Validation and application of a yeast bioassay for screening androgenic activity in calf urine and feed

Abstract

Bioassays are valuable tools for combating the illegal use of steroids in cattle fattening. Previously we described the construction and properties of a rapid and robust yeast androgen bioassay stably expressing the human androgen receptor (hAR) and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. In the present study this yeast androgen bioassay was validated as a qualitative screening method for the determination of androgenic activity in calf urine and animal feed. This validation was performed according to EC Decision 2002/657. 20 blank samples were spiked with testosterone, 17α-methyltestosterone, 19-nortestosterone, 17β-trenbolone, 17β-boldenone or 17α-methylboldenone at 2 or 15 ng mL −1 in urine and 50 or 100 ng g −1 in feed. All blank and spiked samples fulfilled the CCα and CCβ criterions, meaning that all 20 blank samples gave signals below the determined decision limits CCα and were thus classified as compliant ( α = 1%). For each component, at least 19 out of the 20 spiked samples gave a signal above the CCα and were thus classified as suspect ( β = 5%). The method was specific, and high amounts of dexamethasone did not interfere with the outcome of the test. Although high levels of 17α-ethynylestradiol can significantly inhibit the response obtained with low amounts of androgens, that situation is not relevant in veterinary practice. When stored at their specific conditions, the androgens in feed were stable for at least 91 days. Real urine samples from a national control program were screened and a representative part of the compliant and suspect samples were confirmed by gas chromatography–tandem mass spectrometry.