| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥300,000)
Fiscal Year 2007: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
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
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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
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