Design of an electrospun tubular construct combining a mechanical and biological approach to improve tendon repair.

Hand tendon injuries represent a major clinical problem and might dramatically diminish a patient's life quality. In this study, a targeted solution for flexor tendon repair was developed by combining a mechanical and biological approach. To this end, a novel acrylate-endcapped urethane-based polymer (AUP) was synthesized and its physico-chemical properties were characterized. Next, tubular repair constructs were developed using electrospinning of the AUP material with incorporated naproxen and hyaluronic acid (i.e. anti-inflammatory and anti-adhesion compounds, respectively), and with a tubular braid as mechanical reinforcement. Tensile testing of the repair constructs using ex vivo sheep tendons showed that the developed repair constructs fulfilled the required mechanical properties for tendon repair (i.e. minimal ultimate stress of 4 MPa), with an ultimate stress of 6.4 ± 0.6 MPa. Moreover, in vitro biological assays showed that the developed repair tubes and the incorporated bioactive components were non-cytotoxic. In addition, when equine tenocytes and mesenchymal stem cells were co-cultured with the repair tubes, an increased production of collagen and non-collagenous proteins was observed. In conclusion, this novel construct in which a mechanical approach (fulfilling the required mechanical properties) was combined with a biological approach (incorporation of bioactive compounds), shows potential as flexor tendon repair application. Graphical abstract.

Intracellular localisation of platelet-activating factor during mammalian embryo development in vitro: a comparison of cattle, mouse and human.

Platelet-activating factor (PAF) is a well-known marker for embryo quality and viability. For the first time, we describe an intracellular localisation of PAF in oocytes and embryos of cattle, mice and humans. We showed that PAF is represented in the nucleus, a signal that was lost upon nuclear envelope breakdown. This process was confirmed by treating the embryos with nocodazole, a spindle-disrupting agent that, as such, arrests the embryo in mitosis, and by microinjecting a PAF-specific antibody in bovine MII oocytes. The latter resulted in the absence of nuclear PAF in the pronuclei of the zygote and reduced further developmental potential. Previous research indicates that PAF is released and taken up from the culture medium by preimplantation embryos invitro, in which bovine serum albumin (BSA) serves as a crucial carrier molecule. In the present study we demonstrated that nuclear PAF does not originate from an extracellular source because embryos cultured in polyvinylpyrrolidone or BSA showed similar levels of PAF in their nuclei. Instead, our experiments indicate that cytosolic phospholipase A2 (cPLA2) is likely to be involved in the intracellular production of PAF, because treatment with arachidonyl trifluoromethyl ketone (AACOCF3), a specific cPLA2 inhibitor, clearly lowered PAF levels in the nuclei of bovine embryos.

Platelet-activating factor acetylhydrolase 1B3 (PAFAH1B3) is required for the formation of the meiotic spindle during in vitro oocyte maturation.

Platelet-activating factor (PAF) is a well-described autocrine growth factor involved in several reproductive processes and is tightly regulated by its hydrolysing enzyme, PAF acetylhydrolase 1B (PAFAH1B). This intracellular enzyme consists of three subunits: one regulatory, 1B1, and two catalytic, 1B2 and 1B3. PAFAH1B3 has remained uncharacterised until now. Here, we report that PAFAH1B3 is present during the different stages of the first meiotic division in bovine, murine and human oocytes. In these species, the PAFAH1B3 subunit was clearly present in the germinal vesicle, while at metaphase I and II, it localised primarily at the meiotic spindle structure. In cattle, manipulation of the microtubules of the spindle by nocodazole, taxol or cryopreservation revealed a close association with PAFAH1B3. On the other hand, disruption of the enzyme activity either by P11, a selective inhibitor of PAFAH1B3, or by PAFAH1B3 antibody microinjection, caused arrest at the MI stage with defective spindle morphology and consequent failure of first polar body extrusion. In conclusion, our results show that one of the catalytic subunits of PAFAH1B, namely PAFAH1B3, is present in bovine, murine and human oocytes and that it plays a functional role in spindle formation and meiotic progression during bovine oocyte maturation.

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.

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.

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.

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.

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.

Transvaginal ultrasound-guided aspiration of unilateral twin gestation in the mare.

Embryo reduction of unilateral twin vesicles is normally successful before fixation of the vesicles. After fixation, however, it becomes a challenge, and the later in the gestation period that the reduction is performed, the greater the challenge. One therapy to reduce a twin into a singleton pregnancy is the transvaginal ultrasound-guided aspiration (TUGA) of one of the vesicles. The results of 35 unilateral twin reductions by TUGA are discussed in this paper and possible negative influences of age, parity and days pregnant are analysed statistically.