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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. ispartof: JACS AU vol:1 issue:6 pages:750-765 ispartof: location:United States status: published

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 fifteen academic labs 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 labs 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.

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.

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 signal for maternal recognition of pregnancy (MRP) has still not been identified in the horse. High-throughput molecular biology at the embryo&ndash, 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&ndash, maternal interface was prominent, highlighting a potential role of miRNAs in embryo&ndash, maternal communication during early pregnancy in the horse. These data provide a strong basis for future targeted studies.

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.

Urine collection containers used in the doping control collection procedure do not provide a protective environment for urine, against degradation by microorganisms and proteolytic enzymes. An in-house chemical stabilization mixture was developed to tackle urine degradation problems encountered in human sport samples, in cases of microbial contamination or proteolytic activity. The mixture consists of antimicrobial substances and protease inhibitors for the simultaneous inactivation of a wide range of proteolytic enzymes. It has already been tested in lab-scale, as part of World Anti-Doping Agency's (WADA) funded research project, in terms of efficiency against microbial and proteolytic activity. The present work, funded also by WADA, is a follow-up study on the improvement of chemical stabilization mixture composition, application mode and limitation of interferences, using pilot urine collection containers, spray-coated in their internal surface with the chemical stabilization mixture. Urine in plastic stabilized collection containers have been gone through various incubation cycles to test for stabilization efficiency and analytical matrix interferences by three WADA accredited Laboratories (Athens, Ghent, and Rome). The spray-coated chemical stabilization mixture was tested against microorganism elimination and steroid glucuronide degradation, as well as enzymatic breakdown of proteins, such as intact hCG, recombinant erythropoietin and small peptides (GHRPs, ipamorelin), induced by proteolytic enzymes. Potential analytical interferences, observed in the presence of spray-coated chemical stabilization mixture, were recorded using routine screening procedures. The results of the current study support the application of the spray-coated plastic urine container, in the doping control collection procedure. Copyright © 2016 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.

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.

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.

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.

SummaryBackground 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 non-vitrified 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

Een ascenderende infectie van de placenta wordt bij 3 tot 7% van de drachtige merries gezien. Het is een veel voorkomende oorzaak van abortus, premature geboorte en zwak geboren veulens (Troedsson, 2003; LeBlanc, 2010). Gezien de infectie opklimt vanuit de caudale geslachtstractus, zijn de eerste zichtbare en meest uitgesproken letsels te vinden ter hoogte van de caudale pool van het allantochorion waar dit tegen de baarmoedermond aan ligt. De symptomen zijn niet altijd even duidelijk of worden pas heel laat in het ziekteproces opgemerkt. Dit maakt een tijdige diagnose en initiatie van therapie uitermate moeilijk. Experimentele modellen om placentitis te bestuderen zijn moeilijk te managen en dubbel-blindexperimenten met controlegroepen zijn zeldzaam, waardoor het moeilijk is “evidence-based” advies te formuleren. In dit artikel wordt de diagnose, gebaseerd op de symptomatologie en het echografisch en endocrinologisch onderzoek, besproken en worden enkele therapeutische en prognostische aspecten benadrukt.

The epididymis and vas deferens store an important number of fertile spermatozoa called the extragonadal sperm reserves. These stored spermatozoa can be collected in an ultimate attempt to preserve viable spermatozoa of a critically ill or dying stallion. Epididymides are collected via routine castration. After cooled transport of the testicles and epididymides, spermatozoa are collected either by retrograde flushing or by the float-up method. Retrograde flushing usually results in a much higher sperm yield and is considered the method of choice. Epididymal spermatozoa can be frozen using standard freezing protocols.

De diagnose van cryptorchidie bij het paard is vaak een uitdaging aangezien niet altijd definitief uitsluitsel kan gegeven worden op basis van de anamnese, het klinische onderzoek en het rectaal en echografisch onderzoek. Verschillende endocrinologische diagnostische testen zoals de bepaling van het testosteron-, androstenedione-, oestrogenen-, urinaire steroïden- en het antimülleriaans hormoongehalte, die de aanwezigheid van testiculair weefsel aantonen, zijn beschreven. In dit artikel wordt getracht om de voor- en nadelen van deze testen te vergelijken, zodat practici een idee krijgen welke testen in de praktijk gebruikt kunnen worden.

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.

Summary 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.

Toediening van prostagenen voor de instandhouding van de dracht bij merries Verbetering van spermakwaliteit van hengsten Ontworming van grazende koeien

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.

Overdracht van piroplasmose bij paarden Behandeling van cryptosporidiose bij kalveren Melkproductie bij de zeug

This paper reviews the prevalence of legal and illegal stimulants in relation to doping-control analysis. Stimulants are among the oldest classes of doping agents, having been used since ancient times. Despite the ease with which they can be detected and the availability of sensitive detection methods, stimulants are still popular among athletes. Indeed, they remain one of the top three most popular classes of prohibited substances. Because the list of legal and illegal stimulants is extensive only a selection is discussed in detail. The compounds selected are caffeine, ephedrines, amphetamine and related compounds, methylphenidate, cocaine, strychnine, modafinil, adrafinil, 4-methyl-2-hexaneamine, and sibutramine. These compounds are mainly prevalent in sport or are of therapeutic importance. Because stimulants are the oldest doping class the first detection methods were for this group. Several early detection techniques including GC-NPD, GC-ECD, and TLC are highlighted. The more novel detection techniques GC-MS and LC-MS are also discussed in detail. In particular, the last technique has been shown to enable successful detection of stimulants difficult to detect by GC-MS or for stimulants previously undetectable. Because stimulants are also regularly detected in nutritional (food) supplements a section on this topic is also included.

A simple, fast and sensitive method for the quantitative determination of strychnine residues in urine has been developed and validated. The method consists of a liquid–liquid extraction step with ethyl acetate at pH 9.2, followed by LC–MS/MS in positive atmospheric pressure chemical ionization (APCI)-mode. The method is linear in the range of 1–100 ng/mL and allows for the determination of strychnine at sub-toxicological concentrations. The accuracy of the method ranged from 1.3% to 4.4%. The method was used to determine the excretion profile of strychnine after the ingestion of an over-the-counter herbal preparation of Strychnos nux-vomica. Each volunteer ingested a dose equivalent to 380 μg of strychnine. This dose is lower than the prescription dose but results in the detection of strychnine for over 24-h post administration. Maximum detected urinary concentrations ranged from 22.6 to 176 ng/mL. The results of this study show that the use of this type of preparation by athletes can lead to a positive doping case.

Caffeine concentrations were measured in the urines of 11 361 athletes tested for doping control in the Ghent doping control laboratory during the period 1993 - 2002. Determination of these concentrations was done using an alkaline extraction with a mixture of dichloromethane and methanol (9:1; v/v) followed by high performance liquid chromatography and ultraviolet detection (HPLC-UV). The method was validated according to ISO 17 025 standards (International Organisation for Standardisation). Quantification was done by using a calibration curve in the range from 0 to 20 microg/ml. The limit of quantification (LOQ) was 0.10 microg/ml. Most caffeine concentrations were far below 12 microg/ml. Because the results were not normally distributed, transformation of the data was done to evaluate the difference in detected concentrations in several sports. This resulted in an overall average concentration of 1.22 microg/ml +/- 2.45 microg/ml. Comparison of those sports with more than 200 samples being analysed demonstrated that caffeine concentrations in urine samples from bodybuilders are significantly higher in comparison to urines taken in the other sports. Also, a significant difference between caffeine concentrations found in cycling and concentrations found in other sports, including athletics and some ball sports, was observed.

An accurate, reproducible, and validated gas chromatography-mass spectrometry (GC-MS) method for the quantitation of 11 -nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH), the major metabolite of delta9-tetrahydrocannabinol, in urine is described. Extraction was performed with n-hexane/ethyl acetate. Deuterated THC-COOH was used as the internal standard. The GC-MS analysis was done by selected ion monitoring. No interferences were detected in 20 blank urine samples of different origin. The calibration curve was found to be linear over the range of 10-100 ng/mL. The calculated limits of detection and quantitation were 1.0 ng/mL and 1.7 ng/mL, respectively. Results of positive findings for cannabis use in doping control in Flanders and Portugal in the period of 1997-2000 are commented.

Non-steroidal anti-inflammatory drugs (NSAIDs) are prohibited by the International Federation of Horse Racing Authorities but are commonly used in veterinary practice. Plasma and urinary concentrations of the NSAID tolmetin were determined by a high-performance liquid chromatographic procedure with UV detection following oral administration of a dose of 1 g to six fasted untrained standard bred mares. With a limit of quantitation (LOQ) of 0.05 microg/ml tolmetin was present in plasma for 9-12 h post-administration. Maximum concentrations of 2.1+/-0.89 microg/ml were found after 0.7+/-0.25 h. The elimination half-life was 2+/-1.25 h. Plasma protein binding at concentrations of 0.25 and 2.5 microg/ml was 92+/-4.9 and 84+/-4.2%, respectively. As early as 1 h after dosage, tolmetin could be detected in unhydrolysed urine and remained detectable up to 48 h (LOQ=0.5 microg/ml). The maximum concentrations occurred 1.8+/-0.4 h after administration. The percentage of the dose excreted as unchanged tolmetin within 12 h was 58+/-7.9%. Neither conjugates nor metabolites could be detected under the experimental conditions studied. For confirmatory analysis in doping control, an LC-MS method was developed. Analysis was performed on an ion trap LC-MS system equipped with an ESI probe in positive MS(2) mode.

The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid of the four groups was achieved by isobaric tags for relative and absolute quantification (iTRAQ) labelling and HPLC–tandem mass spectrometry. The presence of an embryo in the ipsilateral oviducal fluid of pregnant mares induced upregulation of 11 and downregulation of two proteins compared with the contralateral side, and upregulation of 19 proteins compared with the ipsilateral side of non-pregnant mares. Several of these upregulated proteins are related to early pregnancy in other species. The present study represents the first high-throughput identification of proteins in the oviducal fluid of the mare. The results support the hypothesis that the equine embryo interacts with the oviduct, affecting the maternal secretion pattern of proteins involved in pregnancy-related pathways.

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.

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.

SR9009 and SR9011 are attractive as performance-enhancing substances due to their REV-ERB agonist effects and thus circadian rhythm modulation activity. Although no pharmaceutical preparations are available yet, illicit use of SR9009 and SR9011 for doping purposes can be anticipated, especially since SR9009 is marketed in illicit products. Therefore, the aim was to identify potential diagnostic metabolites via in vitro metabolic studies to ensure effective (doping) control. The presence of SR9009 could be demonstrated in a black market product purchased over the Internet. Via human liver microsomal metabolic assays, eight metabolites were detected for SR9009 and fourteen metabolites for SR9011 by liquid chromatography-high resolution mass spectrometry (LC-HRMS). Structure elucidation was performed for all metabolites by LC-HRMS product ion scans in both positive and negative ionization mode. Retrospective data analysis was applied to 1511 doping control samples previously analyzed by a full-scan LC-HRMS screening method to verify the presence of SR9009, SR9011 and their metabolites. So far, the presence of neither the parent compound nor the metabolites could be detected in routine urine samples. However, to further discourage use of these potentially harmful compounds, incorporation of SR9009 and SR9011 into screening methods is highly recommended.

A new doping control screening method for the analysis of diuretics and stimulants using ultra high pressure liquid chromatography-high resolution Orbitrap mass spectrometry has been developed. The screening was performed in full scan MS with scan-to-scan polarity switching which allowed to detect more than 120 target analytes. Sample preparation was limited to 10-fold dilution of the urine into the internal standard solution followed by injection. Total run time per sample was 10 min. Validation of the method yielded detection limits for diuretics between 25 and 250 ng mL(-1) and for stimulants between 5 and 500 ng mL(-1). The screening method has been implemented in routine doping control.

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.

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(-1) ) and concentrations (≥60 mg ml(-1) ) 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(-1) ). 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.

A fast and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the screening of 18 diuretics and probenecid in human urine is presented. Analyses were performed on a LCQ-Deca instrument equipped with ESI-interface using scan by scan polarity changing. All diuretics and probenecid were separated in less than 20 min after liquid-liquid extraction with ethyl acetate. The LOD for all substances was 100 ng/mL or better. The method was applied to detect diuretics after the oral administration of several drugs including hydrochlorothiazide, bumetanide, spironolactone, furosemide, amiloride, triamterene, chlortalidone and epithizide. All diuretics could be detected for periods up to 96 h after the intake of therapeutic amounts.

The evaluation of the placenta is an inherent part of the post foaling exam in the mare. Findings are important to asses both the health status of the uterus and foal. Initiation of therapy or further inquiries might be based upon this examination. Knowledge of normal, breed specific placental parameters are essential in this regard. It was the aim of the present study to provide normal values for some parameters describing the placenta in Warmblood mares after physiological foaling. From 2010 until 2013, placentae from spontaneous foaling Warmblood mares (aged 11y, STDEV1⁄45.3) giving birth to normal sized, healthy foals were examined for this survey. All mares had, in the last week before parturition, a normal CTUP on ultrasonography, expelled the placenta in time (within 3h pp) and had an adequate colostrum quality on day of foaling. After macroscopic evaluation, only placentae without any abnormality were included. Different univariable linear regression models were fitted to explore the association between different mare related factors and the placenta weight, the foal weight and the length of the body of the placenta, respectively using SPSS 20.0. The different correlation coefficients were calculated and further referred as R. In total 325 placentae were included. On average the Warmblood mares weighed 666.6 kg (range 448-830) and gave birth to a foal of 56.1kg (range 29-75) with a placenta weighing 5.6kg (range 2.8-10). The

A gas chromatographic method for the simultaneous quantitation of ephedrine, pseudoephedrine, norephedrine (phenylpropanolamine), norpseudoephedrine (cathine) and methylephedrine in urine is described. The method consists of a liquid-liquid extraction with tert.-butyl methyl ether at pH 14. The extracts are analysed on a GC system equipped with an Rtx-5 Amine column and a nitrogen-phosphorus detector. Method validation shows excellent separation, linearity, specificity, accuracy, precision, intra-laboratory repeatability and reproducibility, making the method especially suitable for quantitation of ephedrines in urine samples for doping control purposes. A statistical analysis on the abuse of the different ephedrines in urine from athletes controlled in the Flemish doping control laboratory during the period 1993-2000 is included.

A method is described for the determination of anabolic steroids including testosterone, 19-nor-4-androstene-3,17-dione, 4-androstene-3,17-dione and nandrolone in food supplements. Initial clean-up is done by HPLC followed by determination with GC/MS. A ‘contaminated’ food supplement was analysed and appeared to contain 19-nor-4-androstene-3,17-dione and 4-androstene-3,17-dione. One capsule of this nutritional supplement was ingested by five male volunteers. Urine samples were collected and analysed by GC/MS and GC/MS-MS. Neither the ratio testosterone/epitestosterone, nor the ratio androstenedione/epitestosterone increased significantly. Concentrations above 2 ng/ml for norandrosterone, the major metabolite of nandrolone, were detected until 48–144 h after ingestion of the food supplement.

We present a case of xanthogranulomatous cholecystitis associated with a xanthogranulomatous vegetation on the left diaphragm with breakthrough into the thoracic cavity. A similar case has not previously been reported.