【作者】 邹琴；

【导师】 李刘冬； 秦小明；

【作者基本信息】 广东海洋大学 ， 食品科学， 2010， 硕士

【摘要】 本研究采用建立的罗非鱼各组织、鱼苗体内甲基睾丸酮残留的检测方法,对大规模养殖环境条件下,使用甲基睾丸酮后在罗非鱼鱼苗和成鱼体内的残留消除规律进行了系统研究,旨在为行政主管部门制定监督管理措施及罗非鱼药残监督溯源提供理论依据。首先通过比较筛选国内外相关研究,建立了适合于罗非鱼各器官组织及其鱼苗中甲基睾丸酮残留测定的高效液相色谱法。样品经乙醚超声波提取、石油醚净化(血浆、胆汁中辅以固相萃取柱萃取),然后以ODS-C18柱,77%甲醇溶液洗脱分离,紫外检测器检测、外标法定量。该前处理方法能使目标物在色谱图上有合适的保留时间、峰型好、目标峰与杂质峰完全分离,该法甲基睾丸酮浓度在0.040μg/mL~10.0μg/mL范围内线性关系良好(R2=1.0000),样品检出限分别为:背肌、腹肌8μg/kg,鱼皮、肝脏27μg/kg,性腺80μg/kg,血浆、胆汁80μg/L,鱼苗20μg/kg。方法的回收率不低于80.6%,血浆、背肌、腹肌、鱼苗、胆汁、肝脏、鱼皮、性腺的回收率分别为91.4%、88.0%、87.4%、88.9%、87.6%、83.6%、80.9%、80.6%。相对标准偏差不高于5.53%,鱼苗、肝脏、血浆、背肌,胆汁、性腺、腹肌、鱼皮分别为2.51%、3.59%、3.68%、3.96%、4.51%、4.49%、4.91%、5.53%。适用于罗非鱼组织中甲基睾丸酮的检测,同时为开展甲基睾丸酮在罗非鱼体内残留消解规律的研究奠定了基础。接着运用建立好的高效液相色谱法检测鱼苗体内的甲基睾丸酮的方法,研究了养殖场大规模养殖下,甲基睾丸酮用于尼罗罗非鱼苗雄性化后在苗种体内的消解规律。分别采用含200mg/kg、50mg/kg、20mg/kg甲基睾丸酮的饲料持续喂养尼罗罗非鱼苗30d来进行雄性化,随后鱼苗转移到池塘来进行甲基睾丸酮的消解实验,应用高效液相色谱法检测不同时间鱼苗体内甲基睾丸酮的残留量,并用SPSS Statistics 17.0统计软件结合EXCEL 2003进行数据处理和分析,来研究甲基睾丸酮的消解规律。结果表明,两次投喂间甲基睾丸酮在罗非鱼体内是缓慢减少的且出现多峰现象,但是在罗非鱼雄性化期间甲基睾丸酮在罗非鱼体内具有一致性。此外,停药后甲基睾丸酮的消解基本符合指数递减,虽然甲基睾丸酮雄性化效果确实很好,但是甲基睾丸酮后期消解较为缓慢,且甲基睾丸酮浓度越大,在罗非鱼鱼苗体内残留时间也越长。若养殖户私下使用过量甲基睾丸酮来用于罗非鱼的雄性化,或者在鱼苗雄性化之后使用甲基睾丸酮的话,很难在上市之前完全代谢掉,那么消费者食用后就很可能对身体造成一定的危害。因此强烈建议国家有关部门尽快研究出雄性化替代方法,如物理途径温度等来进行罗非鱼的雄性化,从根本上一方面保障养殖户的利益,一方面杜绝使用甲基睾丸酮。鉴于近年来罗非鱼成鱼中屡屡检出甲基睾丸酮药物残留,本文在研究甲基睾丸酮在罗非鱼苗体内的消解规律的基础上,进一步研究了甲基睾丸酮用于罗非鱼成鱼促生长后在鱼体内的消解规律。在现实养殖环境中,采用含甲基睾丸酮200mg/kg的饲料持续投喂罗非鱼成鱼3d,每天定时投喂4次,每次喂药饲料量约为罗非鱼生物量的3%~5%,3d后停止喂药,实验鱼转移到养殖池塘。用先前建立的高效液相色谱法检测用药后不同时间背肌、腹肌、鱼皮、肝脏、性腺、血浆、胆汁中药物浓度,并用DAS 2.1.1药物代谢分析软件结合EXCEL 2003进行数据处理和分析,来研究甲基睾丸酮在各组织中的消解规律。结果发现背肌、腹肌、鱼皮、胆汁、血浆、肝脏、性腺中的甲基睾丸酮残留量趋势为指数递减,其中血浆中甲基睾丸酮为幂指数递减。甲基睾丸酮在各组织中均有分布,组织中甲基睾丸酮浓度分布从高到低依次为:胆汁、肝脏、性腺、血浆、鱼皮、腹肌、背肌,暗示着甲基睾丸酮在鱼体体内的代谢为肝胆代谢。在该条件下各组织中甲基睾丸酮残留量在停药后1164h(49d)时才无检出,其中血浆在804h(34d)时无检出。说明药物代谢需要较长时间,若养殖户私下在成鱼阶段使用甲基睾丸酮,若离上市时间较短,那就很难保证在上市之前能完全代谢完。为了消费者的安全,养殖成鱼阶段应杜绝使用甲基睾丸酮,相关部门应严厉打击惩罚成鱼阶段使用甲基睾丸酮的行为,同时也应该加强对饲料配方的优化来达到促罗非鱼生长的目的,让养殖户从根本上愿意彻底放弃使用甲基睾丸酮。更多还原

【Abstract】 17α-methyltestosterone (MT), a synthetic androgen drugs, has served as a specific medicine on fry culture and gender control in aquaculture. It was found that there were MT residues in tilapia at the market, which would harm the human body as this androgen has great potential hazard on human health. Studies were conducted on the dissipation of MT in Nile tilapia Oreochromis niloticus (L.) fry that received dietary of MT to induce sex reversal and adult Nile tilapia Oreochromis niloticus (L.) that received dietary of MT to promote the growth under aquaculture model, aimed for providing theoretical support for security control of fishery drugs in aquaculture and monitoring of drug residues in tilapia tracing, which was based on the optimized high-performance liquid chromatography method for determination of MT residues in tilapia tissues and tilapia fry.First, by comparing the domestic and foreign studies, a simple high-performance liquid chromatography method for determination of MT residues in tilapia tissues and tilapia fry have been optimized. Samples extracted by ether ultrasonic extraction, purified by petroleum ether (plasma, bile supplemented by SPE extraction), separated by using a ODS-C18 analytical column with methanol water (the ratio was 77%) and UV detector, determined by the external standard method, which had a suitable retention time, good peak shape, target peak and impurity peaks can completely separated, the method got good linear relationship in the range of 0.040μg/mL~10.0μg/mL MT concentration (R2 = 1.0000). The limit of detection for samples were: 8μg/kg for dorsal muscle and abdominal muscle, 27μg/kg for fishskin and liver, 80μg/kg for gonad, 80μg/L for plasma, bile, 20μg/kg for fish fry. The recoveries obtained from these samples were above 80.6%, with the relative standard deviation were below 5.53%. This method was appropriate for MT determination, at the same time laid a foundation for doing the research on the dissipation law of methyl -testosterone residues in tilapia.Based on the method of determination MT residues in fry, studies were conducted on the dissipation of MT in Nile tilapia Oreochromis niloticus (L.) fry that received dietary of MT to induce sex reversal under aquaculture model. Sexually undifferentiated Nile tilapia fry (8mm~10mm, 9mg~13mg) were fed on a diet containing unlabeled MT (200mg/kg, 50mg/kg, 20mg/kg) for 30d to effect masculinization in the concrete tank, daily feeding was regularly, water was changed once a week, shed was used natural to control the temperature. After 30d, the fry were transferred to a pond with normal feed, MT in fry were dissipation in the natural environment. The fry were sampled in different time and frequency in the latter days, then MT residues in fry were measured by high performance liquid chromatography (HPLC), SPSS Statistics 17.0 combines EXCEL 2003 statistical software were applied to the data processing and analysis in order to get the dissipation rule of MT after tilapia sex reversal. The results showed that MT residues in tilapia fry almost maintained consistently during the sex reversal period, although overall trend of the dissipation was still downward while multi-peak phenomenon occurred in the dissipation of MT between two feedings. Besides, though masculinization rate of MT was ideal, following the withdrawal, the dissipation of MT fitted with exponential decline and the dissipation was slowly in the latter. the higher the concentration of MT that fed to tilapia for sex reversal, the longer time MT residues maintained in tilapia fry.If the farmers use excessive MT for sex reversal of tilapia privately, there would be some MT residues in marketing tilapia that can’t eliminated completely before marketing, then it will take some harm to consumers. Strongly recommends that national authorities develope alternative methord for masculinization as soon as possible, which will fundamentally protect the interests of farmers, while eliminate MT use.Recently, MT determination of tilapia often turns up positive results. Studies were conducted on the dissipation of MT in adult Nile tilapia Oreochromis niloticus (L.) that received dietary of MT to promote fish growth under aquaculture model, tilapia weighed approximately 250g were fed on a diet containing unlabeled MT (200mg/kg) for 3d for promote fish growth in the concrete tank, After 3d, the tilapia were transferred to a pond with normal feed, MT in tilapia were dissipation in the natural environment. The tilapia were sampled in the latter days, then MT residues in tilapia tissues were measured by high performance liquid chromatography previously established,DAS 2.1.1 drug metabolism analysis software combines EXCEL 2003 statistical software were applied to the data processing and analysis in order to get the dissipation rule of MT in tilapia tissues. The results showed that the dissipation of MT in dorsal muscle, abdominal muscle, fishskin, bile, plasma, liver, gonad all fitted with exponential decline, of which the dissipation of MT in plasma fitted with index decrease. MT were distributed in various tissues, tissue-MT concentration was down in the order: bile, liver, gonads, plasma, skin, abdominal muscle, dorsal muscle, which suggested that metabolism of MT in fish was hepatobiliary metabolism. Under these conditions, MT residues in tissue is no longer detected following withdrawal 1164h in 49d, in which MT residues in plasma was no longer detected following withdrawal 804h in 34d. Suggested that drug metabolism was slowly, using MT promote fish growth in adults stage, there would be some MT residues in marketing tilapia that can’t eliminated completely before marketing. For consumer safety, MT use must be forbidden in adult fish stage, relevant departments should severely punish farmers that using MT in adults stage privately, also should enhance feed formula optimization for growth of tilapia purpose, so that farmers are willing to completely abandon the use of MT.更多还原