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Summary: Background: The use of commercial chromogenic agar plates for the rapid, easy and correct identification of equine endometritis‐causing bacteria has been proposed. Preliminary tests in our lab revealed undescribed limitations. Therefore, we tested the ability of the Brilliance UTI agar, a commercially available chromogenic agar, to accurately identify bacteria causing equine endometritis. Objectives: To 1) investigate whether bacteria present in the equine uterus are able to grow on this chromogenic agar plate, 2) determine whether these bacteria belong to the genera for which these agar plates were originally designed and 3) consider whether these bacterial genera can be correctly identified, based only on the colour appearance. Study design: In vitro experiments. Methods: Macroscopic growth and colour appearance on the Brilliance UTI agar of 58 bacterial isolates, from previously collected equine uterine samples, were evaluated. Isolates were tentatively identified at the genus level using the manufacturer's guidelines and results were compared with MALDI‐TOF MS as a "gold standard". Results: The study revealed that 1) 77% (N = 45/58) of the bacterial isolates grew well on this chromogenic agar, 2) 83% of the investigated isolates (N = 48/58) belonged to one of the genera for which guidelines for identification were provided by the manufacturer and 3) only 50% of the isolates (N = 29/58) were correctly identified at the genus level, based only on colour appearance. Main limitations: The current study uses purified bacterial isolates to inoculate the chromogenic agar plates, instead of fresh uterine samples. Bacteria were identified to the genus level using MALDI‐TOF MS. Conclusion: This study shows that identification at the genus level based only on colour appearance, without additional tests or the simultaneous use of other media, is not reliable based on the existing identification guidelines. [ABSTRACT FROM AUTHOR]

• Development of a highly sensitive and specific qPCR assay to determine equine sex. • The assay works on trophectoderm biopsies and herniating cells from equine in vitro -produced embryos. • A biopsy did not impair initial pregnancy rates or early pregnancy losses. • Other equine applications that require highly sensitive sex methods may benefit. • In vitro - and in vivo -generated horse embryos can be and characterised genetically. In addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo -flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro -produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro -produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro -produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro -produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications. [ABSTRACT FROM AUTHOR]

Summary: Background: The success rate for vitrification of immature equine oocytes is low. Although vitrified‐warmed oocytes are able to mature, further embryonic development appears to be compromised. Objectives: The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. Study design: Experimental in vitro and in vivo trials. Methods: Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to lower concentrations of CPAs. In Experiment 1, the maturation of oocytes surrounded by multiple layers of cumulus cells (CC oocytes) and oocytes surrounded by only corona radiata (CR oocytes) was investigated. In Experiment 2, spindle configuration was determined for CR oocytes vitrified using the two vitrification protocols. In Experiment 3, further embryonic development was studied after fertilisation and culture. Embryo transfer was performed in a standard manner. Results: Similar nuclear maturation rates were observed for CR oocytes vitrified using the long exposure and nonvitrified controls. Furthermore, a lower maturation rate was obtained for CC oocytes vitrified with the short exposure compared to control CR oocytes (P = 0.001). Both vitrification protocols resulted in significantly higher rates of aberrant spindle configuration than the control groups (P<0.05). Blastocyst development only occurred in CR oocytes vitrified using the short vitrification protocol, and even though blastocyst rates were significantly lower than in the control group (P<0.001), transfer of five embryos resulted in one healthy foal. Main limitations: The relatively low number of equine oocytes and embryo transfer procedures performed. Conclusions: 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. [ABSTRACT FROM AUTHOR]

An 11-year-old gelding presented with a large mass located in the dermis of his preputium that surrounded the penis completely. This mass was first noted years ago but did not increase in size. Histological examination diagnosed a fibroma. No treatment was initiated since no signs of discomfort were noticed and the gelding was performing at a high level in competitive showjumping. Next to skin tumours, tumours of the penis and preputium are most common. Most frequently, squamous cell carcinomas and sarcomas are diagnosed. Although fibromas can develop anywhere in connective tissue, a preputial location of a fibroma in a gelding is rare. Differential diagnosis and possible treatment options are discussed. [ABSTRACT FROM AUTHOR]

Reasons for performing study The therapeutic potential of mesenchymal stromal cells for cellular therapy has generated increasing interest in human as well as veterinary medicine. Considerable research has been performed on the cryopreservation of expanded mesenchymal stromal cells, but little information is available on the cryopreservation of the original mononuclear cell fraction. Objectives The present study describes a protocol to expand equine mesenchymal stromal cells after cryopreserving the mononuclear cells of umbilical cord blood. Methods To this end, mononuclear cells were isolated from 7 umbilical cord blood samples and cryopreserved at a concentration of 1-2 × 109 cells/l cold freezing solution. Cells were cryopreserved and kept frozen for at least 6 months before thawing. Frozen cryotubes were thawed in a 37°C water bath. Putative equine mesenchymal stromal cells were immunophenotyped using multicolour flow cytometry based on a selected 9 marker panel. Results Average cell viability upon thawing was 98.7 ± 0.6%. In 6 out of 7 samples, adherent spindle-shaped cell colonies were observed within 9.0 ± 2.6 days and attained 80% confluency at 12.3 ± 3.9 days. After 3 passages, putative equine mesenchymal stromal cells were successfully immunophenotyped as CD29, CD44 and CD90 positive, and CD45, CD73, CD79α, CD105, MHC II and monocyte-marker negative. Conclusions and potential relevance Equine mesenchymal stromal cells can be cultured after cryopreservation of the isolated mononuclear cells, a time- as well as cost-efficient approach in equine regenerative medicine. [ABSTRACT FROM AUTHOR]

Reasons for performing study: Sperm motility is considered to be one of the key features of semen analysis. Assessment of motility is frequently performed using computer-assisted sperm analysis (CASA). Nevertheless, no uniform standards are present to analyse a semen sample using CASA. Objectives: We hypothesised that the type of counting chamber used might influence the results of analysis and aimed to study the effect of chamber type on estimated concentration and motility of an equine semen sample assessed using CASA. Methods: Commonly used disposable Leja chambers of different depths were compared with disposable and reusable ISAS chambers, a Makler chamber and a World Health Organization (WHO) motility slide. Motility parameters and concentrations obtained with CASA using these different chambers were analysed. The NucleoCounter was used as gold standard for determining concentration. Results: Concentration and motility parameters were significantly influenced by the chamber type used. Using the NucleoCounter as the gold standard for determining concentration, the correlation coefficients were low for all of the various chambers evaluated, with the exception of the 12 µm deep Leja chamber. Filling a chamber by capillary forces resulted in a lower observed concentration and reduced motility parameters. All chambers evaluated in this study resulted in significant lower progressive motility than the WHO prepared slide, with the exception of the Makler chamber, which resulted in a slight, but statistically significant, increase in progressive motility estimates. Conclusions and potential relevance: 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. [ABSTRACT FROM AUTHOR]