The aims of the present study were to investigate the efficacy of measuring bovine urinary zearalenone (ZEN) concentrations by using a commercially available ELISA method in cattle kept under different feeding conditions to monitor the natural contamination of feeds at the farm level, and to investigate the effects of supplementation of a mycotoxin adsorbent (MA) product in the feed based on urinary ZEN concentration. First, Japanese Black cattle herds kept for breeding (4 herds) and fattening (4 herds) purposes were provided with similar feeding conditions. Then, urinary samples from 5 cows in each herd were collected and analyzed. Second, dairy cows from 1 herd fed with total mixed rations (TMR) were selected. After thorough mixing of the MA (40 g/d) with TMR, the supplemented TMR was fed according to the following schedule: with MA for 2 wk, without MA for 3 wk; then with MA for 2 wk and without MA for 6 wk. Urine samples were collected from cows (n = 6 to 7) and examined before and after each interval. Zearalenone concentrations were measured by the ELISA and liquid chromatography-tandem mass spectrometry methods. The concentration of ZEN and its metabolites was expressed after creatinine (Crea) correction [ZEN or metabolites (pg/mL)/Crea (mg/dL); pg/mg of Crea]. In the first experiment, the urinary concentrations of ZEN and its metabolites were variable in all herds, and significant differences were observed between herds. In 1 fattening herd, in particular, urinary ZEN concentrations were greater (P < 0.001) than in the other 3 herds. This might reflect significant natural ZEN contamination of the feed at the farm level. In Exp. 2, urinary ZEN concentrations displayed peculiar trends after supplementation with MA. After 2 wk of supplementation, a significant decrease of ZEN (P < 0.05) was observed. Zearalenone concentrations remained at a reduced amount during 3 wk without MA supplementation and 2 wk with MA supplementation. When MA was not added to the feed for the next 6 wk, the concentrations increased to the original quantity. These findings indicate the usefulness of measuring concentrations of urinary ZEN and its metabolites not only for monitoring the natural ZEN contamination of cattle feed at the farm level but also for in vivo evaluation of MA function after supplementing feeds with MA.

Abstract. The influence of acute exposure to zearalenone (ZEN) on porcine oocyte maturation, fertilization or sperm penetration ability during both in vitro maturation and fertilization was evaluated. First, oocytes were cultured in ZENcontaining (0−1000 μg/l) maturation medium and then fertilized. The oocytes maturing in vitro without ZEN were then fertilized in ZEN-containing fertilization medium. The maturation rates of oocytes and penetration ability of sperm decreased significantly in the presence of 1000 μg/l of ZEN. However, neither increases in the rates of degeneration and DNA fragmentation of oocytes nor reductions in normal and polyspermic fertilization were observed. ZEN did not affect the sperm penetration rates; however, 1000 μg/l ZEN had positive effects on normal and polyspermic fertilization rates. Therefore, it can be suggested that an acute exposure of porcine oocytes during maturation and of oocytes and sperm during fertilization to ZEN up to 1000 μg/l may not affect the fertility of the oocytes.Key words: Oocytes, Porcine, Sperm, Zearalenone (J. Reprod. Dev. 57: [547][548][549][550] 2011) earalenone (ZEN) is a nonsteroidal estrogen-like mycotoxin produced by Fusarium species on several grains. It is an estrogen receptor agonist; its distinct estrogenic and anabolic properties in several animal species exert detrimental effects on the reproductive system resulting in reproductive disorders in domestic animals, particularly in swine [1][2][3][4]. Although in vitro culture systems do not always provide accurate predictions of toxicity in animals, they can be used to assess risks and can help to define the mechanisms by which mycotoxins act on germ cells [5]. Several in vitro culture assays have been employed to determine the effect of ZEN and its metabolites on the reproductive organs of swine. Previous in vitro experiments revealed that exposure to these mycotoxins affects oocyte maturation, pronucleus formation and embryonic development [6,7], as well as viability, motility and acrosome reactions in sperm [8,9]. Nevertheless, the acute effects of exposure to ZEN during in vitro fertilization remain unknown. This study aimed to examine the effects of exposure to ZEN on porcine oocytes and sperm by using an in vitro maturation (IVM) and in vitro fertilization (IVF) systems to assess the state of nuclear DNA damage and fertilization.As shown in Table 1, exposure to 1000 μg/l of ZEN had a negative effect on the meiotic competence of porcine oocytes (P<0.05). However, there were no significant differences among the groups with respect to the percentages of oocytes showing degeneration and DNA damage. Exposure to 100 and 1000 μg/l of ZEN during maturation culture decreased the total rate of sperm penetration (P<0.05) compared with the control group, but did not influence the rates of normal or polyspermic fertilization of oocytes.As shown in Table 2, exposure to ZEN during IVF did not affect total rates of sperm penetration irrespective of the ZEN concentration. However, exposure to 1000 μg/l of...

Abstract. Zearalenone (ZEN) and its metabolites are important nonsteroidal estrogenic mycotoxins that cause reproductive disorders in domestic animals, especially pigs. We aimed to simultaneously detect ZEN and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) in porcine follicular fluid (FF) by liquid chromatographytandem mass spectrometry. ZEN and α-ZOL, but not β-ZOL, were detected in all pooled FF samples collected from coexisting follicles (diameter ≥ 6 mm) within 10 ovaries. Furthermore, ZEN and α-ZOL were detected in samples pretreated with β-glucuronidase/arylsulfatase, but not in those left untreated, suggesting that the FF samples contained glucuronide-conjugated forms of the mycotoxins that may be less harmful to porcine oocytes due to glucuronidation affecting the receptor binding. Nonetheless, the effects of the glucuronide-conjugated forms should be studied, both in vitro and in vivo. earalenone (ZEN) is a nonsteroidal estrogenic mycotoxin that is produced by Fusarium species on several grains. Despite its low acute toxicity and carcinogenicity, ZEN and its metabolites exhibit distinct estrogenic and anabolic properties in several animal species because of their agonistic effect on the estrogenic receptor. Thus, they affect the reproductive system and play important roles in reproductive disorders in domestic animals, particularly swine [1][2][3][4].The presence of environmental pollutants with potential reproductive toxicity in the follicular fluid (FF) of livestock may be of particular importance because the oocyte completes maturation before ovulation within the FF [5]. Recently, we reported that the FF was naturally contaminated with ZEN and its metabolites and showed the in vitro effects of ZEN on oocyte maturation in cattle [6]. However, no reports are available on the contamination of porcine FF by ZEN and its metabolites.The objectives of this preliminary investigation were to determine the concentrations of ZEN and its metabolites, including α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL), in porcine FF by using liquid chromatography-negative ion electrospray tandem mass spectrometry with electrospray ionization (LC/MS/MS).As shown in Table 1, ZEN and α-ZOL were detected in all 10 FF samples supplemented with β-glucuronidase/arylsulfatase solution during preincubation, while the 10 FF samples that were not treated with β-glucuronidase/arylsulfatase solution did not contain ZEN or α-ZOL, even at trace levels. Additionally, β-ZOL was not detected even at trace levels, irrespective of β-glucuronidase/arylsulfatase treatment. Figure 1 shows representative LC/MS/MS chromatograms of the porcine FF samples contaminated with ZEN and α-ZOL. The mean (± SEM) concentrations of ZEN and α-ZOL were 38.9 ± 4.0 pg/ml (max and min: 54.8 and 15.2 pg/ml, respectively) and 17.6 ± 1.7 pg/ml (max and min: 26.4 and 10.0 pg/ ml, respectively), respectively.In the present study, we detected ZEN and α-ZOL but not β-ZOL in porcine FF only after the FF was treated with β-glucuronidase/arylsulfatase solution...

This field study aimed to investigate the relationships between the urinary zearalenone (ZEN) concentration, which reflects dietary ZEN intake, and the numbers of total and transferable embryos in superovulated cattle. A total of 38 cows (Japanese Black, n=16; Holstein, n=22) were superovulated for commercial embryo production. Urine samples were collected from all cows at the time of embryo flushing and the urinary ZEN concentration was measured. The ZEN concentration was corrected for the creatinine (Crea) concentration as follows: ZEN (pg/mL)/Crea (mg/dL); the corrected ZEN concentration was expressed in pg/mg Crea. The cows were divided into two groups according to whether the urinary ZEN level was less than (group 1) or more than (group 2) the mean value for each breed (Japanese Black: 97.4 pg/mg Crea; range 44.5-91.3 pg/mg Crea; Holstein: 155.5 pg/mg Crea; range 32.7-146.9 pg/mg Crea). The embryo flushing results were compared between the two groups within each breed. Overall, the total number of embryos collected and the number of transferable embryos did not differ significantly between the groups. These results suggest that natural ZEN contamination resulting in urine levels below the threshold value (i.e. below the maximal permissible urinary ZEN concentration) does not affect embryo production in Japanese Black and Holstein cows undergoing superovulation.

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