms and transporters of intestinal phosphate transport in equine intestinal tissues with distinct differences between intestinal segments providing a new basis for a better understanding of intestinal phosphate transport in horses.

Developmental expression of B cell molecules in equine lymphoid tissues

Identification and classification of B cell subpopulations has been shown to be challenging and inconsistent among different species. Our study tested aspects of ontogeny, phenotype, tissue distribution, and function of equine CD5hi B cells, which represented a greater proportion of B cells early in development and in the peritoneal cavity. CD5hi and CD5lo B cells differentially expressed B cell markers (CD2, CD21, IgM) measured using flow cytometry, but similar mRNA expression of signature genes (DGKA, FGL2, PAX5, IGHM, IL10) measured using quantitative RT-PCR. Sequencing lambda light chain segments revealed that CD5hi B cells generated diverse immunoglobulin repertoires, and more frequently bound to fluorescence-labeled phosphorylcholine. This study shows developmental characteristics and tissue distribution of a newly described subpopulation of B cells in the horse.

195 EXPRESSION OF MESENCHYMAL STROMAL CELL (MSC) MARKERS IN THE EQUINE ENDOMETRIUM AND IN VITRO INFLUENCE OF STEROID HORMONES ON ENDOMETRIAL-DERIVED MSC

Mesenchymal stromal cell (MSC) are multipotent precursor cells that have been isolated from many tissues, including endometrium in some species. These cells are necessary for tissue homeostasis, which in the cycling equine endometrium is regulated in part by changes in concentration of steroid hormones. The expression of oestrogen and progesterone receptors during the oestrous cycle has been studied before, but MSC gene expression is not reported as well as the effects of steroid hormones on in vitro proliferation of endometrial MSC. This study was designed to investigate the influence of steroid hormones on endometrial MSC proliferation in vitro and to examine mRNA expression of MSC markers (CD29, CD44, CD73, CD90, and CD105) in the healthy equine endometrium during the oestrous cycle. Equine endometrial tissue was collected postmortem (n = 6) and digested using a dissociation medium and mucin-1-bound magnetic beads were utilised to remove epithelial cells from the resulting single-cell solution. The cells were expanded in culture and, at passage 4, incubated with 3 different concentrations of oestradiol and progesterone for 5 days. For the proliferation analysis the Alamar Blue® assay was used according to manufacturer instructions. Endometrial biopsies, for quantitative RT-PCR analysis, were taken from healthy mares (n = 5) on Day 5 and 13 post-ovulation, during oestrus (1 follicle &gt;3.5cm, pronounced uterine oedema), and seasonal anestrous (seasonal anovulation). The ΔCt values were used for statistical analysis using SPSS Statistics 22 (IBM Corp., Armonk, NY). Data for quantitative PCR are presented as gene expression relative to the mean of 18S and GAPDH. No significant differences in proliferation could be detected in the various groups incubated with steroid hormones compared with the controls supplemented with charcoal-stripped fetal bovine serum. Detectable levels of mRNA for all 5 MSC markers analysed were present throughout the oestrous cycle. While the levels of CD73 were consistent, the expression of 3 MSC markers (CD29, CD44, and CD105) was elevated at Day 13. This difference was substantial between Day 13 and oestrus for CD29 (37.6 ± 6.2 and 12.2 ± 3.4; P &lt; 0.01) and CD105 (8.3 ± 0.9 and 4.5 ± 0.6; P &lt; 0.05), and between Day 5 and 13 for CD29 (7.4 ± 2.3 and 37.6 ± 6.2) and CD44 (12.9 ± 1.8 and 4.1 ± 0.3; P &lt; 0.01). In contrast, CD90 expression was higher at oestrus (27.8 ± 3.8) than at Day 5 (6.7 ± 0.9) or 13 (12.0 ± 2.1; P &lt; 0.01). Elevated quantities of MSC marker transcripts during late diestrus might be linked to the preparation of the equine endometrium for the proliferation phase associated with oestrus. However, the in vitro proliferation of endometrial-derived MSC is not influenced by the steroid hormones, although gene expression of steroid hormone receptors is present throughout the oestrous cycle of the mare. In summary, this study shows that the equine endometrium expresses MSC markers, and it does so at variable levels throughout the oestrous cycle; however, cell proliferation in vitro is not influenced by steroid hormones. This information will be useful for future studies aiming to derive endometrial MSC from mares.

Trans- and paracellular calcium transport along the small and large intestine in horses

Intestinal calcium absorption plays a key role in the maintenance of calcium homeostasis and may either occur by paracellular or transcellular mechanisms. The horse has some unique peculiarities in calcium homeostasis compared to other species including a high absorptive capacity for calcium in the intestine, high plasma calcium concentrations, high renal excretion, and low plasma concentrations of vitamin D metabolites. So far, knowledge about the underlying mechanisms and the regulation of intestinal calcium absorption is still limited concerning this species. Several studies have documented that intestinal calcium transport in horses is not as dependent on vitamin D as in other species. However, published data on other potential regulatory mechanisms are still lacking. In the present study, paracellular and transcellular transport mechanisms for intestinal calcium transport along the intestinal axis were identified in horses using the Ussing chamber technique. Furthermore, the expression of respective transport proteins including transient receptor potential vanilloid member 6, calbindin-D9k and calcium ATPase type 1 in line with the determined calcium flux rates was documented. In respect to regulation of transepithelial calcium transport, novel regulatory proteins for maintaining calcium homeostasis such as B-box and SPRY-domain containing protein and calmodulin were investigated for the first time in equine intestinal tissues in this study. This provides the basis for a new approach for a better understanding of equine calcium homeostasis regulation.

Novel equine tissue miRNAs and breed-related miRNA expressed in serum.

BackgroundMiRNAs regulate multiple genes at the post-transcriptional level and therefore play an important role in many biological processes. It has been suggested that miRNA exported outside the cells contribute to inter-cellular communication. Consequently, circulating miRNAs are of particular interest and are promising biomarkers for many diseases. The number of miRNAs annotated in the horse genome is much lower compared to model organisms like human and mouse. We therefore aimed to identify novel equine miRNAs for tissue types and breed in serum.ResultsWe analysed 71 small RNA-seq libraries derived from nine tissues (gluteus medius, platysma, masseter muscle, heart, liver, cartilage, bone, total blood and serum) using miRDeep2 and miRdentify tools. Known miRNAs represented between 2.3 and 62.9 % of the reads in 71 libraries. A total of 683 novel miRNAs were identified. Breed and tissue type affected the number of miRNAs detected and interestingly, affected its average intensity. A total of 50 miRNAs in serum proved to be potential biomarkers to differentiate specific breed types, of which miR-122, miR-200, miR-483 were over-expressed and miR-328 was under-expressed in ponies compared to Warmbloods. The different miRNAs profiles, as well as the differences in their expression levels provide a foundation for more hypotheses based on the novel miRNAs discovered.ConclusionsWe identified 683 novel equine miRNAs expressed in seven solid tissues, blood and serum. Additionally, our approach evidenced that such data supported identification of specific miRNAs as markers of functions related to breeds or disease tissues.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3168-2) contains supplementary material, which is available to authorized users.

Semi-automated International Cartilage Repair Society scoring of equine articular cartilage lesions in optical coherence tomography images.

Arthroscopic optical coherence tomography (OCT) is a promising tool for the detailed evaluation of articular cartilage injuries. However, OCT-based articular cartilage scoring still relies on the operator's visual estimation. To test the hypothesis that semi-automated International Cartilage Repair Society (ICRS) scoring of chondral lesions seen in OCT images could enhance intra- and interobserver agreement of scoring and its accuracy. Validation study using equine cadaver tissue. Osteochondral samples (n = 99) were prepared from 18 equine metacarpophalangeal joints and imaged using OCT. Custom-made software was developed for semi-automated ICRS scoring of cartilage lesions on OCT images. Scoring was performed visually and semi-automatically by five observers, and levels of inter- and intraobserver agreement were calculated. Subsequently, OCT-based scores were compared with ICRS scores based on light microscopy images of the histological sections of matching locations (n = 82). When semi-automated scoring of the OCT images was performed by multiple observers, mean levels of intraobserver and interobserver agreement were higher than those achieved with visual OCT scoring (83% vs. 77% and 74% vs. 33%, respectively). Histology-based scores from matching regions of interest agreed better with visual OCT-based scoring than with semi-automated OCT scoring; however, the accuracy of the software was improved by optimising the threshold combinations used to determine the ICRS score. Images were obtained from cadavers. Semi-automated scoring software improved the reproducibility of ICRS scoring of chondral lesions in OCT images and made scoring less observer-dependent. The image analysis and segmentation techniques adopted in this study warrant further optimisation to achieve better accuracy with semi-automated ICRS scoring. In addition, studies on in vivo applications are required.