Next Article in Journal
Is Toxin-Producing Planktothrix sp. an Emerging Species in Lake Constance?
Previous Article in Journal
A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen
Previous Article in Special Issue
DL-Selenomethionine Alleviates Oxidative Stress Induced by Zearalenone via Nrf2/Keap1 Signaling Pathway in IPEC-J2 Cells
Article

Adsorbents Reduce Aflatoxin M1 Residue in Milk of Healthy Dairy Cow Exposed to Moderate Level Aflatoxin B1 in Diet and Its Exposure Risk for Humans

1
College of Animal Science, Xinjiang Agricultural University, Urumqi 830052, China
2
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
3
College of Animal Science, Hebei Agricultural University, Baoding 071000, China
*
Author to whom correspondence should be addressed.
Received: 1 August 2021 / Revised: 6 September 2021 / Accepted: 13 September 2021 / Published: 17 September 2021
(This article belongs to the Special Issue Remediation Strategies for Mycotoxin in Animal Feed)

Abstract

This study investigated the effect of moderate risk level (8 µg/kg) AFB1 in diet supplemented with or without adsorbents on lactation performance, serum parameters, milk AFM1 content of healthy lactating cows and the AFM1 residue exposure risk in different human age groups. Forty late healthy lactating Holstein cows (270 ± 22 d in milk; daily milk yield 21 ± 3.1 kg/d) were randomly assigned to four treatments: control diet without AFB1 and adsorbents (CON), CON with 8 μg/kg AFB1 (dry matter basis, AF), AF + 15 g/d adsorbent 1 (AD1), AF + 15 g/d adsorbent 2 (AD2). The experiment lasted for 19 days, including an AFB1-challenge phase (day 1 to 14) and an AFB1-withdraw phase (day 15 to 19). Results showed that both AFB1 and adsorbents treatments had no significant effects on the DMI, milk yield, 3.5% FCM yield, milk components and serum parameters. Compared with the AF, AD1 and AD2 had significantly lower milk AFM1 concentrations (93 ng/L vs. 46 ng/L vs. 51 ng/L) and transfer rates of dietary AFB1 into milk AFM1 (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Children aged 2–4 years old had the highest exposure risk to AFM1 in milk in AF, with an EDI of 1.02 ng/kg bw/day and a HI of 5.11 (HI > 1 indicates a potential risk for liver cancer). Both AD1 and AD2 had obviously reductions in EDI and HI for all population groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the suggested tolerable daily intake (TDI) of 0.20 ng/kg bw/day and 1.00 (HI). In conclusion, moderate risk level AFB1 in the diet of healthy lactating cows could cause a public health hazard and adding adsorbents in the dairy diet is an effective measure to remit AFM1 residue in milk and its exposure risk for humans.
Keywords: aflatoxin B1; moderate risk level; adsorbents; aflatoxin M1; transfer rate; dairy cows; exposure risk assessment aflatoxin B1; moderate risk level; adsorbents; aflatoxin M1; transfer rate; dairy cows; exposure risk assessment

1. Introduction

Aflatoxins are a group of toxic secondary metabolites mainly produced by several species of the genus and contaminate animal feeds and products [1,2]. Among approximately 18 identified aflatoxins [3], aflatoxin B1 (AFB1) has been the most widely studied and problematic mycotoxin in dairy cows [4]. AFB1 in the dairy diet is partly bio-transformed into aflatoxin M1 (AFM1) in the liver and then be secreted into the milk [5,6] and further contaminate dairy products, such as fresh milk, cheese, ice cream, powdered milk, yogurt, and baby formula. While acute exposure to a high dose of AFM1 can result in vomiting, abdominal pain and even death, chronic exposure to low doses of AFM1 may lead to liver cancer [7,8], posing a significant human health hazard [9,10]. In particular, children aged 2–4 years old had the highest risk of exposure to AFM1 in milk [11]. Therefore, AFM1 in milk need to be monitored and AFB1 in dairy feed should be limited at the lowest possible levels.
More than 60 countries have set up strict guidelines for maximum residue level (MRL) of AFM1 in milk [12] and more than 100 countries have issued specific regulated or recommended limits for mycotoxin control in products intended for animal feeds [13]. The MRL of AFM1 in raw milk is 0.5 µg /L in the United States and China [14,15], while the European Union set the level at 0.05 µg /L [16]. The maximum permissible amount of AFB1 in dairy feed has also been established, ranging from 20 µg/kg in the United States to 10 µg/kg in China and 5 µg/kg in the European Union [17]. However, these legal regulations have not eradicated milk AFM1 successfully [3,18,19]. The estimated daily intake (EDI) of milk AFM1 in previous studies was reported to exceed the tolerable daily intake (TDI) limit of 0.20 ng/kg bw/day in several countries [11,20,21,22,23].
Based on field experience and laboratory investigation, we defined the dietary AFB1 risk of dairy cows into four levels: critical (>20 µg/kg), high (10–20 µg/kg), moderate (5–10 µg/kg) and low (<5 µg/kg). Many surveys about AFB1 contamination in feedstuffs have been done worldwide, the overall data showed that most of the AFB1 risk in the dairy feed is low and moderate levels [13,24,25,26]. Biomin Inc. (Ferndale, MI, USA) conducted a worldwide survey of mycotoxin contamination in feed ingredients in 2018 [27] and 2019 [28], the results showed that the aflatoxin positive rates of finished feed in Asia were 44% and 30%, respectively, and the median of positive samples were both 8 µg/kg [29]. Meanwhile, most researchers conducted the field trails in a critical high level of (>20 µg/kg) AFB1 dosages from 20 µg/kg [19,30,31], 22.28 µg/kg (naturally contaminated) [32], 40 µg/kg [31], 63 µg/kg [17], 76 µg/kg (1725 µg/d) [33], 100 µg/kg [34,35], 120 µg/kg [36] and up to 300 µg/kg in diet to investigate their negative effects to the cows [34]. Furthermore, there were no field trials that estimated the human exposure risk to milk AFM1 residue before. Although previous epidemiological surveys on AFM1 in commercial and raw milk have assessed their exposure risk for humans, data on AFB1 content in the diets were usually unusable. Meanwhile, when the prevention of aflatoxin contamination with crops and grains during pre-harvest and storage fails, adding AFB1 adsorbents to dairy diets was proved to be a very effective option to mitigate the negative impact of AFB1 [29]. However, few studies have determined the effects of moderate risk AFB1 and adsorbents in the diet on production performance and milk AFM1 concentration of lactating dairy cows and the risk assessment of milk AFM1 residual for different populations, although most of the dairy cows are likely facing moderate risk AFB1 exposure every day.
Thus, in the present study, we collaborated with the European Horizon Project, set up a moderate risk level (8 µg/kg) of AFB1 to fill up the MRL gap between China and European Union. The objectives of this study were to investigate the effects of supplemental moderate risk level AFB1 and two adsorbents on lactation performance, serum parameters, milk AFM1 content of dairy cows and estimate human exposure risk to current milk AFM1 residue.

2. Results

2.1. Feed Intake and Lactation Performance

All the cows in the four dietary treatments behaved normally and there were not any clinical signs of aflatoxicosis observed throughout the entire feeding trail. Meanwhile, there were no significant differences in the DMI, milk yield, 3.5% FCM yield and milk components (milk fat, protein, lactose and somatic cell count (SCC)) of the dairy cows fed with the moderate risk level of (8 μg/kg of diet dry matter) AFB1 with or without adsorbents, as shown in Table 1.

2.2. Serum Parameters

The effects of moderate risk level (8 µg/kg) of AFB1 with or without adsorbents on serum metabolite parameters are shown in Table 2. No significant difference was observed in the parameters of energy metabolism (GLU, NEFA and BHBA), liver function (ALT, AST and TP), oxidative stress (SOD, GSH-Px, TAOC and MDA) and gastrointestinal permeability (DAO, D-LA and LPS) (p > 0.05).

2.3. AFM1 Content in Milk

The effect of the moderate risk level of AFB1 with or without adsorbents on milk AFM1 content of dairy cows at steady state (day 7 to 14) is shown in Table 3. The AFM1 content in CON was below the detection limits (10 ng/L). The average AFM1 concentration in milk at the platform was 93 ng/L in the AF treatment, which was below the level of AFM1 MRL set by the United States and China, but it was 1.86 times higher than the legal limit of the European Union. The transfer rate of AFB1 from the diet into AFM1 in milk was 1.16% in the AF treatment. Compared to AF, AD1 and AD2 had significantly lower AFM1 concentrations in milk (93 ng/L vs. 46 ng/L vs. 51 ng/L), AFM1 excretion (1.94 μg/d vs. 0.96 μg/d vs. 1.06 μg/d) and transfer rate (1.16% vs. 0.57% vs. 0.63%) (p < 0.05). Meanwhile, AD1 had a greater reduction in AFM1 concentration (50.54% vs. 45.16%), AFM1 excretion (50.52% vs. 45.36%) and the transfer rate (50.86% vs. 45.69%) compared to AD2, but not statistically significant (p > 0.05).
The milk AFM1 concentrations in AF, AD1 and AD2 treatments throughout the entire experimental period are shown in Figure 1. The milk AFM1 concentrations of AF, AD1 and AD2 treatments reached a mean of 66, 49 and 56 ng/kg at 24 h after the first AFB1 administration, then they were maintained up to a relatively stable level at day 7 (93, 50 and 47 ng/L) and day 14 (93, 43 and 55 ng/L). Therefore, the steady-state (day 7–14) was defined with the average milk AFM1 concentrations of 93, 46 and 51 ng/L, respectively, in AF, AD1 and AD2 treatments. The milk AFM1 concentrations dropped to 43, 41 and 33 ng/L at 24 h after withdrawal AFB1 (day 15), continued decreasing in the following days and were undetectable on 5 days after withdrawing AFB1 administration (day 19).
Comparison of the transfer rate under different risk levels of dietary AFB1 with or without adsorbents or other detoxification agents in previous studies and the present study is shown in Table 4. In previous studies, offering detoxification agents to dairy cows challenged with different AFB1 dosages in the diet has shown a reduction in the transfer rate of AFB1 from diet to AFM1 in milk regarding production variables.

2.4. Exposure Risk Assessment

Based on the average AFM1 concentrations of milk in this study, the risk assessment of AFM1 exposure in different populations is calculated and shown in Table 5. It can be seen that EDI values for AF, AD1 and AD2 ranged from 0.17 to 1.02, 0.08 to 0.51 and 0.09 to 0.56 ng/kg bw/day, respectively, in different human age groups. HI values for AF, AD1 and AD2 ranged from 0.84 to 5.11, 0.41 to 2.52 and 0.46 to 2.80, respectively. Compared to AF, both AD1 and AD2 had reductions in EDI and HI in all age groups, whereas, the EDI (≥0.25 ng/kg bw/day) and HI (≥1.23) of children aged 2–11 years old were still higher than the TDI and 1.00 (HI). It is worth noting that the risk of AFM1 exposure was highest in milk consumers aged 2–4 years old, with an EDI of 1.02, 0.51 and 0.56 ng/kg bw/day and a HI of 5.11, 2.52 and 2.80 in AF, AD1 and AD2, respectively. Meanwhile, milk consumers aged 30–45 years old were found to have the lowest risk of AFM1 exposure, with an EDI of 0.17, 0.08 and 0.09 ng/kg bw/day and a HI of 0.84, 0.41 and 0.46 in AF, AD1 and AD2, respectively.

3. Discussion

All the cows in the four dietary treatments were in apparently healthy condition and there were not any clinical signs of aflatoxicosis observed throughout the entire feeding trail. The previous studies indicated that critical level of (≥20 µg/kg) AFB1: 20 µg/kg [2,19], 40µg/kg [2], 63 µg/kg (1197 µg/d) [17], 75 µg/kg (1725 µg/d) [33], 100 µg/kg [41], 117 µg/kg [37], 112 µg/kg [38] and adsorbents administration in diet had no significant effects on the production performance of dairy cows. However, Queiroz et al. [43] and Malinee et al. [32] reported that cows were exposed to naturally contaminated diets containing 22.28 µg/kg AFB1 resulted in a significant reduction of milk protein concentration and milk fat yield. It is noteworthy that the cows were exposed to naturally contaminated TMR diets may co-exposure to the mycotoxin combinations, which led to more adverse effects on the cows than purified AFB1.
The moderate risk level of (8 µg/kg) AFB1 with or without adsorbents in diet did not affect the GLU, NEFA and BHBA in the serum, indicating that moderate risk level AFB1 did not affect the energy metabolism of lactating dairy cows. It is well known that the liver is the main organ for AFB1 metabolism and the target organ for aflatoxicosis. While alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total protein (TP) are the main liver function parameters of dairy cows, there were no statistically significant differences in ALT, AST and TP content among each dietary treatment in the current study. Likewise, both short-term addition of critical risk level (63 µg/kg) and long-term addition of critical risk level (20 µg/kg) AFB1 in the diet of cows did not cause statistically significant changes in ALT, AST and TP [17,19]. Meanwhile, Keller et al. (2015) reported that the yeast cell wall extracts and sodium alginate in adsorbent might stimulate both nonspecific and specific immunological responses, thus improving the performance of cows [51]. Further study is needed to understand the interaction between liver function parameters and AFB1 challenge to the healthy dairy cows. Xiong et al. reported that long-term critical high level (20 µg/kg) AFB1 significantly decreased serum concentrations of SOD, GSH-Px and TAOC of cows, meanwhile increased the serum MDA concentration [19]. In addition, dietary addition of vitamin E, yeast extract and sodium montmorillonite could alleviate oxidative stress in cows with the AFB1 challenge [2]. Diamine oxidase (DAO), D-lactic acid (D-LA) and Lipopolysaccharide (LPS) are the key indicators for the barrier function of the gastrointestinal mucosa, reflects the integrity of the intestinal mechanical barrier and the degree of damage [52,53]. The present study was consistent with previous results [2,19], cows that consumed diet contaminated with AFB1 did not affect their gastrointestinal permeability. In summary, moderate risk level (8 µg/kg) AFB1 with or without adsorbents in diet do not affect energy metabolism, liver function, oxidative stress and gastrointestinal permeability of the healthy dairy cows.
The aflatoxin B1, B2, G1, G2, deoxynivalenol, T-2 toxin, zearalenone and ochratoxin A contents in the basal TMR diet were below the detection limits (0.01 µg/kg). AFM1 was not detected in the milk samples of all cows during the 7 days before the experiment started as well as in the milk of control cows during the entire experimental period. The milk AFM1 contents (66, 49 and 56 ng/kg) of AF, AD1 and AD2 treatments exceeded or were at risk of exceeding the MRL of the European Union (0.05 µg/L). A previous study on lactating dairy cows reported that the plasma AFM1 was detectable at 5 min (10.4 ng/L) and peaked at 25 min (136.3 ng/L) after a single oral intake of 4.9 mg AFB1 [54]. Furthermore, the study of Frobish et al. disclosed that AFM1 appeared in the milk within 12 h after dairy cows receiving AFB1 contaminated feed [55]. A plateau of AFM1 concentration in the milk was observed on day 7 after AFB1 administration and the steady-state condition was maintained up to the last day of the AFB1-dosing period (day 14). Likewise, previous studies have confirmed that the plateau of AFM1 concentration in the milk was observed at day 1 [33,55] to day 4 [17] after AFB1 administration with or without adsorbents in the diet. The present study was consistent with previous results [2,33,43], a sharp decrease of AFM1 level in milk was detected within 24 h after withdrawal AFB1. The difference was the milk AFM1 concentrations (43, 41 and 33 ng/kg) of AF, AD1 and AD2 treatments dropped below the MRL of the European Union. The milk AFM1 content continued decreasing and was ultimately undetectable by 5 days after stop administrating AFB1. This was consistent with previous studies that the duration of AFM1 clearance in the milk of dairy cows could be 3 [2,33,43] to 4 days [17] after the last critical risk (>20 µg/kg) AFB1 administration. These findings suggested that moderate risk AFB1 (8 µg/kg) administration has a similar effect tendency to AFM1 content in milk of apparently healthy dairy cows with a critical level (>20 µg/kg) AFB1 administration. The average AFM1 concentration in milk at steady state was 93 ng/L in the AF treatment, which was below the AFM1 MRL set by the United States and China, but it was 1.86 times higher than the legal limit of the European Union.
The transfer rate of dietary AFB1 into milk AFM1 is highly correlated with milk yield, the transfer rate usually is 1–2% for late lactating dairy cows (yield < 30 kg/d) and up to 6% for high-yielding cows (yield > 30 kg/d) [56]. Furthermore, the cow species difference, general health, hepatic biotransformation capacity, rate of ingestion and the integrity of the mammary alveolar cell membranes have been shown to affect the transfer rate [56]. According to the transfer rate equation above, 5–10 µg/kg AFB1 in the diet of lactating cows converted to 40–430 ng/L AFM1 in raw milk, which is below the AFM1 MRL set by the United States and China, but at risk of exceeding the legal limit of the European Union. However, the feedstuffs for dairy cows are normally co-exposure to the mycotoxin combinations, due to the possible additive or synergic effect, which may lead to more adverse effects than purified AFB1. Thus, the dietary AFB1 risk of dairy cows was defined into four levels: critical (>20 µg/kg), high (10–20 µg/kg), moderate (5–10 µg/kg) and low (<5 µg/kg).
The transfer rates of AFB1 from the diet into AFM1 in milk were 1.16, 0.57 and 0.63% in the AF, AD1 and AD2 treatments. In accordance with the results of our study, the reports of Guo et al. [17], Maki et al. [37] and Ogunade et al. [33] showed the carry-over rates were 1.06%, 1.13% and 1.07% when cows were challenged with critical AFB1 dosing of 63 µg/kg, 75 µg/kg and 100 µg/kg, respectively. The highest transfer rate of 7.26% was observed in the report of Malinee et al., while the cows (milk yield of 10 kg per day) were fed with TMR diet contained 22.28 µg/kg AFB1 [32]. Furthermore, the inclusion of adsorbents in the AFB1 contaminated diet significantly reduced the transfer rates in previous studies [19,37,38,41,42,46] regardless of milk production and dietary AFB1 dosage variables (Table 4). In addition to adsorbents, biodegradation products such as Bacillus subtilis ANSB060 [17], Kluyveromyces marxianus and Pichia kudriavzevii which isolated from the ruminal fluid of dairy cows [32] also observably reduced the milk AFM1 content and transfer rates. Masoero et al. proposed a linear regression equation to describe the relationship between the carry-over rate of diet AFB1 to milk AFM1 and the milk yield as follows: carry-over% = −0.326 + 0.077 × milk yield; r2 = 0. 58) [57]. Our current data fitted the equation well, with the actual and estimated carry-over rate of 1.16% and 1.28%, respectively. Compared to the AF, the AD1 and AD2 significantly decreased the mean AFM1 concentration (93 ng/L vs. 46 ng/L vs. 51 ng/L), AFM1 excretion (1.94 μg/d vs. 0.96 μg/d vs. 1.06 μg/d) and the transfer rate (1.16% vs. 0.57% vs. 0.63%), respectively. Although the mean milk AFM1 concentrations of AD1 and AD2 treatments significantly decreased to 46 and 51 ng/L, which were below the AFM1 MRL set by the United States and China, still at risk of exceeding the legal limit of the European Union.
To assess the risk of AFM1 exposure in Chinese populations due to milk AFM1 intake under moderate risk level AFB1 and adsorbents in the diet of apparently healthy lactating cows, we calculated the estimated daily intake (EDI) and the hazard index (HI) values in different human age groups. Kuiper-Goodman [23] determined a No Observed Effect Level (NOEL) for AFM1 of <2.5 g/kg bw/day and proposed the AFM1 tolerable daily intake (TDI) of 0.20 ng/kg body weight/day as a “safe dose”, i.e., 50% of the animals would have developed tumors (TD50) dividing by a large safety factor of 50,000. HI above 1.00 indicates that milk AFM1 intake is considered a potential risk for liver cancer in consumers [58]. In this study, the EDI values ranged from 0.17 to 1.02, 0.08 to 0.51 and 0.09 to 0.56 ng/kg bw/day, with the HI values, ranged from 0.84 to 5.11, 0.42 to 2.53 and 0.46 to 2.80 in AF, AD1 and AD2, respectively. The HI values of the youth population aged 2–18 years old and the elderly population aged >60 years old in AF were above 1.00, indicates that milk AFM1 intake is a potential risk for liver cancer in the public, expressly for youth and elderly consumers. Compared to AF, both AD1 and AD2 had obvious reductions in the HI values in all age groups, which proves that adding adsorbents in the diet of cows is an effective measure to remit milk AFM1 exposure risk for humans. However, the HI values of youth consumers aged 2–11 years old were still above 1.00, indicates that adding adsorbents is not a guaranteed measure to eliminate milk AFM1 residue, which is still presenting exposure risk for youth consumers aged 2–11 years old. Previous epidemiological surveys have also assessed people’s risk of exposure to AFM1 in milk and found that the EDI was 0.242 ng/kg bw/day in Iran [59], was 0.025–0.328 ng/kg bw/day in Italy [60], 0.495 ng/kg bw/day in Lebanon [22], 0.22 ng/kg bw/day [11] and 0.263 ng/kg bw/day [20] in China. Furthermore, the risk of AFM1 exposure was highest in milk consumers aged 2–4 years old, with an EDI of 1.02, 0.51 and 0.56 ng/kg bw/day and a HI of 5.11, 2.53 and 2.80 in AF, AD1 and AD2, respectively. These were lower than the EDI of 3.7 ng AFM1/kg bw/day for a four-month-old infant weighing 6 kg, representing a daily intake of 22 ng of AFM1 reported by Oliveira et al. in Brazil [61]. Peng and Chen conducted a Monte Carlo simulation to estimate the AFM1 intake of different population groups in Taiwan and found a mean AFM1 intake of 3.25 ng/day for 19 to 44 years old women to 5.67 ng/day for 19 to 44 years old men [62]. Meanwhile, the lowest exposure risk was observed in the population aged 30–45 years old, with the EDI of 0.17 and the HI of 0.84 and increased gradually in people aged above 45 years old. The EDI values were 0.21 and 0.25 ng/kg bw/day and HI values were 1.04 and 1.24 for the elderly population aged 60–70 and >70 years old, respectively. The elderly population may also be sensitive to the adverse effects of AFM1 due to decreased immunity and poor physical condition.
According to the TDI of 0.20 ng/kg bw/day and based on the body weight and milk consumption of children aged 2–4 years old in this study, the maximum average concentration of AFM1 in milk consumed by these children was calculated to be 18.2 ng/L [11], which is below the AFM1 MRL set by the United States, China and the European Union. According to the carry-over equation proposed by Britzi et al. [56], moderate risk level (5–10 µg/kg) AFB1 in the diet of apparently healthy lactating cows converted to 40–430 ng/L AFM1 in raw milk, posing a significant human health hazard, expressly for youth and elderly population. To our knowledge, this is the first study to assess the human risk of exposure to milk AFM1 from cows fed with moderate risk level AFB1 and adsorbents. Our results suggested that the inclusion of mycotoxin adsorbents in the dairy diet could decrease AFM1 residual in raw milk and reduce the exposure risk for the public.

4. Conclusions

Supplemental moderate risk level (8 µg/kg) AFB1 and adsorbents in the diet of healthy lactating cows did not affect the behaviors, dry matter intake (DMI), milk yield, milk compositions and serum parameters of the dairy cows. Moderate risk level AFB1 significantly increased the AFM1 residual in raw milk and the transfer rates of AFB1 from the diet into AFM1 in milk of apparently healthy cows, posing a significant human health hazard, expressly for the youth and elderly population. The inclusion of mycotoxin adsorbents in the AFB1 contaminated diet proved to be an effective measure to remit milk AFM1 residue and its exposure risk for humans.

5. Materials and Methods

All cow feeding and management in this study were performed according to the China Agricultural University animal research committee protocol (Protocol number: 2013-5-LZ) and all the protocols in present study were approved by the Ethical Committee of China Agricultural University (Protocol number: CAU20180825-2; Date: 25 August 2018).

5.1. Experimental Design, Diets and Cow Management

Forty healthy lactating multiparous Holstein cows (parity (mean ± SD) = 3.1 ± 0.3, days in milk = 270 ± 22 d, daily milk yield= 21 ± 3.1 kg/d, bodyweight = 650 ± 25 kg) from the Aomei dairy farm (Xinxiang, Henan province, China) were randomly assigned into one of four treatments: (1) control diet (CON), basal total mixed ration (TMR) without AFB1 and adsorbents; (2) aflatoxin diet (AF), CON diet + 168 µg/d AFB1 (resulted in 8 µg/kg AFB1 of diet dry matter); (3) adsorbent 1 diet (AD1), AF diet + 15 g/d adsorbent 1 (0.07% of diet dry matter); (4) adsorbent 2 diet (AD2), AF diet + 15 g/d adsorbent 2 (0.07% of diet dry matter). The experiment lasted for 19 days, AFB1-dosing for 14 days as the AFB1-challenge period (day 1 to 14), then following as the AFB1-withdraw period (day 15 to 19). Diet was formulated to meet NRC requirements of a dairy cow producing 21 kg/d milk [63]. The ingredients and chemical compositions of the diet are present in Table 6. Adsorbent 1 (Patent ID: CN111296722A, a patented product developed by our laboratory) consists of montmorillonite and diatomite in a ratio of 50:50; adsorbent 2 is a commercial product that consists of montmorillonite, diatomite, yeast cell wall extracts and sodium alginate. Before the trial, we continually measured the daily DMI for 7 days and then calculated the average AFB1 intake of cows according to the average DMI. The DMI and AFB1 intakes of cows were 21 kg/d and 8 µg/kg/d, respectively. Pure AFB1 (purity: 99.5%, Shanghai Yuduo Biotechnology Co., Ltd., Shanghai, China) was dissolved in methanol. The AFB1 was administrated daily to each cow in the treatment groups by top-dressing and the adsorbent was manually mixed with TMR. All of the cows in the four treatments were only fed the basal TMR during the AFB1-withdraw period. Cows were fed twice daily (07:00 and 17:00). All cows were access to feed and water ad libitum. Two experienced veterinarians assessed and recorded the health condition of the dairy cows every day during the entire trial period.

5.2. Sample Collection and Analysis

Samples for TMR in each group were collected and stored at −20 °C. The TMR samples were dried at 65 °C for 48 h in a forced-air oven and ground to pass through a 1 mm screen using a feedstuff mill (KRT-34; KunJie, Beijing, China) subsequently. In addition, the samples were then divided into two portions and stored at −20 °C until the analysis of chemical composition and mycotoxins. The DM, CP of TMR samples were determined according to the methods described by the Association of Official Analytical Chemists (AOAC) [64]. The content of NDF and ADF were analyzed by the Ankom fiber analyzer (A2000i; Ankom Technology, Fairport, NY, USA) following the procedures of Van Soest et al. [65]. The quantification for mycotoxins (AFB1, AFB2, AFG1, AFG2, deoxynivalenol, T-2 toxin, zearalenone and ochratoxin A) in diet were determined as previously described by Li et al. [66]. The aflatoxin B1, B2, G1, G2, deoxynivalenol, T-2 toxin, zearalenone and ochratoxin A contents in the experimental diets were below the detection limits (0.01 µg/kg).
The cows were milked twice daily (06:30 and 16:30) using a DeLaval milking system and milk yield was recorded at each milking time. Milk samples were collected at each milking time on days 0, 1, 7, 14, 15, 18 and 19 and approximately 100 mL of milk was collected into two 50 mL tubes. Milk samples from one tube were sent to Henan Dairy Herd Improvement (DHI) Testing Center (Zhengzhou, China) for the analysis of milk fat, protein, lactose and somatic cell count (SCC) by an automated near-infrared milk analyzer (Seris300 CombiFOSS; Foss Electric, Hillerød, Denmark). Milk from another tube was stored at −20 °C for mycotoxins analysis. The quantification of AFM1 in milk samples was conducted by the Romer Laboratory (Wuxi, China) following the LC-MS/MS method from the Ministry of Health, China [67].
Blood samples were collected from the coccygeal vein before the morning feeding on days 7 and 14 and centrifuged at 3000 rpm for 15 min at 4 °C to obtain the serum. All serum samples were submitted to Huaying Biotechnology Co., Ltd. (Beijing, China). The glucose (Glu), total protein (TP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), nonestesterified fatty acid (NEFA), β-hydroxybutyric acid (BHBA), malondialdehyde (MDA), glutathione peroxidase (GSHPx), total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and total bilirubin (TBIL) were analyzed using Hitachi 7160 automatic biochemical analyzer (Hitachi 7160; Hitachi Incorporated, Tokyo, Japan) through a colorimetric kit (DiaSys Diagnostics Systems GmbH, Frankfurt, Germany). Serum diamine oxidase (DAO), D-lactic acid (D-LA), Lipopolysaccharide (LPS) concentrations were detected using an enzyme-labeled instrument (Thermo Multiskan Ascent, American) with enzyme-linked immunosorbent assay (ELISA).

5.3. Risk Assessment of Exposure to AFM1

The estimated daily intake (EDI) and the hazard index (HI) of the average AFM1 concentration in milk during the platform in the current study were calculated according to the equations as follows:
EDI   ( ng / kg   bw / day )   =   AFM 1   concentration   in   milk   ×   ( daily   milk   consumption ) average   body   weight
where data on daily milk consumption and average body weight of different ages in China were found in previous studies [11,50,59] and the AFM1 contents in the milk of dairy cows fed different diets in the current study were used as the AFM1 concentration in milk in this equation.
HI   =   estimated   daily   intake   ( EDI ) tolerable   daily   intake   ( TDI )
where TDI as the safe dose, was set as 0.20 ng/kg bw/day as suggested by Kuiper-Goodman (1990), it was determined by dividing the TD50 (the dose at which 50% of the animals would have developed tumors) by a safety factor of 50,000 [23]. A HI value higher than 1 indicates that milk AFM1 intake is considered a potential risk for liver cancer in consumers [58].

5.4. Calculations

3.5% FCM yield = 0.4324 × milk yield + 16.218 × milk fat yield [63].
AFM1 excretion (μg/d) = concentration of AFM1 in milk (μg/kg) × milk yield (kg/d).
Transfer rate (%) = excretion of AFM1(μg/d)/AFB1 consumption (μg/d) × 100%.

5.5. Statistical Analysis

The milk yield, milk components, serum parameters and the AFM1 content in milk were analyzed by one-way analysis of variance (ANOVA) using SAS (version 9.2; SAS Institute, Cary, NC, USA). A significant difference among the treatments was determined by Duncan’s multiple range tests. The significance level was set at 0.05.

Author Contributions

Conceptualization, M.C., Y.W., and S.L.; methodology, M.C., Y.H., S.L.; software, E.W.; validation, S.J., W.S.; formal analysis, M.C., Y.H.; investigation, M.C., S.H.; resources, Y.W., S.L; data curation, E.W.; writing—original draft preparation, M.C.; writing—review and editing, L.Z., S.H, E.W., S.J., W.W.; visualization, E.W.; supervision, Y.W.; project administration, S.L.; funding acquisition, S.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by China Agriculture Research System (CARS-36) and Guiding Local Science and Technology Development Projects by the Central Government (GKZY20111004).

Institutional Review Board Statement

All cow feeding and management in this study were performed according to the China Agricultural University animal research committee protocol (Protocol number: 2013-5-LZ) and all the protocols in present study were approved by the Ethical Com-mittee of China Agricultural University (Protocol number: CAU20180825-2; Date: 25 August 2018).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Battacone, G.; Nudda, A.; Rassu, S.P.G.; Decandia, M.; Pulina, G. Excretion of aflatoxin M1 in milk of goats fed a single dose of aflatoxin B1. J. Dairy Sci. 2012, 95, 2656–2661. [Google Scholar] [CrossRef]
  2. Xiong, J.L.; Wang, Y.M.; Nennich, T.D.; Li, Y.; Liu, J.X. Transfer of dietary aflatoxin B1 to milk aflatoxin M1 and effect of inclusion of adsorbent in the diet of dairy cows. J. Dairy Sci. 2015, 98, 2545–2554. [Google Scholar] [CrossRef]
  3. Nina, B.; Đurđica, B.; Maja, Đ.; Sedak, M.; Kolanović, B.S. Assessment of aflatoxin M1 contamination in the milk of four dairy species in Croatia. Food Control 2014, 43, 18–21. [Google Scholar]
  4. Campagnollo, F.B.; Ganev, K.C.; Khaneghah, A.M.; Portella, J.; Cruz, A.G.; Granato, D.; Corassin, C.H.; Oliveira, C.A.F.; de Souza Sant’Ana, A. The occurrence and effect of unit operations for dairy products processing on the fate of aflatoxin M1: A review. Food Control 2016, 68, 310–329. [Google Scholar] [CrossRef]
  5. Diaz, D.E.; Hagler, W.M.; Blackwelder, J.T.; Eve, J.A.; Whitlow, L.W. Aflatoxin binders Ⅱ: Reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed. Mycopathologia 2004, 157, 233–241. [Google Scholar] [CrossRef]
  6. Battacone, G.; Nudda, A.; Palomba, M.; Pascale, M.; Nicolussi, P.; Pulina, G. Transfer of aflatoxin B1 from feed to milk and from milk to curd and whey in dairy sheep fed artificially contaminated concentrates. J. Dairy Sci. 2005, 88, 3063–3069. [Google Scholar] [CrossRef]
  7. Etzel, R.A. Mycotoxins. JAMA J. Am. Med. Assoc. 2002, 287, 425–427. [Google Scholar] [CrossRef]
  8. Sherif, S.O.; Salama, E.E.; Abdel-Wahhab, M.A. Mycotoxins and child health: The need for health risk assessment. Int. J. Hyg. Environ. Health 2009, 212, 347–368. [Google Scholar] [CrossRef]
  9. Ismail, A.; Gonçalves, B.L.; de Neeff, D.V.; Ponzilacqua, B.; Coppa, C.F.S.C.; Hintzsche, H.; Sajid, M.; Cruz, A.G.; Corassin, C.H.; Oliveira, C.A.F. Aflatoxin in foodstuffs: Occurrence and recent advances in decontamination. Food Res. Int. 2018, 113, 74–85. [Google Scholar] [CrossRef]
  10. Peraica, M.; Radi, B.; Luci, A.; Pavlovi, M. Toxic effects of mycotoxins in human. Bull. World Health Organ. 1999, 77, 754–766. [Google Scholar]
  11. Xiong, J.L.; Zhang, X.F.; Zhou, H.L.; Lei, M.Q.; Liu, Y.L.; Ye, C.; Wu, W.X.; Wang, C.; Wu, L.Y.; Qiu, Y.S. Aflatoxin M1 in pasteurized, ESL and UHT milk products from central China during summer and winter seasons: Prevalence and risk assessment of exposure in different age groups. Food Control 2021, 125, 6. [Google Scholar] [CrossRef]
  12. Sadia, A.; Jabbar, M.A.; Deng, Y.; Hussain, E.A.; Riffat, S.; Naveed, S.; Arif, M. A survey of aflatoxin M1 in milk and sweets of Punjab, Pakistan. Food Control 2012, 26, 235–240. [Google Scholar] [CrossRef]
  13. Gallo, A.; Giuberti, G.; Frisvad, J.C.; Bertuzzi, T.; Nielsen, K.F. Review on mycotoxin issues in ruminants: Occurrence in forages, effects of mycotoxin ingestion on health status and animal performance and practical strategies to counteract their negative effects. Toxins 2015, 7, 3057–3111. [Google Scholar] [CrossRef]
  14. Food and Drug Administration (FDA). Guidance for Industry: Action Levels for Poisonous or Deleterious Substances in Human Food and Animal Feed; FDA: Silver Spring, MD, USA, 2000. [Google Scholar]
  15. China Ministry of Health. Maximum Residue Level of Mycotoxin in Food—National Regulations for Food Safety; National Standard No. 2761-2011; China Ministry of Health: Beijing, China, 2011.
  16. European Food Safety Authority (EFSA). Opinion of the scientific panel on contaminants in the food chain on a request from the Commission related to aflatoxin B1 as undesirable substance in animal feed. EFSA J. 2004, 2, 39. [Google Scholar] [CrossRef]
  17. Guo, Y.; Zhang, Y.; Wei, C.; Ma, Q.; Ji, C.; Zhang, J.; Zhao, L. Efficacy of Bacillus subtilis ANSB060 biodegradation product for the reduction of the milk aflatoxin M1 content of dairy cows exposed to aflatoxin B1. Toxins 2019, 11, 161. [Google Scholar] [CrossRef]
  18. Xiong, J.L.; Wang, Y.M.; Ma, M.R.; Liu, J.X. Seasonal variation of aflatoxin M1 in raw milk from the Yangtze River Delta region of China. Food Control 2013, 34, 703–706. [Google Scholar] [CrossRef]
  19. Xiong, J.L.; Wang, Y.M.; Zhou, H.L.; Liu, J.X. Effects of dietary adsorbent on milk aflatoxin M1 content and the health of lactating dairy cows exposed to long-term aflatoxin B1 challenge. J. Dairy Sci. 2018, 101, 8944–8953. [Google Scholar] [CrossRef]
  20. Guo, Y.; Han, X.; Peng, S.; Yue, T.; Wang, Z. Occurrence and risk assessment of aflatoxin M1 in commercial milk in Shaanxi. J. Northwest Univ. Nat. Sci. Ed. 2020, 48, 131–137. [Google Scholar]
  21. Bogalho, F.; Duarte, S.; Cardoso, M.; Almeida, A.; Cabecas, R.; Lino, C.; Pena, A. Exposure assessment of Portuguese infants to Aflatoxin M1 in breast milk and maternal social-demographical and food consumption determinants. Food Control 2018, 90, 140–145. [Google Scholar] [CrossRef]
  22. Daou, R.; Afif, C.; Joubrane, K.; Khabbaz, L.R.; Maroun, R.; Ismail, A.; El Khoury, A. Occurrence of aflatoxin M-1 in raw, pasteurized, UHT cows’ milk, and dairy products in Lebanon. Food Control 2020, 111, 107055. [Google Scholar] [CrossRef]
  23. Kuiper-Goodman, T. Uncertainties in the risk assessment of three mycotoxins: Aflatoxin, ochratoxin, and zearalenone. Can. J. Physiol. Pharmacol. 1990, 68, 1017–1024. [Google Scholar] [CrossRef] [PubMed]
  24. Rodrigues, I.; Naehrer, K. A three-year survey on the worldwide occurrence of mycotoxins in feedstuffs and feed. Toxins 2012, 4, 663–675. [Google Scholar] [CrossRef]
  25. Han, R.W.; Zheng, N.; Wang, J.Q.; Zhen, Y.P.; Xu, X.M.; Li, S.L. Survey of aflatoxin in dairy cow feed and raw milk in China. Food Control 2013, 34, 35–39. [Google Scholar] [CrossRef]
  26. Selvaraj, J.N.; Wang, Y.; Zhou, L.; Zhao, Y.; Liu, Y. Recent mycotoxin survey data and advanced mycotoxin detection techniques reported from China: A review. Food Addit. Contam. Part A-Chem. 2015, 32, 440–452. [Google Scholar] [CrossRef]
  27. Biomin Mycotoxin Survey. 2018. Available online: https://engage.biomin.net/hubfs/BIOMIN/Mycotoxin%20Survey%20Report/MAG_MTX-Survey-Report_2018_EN.pdf (accessed on 10 May 2019).
  28. Biomin Mycotoxin Survey. 2019. Available online: https://cdn2.hubspot.net/hubfs/5480243/BIOMIN/Downloads/MAG_MTXSurveyReport_2019_EN.pdf (accessed on 11 October 2019).
  29. Jiang, Y.; Ogunade, I.M.; Vyas, D.; Adesogan, A.T. Aflatoxin in Dairy Cows: Toxicity, Occurrence in Feedstuffs and Milk and Dietary Mitigation Strategies. Toxins 2021, 13, 23. [Google Scholar] [CrossRef]
  30. Jones, M.G.S.; Ewart, J.M. Effects on milk-production associated with consumption of decorticated extracted froundnut meal contaminated with aflatoxin. Vet. Rec. 1979, 105, 492–493. [Google Scholar] [CrossRef]
  31. Wang, Q.; Zhang, Y.D.; Zheng, N.; Guo, L.Y.; Song, X.M.; Zhao, S.G.; Wang, J.Q. Biological system responses of dairy cows to aflatoxin B1 exposure revealed with metabolomic changes in multiple biofluids. Toxins 2019, 11, 77. [Google Scholar] [CrossRef] [PubMed]
  32. Intanoo, M.; Kongkeitkajorn, M.B.; Suriyasathaporn, W.; Phasuk, Y.; Bernard, J.K.; Pattarajinda, V. Effect of supplemental Kluyveromyces marxianus and Pichia kudriavzevii on aflatoxin M1 excretion in milk of lactating dairy cows. Animals 2020, 10, 709. [Google Scholar] [CrossRef]
  33. Ogunade, I.M.; Arriola, K.G.; Jiang, Y.; Driver, J.P.; Staples, C.R.; Adesogan, A.T. Effects of 3 sequestering agents on milk aflatoxin M1 concentration and the performance and immune status of dairy cows fed diets artificially contaminated with aflatoxin B1. J. Dairy Sci. 2016, 99, 6263–6273. [Google Scholar] [CrossRef]
  34. Mertens, D.R.; Wyatt, R.D. Acute aflatoxicosis in lactating dairy-cows. J. Dairy Sci. 1977, 60, 153–154. [Google Scholar]
  35. Patterson, D.S.P.; Anderson, P.H. Recent aflatoxin feeding experiments in cattle. Vet. Rec. 1982, 110, 60–61. [Google Scholar] [CrossRef] [PubMed]
  36. Guthrie, L.D. Effects of aflatoxin in corn on production and reproduction in dairy cattle. J. Dairy Sci. 1979, 62, 134. [Google Scholar]
  37. Maki, C.R.; Thomas, A.D.; Elmore, S.E.; Romoser, A.A.; Harvey, R.B.; Ramirez-Ramirez, H.A.; Phillips, T.D. Effects of calcium montmorillonite clay and aflatoxin exposure on dry matter intake, milk production, and milk composition. J. Dairy Sci. 2016, 99, 1039–1046. [Google Scholar] [CrossRef]
  38. Kutz, R.E.; Sampson, J.D.; Pompeu, L.B.; Ledoux, D.R.; Spain, J.N.; Vázquez-Añón, M.; Rottinghaus, G.E. Efficacy of solis, NovasilPlus, and MTB-100 to reduce aflatoxin M1 levels in milk of early to mid lactation dairy cows fed aflatoxin B1. J. Dairy Sci. 2009, 92, 3959–3963. [Google Scholar] [CrossRef]
  39. Weatherly, M.E.; Pate, R.T.; Rottinghaus, G.E.; Roberti Filho, F.O.; Cardoso, F.C. Physiological responses to a yeast and clay-based adsorbent during an aflatoxin challenge in Holstein cows. Anim. Feed Sci. Technol. 2018, 235, 147–157. [Google Scholar] [CrossRef]
  40. Pate, R.T.; Paulus Compart, D.M.; Cardoso, F.C. Aluminosilicate Clay Improves Production Responses and Reduces Inflammation During an Aflatoxin Challenge in Lactating Holstein Cows. J. Dairy Sci. 2018, 101, 11421–11434. [Google Scholar] [CrossRef]
  41. Sulzberger, S.A.; Melnichenko, S.; Cardoso, F.C. Effects of clay after an aflatoxin challenge on aflatoxin clearance, milk production, and metabolism of Holstein cows. J. Dairy Sci. 2017, 100, 1856–1869. [Google Scholar] [CrossRef]
  42. Rodrigues, R.O.; Rodrigues, R.O.; Ledoux, D.R.; Rottinghaus, G.E.; Borutova, R.; Averkieva, O.; McFadden, T.B. Feed additives containing sequestrant clay minerals and inactivated yeast reduce aflatoxin excretion in milk of dairy cows. J. Dairy Sci. 2019, 102, 6614–6623. [Google Scholar] [CrossRef]
  43. Queiroz, O.C.M.; Han, J.H.; Staples, C.R.; Adesogan, A.T. Effect of adding a mycotoxin-sequestering agent on milk aflatoxin M1 concentration and the performance and immune response of dairy cattle fed an aflatoxin B1 contaminated diet. J. Dairy Sci. 2012, 95, 5901–5908. [Google Scholar] [CrossRef]
  44. Maki, C.R.; Allen, S.; Wang, M.; Ward, S.; Phillips, T. Calcium Montmorillonite Clay for the Reduction of Aflatoxin Residues in Milk and Dairy Products. J. Dairy Vet. Sci. 2017, 2, 555587. [Google Scholar]
  45. Sumantri, I.; Murti, T.W.; Van der Poel, A.F.B.; Boehm, J.; Agus, A. Carry-over of aflatoxin B1-feed into aflatoxin M1-milk in dairy cows treated with natural sources of aflatoxin and bentonite. J. Indones. Trop. Anim. Agric. 2012, 37, 271–277. [Google Scholar] [CrossRef]
  46. Masoero, F.; Gallo, A.; Diaz, D.; Piva, G.; Moschini, M. Effects of the procedure of inclusion of a sequestering agent in the total mixed ration on proportional aflatoxin M1 excretion into milk of lactating dairy cows. Anim. Feed Sci. Technol. 2009, 150, 34–45. [Google Scholar] [CrossRef]
  47. Polat, F.; Aksu, T. Determination of Aflatoxin Levels of Feeds Used in Dairy Cow Farms and Their Effects on Blood Parameters and Milk Aflatoxin Levels in Hatay Province. Ataturk Univ. J. Vet. Sci. 2015, 10, 146–155. [Google Scholar] [CrossRef]
  48. Mojtahedi, M.; Mesgaran, M.D.; Vakili, S.A.; Ghezeljeh, E.A. Effect of esterified glucomannan on carryover of aflatoxin from feed to milk in lactating Holstein dairy cows. Ann. Rev. Res. Biol. 2013, 3, 76–82. [Google Scholar]
  49. Costamagna, D.; Gaggiotti, M.; Chiericatti, C.A.; Costabel, L.; Audero, G.M.L.; Taverna, M.; Signorini, M.L. Quantification of aflatoxin M1 carry-over rate from feed to soft cheese. Toxicol. Rep. 2019, 6, 782–787. [Google Scholar] [CrossRef] [PubMed]
  50. Guo, Y.; Yuan, Y.; Yue, T. Aflatoxin M-1 in Milk Products in China and Dietary Risk Assessment. J. Food Prot. 2013, 76, 849–853. [Google Scholar] [CrossRef]
  51. Keller, L.; Abrunhosa, L.; Keller, K.; Rosa, C.A.; Cavaglieri, L.; Venancio, A. Zearalenone and its derivatives alpha-zearalenol and beta-zearalenol decontamination by saccharomyces cerevisiae strains isolated from bovine forage. Toxins 2015, 7, 3297–3308. [Google Scholar] [CrossRef] [PubMed]
  52. Chen, L.; Tian, Y.; Chen, S.; Liesenfeld, O. Performance of the Cobas® influenza A/B assay for rapid Pcr-based detection of influenza compared to prodesse ProFlu+ and viral culture. Eur. J. Microbiol. Immunol. 2015, 5, 236–245. [Google Scholar] [CrossRef]
  53. Shojaei, T.R.; Tabatabaei, M.; Shawky, S.; Salleh, M.A.M.; Bald, D. A review on emerging diagnostic assay for viral detection: The case of avian influenza virus. Mol. Biol. Rep. 2015, 42, 187–199. [Google Scholar] [CrossRef] [PubMed]
  54. Moschini, M.; Masoero, F.; Gallo, A.; Diaz, D. Mucosal absorption of aflatoxin B1 in lactating dairy cows. Ital. J. Anim. Sci. 2007, 6, 324–326. [Google Scholar] [CrossRef]
  55. Frobish, R.A.; Bradley, B.D.; Wagner, D.D.; Long-Bradley, P.E.; Hairston, H. Aflatoxin residues in milk of dairy cows after ingestion of naturally contaminated grain. J. Food Prot. 1986, 49, 781–785. [Google Scholar] [CrossRef] [PubMed]
  56. Britzi, M.; Friedman, S.; Miron, J.; Solomon, R.; Cuneah, O.; Shimshoni, J.A.; Soback, S.; Ashkenazi, R.; Armer, S.; Shlosberg, A. Carry-over of aflatoxin B1 to aflatoxin M1 in high yielding israeli cows in mid- and late-lactation. Toxins 2013, 5, 173–183. [Google Scholar] [CrossRef] [PubMed]
  57. Masoero, F.; Gallo, A.; Moschini, M.; Piva, G.; Diaz, D. Carryover of aflatoxin from feed to milk in dairy cows low or high somatic cell counts. Animal 2007, 1, 1344–1350. [Google Scholar] [CrossRef] [PubMed]
  58. Ishikawa, A.T.; Takabayashi-Yamashita, C.R.; Ono, E.Y.S.; Bagatin, A.K.; Rigobello, F.F.; Kawamura, O.; Hirooka, E.Y.; Itano, E.N. Exposure Assessment of Infants to Aflatoxin M-1 through Consumption of Breast Milk and Infant Powdered Milk in Brazil. Toxins 2016, 8, 246. [Google Scholar] [CrossRef] [PubMed]
  59. Bahrami, R.; Shahbazi, Y.; Nikousefat, Z. Aflatoxin M-1 in milk and traditional dairy products from west part of Iran: Occurrence and seasonal variation with an emphasis on risk assessment of human exposure. Food Control 2016, 62, 250–256. [Google Scholar] [CrossRef]
  60. Serraino, A.; Bonilauri, P.; Kerekes, K.; Farkas, Z.; Giacometti, F.; Canever, A.; Zambrini, A.V.; Ambrus, A. Occurrence of Aflatoxin M1 in Raw Milk Marketed in Italy: Exposure Assessment and Risk Characterization. Front. Microbiol. 2019, 10, 2516. [Google Scholar] [CrossRef]
  61. Oliveira, C.A.F.; Germano, P.M.L.; Bird, C.; Pinto, C.A. Immunochemical assessment of aflatoxin M(1) in milk powder consumed by infants in Sao Paulo, Brazil. Food Addit. Contam. 1997, 14, 7–10. [Google Scholar] [CrossRef] [PubMed]
  62. Peng, K.-Y.; Chen, C.-Y. Prevalence of Aflatoxin M-1 in Milk and Its Potential Liver Cancer Risk in Taiwan. J. Food Prot. 2009, 72, 1025–1029. [Google Scholar] [CrossRef]
  63. NRC. Nutrient Requirements of Dairy Cattle, 7th ed.; National Academies Press: Washington, DC, USA, 2001. [Google Scholar]
  64. Association of Official Analytical Chemists (AOAC). Official Methods of Analysis, 16th ed.; AOAC Int.: Washington, DC, USA, 1995. [Google Scholar]
  65. Vansoest, P.J.; Robertson, J.B.; Lewis, B.A. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 1991, 74, 3583–3597. [Google Scholar] [CrossRef]
  66. Li, X.Y.; Zhao, L.H.; Yu, F. Occurrence of Mycotoxins in Feed Ingredients and Complete Feeds Obtained From the Beijing Region of China. J. Anim. Sci. Biotechnol. 2014, 5, 37. [Google Scholar] [CrossRef]
  67. Ministry of Health (MoH), China. National Food Safety Standard Determination of Aflatoxin M1 in Milk and Milk Products; National Standard No. 5009.24-2016; Ministry of Health: Beijing, China, 2016.
Figure 1. Effects of the moderate risk level of AFB1 with or without adsorbents on milk AFM1 concentration of dairy cows. AFB1− challenge period: day 1 to 14; AFB1− withdraw period: day 15 to 19. AF: the basal diet + 8 μg/kg AFB1; AD1: AF + 15 g/d adsorbent 1; AD2: AF + 15 g/d adsorbent 2. EU MRL: maximum residue level (MRL) of the European Union (50 ng/L).
Figure 1. Effects of the moderate risk level of AFB1 with or without adsorbents on milk AFM1 concentration of dairy cows. AFB1− challenge period: day 1 to 14; AFB1− withdraw period: day 15 to 19. AF: the basal diet + 8 μg/kg AFB1; AD1: AF + 15 g/d adsorbent 1; AD2: AF + 15 g/d adsorbent 2. EU MRL: maximum residue level (MRL) of the European Union (50 ng/L).
Toxins 13 00665 g001
Table 1. Effect of the moderate risk level of AFB1 with or without adsorbents on the performance of cows at steady state (days 7 to 14) (n = 10).
Table 1. Effect of the moderate risk level of AFB1 with or without adsorbents on the performance of cows at steady state (days 7 to 14) (n = 10).
Item 1Dietary Treatment 2SEMp-Value
CONAFAD1AD2
DMI (kg/d)20.8820.7320.8620.821.840.65
Milk yield (kg/d)20.8520.9120.9720.820.250.15
3.5% FCM (kg/d)23.7925.1324.1125.790.710.98
Fat (%)4.874.884.784.850.090.98
Protein (%)4.204.364.224.160.060.68
Lactose (%)4.814.704.774.810.020.25
Solid (%)14.3614.4714.2614.260.150.96
SCC (× 1000/mL)196.20188.50249.65190.7520.520.73
1 DMI: dry matter intake; 3.5% FCM (kg/d) = 0.432 × milk yield + 16.23 × fat yield; SCC: somatic cell count. SEM: standard error of the mean. 2 CON: the basal diet without AFB1 and adsorbents; AF: CON + 8 μg/kg AFB1; AD1: AF + 15 g/d adsorbent 1; AD2: AF + 15 g/d adsorbent 2.
Table 2. Effect of the moderate risk level of AFB1 with or without adsorbents on serum metabolite parameters of dairy cows (n = 10).
Table 2. Effect of the moderate risk level of AFB1 with or without adsorbents on serum metabolite parameters of dairy cows (n = 10).
Item 1Dietary Treatment 2SEMp-Value
CONAFAD1AD2
Energy metabolism
GLU (mmol/L)4.153.954.334.400.060.31
NEFA (μmol/L)73.0360.5269.6873.033.490.19
BHBA (mmol/L)0.620.410.480.590.050.12
Liver function
ALT (U/L)21.8026.1625.7622.010.570.12
AST (U/L)54.3459.8161.8254.071.090.70
TP (g/L)73.1372.4274.1475.540.600.53
Oxidative stress
T-AOC (U/mL)9.4910.458.848.640.370.61
GSHPx (μmol/L)385.86409.89402.73401.439.600.29
SOD (U/mL)40.4637.8541.5141.141.020.99
MDA (nmol/mL)3.132.773.133.170.190.58
Gastrointestinal permeability
DAO (ng/mL)5.374.523.994.680.310.56
D-LA (μmol/mL)16.5913.3413.9715.831.130.18
LPS (EU/L)352.72311.95331.15348.7423.250.99
1 GLU, Glucose; NEFA, non-esterified fatty acid; BHBA, β-hydroxybutyric acid; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TP, total protein; TAOC, total antioxidant capacity; SOD, superoxide dismutase; GSHPx, glutathione peroxidase; DAO, diamine oxidase; D-LA, D-lactic acid; LPS, Lipopolysaccharide. SEM: standard error of the mean. 2 CON: the basal diet without AFB1 and adsorbents; AF: CON + 8 μg/kg AFB1; AD1: AF + 15 g/d adsorbent 1; AD2: AF + 15 g/d adsorbent 2.
Table 3. Effect of the moderate risk level of AFB1 with or without adsorbents on the concentration, excretion and transfer rate of AFM1 in the milk of dairy cows at steady state (day 7 to 14) (n = 10).
Table 3. Effect of the moderate risk level of AFB1 with or without adsorbents on the concentration, excretion and transfer rate of AFM1 in the milk of dairy cows at steady state (day 7 to 14) (n = 10).
ItemDietary Treatments 1SEMp-Value
CONAFAD1AD2
AFB1 intake (μg/d)ND168168168
AFM1 concentration in milk (ng/L)ND93 a46 b51 b70.04
AFM1 excretion 2 (μg/d)ND1.94 a0.96 b1.06 b0.14<0.01
Transfer rate 3 (%)\1.16 a0.57 b0.63 b0.01<0.01
a,b Values in the same row with no common superscript differ significantly(p < 0.05). 1 CON: the basal diet without AFB1 and adsorbents; AF: CON + 8 μg/kg AFB1; AD1: AF + 15 g/d adsorbent 1; AD2: AF + 15 g/d adsorbent 2. 2 AFM1 excretion (μg/d) = concentration of AFM1 in milk (μg/L) × milk yield (kg/d). 3 Transfer rate (%) = excretion of AFM1 (μg/d)/AFB1 consumption (μg/d) × 100. SEM: standard error of the mean.
Table 4. Comparison of AFM1 transfer rate under different risk levels of dietary AFB1 with or without adsorbents in previous studies and the present study.
Table 4. Comparison of AFM1 transfer rate under different risk levels of dietary AFB1 with or without adsorbents in previous studies and the present study.
StudyDIM 1
(days)
AFB1 Source 2AFB1 Dosage
(μg/kg)
Milk Yield
(kg/day)
Detoxification
Agent
Agent Dosage (%) 3Transfer Rate (%)
Maki et al., 2016 [37]114 ± 14Ap (NRRL-2999) culture
(758 mg/kg)
11721.30\\1.07
21.20NovaSil Plus0.5%0.52
20.60NovaSil Plus1%0.32
Kutz et al., 2009 [38]163 ± 54Ap (NRRL-2999) culture
(760 mg/kg)
112.234.19\\2.65
34.13Solis0.56%1.48
33.73NovasilPlus0.56%1.42
34.43MTB-1000.56%2.52
Weatherly et al., 2018 [39]153 ± 83Ap (NRRL-2999) culture
(102 mg/kg)
10032.3\\\
35.0adsorbent30 g/day\
32.1adsorbent60 g/day\
33.7PROT60 g/day\
Pate et al., 2018 [40]157 ± 43Ap (NRRL-2999) culture
(102 mg/kg)
10035.59\\0.45
38.14FloMatrix113 g/day0.49
37.17FloMatrix227 g/day0.39
Sulzberger et al., 2017 [41]146 ± 69Ap (NRRL-2999) culture
(102 mg/kg)
10037.83\\1.37
37.57Clay0.5%1.01
37.28Clay 1%0.98
36.44Clay2%0.74
Rodrigues et al., 2019 [42]183 ± 70Ap (NRRL-2999) culture
(650 mg/kg)
76.8737.1\\2.70
77.6536.1Toxy-Nil0.4%1.00
73.9737.8Unike Plus0.4%1.30
Ogunade et al., 2016 [33]150–200Ap (NRRL-2999) culture
(Not described)
7526.6\\1.13
26.5SEQ120 g/day1.14
26.7SEQ220 g/day1.11
26.1SEQ320 g/day1.08
Queiroz et al., 2012 [43]295 ± 45Ap (NRRL-2999) culture
(640 mg/kg)
7518.9\\0.61
19.9Calibrin A0.2%0.75
19.1Calibrin A1%0.51
Guo et al., 2019 [17]254 ± 19Pure AFB16320\\1.06
6420BDP (ANSB060)0.2%0.76
Maki et al., 2017 [44]Not describedAp (NRRL 2999) culture
(758 mg/kg)
5036.45\\1.78
36.27Novasil Plus0.125%1.50
36.18Novasil Plus0.25%1.46
Xiong et al., 2015 [2]271 ± 29Af (No. 3.4409) culture
(28.8 mg/kg)
2021.3\\0.56
21.3Solis Mos0.25%0.46
4022.4\\0.59
22.6Solis Mos0.25%0.57
Sumantri et al., 2012 [45]84–98Naturally contaminated ground
peanut meal
(1358 and 13 μg/kg)
0.306.75\\0.12
30.626.72\\0.10
30.816.85Bentonite0.5%0.10
30.657.27Bentonite2.0%0.10
Intanoo et al., 2020 [32]180 ± 21Naturally contaminated diet
(22.28 μg/kg)
22.2810.03\\7.26
10.23CPY12 g/day1.18
10.18RSY5 2 g/day1.44
10.10YSY22 g/day1.69
Xiong et al., 2018 [19]33 ± 7Af (No. 3.4409) culture
(28.8 mg/kg)
2035.7\\1.38
35.5Solis Mos 0.25%0.89
Masoeroa et al., 2009 [46] 120 ± 22Naturally contaminated corn meal (32.13μg/kg) and Pmx (4.13 μg/kg) 7.3131.03\\3.80
7.4733.25Cay SA 0.83%2.10
Polat et al., 2015 [47]Passed peakNaturally contaminated diet from 20 dairy farms5.77819.9\\2.66
Mojtahedi et al., 2013 [48]95 ± 17Naturally contaminated diet
(4.6 μg/kg)
4.637.8\ 1.30
37.3EG18 g/day1.47
37.6EG27 g/day1.86
37.6EG36 g/day1.24
Costamagna et al., 2019 [49] <90Naturally contaminated diet
(3.4μg/kg)
3.434.12\\0.88
90–15030.54\\1.09
>15020.15\\0.56
Present study270 ± 22Pure AFB1820.85\\1.16
20.91adsorbent 115 g/day0.57
20.97adsorbent 215 g/day0.63
1 DIM: Days in milk of the cows used in trails. 2 Ap: Aspergillus parasiticus; Af: Aspergillus flavus; AFB1 concentration of the AFB1 source. 3 % of Diet DM.
Table 5. Effect of the moderate risk level of AFB1 with or without adsorbents on the estimated daily intake (EDI) and the hazard index (HI) in different human age groups.
Table 5. Effect of the moderate risk level of AFB1 with or without adsorbents on the estimated daily intake (EDI) and the hazard index (HI) in different human age groups.
AgeMilk Consumption 1 (mL/d)Average Body Weight 2 (kg)EDI 3 HI 4
AFAD1AD2AFAD1AD2
2–4151.713.81.020.510.565.112.532.80
4–7130.217.90.680.330.373.381.671.85
7–11136.825.60.500.250.272.491.231.36
11–14141.036.30.360.180.201.810.890.99
14–18133.849.20.250.130.141.260.630.69
18–30120.557.70.190.100.110.970.480.53
30–45109.060.10.170.080.090.840.420.46
45–60118.959.70.190.090.100.930.460.51
60–70127.257.00.210.100.111.040.510.57
>70142.453.60.250.120.141.240.610.68
1,2 Data on Milk consumption and Average body weight are from the previous studies [11,50]. 3 EDI: estimated daily intake (ng/kg bw/day). Milk AFM1 concentrations were 93 ng/L, 46 ng/L and 51 ng/L for AF, AD1 and AD2, respectively, which were used to calculate the EDI values. 4 HI: hazard index, which was calculated as follows: HI = EDI/tolerable daily intake (TDI), where TDI was set as 0.20 ng/kg bw/d suggested by Kuiper-Goodman [23].
Table 6. Ingredients and chemical compositions of the basal diet.
Table 6. Ingredients and chemical compositions of the basal diet.
Ingredients 1Amount (% of DM)
Corn silage41.72
Alfalfa silage8.83
Oat hay4.07
Corn-steam flaked7.94
Soybean meal5.96
Ground Corn12.35
Wheat bran1.99
Cottonseed meal7.62
Extruded soybean1.53
DDGS2.8
Bicarb1.48
Premix 22.87
Magnesium oxide0.48
Yeast0.36
Chemical levels (% of DM)
CP16.03
EE3.04
NDF31.85
ADF18.5
Ash8.16
NEL 3 (MJ/kg)1.59
Ca (g/kg)0.8
P (g/kg)0.4
Aflatoxin B1, B2, G1, G2 (μg/kg)ND 4
Deoxynivalenol (μg/kg)ND
T-2 toxin (μg/kg)ND
Zearalenone (μg/kg)ND
Ochratoxin A (μg/kg)ND
1 DM: dry matter; DDGS: dry distilled grain soluble; CP: crude protein; EE: Ether extract; NDF: neutral detergent fiber; ADF: acid detergent fiber; NEL: net energy for lactation. 2 Premix was Formulated with 20% salt, 18% Ca, 10% P, 800 mg/kg Cu, 700 mg/kg Mn, 800 mg/kg Zn, 20 mg/kg Fe, 125 mg/kg I, 80 mg/kg Se; 70 mg/kg Co, 300,000 IU/kg vitamin A, 7600 IU/kg vitamin D3, 10,000 IU/kg Vitamin E. 3 NEL was a calculated value, while the others were measured values. 4 ND: not detected.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Back to TopTop