Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS)

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g., Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine, having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96 h. Liquid samples were frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at RT until analysis 1 week after. The specimens were analyzed by LC-ESI-MS/MS. As expected, degradation of EtG, but not of EtS, was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in the studied concentration range, good precision, accuracy and selectivity.

Effects of ground semen collection on weight bearing on hindquarters, libido, and semen parameters in stallions

Collection of semen on the ground from the standing stallion represents an alternative method to dummy mount semen collection and is of increasing popularity for sport stallions, males suffering from health problems, or in studs without a dummy or suitable mare at disposal. Our aim was to collect and compare spermatological and physiological data associated with traditional and ground semen collection. Twelve of 23 Franches-Montagnes stallions were selected to carry out semen collection on a dummy and while standing in a crossed experimental protocol. Semen quantity and quality parameters, weight bearing on hindquarters, and behavioral and libido data were recorded. Ground versus dummy mount semen collection was accompanied by lower seminal volume (15.9 ± 14.6 vs. 22.0 ± 13.3 mL; P < 0.01) and lower total sperm count (4.913 ± 2.721 × 10(9) vs. 6.544 ± 2.856 × 10(9) sperm; P < 0.001). No significant differences were found concerning sperm motility and viability. Time to ejaculation was longer, and the number of attempts to ejaculation was higher (P = 0.053) in the standing position compared with the mount on the dummy. A higher (P < 0.01) amount of tail flagging was manifested by the stallions during ejaculation on the dummy compared to when standing. There was no difference in weight bearing on hindquarters when comparing dummy collection (51.2 ± 2.5%) and standing collection (48.9 ± 5.5%). Ground semen collection can be considered as a viable option for stallions that cannot mount a dummy or a mare. However, it requires training and may be not easily accepted by all stallions. Owners should be advised that ground semen collection is associated with significantly lower sperm numbers than with dummy mount semen collection.