During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross-reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79a, CD90, CD105, MHC-II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood-derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine-marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7-aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79a, MHC-II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine-marker panel can be used for the adequate immunophenotyping of equine MSC. '

During maternal recognition of pregnancy (MRP), a conceptus-derived signal leads to the persistence of the corpus luteum and the maintenance of gestation. In the horse, the nature of this signal remains to be elucidated. Several studies have focused on the changes in gene expression during MRP, but little information exists at the protein level. The aim of this study was to identify the proteins at the embryo-maternal interface around signalling of MRP in the horse (day 13) by means of mass spectrometry. A distinct influence of pregnancy was established, with 119 proteins differentially expressed in the uterine fluid of pregnant mares compared to cyclic mares and with upregulation of several inhibitors of the prostaglandin synthesis during pregnancy. By creating an overview of the proteins at the embryo-maternal interface in the horse, this study provides a solid foundation for further targeted studies of proteins potentially involved in embryo-maternal interactions, MRP and pregnancy loss in the horse.

Computer-assisted sperm analysis can only provide a rough estimate of sperm concentration and overestimation is likely when drop-filled slides with a coverslip are used. Motility estimates using CASA are highly influenced by the counting chamber; therefore, a complete description of the chamber type used should be provided in semen reports and in scientific articles.

The development of a healthy intestinal immune system requires early microbial exposure. However, it remains unclear whether microbial exposure already begins at the prenatal stage. Analysis of such low microbial biomass environments are challenging due to contamination issues. The aims of the current study were to assess the bacterial load and characterize the bacterial composition of the amniotic fluid and meconium of full-term calves, leading to a better knowledge of prenatal bacterial seeding of the fetal intestine. Amniotic fluid and rectal meconium samples were collected during and immediately after elective cesarean section, performed in 25 Belgian Blue cow-calf couples. The samples were analyzed by qPCR, bacterial culture using GAM agar and 16S rRNA gene amplicon sequencing. To minimize the effects of contaminants, we included multiple technical controls and stringently filtered the 16S rRNA gene sequencing data to exclude putative contaminant sequences. The meconium samples contained a significantly higher amount of bacterial DNA than the negative controls and 5 of 24 samples contained culturable bacteria. In the amniotic fluid, the amount of bacterial DNA was not significantly different from the negative controls and all samples were culture negative. Bacterial sequences were identified in both sample types and were primarily of phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, with some individual variation. We conclude that most calves encounter in utero maternal-fetal transmission of bacterial DNA, but the amount of bacterial DNA is low and viable bacteria are rare.

For vitrification of immature equine oocytes, the use of 1) CR oocytes, 2) a high concentration of CPAs, and 3) a short exposure time may be key factors for maintaining developmental competence.