Measurement of urinary concentrations of the mycotoxins zearalenone and sterigmatocystin as biomarkers of exposure in mares.

Mycotoxins may affect animal health, including reproduction. Little is known about the clinical relevance of exposure of horses to contaminated feed. This study aimed at (i) monitoring the levels of the mycotoxins zearalenone (ZEN), with its metabolites α- and ß-zearalenol (α- and ß-ZOL), and sterigmatocystin (STC) in urine samples from thoroughbred mares in Japan and (ii) relating these findings to the potential effects on reproductive efficacy of breeding mares. Sixty-three urine samples of breeding mares from 59 breeding farms were used. Urine samples and reproductive records were collected from each mare when it was presented to the stallion station. Urinary concentrations of ZEN, α- and ß-ZOL, and STC were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). ZEN, α- and ß-ZOL were measurable in the urine of all examined mares, indicating the prevalence of ZEN in equine feeds. In seven of the 63 samples, STC was also detected at levels ranging from 1.3 to 18.0 pg/mg creatinine. No significant correlation between the concentrations of mycotoxins and pregnancy status was observed. In conclusion, measurement of mycotoxins in urine samples is a useful non-invasive method for monitoring the systemic exposure of mares to multiple mycotoxins.

Effects of Dietary Contamination by Zearalenone and Its Metabolites on Serum Anti-Müllerian Hormone: Impact on the Reproductive Performance of Breeding Cows.

We investigated the effects of in vivo exposure to low zearalenone levels on the anti-Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (<1 ppm) at 30 days after calving. Urinary zearalenone, α-zearalenol and ß-zearalenol concentrations were measured by chromatography-tandem mass spectrometry, and serum anti-Müllerian hormone concentrations were determined along with serum biochemical parameters. Urinary concentrations of α-zearalenol were significantly higher (p < 0.05) in cattle in Herd 1 than in cattle in Herd 2, reflecting the different amounts of zearalenone in the diet of the two herds. Although the number of 5-mm and 10-mm follicles of the herds and their fertility after artificial insemination were similar, the serum anti-Müllerian hormone concentrations in herds 1 and 2 were 438.9 ± 48.6 pg/ml and 618.9 ± 80.0 pg/ml, respectively, with a trend towards a significant difference (p = 0.053), which may indicate differences in the antral follicle populations between herds. Thus, zearalenone intake from dietary feed, even when below the threshold zearalenone contamination level permitted in Japan, may affect the ovarian antral follicle populations, but not the fertility, of post-partum cows.

Natural contamination of dietary rice straw with zearalenone and urinary zearalenone concentrations in a cattle herd.

The present study was conducted to 1) identify the natural source of feed contamination by zearalenone (ZEN), which was suspected to have caused persistently increased urinary ZEN concentrations in one of our experimental cattle herds, and 2) evaluate the effects of intervention against this source of contamination. As an experimental model, a fattening Japanese Black cattle herd showing persistently increased urinary ZEN concentrations was identified. Urinary ZEN concentrations of cows fed with new rice straw (experimental group, n = 6) vs. cows that continued to feed on the old rice straw (control group, n = 4) were measured at the start (d 1) and at 2 wk (d 14) after the onset of feeding with straw. In addition, the ZEN concentration in feed and water samples was measured by using both the ELISA and HPLC methods. Furthermore, isolation and identification of fungi from rice straw and concentrate feed samples were performed. The urinary ZEN concentration [ZEN (pg/mL)/creatinine (mg/mL) = pg/mg of creatinine] of cows fed with new rice straw was significantly (P < 0.05) less (843 pg/mg of creatinine) than that of cows fed with old rice straw (15,951 pg/mg of creatinine). On both d 1 and 14, the ZEN concentrations of old rice straw were greater than those of new rice straw. In addition, fungal colonies were observed in the culture media that was obtained from the old rice straw suspected of ZEN contamination, but not in the culture media from new rice straw or other feed samples. In conclusion, our field trials clearly indicate that the rice straw fed to the cows was naturally contaminated with ZEN, and that the monitoring of urinary ZEN concentrations could prove to be a useful tool for detecting the exposure of cattle to ZEN contamination at the farm level.

Measurement of urinary zearalenone concentrations for monitoring natural feed contamination in cattle herds: On-farm trials.

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.