*4.2. Screening by Urinary ZEN Monitoring to Detect Cattle Herds Fed with Dietary Roughage with Elevated ZEN Contamination*

Before the rice harvest period in September 2019, this screening was conducted at the Japanese Black (JB) breeding cattle production site to monitor the extent of ZEN contamination of rice straw and/or whole crop silage (WCS) stored by cattle farmers in the summer season when the mean temperature of daytime is higher than 30 ◦C. At the request of the managing veterinarian, urinary ZEN monitoring was performed in four herds (A, B, C, and D) of JB cows kept for breeding in the neighborhood in the Kyushu area, Japan, for which the veterinarian routinely provides veterinary treatments and consults with four farmers. All animals were housed indoors, and roughage and concentrates were fed separately. Feeding and management systems were similar in each herd and the dates of sampling and contents of the feeds in each herd are detailed in Table 8. As feed intake may reflect the ZEN exposure, urine samples were collected from two cows with similar body weight within each herd during natural urination after softly massaging the perineum. Regarding the number of cows to be sampled for urine in each herd, referring to our previous report [10], we considered samples from two cows to be sufficient to evaluate and estimate the contamination status of feed fed the same amount and same lot of feed. In addition, samples of all roughages, such as rice straw and WCS, were obtained from each herd to measure both ZEN and STC concentrations in the roughage. All concentrates fed to cattle in each herd were purchased from feed companies and are generally tested for mycotoxin contamination during the manufacturing stage. The urine and roughage samples were immediately placed into a cooler, protected from light, transported to the clinic office, and frozen. The frozen samples were sent to our laboratory and stored at −30 ◦C until our analysis of ZEN and creatinine (Crea) concentrations in the urine, and ZEN and STC concentrations in the roughage.


**Table 8.** Composition of feeds provided to the monitored herds kept for breeding purposes.

\* Age of the breeding cattle at sampling, y; years old, m; month old. WCS: whole crop silage.

*4.3. Follow-up Monthly Monitoring on the Breeding Cattle Herd with Known Feed Contamination*

Since contamination of rice straw/WCS from herd C collected in August 2019 exceeded the standard value of ZEN ≥ 1 mg/kg concomitant with STC was detected, this herd was

zearalenone.

*4.4. Reproductive Records* 

tem on herd fertility.

selected for further monitoring. Therefore, monthly regular urinary ZEN monitoring of herd C was performed from July 2020 to help determine whether similar ZEN and STC exposure from the rice straw/WCS occurred year to year. For monitoring, five cows (Cows 1 to 5: mean 5.0 y: 3.6–6.3 y) in Herd C with similar body weight (approximately 500 kg) fed with the same roughage and concentrated feed were selected and monthly urine, blood, and roughage were sampled. We collected both urine and blood samples from the five cows at the beginning of each month, approximately 2 h after the morning feed, as per our previous methodology [10], and we also collected roughage samples fed to these cows. Both urine and blood samples were immediately stored on ice, protected from light, and transported to the laboratory, and were stored at –30 ◦C after centrifugation as dispensed urine and serum in microtubes until analysis. The collected roughages were also stored at –30 ◦C until measurement of both ZEN and STC concentrations.

Zearalenone concentrations in the collected urine samples were measured by ELISA every two months, as described below, and urine samples were measured monthly when deemed necessary by the herd manager monitoring the condition of the roughage being fed or by contamination status of the roughage by fungi at the monthly sampling. During the follow-up period, daily feeding was performed while sharing the urinary ZEN concentration measurement results with the herd manager and the managing veterinarian. When a high urinary ZEN concentration was confirmed, the roughage lot fed at the time of sampling was changed, and the urinary ZEN concentration was measured again in the following month for follow-up purposes, concomitant with measurement of both ZEN and STC concentrations of roughage samples by LC-MS/MS as mentioned below. Concentrations of urinary ZEN, its metabolites, α-ZEL, β-ZEL, and STC of all collected urine samples during the follow-up period were measured by LC-MS/MS within one assay for reconfirmation of results by the ELISA assay and urinary STC measurement. A schematic representation of the experimental design is shown in Figure 2. *Toxins* **2022**, *14*, x FOR PEER REVIEW 12 of 17 reconfirmation of results by the ELISA assay and urinary STC measurement. A schematic representation of the experimental design is shown in Figure 2.

**Figure 2.** Schematic representation of the experimental design. C\*, herd with high contamination: urinary ZEN concentrations exceeding the standard concentration of the ELISA kit, and ZEN-contaminated roughage exceeding the standard value in Japan (>1 mg/kg). α-ZEL: α-zearalenol; β-ZEL: β-zearalenol; AMH: anti-Müllerian hormone; SAA: serum amyloid A; STC: sterigmatocystin; ZEN: **Figure 2.** Schematic representation of the experimental design. C\*, herd with high contamination: urinary ZEN concentrations exceeding the standard concentration of the ELISA kit, and ZENcontaminated roughage exceeding the standard value in Japan (>1 mg/kg). α-ZEL: α-zearalenol; β-ZEL: β-zearalenol; AMH: anti-Müllerian hormone; SAA: serum amyloid A; STC: sterigmatocystin; ZEN: zearalenone.

*4.5. Analytical Methods of ZEN in Urine and Feed Samples* 

year from 2017 to 2021. Additionally, the reproductive records from the five cows examined between pre-ZEN monitoring (2017 to 2019) and post-ZEN monitoring periods (2020 and 2021) were evaluated to confirm the impact of introducing the ZEN monitoring sys-

Zearalenone concentration in urine was determined using a commercially available kit (RIDASCREEN Zearalenon; R-Biopharm AG, Garmstadt, Germany) according to the manufacturer's instructions, with minor modifications. Briefly, a urine sample (0.1 mL: 5 fold dilution of the kit) was added into 3 mL of 50 mM sodium acetate buffer (pH 4.8) and the solution was incubated for 15 h at 37 °C in the presence of 10 μL of β-glucuronidase/arylsulfatase solution. Thereafter, the samples were loaded onto a C18 solid-phase extraction (SPE) column (Strata; Phenomenex, Torrance, CA, USA), which had been preconditioned with 3 mL of methanol, followed by 2 mL of 20 mM Tris buffer (pH 8.5)/methanol (80:20). After washing the SPE column with 2 mL of 20 mM Tris buffer (pH 8.5)/methanol (80:20) and 3 mL of methanol (40%), the column was centrifuged for 10 min at 500× *g* to dry the column. The analytes were then eluted slowly (flow rate: 15 drops/min) with 1 mL of methanol (80%). The eluate was evaporated to dryness at 60 °C using a centrifugation evaporator. The dried residue was redissolved in 50 μL of methanol, 450 μL of sample dilution buffer was added, the solution was mixed thoroughly, and an aliquot of 50 μL was used for the ELISA assay. To determine the ZEN concentration in the urine sample, RIDA SOFT Win (R-Biopharm) was used to calculate the absorbance at 450 nm using a microplate spectrophotometer. The cross-reactivity rates using this particular ELISA kit for α-ZEL, β-ZEL, and Zeranol were 41.6, 13.8%, and 27.7%, respectively, based on the

#### *4.4. Reproductive Records*

As a reproductive record, the calving intervals of the herd were compared for each year from 2017 to 2021. Additionally, the reproductive records from the five cows examined between pre-ZEN monitoring (2017 to 2019) and post-ZEN monitoring periods (2020 and 2021) were evaluated to confirm the impact of introducing the ZEN monitoring system on herd fertility.

#### *4.5. Analytical Methods of ZEN in Urine and Feed Samples*

Zearalenone concentration in urine was determined using a commercially available kit (RIDASCREEN Zearalenon; R-Biopharm AG, Garmstadt, Germany) according to the manufacturer's instructions, with minor modifications. Briefly, a urine sample (0.1 mL: 5-fold dilution of the kit) was added into 3 mL of 50 mM sodium acetate buffer (pH 4.8) and the solution was incubated for 15 h at 37 ◦C in the presence of 10 µL of βglucuronidase/arylsulfatase solution. Thereafter, the samples were loaded onto a C18 solid-phase extraction (SPE) column (Strata; Phenomenex, Torrance, CA, USA), which had been preconditioned with 3 mL of methanol, followed by 2 mL of 20 mM Tris buffer (pH 8.5)/methanol (80:20). After washing the SPE column with 2 mL of 20 mM Tris buffer (pH 8.5)/methanol (80:20) and 3 mL of methanol (40%), the column was centrifuged for 10 min at 500× *g* to dry the column. The analytes were then eluted slowly (flow rate: 15 drops/min) with 1 mL of methanol (80%). The eluate was evaporated to dryness at 60 ◦C using a centrifugation evaporator. The dried residue was redissolved in 50 µL of methanol, 450 µL of sample dilution buffer was added, the solution was mixed thoroughly, and an aliquot of 50 µL was used for the ELISA assay. To determine the ZEN concentration in the urine sample, RIDA SOFT Win (R-Biopharm) was used to calculate the absorbance at 450 nm using a microplate spectrophotometer. The cross-reactivity rates using this particular ELISA kit for α-ZEL, β-ZEL, and Zeranol were 41.6, 13.8%, and 27.7%, respectively, based on the manufacturer's instruction, and the mean recovery rate of the ELISA assay based on the three trials was 84% ± 14%.

Urine creatinine concentrations were determined using a commercial kit (Sikarikit-S CRE, Kanto Chemical, Tokyo, Japan), according to the manufacturer's instructions, and were measured using a 7700 Clinical Analyzer (Hitachi High-Tech, Tokyo, Japan). All urine concentrations were expressed as a ratio of creatinine (pg/mg creatinine), as described previously [10].

Based on the results of the first screening and measurement of urinary ZEN concentrations by ELISA, both the urine and roughage samples in herds expected to have high ZEN infiltration in the feed were retested using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurement system not only for the confirmation of the ELISA results but also for measuring the ZEN metabolites, α-ZEL and β-ZEL. Additionally, as per our previous reports, urinary STC levels were concomitantly higher in cattle fed ZEN-contaminated rice straw in Japan; thus, it was speculated that co-contamination of both ZEN and STC was observed. Therefore, in the retest, the STC concentration in urine and roughage was also measured according to our previous reports [10,12].

The LC-MS/MS method and validation have been described in our previous report [10]. Briefly, each urine sample (0.5 mL) was mixed with 3.0 mL of 50 mM ammonium acetate buffer (pH 4.8) and 8 µL of glucuronidase/arylsulfatase solution and incubated for 12 h at 37 ◦C. The solution was loaded onto a C18 SPE column, which was preconditioned with 3 mL 100% methanol and 2 mL Tris buffer, followed by the addition of 2 mL Tris buffer and 3 mL of 40% methanol. After washing the SPE column with approximately 1 mL of 80% methanol, the volume of the eluted solution was adjusted to 1 mL. Then, 20 µL of the reconstituted solution was injected into the LC-MS/MS system. The LC-MS/MS analyses were performed on an API 2000 MS/MS system (Applied Biosystems, Foster City, CA, USA) equipped with an electrospray ionization (ESI) interface and a 1200 Infinity Series HPLC system (Agilent Technologies, Santa Clara, CA, USA). The detection limits for ZEN, α-ZEL, and β-ZEL were 0.04 ng/mL, 0.05 ng/mL, and 0.05 ng/mL, respectively, while the mean recovery rates for ZEN, α-ZEL, and β-ZEL were 90%, 109%, and 90%, respectively. STC concentrations of the same eluted solution described above were also determined by LC-MS/MS using an API 2000 system equipped with an ESI as previously described [12]. Briefly, after elution with approximately 1 mL of 80% methanol, the volume was adjusted to exactly 1 mL, and 20 µL of the solution was injected into the LC-MS/MS system. Chromatographic separation was performed on an Inertsil ODS-3 column (4.6 i.d. × 100 mm, 5 µm; GL Sciences, Tokyo, Japan) at 40 ◦C. A mobile phase consisting of methanol/water/acetic acid (97:3:0.01, v:v:v) was used (200 µL/min) to separate the analyte in isocratic mode. Measurements were performed for 15 min. The limit of detection (LoD) was 0.2 ng/mL. ZEN, α-ZEL, β-ZEL, and STC concentrations in the urine are expressed as a ratio to creatinine (pg/mg creatinine).

Both STC and ZEN concentrations in the roughage samples were measured using an API 3200 LC-MS/MS system (AB Sciex, Tokyo, Japan) equipped with an electrospray ionization (ESI) interface and a Prominence HPLC system (Shimadzu Corp., Kyoto, Japan), according to the Food and Agricultural Materials Inspection Center [45] at Shokukanken Inc., Gunma, Japan. In brief, representative samples of stored straw (2 g) and concentrate (10 g) were homogenized and chopped into small pieces. Each sample was placed in a sample tube, to which 20 mL of 84% acetonitrile was added. The tubes were shaken for 1 h and centrifuged for 10 min at 500× *g* at room temperature. The supernatant (10 mL) was loaded onto a MultiSep 226 Aflazon + multifunctional column (Romer Labs, Union, MO, USA). Subsequently, 1 mL of the eluent was mixed with 1 mL acetic acid (1 + 100) and centrifuged for 5 min at 500× *g*. Next, 10 µL of supernatant was injected into the LC-MS/MS system under the following conditions: column, Synergi 4 µm Polar-RP 80 A (2 mm × 150 mm, 4 µm); oven temperature, 40 ◦C; eluent flow, 200 µL/min; and solvent, methanol (A) + 1 mM Ammonium acetate in 0.1% aqueous acetic acid (B). An ESI probe was used in the positive mode for the STC analysis and the negative mode for the ZEN analysis. The detection limit for each analyte was 0.01 mg/kg. The mean STC and ZEN recovery rates were 90.5%–93.5% and 95.3%–98.5%.

#### *4.6. Analytical Methods of AMH and SAA in Serum Samples*

Serum AMH concentration was measured using a bovine AMH ELISA kit (AnshLabs, Webster, TX, USA), according to a previous report [46] to monitor the ovarian AFC of the examined cows during the follow-up period. Briefly, undiluted plasma (50 µL) was used for the assay, which had a limited detection of 11 pg/mL and a coefficient of variation of 2.9%, according to the manufacturer's instructions. Based on our previous studies [19], it is clear that the blood AMH concentration in cattle is lower than usual during the peripartum period; thus, in this study, the AMH concentration in each cow's calving month during the monitoring period was evaluated with particular care. Additionally, SAA concentrations were measured using an automated biochemical analyzer (Pentra C200; HORIBA ABX SAS, Montpellier, France) with a special SAA reagent for animal serum or plasma (VET-SAA 'Eiken' reagent; Eiken Chemical Co. Ltd., Tokyo, Japan) to monitor the inflammation status of each cow during sampling. The SAA concentration was calculated using a standard curve generated using a calibrator (VET-SAA calibrator set; Eiken Chemical Co. Ltd., Tokyo, Japan).

#### *4.7. Data Management and Statistical Analysis*

Monthly estimates for ZEN, AMH, and SAA were calculated using mixed model analysis with subject as a variable factor, because they contain missing data due to calving of the examined cows. Because the ZEN and SAA values approximate a lognormal distribution, the geometric mean estimate was calculated. The AMH value approximates a normal distribution; therefore, the arithmetic mean estimate was calculated. The effects of ZEN and AMH values were evaluated by calculating the simple regression of ZEN values with AMH and the time-lagged regression, which examines the effect of ZEN values one month earlier (lag 1 month), using a linear mixed model. Furthermore, the effects

of ZEN on AMH change were evaluated by defining the change in AMH value over one month as the change from the previous month. The analysis was similarly evaluated by calculating the simple regression of ZEN value to AMH change (lag 0 model) and the time-lag regression to examine the effect of ZEN value one month earlier using a linear mixed model. In other words, the lag 1-month model evaluates the effect of the ZEN value of the current month on the AMH change until the next month. In addition, the effect of ZEN on SAA was also evaluated using the same linear mixed model as described above. A two-sided *p*-value ≤ 0.05 was considered statistically significant. All statistical analyses were performed using SPSS for Windows (version 24.0; IBM Japan, Tokyo, Japan).

All results of the reproductive records of the herds obtained are expressed as the mean ± SEM. Statistical analyses were performed using BellCurve for Excel software (Social Survey Research Information Co., Ltd., Tokyo, Japan). Calving intervals of the herd from 2017 to 2021 were compared using a one-way analysis of variance, followed by a post-hoc test (Tukey-Kramer). Additionally, calving intervals during the pre- (2017 and 2019) and post-ZEN monitoring (2020 and 2021) periods of the examined five cows were compared between the groups using Student's t-test to determine the effects of introducing the monthly urinary ZEN monitoring system on the reproductive efficacies of the breeding herd. Statistical significance was set at *p* ≤ 0.05, whereas *p*-values ranging between 0.05 and 0.1 were considered to indicate a trend toward significance.

**Author Contributions:** Conceptualization, O.S.W., M.E., M.L. and M.T. (Mitsuhiro Takagi); formal analysis, O.S.W., E.K., S.U., O.Y., D.P., H.O., and M.T. (Mitsuhiro Takagi); investigation, O.S.W., M.E., and M.T. (Mitsuhiro Takagi); methodology, E.K., S.U., O.Y., M.T. (Masayasu Taniguchi) and M.T.(Mitsuhiro Takagi); project administration, M.T. (Mitsuhiro Takagi); resources, M.T.(Masayasu Taniguchi) and M.T. (Mitsuhiro Takagi); supervision, M.T. (Masayasu Taniguchi), M.L. and M.T. (Mitsuhiro Takagi); visualization, O.S.W.; writing—original draft, O.S.W., D.P. and M.T.(Mitsuhiro Takagi); writing—review and editing, O.S.W., M.E., E.K., S.U., O.Y., D.P., H.O., M.T. (Masayasu Taniguchi), M.L. and M.T. (Mitsuhiro Takagi) All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by JSPS KAKENHI Grant Number JP21K05920.

**Institutional Review Board Statement:** All experiments were conducted according to the guidelines and regulations for the protection of experimental animals and guidelines stipulated by Yamaguchi University, Japan (no. 40, 1995; approved on 27 March 2017).

**Informed Consent Statement:** Informed consent was obtained from all farmers involved in this study.

**Data Availability Statement:** The original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author.

**Acknowledgments:** The authors would like to express our sincerely gratitude to J. Fink-Gremmels, Utrecht University, The Netherlands, for her critical reading of this manuscript and encouragement to promote our research activity.

**Conflicts of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

#### **References**

