Characterization of In Vitro Synthesized Equine Metabolites of the Selective Androgen Receptor Modulators S24 and S4

Several selective androgen receptor modulators (SARMs) have been synthesized and investigated in humans, rats, and dogs in the past, but no data are yet available concerning the metabolism of SARMs in horses. The aryl-propionamide-derived drug candidates S24 and S4 (andarine) have a strong androgen receptor binding affinity and show distinctive specific cell answers. Although no SARM drug candidate (aiming for testosterone replacement therapy) has completed clinical trials yet, S4 has been illicitly available via the Internet. These facts led to the prohibition of SARMs by the German equestrian federation, and the (mis)use of such compounds would further represent a doping rule violation in horse racing. In this study, the drug candidates S24 and S4 were subjected to in vitro metabolism experiments with equine liver microsomal preparations from a female Quarter Horse to obtain information about potential target analytes in equine doping control analysis. The enzymatically synthesized metabolites were characterized by liquid chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry. All observed S24 and S4 equine metabolites are in agreement with earlier in vitro and in vivo studies in humans and dogs. Nevertheless, the relative percentage of generated equine metabolites (as determined from the analytes’ response in full-scan chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry measurements) differs considerably from the reported profiles. Although the S24 metabolite pattern is comparably balanced concerning glucuronidated and sulfonated conjugates, the major S4 metabolite was found to be the unconjugated dephenylated compound, with a proportion of more than 90%.     

Anthelmintic resistance in equine parasites - detection, potential clinical relevance and implications for control

During the past two decades anthelmintic resistance in equine parasites has been found in the group of small strongyle species (cyathostomins) and in the ascarid species Parascaris equorum. The ubiquitous nature and possible severe consequences of disease with these nematodes make them the prime targets of current worm control programmes. Traditional control strategies mainly rely on the strategic application of anthelmintics, currently represented by three major drug classes: benzimidazoles (BZ), the tetrahydropyrimidine pyrantel (PYR) and macrocyclic lactones (ML). Following decades of routine and frequent anthelmintic applications, many cyathostomin populations on horse farms in industrialised countries must be considered as resistant to BZ anthelmintics. However, to date no published cases of cyathostomin disease specifically associated with anthelmintic resistance were reported. Possibly this is due to the generally subclinical and unspecific symptoms associated with cyathostomin infections. Nevertheless, exclusive reliance on the ML drug class may increase the threat of clinical disease due to drug-resistant cyathostomins. More recently, P. equorum has been reported as having developed resistance against ivermectin and moxidectin, two representatives of the ML-class. These anthelmintics are currently the most frequently used drug class in horses. This nematode species is mainly found in foals and in younger horses due to the development of immunity following exposure to infection. Infection with P. equorum can result in clinically drastic consequences such as obstruction and/or penetration of the small intestine, the latter usually leading to death. In conclusion, on horse farms the efficacy of anthelmintic treatments should be examined routinely for each drug class. Several factors can influence the rate at which anthelmintic resistance develops; high frequency of treatment being one of the most important. Modern control strategies should therefore attempt to significantly reduce anthelmintic treatments. Several pasture and farm management practices found to be negatively associated with nematode and anthelmintic resistance prevalence will be discussed in the review presented here.     

Positive nitrogen balance of <Emphasis Type="Italic">Acacia mangium</Emphasis> woodlots as fallows in the Philippines based on <Superscript>15</Superscript>N natural abundance data of N<Subscript>2</Subscript> fixation

Nitrogen inputs from biological nitrogen fixation contribute to productivity and sustainability of agroforestry systems but they need to be able to offset export of N when trees are harvested. This study assessed magnitudes of biological nitrogen fixation (natural ¹⁵N abundance) and N balance of Acacia mangium woodlots grown in farmer’s fields, and determined if N₂ fixation capacity was affected by tree age. Tree biomass, standing litter, understory vegetation and soil samplings were conducted in 15 farmer’s fields growing A. mangium as a form of sequential agroforestry in Claveria, Misamis Oriental, Philippines. The trees corresponded to ages of 4, 6, 8, 10 and 12 years, and were replicated three times. Samples from different plant parts and soils (0–100 cm) were collected and analyzed for δ¹⁵N and nutrients. The B-value, needed as a reference of isotopic discrimination when fully reliant on atmospheric N, was generated by growing A. mangium in an N₂-free sand culture in the glasshouse. Isotopic discrimination occurring during N₂ fixation and metabolic processes indicated variation of δ¹⁵N values in the order of nodules > old leaves > young leaves > stems > litterfall and roots of the trees grown in the field, with values ranging from −0.8 to 3.5‰ except nodules which were enriched and significantly different from other plant parts (P < 0.0001). Isotopic discrimination was not affected by tree age (P > 0.05). Plants grown in N free sand culture exhibited the same pattern of isotopic discrimination as plants grown in the field. The estimated B-value for the whole plant of A. mangium was −0.86‰. Mature tree stands of 12 years accumulated up to 1994 kg N ha⁻¹ in aboveground biomass. Average proportion of N derived from N₂ fixation of A. mangium was 54% (±22) and was not affected by age (P > 0.05). Average yearly quantities of N₂ fixed were 128 kg N ha⁻¹ in above-ground biomass amounting to 1208 kg N fixed ha⁻¹ over 12 years. Harvest of 12-year old trees removed approximately 91% of standing aboveground biomass from the site as timber and fuel wood. The resulting net N balance was +151 kg N ha⁻¹ derived from remaining leaves, twigs, standing litter, and +562 kg N ha⁻¹ when tree roots were included in the calculation. The fast growing A. mangium appears to be a viable fallow option for managing N in these systems. However, other nutrients have to be replaced by using part of the timber and fuel wood sales to compensate for large amounts of nutrient removed in order for the system to be sustainable.     

Alopecia areata in Eringer cows

Alopecia areata is a hair loss disorder in humans, dogs and horses with a suspected autoimmune aetiology targeting anagen hair follicles. Alopecia areata is only sporadically reported in cows. Recently, we observed several cases of suspected alopecia areata in Eringer cows. The aim of this study was to confirm the presumptive diagnosis of alopecia areata and to define the clinical phenotype and histopathological patterns, including characterization of the infiltrating inflammatory cells. Twenty Eringer cows with alopecia and 11 Eringer cows without skin problems were included in this study. Affected cows had either generalized or multifocal alopecia or hypotrichosis. The tail, forehead and distal extremities were usually spared. Punch biopsies were obtained from the centre and margin of alopecic lesions and normal haired skin. Histological examination revealed several alterations in anagen hair bulbs. These included peri‐ and intrabulbar lymphocytic infiltration, peribulbar fibrosis, degenerate matrix cells with clumped melanosomes and pigmentary incontinence. Mild lymphocytic infiltrative mural folliculitis was seen in the inferior segment and isthmus of the hair follicles. Hair shafts were often unpigmented and dysplastic. The large majority of infiltrating lymphocytes were CD3⁺ T cells, whereas only occasional CD20⁺ lymphocytes were present in the peribulbar infiltrate. Our findings confirm the diagnosis of T‐cell‐mediated alopecia areata in these cows. Alopecia areata appears to occur with increased frequency in the Eringer breed, but distinct predisposing factors could not be identified.     

Die Bestimmung der Tiefgefriertauglichkeit von Bullensperma mit Hilfe des Objektträgertestes

Inhalt Es Wurde versucht, 276 Ejakulate von 50 Bullen der Rasse Deutsche. Fleckvich mit Hilfe eines einfachen Objekträgertestes ohne die im Ruutineverfahren üblichen Maβnahmen (Adaption, Einfrieren) auf ihre Gefriertauglichkeit zu testen. Dies erfolgte durch einen Vergleich der Auftauraten von verdünnten Spermaproben gleich nach dem Tiefgefrieren ohne Adaption auf Objektträgern und von solchen die nach 6–8stündigcr Adaption routinemäβig in Ampullen bzw. Pailletten eingefroren wurden. Die Untersuchungen führten zu folgenden Ergebnissen: 1. Die Auftaurate von Spermaproben, die ohne Adaption auf Objektträgern im Stickstoffdampf eingefroren wurden, lassen bis zu einem gewissen Grade eine Vorausbestimmung des Einfrierergebnisses und damit der Tiefgefriertauglichkeit eines Ejakulates zu. Bullen, deren Sperma im Objektträgertest ein Auftaurate von über 20% zeigt, werden als gefriertauglich, jene mit einer Spermienmotilität nach dem Auftauen der Objektträgerproben von unter 5% als gefrieruntauglich und solche, deren aufgetautes Sperma 5% bis 20 % motile Spermien aufweisen, als bedingt tauglich angesprochen. 2. Zur Beurteilung eines Bullen hinsichtliclz der Gefriertauglichkeit seines Spermas reicht die Testung von 3 Ejakulaten aus. 3. Der Objektträgertest ist leicht durchführbar und zeitsparend. Er ist für die Auswahl der Bullen hinsichtlich der Gefriertauglichkeit ihres Spermas für die Praxis allerdings nur bedingt einsatzfähig, da ein groβer Teil der Stiere doch noch routinemäβig nachgetestet werden muβ. Er bringt eindeutige Ergebnisse nur dann, wenn die Auftaurate höchstens 5% und deutlich mehr als 20 % beträgt. Contents (The determination of the suitability for deep freezing of bull semen using a slide test) . 276 ejaculates from 50 bulls of ther German Fleckvieh breed were studied for deep freezing suitability using a simple slide test without the routinely used mea.rures (adaptation, freezing). A comparison was made of the thawing rates of diluted semen samples immediately after deep-freezing without adaptation on slides and after 6–8 hours adaptntion in ampoules or paillettes. The following results were obtained: 1. The thawing rate of semen samples frozen without adaptation on slides in the vapour phase of nitrogen allow a certain degree of predetermination of the freezing result and therefore of the suitability of an ejaculate for deep freezing. Bulls whose semen shows a thawing rate of over 20% by the slide test are taken as being suitable for freezing; those with a sperm motility after the slide test of less than 5% are unsuitable; those with a sperm motility of 5–20% are considered as conditionally suitable. 2. 3 ejaculates are sufficient to test a bull for suitability of semen for deep-freezing. 3. The slide test is easy to carry out and saves time. However, for the selection of bulls on the basis of deep freezing of semen in practice it is limited, since a large proportion of bulls still have to be tested further. The test only gives unequivocal results if the thawing rate is less than 5% or more than 20%.     

Ein Beitrag zur Beurteilung der täglichen Spermienproduktion bei Ebern der Deutschen Landrasse

Inhalt Bei 8 Ebern der Deutschen Landrasse im Alter von 1 bis 4 Jahren wurde die spermatogenetische Leistung durch Entsamungen mit unterschiedlichen Intervallen untersucht. Dabei wurde festgestellt, daβ die tägliche Spermienproduktion des 1jährigen Ebers bei 10 · 10⁹ Spermien liegt und beim 4jährigen Eber 20 · 10⁹übersteigt. Die Spermienproduktion von Ebern ist, wie bei anderen Tierarten, von Alter und damit vom steigenden Hodengewicht der Tiere abhängig. Mit einer Vergröβerung der Entsamungspausen steigen die Spermienverluste an und der errechnete tägliche Spermien-“output” nimmt ab. Contents (A contribution to the evaluation of the daily sperm production of boars of German Landrace breed). The “Daily Sperm Output” in boars was examined by means of various semen collection intervals. Eight boars–German landrace type, age one to four years-served as test animals. The investigations established a daily sperm production of 10·10⁹ sperms with one year old boars whereas 4 years old boars produce more than 20 · 10⁹ sperms. The sperm production depends on age and growing testis weight as is observed in other animal species With increased pauses of semen collection sperm losses are likewise increased, and the calculated “Daily Sperm Output” is diminished.     

Untersuchungen uber den Einfluß von Prolan A1) und Norethisteronazetat2) auf die Spermaqualität von Ziegenböcken

Inhalt: Die Ejakulate von 10 Ziegenböcken wurden dahingehend geprüft, ob und in welcher Weise durch die Zufuhr von Prolan A (parenteral) bzw. Norethisteronazetat (peroral) die Samenbeschaffenheit verändert wird. Die Auswertung der Versuchsergebnisse erfolgte auf rechnerischem Wege und an Hand graphischer Darstellungen. Contents: The effect of parenteral application of Prolan A and of oral application of Norethisteronacetate, on quality of sperma, were tested on 10 male goats. Results were evaluated both statistially and graphically.