Background Sex steroids administration in meat producing animals is forbidden within the EU to preserve consumers’ safety, but continuous monitoring to identify resurgence of their misuse is needed. Among biomarkers related to sex steroids abuse in veal calves the regucalcin (RGN) mRNA perturbations in testis have been described in RNAlater samples. To setup novel diagnostic method, to update current tests available in National Residue Control Plans (NRCPs) and in legal dispute when illicit practices on farm animals are suspected, the reliability of RGN profiling was assessed by histological and molecular techniques. Methods Formalin fixed paraffin embedded (FFPE) testis samples, chosen being the most effective preservation strategy adopted by histological NRCPs and allowing easier retrospective analysis if required by legal disputes, were analyzed from veal calves treated with nandrolone, 17β-estradiol and a cocktail of the two hormones. RGN levels were determined by quantitative Real Time PCR and Immunohistochemistry assays. Test performances were assessed and compared by multiple ROC curves. Results Both tests resulted sensitive and specific, allowing to enrich, in future field investigation, novel integrated diagnostic protocols needed to unveil sex steroid abuse. Discussion Developed RT-qPCR and IHC methods confirmed RGN as a useful and robust biomarker to detect illegal administration of sex steroid hormones in veal calves. The developed methods, successfully applied to ten years old FFPE blocks, could allow both retrospective analysis, when supplementary investigations are requested by authorities, and future implementation of current NRCPs.

中文翻译：

---

小牛睾丸中 Regucalcin 的表达谱：组织学和分子测试的验证以检测性类固醇非法给药

背景 为了保护消费者的安全，欧盟禁止在肉类生产动物中使用性类固醇，但需要持续监测以识别其滥用的再次出现。在与小牛性类固醇滥用相关的生物标志物中，已在 RNAlater 样本中描述了睾丸中的 regucalcin (RGN) mRNA 扰动。为了建立新的诊断方法，更新国家残留控制计划 (NRCPs) 中可用的当前测试，并在怀疑农场动物非法行为时的法律纠纷中，通过组织学和分子技术评估 RGN 分析的可靠性。方法 福尔马林固定石蜡包埋 (FFPE) 睾丸样本，被选择为组织学 NRCP 采用的最有效的保存策略，如果法律纠纷需要，可以更轻松地进行回顾性分析，从用诺龙、17β-雌二醇和两种激素的混合物处理的小牛肉中分析。RGN 水平通过定量实时 PCR 和免疫组织化学测定法确定。通过多条 ROC 曲线评估和比较测试性能。结果 两项测试均具有敏感性和特异性，可以在未来的实地调查中丰富揭示性类固醇滥用所需的新型综合诊断方案。讨论 开发的 RT-qPCR 和 IHC 方法证实 RGN 是一种有用且强大的生物标志物，可用于检测小牛中性类固醇激素的非法施用。开发的方法成功应用于十年前的 FFPE 区块，可以在当局要求进行补充调查时进行回顾性分析，也可以在未来实施当前的 NRCP。