Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos.

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.

Limitations of a chromogenic agar plate for the identifying bacteria isolated from equine endometritis samples.

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.

Blastocyst production after intracytoplasmic sperm injection with semen from a stallion with testicular degeneration.

In horse breeding, intracytoplasmic sperm injection (ICSI) has gained interest to obtain offspring from subfertile individuals. This paper presents a case report of a stallion with severe testicular degeneration. Semen analysis showed very low motility and 83.5% of detached heads. Histology of a testicular biopsy showed severely decreased spermatogenesis, while transmission electron microscopy of the sperm cells revealed no significant abnormalities. A total of 39 oocytes were fertilized by ICSI with frozen-thawed spermatozoa of this stallion: 25 oocytes with intact spermatozoa and 24 with detached heads. When using intact sperm cells, 8 out of the 25 oocytes cleaved, and 1 developed to the blastocyst stage 9 days after ICSI. None of the oocytes injected with a detached sperm head cleaved. Studies on the paternal influence on ICSI outcome are limited in the horse and further research is needed to define which stallion factors may influence ICSI results. Here, we report the possibility to produce a blastocyst by ICSI of a stallion suffering from testicular degeneration with a poor spermiogram, as long as an intact sperm cell containing a centriole is selected.

An improved vitrification protocol for equine immature oocytes, resulting in a first live foal.

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.

Malignant seminoma in two unilaterally cryptorchid stallions.

Two unilateral cryptorchid stallions were referred to the clinic because of chronic debilitating condition with emaciation. Rectal examination, and ultrasound and gross examination revealed in both animals an abdominal mass, caudally of the kidney, and multiple nodules spread over the abdomen. Histologic analysis revealed an intra-abdominal malignant seminoma with intraperitoneal and renal metastasis. Interestingly, a seminoma was also present in the descended testis of the draught horse.

Effect of artificial insemination protocol and dose of frozen/thawed stallion semen on pregnancy results in mares.

Deep intra-uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra-uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen-thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi-flexible Minitube pipette was compared with a newly designed insemination device with a more flexible telescopic insemination catheter (Ghent device). The semi-flexible Minitube pipette performed better than the newly designed insemination device with respect to pregnancy outcome (p = 0.008). The superiority of deep horn insemination over uterine body insemination was reflected by the better pregnancy rates obtained after deep insemination using the same low doses (30.6% better pregnancy rates) (p = 0.0123).

Hydrallantois in the mare--a report of five cases.

Hydrallantois in the mare is a very rare condition, and clinical reports help to gather information to elucidate its pathogenesis, treatment options and prognosis. Five different cases of hydrallantois in the mare are reported in this article, all with the involvement of placentitis. The five mares were presented because of acute distention of the abdomen, dyspnoea, stiff gait and a lack of appetite. After a gradual release of the excessive amount of allantoic fluid, an abortion was induced in all five mares. The foals were either born dead or euthanized. The mares recovered quickly. One mare conceived within the same season, one remained barren despite several cycles of natural breeding, and no data were available on the other three mares. In this series, the condition is reported for the first time in two Shetland ponies, both pregnant with foals sharing a close genetic background. In both cases, the condition led to hyperlipidemia. The condition as it occurs in nulliparous mares is also discussed. Finally, the possible involvement of placentitis in the pathogenesis is emphasized.

Congenital ascites due to hepatoblastoma with extensive peritoneal implantation metastases in a premature equine fetus.

A premature dead equine fetus with excessive fluctuating distension of the abdomen was delivered by extraction. Post-mortem examination revealed ascites and a solitary, irregular, bulging, multinodular, firm, yellow mass of 25 cm in diameter in the right liver lobe. Extensive peritoneal implantation metastases were present. The masses were composed of polygonal embryonal cells arranged in sheets and nests. Based on the immunohistochemical expression of Ki67, low molecular weight cytokeratin and alpha-1 fetoprotein, a diagnosis of hepatoblastoma with peritoneal implantation metastases was made.

Successful isolation of equine mesenchymal stromal cells from cryopreserved umbilical cord blood-derived mononuclear cell fractions.

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 × 10(9) 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.

A pilot comparison of laser-assisted vs piezo drill ICSI for the in vitro production of horse embryos.

Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore an alternative method avoiding oocyte trauma, namely laser-assisted ICSI (LAI), which involves creating a hole in the ZP prior to ICSI. In this pilot study, PDAI and LAI were compared for ICSI in the horse. No significant influences on subsequent embryonic development were observed.

Influence of counting chamber type on CASA outcomes of equine semen analysis.

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.

Sperm selection using single layer centrifugation prior to cryopreservation can increase thawed sperm quality in stallions.

REASONS FOR PERFORMING STUDY: The increasing use of modern reproductive techniques in human medicine has led to a higher demand for isolation of motile sperm. Several of these isolation techniques have been adapted for veterinary use and can be applied for the selection of a superior sperm sample from stallion semen. Until recently a major disadvantage of such isolation techniques was the limitation in sperm volume that could be handled. Androcoll-E had been shown to be successful for processing large volumes of equine semen but there are few data to substantiate the potential beneficial effect of freezing an Androcoll-E selected equine sperm sample to obtain higher quality following thawing. OBJECTIVES AND METHODS: In this study, the effect of Androcoll-E treatment of sperm prior to cryopreservation was compared with cushioned centrifugation using ejaculates from 8 different stallions selected because they were known to have semen of differing quality following freezing. RESULTS: Androcoll-E treatment increased measures of semen quality prior to freezing. However, Androcoll-E treatment reduced the yield of sperm following centrifugation when compared with the cushion centrifuged control group (50.9 ± 14.2% vs. 97.1 ± 9.0%, respectively). Quality analysis following thawing showed an overall improved sperm quality for Androcoll-E treated samples and average post thaw progressive motility (PM) was 41.6% compared with 30.5% for the cushion centrifuged group. CONCLUSIONS AND POTENTIAL RELEVANCE: Androcoll-E can be used with good results to select a superior sperm population prior to cryopreservation, in order to produce good-quality frozen thawed semen.

Two atypical cases of granulosa cell tumours in Belgian Blue heifers.

This paper describes two atypical cases of granulosa cell tumours (GCTs) in Belgian Blue heifers. In the first case, an 18-month-old heifer died after ovariectomy, and a granulosa-theca cell tumour was diagnosed post-mortem. The second heifer (31 months of age) was near term and was successfully treated surgically for a malignant granulosa cell tumour. Unfortunately, no further reproductive data are available, as the heifer was sold for slaughter. This is the first report describing GCTs in Belgian Blue beef cattle.

Uterine fibrosarcoma in a Warmblood mare.

This paper describes a case of uterine fibrosarcoma in an 18-year-old Warmblood mare. The mare had exhibited bloody fluid accumulation inside the uterus and vaginal haemorrhagic discharge since the previous foaling. The mare was euthanized, and on pathological examination, in addition to the uterine neoplasia, multiple metastases were found in the lungs, liver and spleen. The histological and immunohistochemical examination determined that the tumour was a fibrosarcoma. To our knowledge, this is the first paper to describe a uterine fibrosarcoma in a mare.

Validation and usefulness of the Sperm Quality Analyzer V equine for equine semen analysis.

Routine semen analysis includes evaluation of concentration combined with seminal volume, morphology and motility. Subjective analysis of these parameters is known to be inaccurate, imprecise and subject to variability. Automated semen analysis could lead to an increased standardization in and between laboratories but for that to happen automated devices need to be validated. A new device, the sperm quality analyzer V equine (SQA-Ve) version 1.00.43, was evaluated for its repeatability and agreement with light microscopy (LM), for raw and extended equine semen. Results were compared with computer assisted sperm analysis (CASA), which was also tested for its repeatability and agreement with LM. The SQA-Ve showed a good repeatability and fine agreement for assessing sperm concentration of raw semen based on scatter and Bland-Altman plots. This was in contrast with the motility parameters, which had a low repeatability. Morphology assessment with SQA-Ve was poorly repeatable as well as in poor agreement with LM. For extended semen, the findings were comparable. The SQA-Ve did well for concentration, whereas for the motility parameters repeatability was only just acceptable, with no agreement with LM. This sharply contrasted the CASA findings that were highly repeatable and almost in perfect agreement with LM. Based on these findings, the tested version of the SQA-Ve is insufficiently accurate to be used for analyzing raw or extended equine semen.

Case of bilateral seminoma in a trotter stallion.

This report describes a bilateral seminoma in a stallion. After slaughter, histological examination revealed that the tumour consisted predominantly of polyhedral tumour cells with large nuclei, obvious nucleoli and a small border of cytoplasm. The mitotic index was low and Ki67 staining revealed 4% nuclear staining. To our knowledge, this paper is the first using Ki67 staining as a method to evaluate the mitotic rate in a testicular seminoma in the stallion.

An abortion of monozygotic twins in a warmblood mare.

Naturally occurring monozygotic twins are extremely rare in the horse. This paper describes an abortion in a mare after 260 days of pregnancy with monozygotic twins, one a fresh foal and the other a mummified foal.