Monitoring of naturally contaminated zearalenone concentrations in feeding environment of cattles.especially the effects for reproductive efficacy

牛饲养环境中自然污染的玉米赤霉烯酮浓度的监测，特别是对繁殖功效的影响

• 批准号: 18580318
• 负责人: TAKAGI Mitsuhiro
• 金额: $ 2.24万
• 依托单位: Kagoshima University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18580318/
• 关键词: Cattle; Zearalenone; LC; MS; Oocyte maturation; 尿

It is well known that Zearalenone (Zen) and its metabolites possess estrogenic activity, indicating their important role in reproductive disorders in domestic animals. In the practical field of cattle, however, information concerning the degree of animal exposure to Zen under feeding conditions on farms remains obscure. As a preliminary investigation, the objectives of the present study were to : 1) simultaneous determination of Zen and metabolites in bovine follicular fluids (FFs) by liquid chromatography/coupled to tandem mass spectrometry (LC/MS/MS) (Exp. 1), and 2) examine the in vitro effects of Zen exposure to bovine oocytes (Exp. 2). Preliminary experiments revealed that cleaning-up of FF samples with combination of C18 and immuno-affinity columns were most suitable for analysis of Zen and its metabolites using LC/MS/MS. Those recovery rates more than 90% were achieved in case of 0.1 μg/L additive in the FF Pool. In Exp. 1, Bovine ovaries were collected from a slaughterhouse, an … More d the FF aspirated from follicles. The follicles were divided into 2 groups, normal follicle (10 to 20 mm in diameter ; n= 32) and cystic follicles (> 25 mm ; n=20). Zen and its metabolites were detected from 6 out of 32 (18.8%) normal follicles and 7 out of 20 (35%) cystic follicles. In Exp. 2, bovine oocytes aspirated from the follicles (2 to 7 mm in diameter) of ovaries were cultured in maturation medium supplemented with various concentrations[0 μg/L (control), 1μg/L, 10μg/L, 100μg/L, and 1000 μg/L]of Zen. After 21 h of maturation culture, the oocytes were fertilized with frozen-thawed spermatozoa and then cultured for 7 days, in order to evaluate the effects of Zen on meiotic competence and the subsequent development of oocytes. A dose-dependent decrease of maturation rates was observed (control:84.5%, 1μg/L:85.5%, 10μg/L:81.0 %, 100μg/L:78.7% and 1000μg/L:36.3%), with a significant decrease (P < 0.01) in the 1000μg/L group. Fifty percent (62/124) of the examined oocytes in the 1000μg/L group were arrested at metaphase I. However, the fertilization rate in 1000μg/L group was similar to that in control (66.7% vs 73.2%, respectively, P>0.05), and no significant differences in blastocyst formation rates after in vitro fertilization and culture were observed among the groups (control:23.4%,1μg/L:23.3%, 10μg/L:16.3%, 100 μg/L:33.0% and 1000μg/L:26.2%). In conclusion, although the concentrations were extremely low level, our results indicate that Zen and its metabolites could be detected in bovine FF. A high concentration of Zen may have a detrimental effect on the meiotic competence of oocytes, but not their both fertilization and development rates after in vitro fertilization. Less

众所周知，玉米赤霉烯酮（Zen）及其代谢产物具有雌激素活性，这表明它们在家畜生殖障碍中具有重要作用。然而，在牛的实际领域，有关动物在农场饲养条件下接触禅宗的程度的信息仍然模糊不清。作为初步研究，本研究的目的是：1)通过液相色谱/串联质谱（LC/MS/MS）同时测定牛卵泡液（FFs）中的Zen及其代谢物（实验1），以及2)检测Zen暴露于牛卵母细胞的体外影响（实验2）。初步实验表明，C18和免疫亲和柱联合清洗FF样品最适合LC/MS/MS分析Zen及其代谢物。当添加量为0.1 μg/L时，回收率可达90%以上。在实验1中，从屠宰场收集牛卵巢，并从卵泡中抽取更多的FF。将卵泡分为正常卵泡（直径10 ~ 20mm， n= 32）和囊性卵泡（直径25mm， n=20）两组。32个正常卵泡中有6个（18.8%）和20个囊性卵泡中有7个（35%）检测到Zen及其代谢物。在实验2中，从卵巢卵泡（直径2 ~ 7mm）中抽取牛卵母细胞，在添加不同浓度[0 μg/L（对照）、1μg/L、10μg/L、100μg/L和1000 μg/L] Zen的成熟培养基中培养。成熟培养21 h后，用冻融精子与卵母细胞受精，再培养7 d，以评价Zen对卵母细胞减数分裂能力及后续发育的影响。成熟率呈剂量依赖性降低（对照组：84.5%,1μg/L:85.5%, 10μg/L: 81.0%, 100μg/L:78.7%, 1000μg/L:36.3%），其中1000μg/L组显著降低（P < 0.01）。1000μg/L组50%（62/124）的受精率与对照组相似（分别为66.7%和73.2%,P < 0.05），各组体外受精和培养后囊胚形成率（对照组：23.4%、1μg/L:23.3%、10μg/L:16.3%、100 μg/L:33.0%和1000μg/L:26.2%）无显著差异。综上所述，虽然浓度极低，但我们的结果表明，在牛FF中可以检测到Zen及其代谢物。高浓度的Zen可能对卵母细胞减数分裂能力有不利影响，但对卵母细胞体外受精后的受精率和发育率没有不利影响。少

项目成果

Monoitoring of naturally contaminated zearalenone concenadons in urine and follicular fluid and in vitro effects of zeazalen on oocyte maturation in cattle

尿液和卵泡液中天然污染的玉米赤霉烯酮浓缩物的监测以及玉米赤霉烯酮对牛卵母细胞成熟的体外影响

• 发表时间: 2007
• 作者: M.Takagi;et. al.
• 通讯作者: et. al.

Monoitoring of naturally contaminated zearalenone concentrations in urine and follicular fluid and in vitro effects of zearalenone on oocyte maturation in cattle.

监测尿液和卵泡液中天然污染的玉米赤霉烯酮浓度以及玉米赤霉烯酮对牛卵母细胞成熟的体外影响。

• 发表时间: 2007
• 作者: M. Takagi;K. Takagaki;T. Kuriyagawa. S. Uno;T. Otoi;A. Budiyanto;K. Okamoto;E. Deguchi
• 通讯作者: E. Deguchi

「研究成果報告書概要(欧文)」より

摘自《研究结果报告摘要（欧洲）》

• 发表时间: 2006
• 期刊: Seibutsu Butsuri 46(1)
• 作者: Yasushi Shigeri;Keiko Shimamoto
• 通讯作者: Keiko Shimamoto