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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]

Plasma volume expanders (PVEs) such as hydroxyethyl starch (HES) and dextran are misused in sports because they can prevent dehydration and reduce haematocrit values to mask erythropoietin abuse. Endogenous hydrolysis generates multiple HES and dextran oligosaccharides which are excreted in urine. Composition of the urinary metabolic profiles of PVEs varies depending on post-administration time and can have an impact on their detectability. In this work, different mass spectrometry data acquisition modes (full scan with and without in-source collision-induced dissociation) were used to study urinary excretion profiles of HES and dextran, particularly by investigating time-dependent detectability of HES and dextran urinary oligosaccharide metabolites in post-administration samples. In-source fragmentation yielded the best results in terms of limit of detection (LOD) and detection times, whereas detection of HES and dextran metabolites in full scan mode with no in-source fragmentation is related to recent administration (< 24 hours). Urinary excretion studies showed detection windows for HES and dextran respectively of 72 and 48 hours after administration. Dextran concentrations were above the previously proposed threshold of 500 µg · mL-1 for 12 hours. A "dilute-and-shoot" method for the detection of HES and dextran in human urine by ultra-high-pressure liquid chromatography-electrospray ionization-high resolution Orbitrap™ mass spectrometry was developed for this study. Validation of the method showed an LOD in the range of 10-500 µg · mL-1 for the most significant HES and dextran metabolites in the different modes. The method allows retrospective data analysis and can be implemented in existing high-resolution mass spectrometry-based doping control screening analysis. [ABSTRACT FROM AUTHOR]

Contents 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. [ABSTRACT FROM AUTHOR]

Unscheduled mortality preceded by adverse respiratory clinical signs in rats dosed by oral gavage may not only be caused by technical gavage error or systemic toxicity but may also be caused by gastro-esophageal reflux and subsequent aspiration of high concentrations of drug formulation. In a 3 week oral gavage rat toxicity study for an early drug development compound, preterminal deaths (approximately 20% of animals) at high doses (≥1000 mg kg) and concentrations (≥60 mg ml) were preceded by recurrent dyspnea, rales or excessive salivation, without evidence of accidental intrapulmonary gavage error. Histological evaluation revealed extensive necrosis and inflammatory changes in the upper respiratory tract, especially in the nasal turbinates and/or nasopharynx. The presence of food particles in inflammatory exudates suggested a retrograde aspiration of stomach content with test formulation via the nasopharyngeal duct into the posterior region of the nose. In contrast, no mortality or adverse respiratory effects were observed in rats following 2 week intravenous administration at comparable exposures or oral gavage administration at lower concentrations (≤20 mg ml). In a pharmacology study, the compound caused a dose-dependent increase in gastric content (partly due to inhibition of gastric emptying), providing a pharmacological basis for the suspected gavage-mediated gastroesophageal reflux. Reducing the dose volume and dosing fasted animals substantially reduced or eliminated the respiratory effects and mortality at the high test article concentrations, demonstrating that the adverse effects are related to the gavage method. Copyright © 2010 John Wiley & Sons, Ltd. Unscheduled mortality preceded by adverse respiratory clinical signs in rats dosed by oral gavage may not only be caused by technical gavage error or systemic toxicity but may also be caused by gastro-esophageal reflux and subsequent aspiration of high concentrations of drug formulation. Reducing the dose volume and dosing fasted animals may substantially reduce or eliminated the respiratory effects and mortality at the high test article concentrations, demonstrating that the adverse effects are related to the gavage method and reflux. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Focuses on the quantitation of 11-nor-&delta:[sup9]-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine by an accurate, reproducible and validated gas chromatography-mass spectrometry method. Validity of the method; Statistical evaluation of a calibration curve; Determination of the theoretical concentrations of calibrators.

Stimulants are often used to treat attention deficit disorders and nasal congestion. As they can be misused and overdosed, the detection of stimulants is relevant in the toxicological field as well as in the doping control field. The effects of stimulants can indeed be beneficial for athletes. Therefore, their in-competition use is prohibited by the World Anti-Doping Agency (WADA). As stimulants represent one of the most detected categories of prohibited substances, automation of methods to detect and confirm their presence is desirable. Previous work has shown the advantages of using turbulent flow online solid-phase extraction liquid chromatography-tandem mass spectrometry (online SPE LC-MS-MS) for the detection and confirmation of diuretics and masking agents. Hence, a turbulent flow online SPE LC-MS-MS method, compliant with the WADA's identification criteria, was developed and validated for the detection and confirmation of 80 stimulants or metabolites with limits of identification varying between 10 (or possibly lower) and 100 ng/mL. As several metabolites are common metabolites for multiple administered stimulants, this means that with this method, misuse of well over 100 compounds can be detected. As the developed method uses the same columns and mobile phases as our turbulent flow online SPE LC-MS-MS method for the confirmation of diuretics and masking agents, there is no need to change the configuration of the instrument when switching between the diuretics method and the developed stimulants method., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550., Competing Interests: The authors declare the following competing financial interest(s): L. Van Oudenhove, J. Claereboudt, S. Oehrle, N. Tanna, and J. P. C. Vissers are employed by Waters Corporation. C. S. Lane, S. El Ouadi, J.-B. Vincendet, and N. Morrice are employed by Sciex. F. Sigloch and A. Bhangu-Uhlmann are employed by Polyquant GmbH. M. Razavi and L. Anderson are employed by SISCAPA., (© 2021 The Authors. Published by American Chemical Society.)

As hypoxia-inducible factor stabilizers (HIFs) can artificially enhance an athlete's erythropoiesis, the World Anti-Doping Agency prohibits their use at all times. Every urine sample for doping control analysis has to be evaluated for the presence of HIFs and therefore sensitive methods that allow high sample throughput are needed. Samples suspicious for the presence of HIFs need to be confirmed following the identification criteria established by the World Anti-Doping Agency. Previous work has shown the advantages of using turbulent flow online solid-phase extraction (SPE) procedures to reduce matrix effects and retention time shifts. Furthermore, the use of online SPE allows for automation and high sample throughput. Both an initial testing procedure (ITP) and a confirmation method were developed and validated, using online SPE liquid chromatography-tandem mass spectrometry (LC-MS/MS), with limits of detection between 0.1 ng/ml (or possibly lower) and 4 ng/ml (or higher for GSK360a) and limits of identification between 0.1 ng/ml (or possibly lower) and 1.17 ng/ml. The ITP only takes 6.5 min per sample. To the best of our knowledge, these are the first ITP and confirmation methods that include more than three HIFs without the need for manual sample preparation., (© 2020 John Wiley & Sons, Ltd.)

Detection of endogenous anabolic androgenic steroids (EAAS) misuse is a major challenge in doping control analysis. Currently, a number of endogenous steroids, which constitute the steroid profile, are quantified using gas chromatography (GC). With this methodology, only the sum of the free and glucuronidated steroids is measured together. A dilute-and-shoot LC-MS method, which is compliant with the quality requirements for measuring EAAS established by the World Anti-Doping Agency (WADA), was developed and validated containing glucuronidated and sulfated steroids in order to gain some extra information and to expand the existing steroid profile. The developed method is, to the best of our knowledge, the first method to combine both steroid glucuronides and sulfates, which is compliant with the quality standards of the technical document on EAAS, established by WADA. The first advantage of this new steroid profile is the reduced sample preparation time, as it is a direct injection method of diluted urine. A second advantage is the ability of the used gradient to separate 5α-androstane-3α,17β-diol-3-glucuronide (5ααβdiol3G), 5α-androstane-3α,17β-diol-17-glucuronide (5ααβdiol17G), 5β-androstane-3α,17β-diol-3-glucuronide (5βαβdiol3G) and 5β-androstane-3α,17β-diol-17-glucuronide (5βαβdiol17G) allowing to gain specific information on these isomers, which cannot be accomplished in GC-MS screening due to hydrolysis. This steroid profile also contains free testosterone, 5α-androstane-3,17-dione and 5β-androstane-3,17-dione as markers of degradation. In total, 17 compounds and 10 isotopically labelled internal standards are included in this method., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo-maternal interface has substantially contributed to the knowledge on pathways affected during MRP, but an integrated study in which proteomics, transcriptomics and miRNA expression can be linked directly is currently lacking. The aim of this study was to provide such analysis. Endometrial biopsies, uterine fluid, embryonic tissues, and yolk sac fluid were collected 13 days after ovulation during pregnant and control cycles from the same mares. Micro-RNA-Sequencing was performed on all collected samples, mRNA-Sequencing on the same tissue samples and mass spectrometry was conducted previously on the same fluid samples. Differential expression of miRNA, mRNA and proteins showed high conformity with literature and confirmed involvement in pregnancy establishment, embryo quality, steroid synthesis and prostaglandin regulation, but the link between differential miRNAs and their targets was limited and did not indicate the identity of an unequivocal signal for MRP in the horse. Differential expression at the embryo-maternal interface was prominent, highlighting a potential role of miRNAs in embryo-maternal communication during early pregnancy in the horse. These data provide a strong basis for future targeted studies.

Diuretics can be misused to force diuresis to achieve weight loss or to mask the intake of a prohibited substance and are therefore prohibited by the World Anti-Doping Agency (WADA). For similar reasons other masking agents (vaptans, probenecid, etc.) are also prohibited by the WADA. The currently employed methods to detect diuretics in urine use extraction or dilute-and-shoot, combined with 1D- liquid chromatography (LC) high resolution mass spectrometry (MS) or LC-triple quadrupole MS. Dilute-and-shoot methods save time and work, but these methods encounter some problems (e.g., peak drift and matrix effect). Therefore, a 2D-LC-MS/MS application was developed, validated and evaluated as an alternative. The effect of a turbulent flow rate was studied by loading samples under different conditions and the turbulent flow rate was found to be more effective in removing matrix interferences. A correlation with the specific gravity was observed. A turbulent flow online solid phase extraction (SPE) method combined with LC-MS/MS for the detection of 50 diuretics and masking agents was developed and validated for identification purposes. This method combines the advantages of dilute-and-shoot while solving the issues of matrix effect and retention time shift. Furthermore, the presented method is compliant with WADA's identification criteria and can hence be used for screening and/or confirmation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In recent years, the interest in equine foetal gender determination (FGD) during gestation increased remarkably. Ultrasonographic FGD can be performed in two different periods during gestation. The earliest examination can take place at a gestational age of 60-70 days, whereby the genital tubercle is used to differentiate between male and female foeti. The time window of the second approach is wider (120-210 days), and there are more characteristics to take into consideration. In this article, the feasibility and accuracy of ultrasonographic FGD in mid- to late gestation are evaluated. One hundred twenty-one mares from different breeds with a pregnancy stage between 120 and 270 days were examined once, using B-scale ultrasonography (Esaote MyLab™ClassC). None of the mares were sedated nor shaved, and the procedure was completed within 15 min. Diagnosis was firstly based on the gonads. The final judgement was made based on all visible foetal reproductive organs. In three cases with a pregnancy stage beyond 257 days, FGD was not possible. All of the examined mares in which a FGD could be performed gave birth to a healthy foal. In 98% of the examinations (116/118), the diagnosis was correctly made. In both cases of misdiagnosis, only one characteristic was seen during the procedure and wrongly interpreted. Beyond 210 days of pregnancy, the extremities can preclude a good visualization of the inguinal region. In conclusion, equine FGD in mid- to late gestation is an accessible and accurate technique, although a good ultrasound device is a prerequisite and experience and expertise is necessary., (© 2018 Blackwell Verlag GmbH.)

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.