Effect of experimental feed additives on aflatoxin in milk of dairy cows fed aflatoxin-contaminated diets

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1 DOI: /j x ORIGINAL ARTICLE Effect of experimental feed additives on aflatoxin in milk of dairy cows fed aflatoxin-contaminated diets L. Kissell 1,2, S. Davidson 1, B. A. Hopkins 1, G. W. Smith 2 and L. W. Whitlow 1 1 Department of Animal Science, College of Agriculture and Life Sciences, Raleigh, NC, USA, and 2 Department of Population Health and Pathobiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA Keywords aflatoxin, milk, binder, feed additive Correspondence G. Smith, DVM, PhD, Department of Population Health and Pathobiology, North Carolina State University, 1060 William Moore Drive, Raleigh, NC 27607, USA. Tel: +(919) ; Fax: +(919) ; geoffrey_smith@ncsu.edu Received: 11 January 2012; accepted: 8 May 2012 Summary Three studies were conducted to determine the potential of experimental feed additives (EFAs), clays or non-digestible yeast oligosaccharides, to reduce milk aflatoxin (AFM 1 ) concentrations in lactating Holstein cows consuming aflatoxin-contaminated diets. All studies included a pre-treatment period and a 2-week experimental period in a randomized block design. During the pre-treatment period, cows received a total mixed ration (TMR) with no aflatoxin contamination. During both experimental weeks, all cows were fed a TMR containing aflatoxincontaminated corn. During experimental week 1, cows received no EFA s in the TMR, but EFA s were included in the TMR for the second experimental week. In studies 1 and 2, the experimental period consisted of 2 weeks each lasting 7 days with 12 cows per treatment. Aflatoxin M 1 concentrations were analysed by HPLC for milk samples collected on days 5 7 and days In various experiments, treatments included control (no EFA), 100 g/cow daily of experimental Lallemand Ò product, 10 g/cow daily of MTB-100 Ò -2004, (Alltech, Inc.), 10 g/cow daily of MTB-100 Ò -2006, (Alltech, Inc.), 10 g/cow daily of experimental Alltech Ò product (Alltech, Inc.) and 227 g/cow daily of Astra-Ben 20 Ò (AB-20 Ò ; Prince Agri Products, Inc.). In study 3, the experimental period of 2 weeks each lasting 8 days and milk samples were collected from day 4 to 8 and day 11 to 16. Milk samples from study 3 were analysed for AFM 1 concentrations by ELISA. For all experiments, changes in AFM 1 concentrations because of the addition of EFA s were calculated. Four of the five EFAs tested in this study had no significant effect on AFM 1 concentrations. However, the addition of AB- 20 Ò resulted in a significant decrease in AFM 1 concentrations (60.4%). In summary, the addition of AB-20 Ò to the diet of cattle appears to be effective for significantly reducing AFM 1 concentrations in the milk of cows fed an aflatoxin-contaminated diet. Introduction Aflatoxins are a group of mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus. They are naturally occurring contaminants that can form during all stages of growth, harvest, storage and feeding. This group of mycotoxins are known carcinogens that routinely occur in various feedstuffs including corn, peanuts and cottonseed. Aflatoxin B 1 (AFB 1 ) is the most common form of aflatoxin that contaminates Journal of Animal Physiology and Animal Nutrition ª 2012 Blackwell Verlag GmbH 1

2 Efficacy of feed additives to bind aflatoxin L. Kissell et al. feeds. When diets containing AFB 1 are fed to lactating animals, a metabolite, aflatoxin M 1 (AFM 1 ) is secreted in milk (Van Egmond and Paulsch, 1986). The formation of AFM 1 is via hydroxylation of AFB 1 primarily in the liver. The toxicity and carcinogenicity of AFM 1 is less than AFB 1 ; however, it remains a significant contaminant of concern for food products (Hsieh et al., 1984; Sinnhuber et al., 1974). The transfer of aflatoxin from feed to milk is of critical importance because it is regulated in most countries. In the United States, the Food and Drug Administration (FDA) has set an action level of 0.5 ppb for AFM 1 in milk (Code of Federal Regulations Part 109). Milk containing AFM 1 concentrations above the action level must be discarded, causing significant economic loss for the dairy producer. Similar regulations exist in most developed countries. Certain feed additives are considered sequestering agents that are capable of attaching other substances to their surface without any chemical action and have been used to reduce the absorption of aflatoxin from the gastrointestinal tract Diaz et al. (2004). These products can also protect animals from the toxic effects of AFB 1 (Ramos et al., 1996). Examples of these feed additives include products such as, clays (montmorrilonite and bentonite) activated carbons and yeast derivatives (non-digestible yeast oligosaccharides). In vitro studies have shown many of these products to be effective for the adsorption of aflatoxin (Phillips et al., 1988; Maryamma et al., 1991; Diaz et al., 2004). However, the in vitro binding ability of the feed additives does not necessarily predict the in vivo binding of the experimental feed additives (Dwyer et al., 1997; Diaz et al., 2002; Stroud, 2006). The primary objective of this study was to determine the effect of several experimental feed additives (EFAs) on AFM 1 concentrations when lactating dairy cattle were fed aflatoxin-contaminated diets. Materials and methods Diet Corn naturally contaminated with approximately 800 ppb of aflatoxin was used as a source of AFB 1 for the aflatoxin-contaminated total mixed ration (TMR). Aflatoxin-contaminated corn was blended with molasses and corn oil to eliminate dust during feed mixing. Aflatoxin-contaminated corn was ground and mixed thoroughly to ensure uniform distribution of aflatoxin. Aflatoxin-contaminated corn was mixed with uncontaminated corn in a 1 10 dilution so that total aflatoxin level in the feed would be approximately 80 ppb. Corn was blended into the TMR using a mixer wagon. Each treatment diet was mixed into an appropriate TMR using a Data Ranger (American Calan Inc., Northwood, NH, USA). At each feeding, one of the EFAs was initially blended with soybean meal and then mixed with the appropriate TMR. Diets were fed through Calan (American Calan Inc.) feeding stations. After each treatment was fed, the mixer was flushed with 28 kg of silage to avoid cross contamination of EFAs. Experimental feed additives The experimental Lallemand Ò (Milwaukee, WI, USA) product is characterized as a blend of yeast cell wall extract (glucomannan) and aluminosilicate. MTB-100 Ò (Alltech, Inc., Nicholasville, KY, USA) is a modified glucomannan product (also known as Integral Ò or Mycosorb Ò in some countries). MTB- 100 Ò and MTB-100 Ò were commercially available formulations during the years 2004 and Astra-Ben 20 Ò (AB-20 Ò ) (Prince Agri Products, Inc., Quincy, IL, USA) is a sodium bentonite. Experimental design All protocols were approved by the North Carolina State University Institutional Animal Care and Use Committee. For all three studies, lactating Holstein cows from the Piedmont Research Station in Salisbury, North Carolina were randomly assigned to treatment groups. Diets were formulated to meet the nutrient requirements for the average milk production of the group (NRC, 2001). Ingredient composition and chemical analysis of the TMR are listed in Tables 1 and 2 respectively. To ensure total intake of the EFAs, feed consumption was measured and then diets were allocated to minimize refusal (orts), resulting in a dry matter intake (DMI) of approximately 26 kg daily. Feed samples were collected daily to determine dry matter (DM). Dry matter analysis was performed using the National Forage Testing Association method (oven drying for 3 h at 105 o C). In study 1, a total of 24 cows, with a mean weight of 603 ± 102 kg, were assigned randomly to the following treatments: (i) control (no EFAs), n = 12 and (ii) 100 g/cow daily of the experimental Lallemand Ò product, n = 12. Cows were in late lactation [>200 days in milk (DIM)] and produced a mean of 21.0 ± 5.83 kg of milk per day. Diet DM contained 91.0 ppb of AFB 1. For study 2, a total of 48 cows, with a mean weight of 610 ± 80 kg, were randomly assigned to 2 Journal of Animal Physiology and Animal Nutrition ª 2012 Blackwell Verlag GmbH

3 L. Kissell et al. Efficacy of feed additives to bind aflatoxin Table 1 Ingredient composition of total mixed ration fed to all cows in experiments 1, 2 and 3 Ingredients % of diet Corn silage 64 Corn grain 5.8 Aflatoxin corn grain % Soybean meal 10.5 Cottonseed hulls 3.7 Whole cottonseed 6.1 Lime 0.58 Dicalcium phosphate 0.36 Salt 0.22 Bicarbonate 0.40 Trace mineral-vitamin 0.06 Dynamate 0.10 Table 2 Chemical analysis of ration fed to all cows in experiments 1, 2 and 3 DM% 93.9 CP, % DM 16.3 TDN, % DM 69.9 NE L, Mcal/lb 0.73 ADF, % DM 23.9 Ca, % DM 0.46 P, % DM 0.50 Mg, % DM 0.18 Fe, ppm 160 each of the following treatment groups: (i) control (no EFA), n = 12, (ii) 10 g/cow daily of MTB-100 Ò , (Alltech, Inc.), n = 12. (iii) 10 g/cow daily of MTB-100 Ò -2006, (Alltech, Inc.), n = 12, and (iv) 10 g/cow daily of a new experimental Alltech Ò product, (Alltech, Inc.), n = 12. Cows were again in later lactation (>200 DIM) and produced a mean of 21.2 ± 6.29 kg of milk per day. Diet DM contained 94.0 ppb of AFB 1. For study 3, a total of 14 cows were randomly assigned to the following treatments groups: (i) control (no EFA), n = 4, 2) 50 g/cow daily of MTB- 100 Ò -2006, n = 5 and 3) 227 g/cow daily of AB-20 Ò (Prince Agri Products, Inc.), n = 5. Cows in this study were in later lactation (>200 DIM) and produced a mean of 20.0 ± 5.28 kg of milk/day. Diet DM contained 86.0 ppb of AFB 1. Previous studies by our research group have shown AB-20 Ò to consistently reduce AFM 1 concentrations in milk (Diaz et al., 2004; Stroud, 2006) and it was therefore chosen as a positive control. A randomized block design was used for studies 1, 2 and 3. Cows were assigned within milk production and parity blocks. All three studies included two periods: period 1 (pre-treatment period), and period 2 which was comprised of experimental week 1 and 2. Period 1 was defined as the pre-treatment period (1 2 days) during which all cows received a TMR that contained corn that was not contaminated with aflatoxin. Milk samples were collected prior to beginning all studies to determine background AFM 1 concentrations. For studies 1 and 2, milk samples were collected for 2 days and for study 3 milk samples were collected for 1 day prior to beginning. For all studies, cows were fed an aflatoxin-contaminated TMR during period 2 (experimental weeks 1 and 2). During period 2, experimental week 1, cows received aflatoxin-contaminated corn, but no EFAs in the TMR. During period 2, experimental week 2, both aflatoxin-contaminated corn and EFAs were included in the TMR. For all periods, daily milk samples were collected in the evening (1400 h) and the following morning (0200 h). In experiment 1 and 2, period 2 consisted of two experimental weeks each lasting 7 days where milk samples were collected on day 5 7 and day In experiment 3, period 2 consisted of two experimental weeks each lasting 8 days and milk samples were collected on days 4 8 and days At the conclusion of each study, feeding of the AFB 1 contaminated TMR was discontinued and milk was discarded from all cows until AFM 1 was cleared from the milk. Aflatoxin M 1 analysis For all three studies, milk samples were composited from each cow to represent 1 day s production. A portion of milk from the evening sampling was combined with a portion of milk from the following morning sampling. The amount of milk composited from each sampling was determined based on the individual cow s milk yield for the two milking times. For studies 1 and 2, milk samples were analysed for AFM 1 using HPLC (Trilogy Analytical Laboratory, Washington, MO, USA). For study 3, milk samples were analysed for AFM 1 using ELISA (Ridascreen Ò Fast Aflatoxin M 1 ELISA; R-Biopharm AG, Darmstadt, Germany). Statistical analysis The objective was to statistically measure changes in AFM 1 concentrations >25%. The 25% difference in AFM 1 concentrations that could be statistically measured was dictated by the power analysis. For all Journal of Animal Physiology and Animal Nutrition ª 2012 Blackwell Verlag GmbH 3

4 Efficacy of feed additives to bind aflatoxin L. Kissell et al. three studies, a randomized block design was used. Cows were blocked based on the milk production of the previous lactation and parity. The efficacy of each EFA was determined based on the reduction in AFM 1 concentrations. For all three studies, statistical analyses were conducted using the MIXED procedure of SAS (SAS Ò, 2004) (Statistical Analysis Service, Cary, NC, USA). The trial was divided into period 1 (pre-treatment period), where cows received a TMR with no aflatoxin contamination and period 2 which consisted of experimental week 1 (cows received diets containing aflatoxin, but no EFA) and experimental week 2 (cows received diets containing aflatoxin plus EFAs). The effects of the EFAs on AFM 1 concentrations were calculated as the percentage difference in AFM 1 concentrations between experimental weeks 1 and 2 normalized to the control (the control was defined as having zero change) (Equations 1, 2 and 3). The control was assumed to have no reduction in AFM 1 concentrations because the diet did not change during experimental weeks 1 and 2 of period 2. Normalizing the data was necessary to reduce noise from the control data to prevent skewed comparisons between treatments. After normalizing the data, the percent reduction in each treatment was able to be compared with the percent reduction in the control. The changes in AFM 1 concentrations because of the EFAs were considered significant at p < The model included the effects of treatment and error. Equation 1: Mean change in AFM 1 concentration for thecontrol group ¼ mean AFM 1 concentrations in period 2;experimental week 1 mean AFM 1 concentration inperiod 2;experimental week 2: Equation 2: Change inmean AFM 1 concentration for treatment group ¼ mean AFM 1 concentration in period 2; experimental week 1 mean AFM 1 concentration inperiod 2; experimental week 2: Equation 3: Adjusted mean AFM 1 concentration for treatment group ¼ change in mean AFM 1 concentration for treatment group mean change in AFM 1 concentration for the control group: Differences in DMI and milk production across treatment groups were compared using the MIXED procedure of SAS Ò. The effect of aflatoxin on DMI or milk yield was analysed using the MIXED procedure of SAS Ò (SAS, 2004). The DMI or milk yield for experimental week 1 of period 2, when cows were fed only an aflatoxin-contaminated TMR, was covariately adjusted using that of period 1. The effect of the EFAs on DMI or milk yield was analysed using the MIXED procedure of SAS Ò (SAS, 2004). The DMI or milk yield for experimental week 2 of period 2, when cows were fed an aflatoxin-contaminated TMR and an EFA, was covariately adjusted using experimental week 1 of period 2, when cows were fed only an aflatoxin-contaminated TMR. Results These studies were designed to detect changes in AFM 1 concentrations of >25%. Mean AFM 1 concentrations by experimental period for each treatment are shown in Table 3. Of the EFAs used in the three experiments, only AB-20 Ò resulted in a significant decrease in AFM 1 concentrations (60.4%). In study 1, there was no significant difference in AFM 1 concentrations between the Lallemand product and the control (Table 4). In study 2, there was no significant difference in AFM 1 concentrations between MTB-100 Ò -2004, MTB-100 Ò -2006, or the experimental Alltech Ò product and the control. In study 3, MTB-100 Ò did not significantly reduce AFM 1 concentrations in relation to the control. However, addition of AB-20 Ò resulted in a significant decrease (60.4%) in AFM 1 concentrations in relation to the control (Table 4). Mean DMI and milk production by period are presented in Tables 5 and 6. Feed intake and milk yield were not significantly different between groups in any of the three trials. Discussion The effect of feed additives on AFM 1 concentrations in vivo has not been accurately predicted in vitro (Dwyer et al., 1997; Diaz et al., 2004; Stroud, 2006). However, efficacy of the EFAs to bind AFB 1 has been evaluated numerous times in vitro and in vivo. Diaz et al. (2004) reported that MTB-100 Ò bound 96.6% of AFB 1 in vitro and reduced AFM 1 concentrations by 59% in vivo when fed to lactating Holstein cows at 10 g/cow daily. This study also reported that AB-20 Ò bound 98% of AFB 1 in vitro; however, in vivo milk AFM 1 concentrations were reduced by 61% when lactating Holstein cows were fed AB-20 Ò at 227 g per cow daily. Stroud (2006) conducted both in vitro and in vivo studies using both 4 Journal of Animal Physiology and Animal Nutrition ª 2012 Blackwell Verlag GmbH

5 L. Kissell et al. Efficacy of feed additives to bind aflatoxin Table 3 Milk aflatoxin concentrations for each group during experiments 1, 2 and 3 (mean ± standard deviation) Milk aflatoxin concentrations (ppb) Table 5 Mean dry matter intake (kg) for each group during experiments 1, 2 and 3 (mean ± standard deviation) Dry matter intake (kg) Experimental group Pre-treatment Aflatoxin only Aflatoxin + EFA Experimental group Pre-treatment Aflatoxin only Aflatoxin + EFA Control group 0.11 ± ± ± 0.75 Experimental 0.11 ± ± ± 0.85 Lallemand group Control group 0.11 ± ± ± 0.75 Experimental Alltech group 0.13 ± ± ± 1.31 MTB-100 Ò group 0.09 ± ± ± 0.72 MTB-100 Ò group 0.11 ± ± ± 0.95 Control group 0.27 ± ± ± 0.83 MTB-100 Ò group 0.26 ± ± ± 0.91 Astra-Ben 20A Ò group 0.25 ± ± ± 0.21 Control group 26.2 ± ± ± 4.57 Experimental 26.4 ± ± ± 3.34 Lallemand group Control group 26.2 ± ± ± 4.57 Experimental Alltech group 26.7 ± ± ± 5.44 MTB-100 Ò group 24.9 ± ± ± 4.96 MTB-100 Ò group 27.1 ± ± ± 3.29 Control group 24.3 ± ± ± 3.48 MTB-100 Ò group 23.8 ± ± ± 2.46 Astra-Ben 20A Ò group 26.7 ± ± ± 2.41 Table 4 Percentage reductions in milk aflatoxin concentration owing to the addition of experimental feed additives of cows fed 80 ppb aflatoxin-contaminated diet Experimental feed additive Experimental Lallemand +5.2 product, 100 g/day MTB-100 Ò -2004, 10 g/day +8.0 MTB-100 Ò -2006, 10 g/day +6.2 Experimental Alltech +9.5 product, 10 g/day MTB-100 Ò -2006, 50 g/day )5.1 Astra-Ben 20A Ò, 227 g/day )60.4* *Values are different from control (p < 0.05). Change in milk aflatoxin concentration (%) MTB-100 Ò and AB-20 Ò. They found that in vitro, MTB-100 Ò and AB-20 Ò bound 96.2% and 43.4% of AFB 1 respectively. Subsequent in vivo studies reported that MTB-100 Ò did not reduce AFM 1 concentrations; however, AB-20 Ò reduced AFM 1 concentrations in milk by 48.9% when lactating Holstein cows were fed 100 g per cow daily of either EFA. Recently, Kutz et al. (2009) found that MTB- 100 Ò fed at 0.5% of the diet was not effective at reducing milk AFM1 concentrations, AFM1 excretion, or AF transfer from feed to milk. For AB-20 Ò, the results from this study confirms the work of Diaz et al. (2004) and Stroud (2006) showing that this feed additive decreases AFM 1 Table 6 Mean milk production (kg) for each group during experiments 1, 2 and 3 (mean ± standard deviation) Experimental group Milk production (kg) Pre-treatment Aflatoxin only Aflatoxin + EFA Control group 20.8 ± ± ± 6.0 Experimental 21.5 ± ± ± 3.86 Lallemand group Control group 20.8 ± ± ± 6.0 Experimental Alltech group 21.8 ± ± ± 5.86 MTB-100 Ò group 19.3 ± ± ± MTB-100 Ò group 22.0 ± ± ± 4.22 Control group 19.3 ± ± ± 6.51 MTB-100 Ò group 19.6 ± ± ± 5.76 Astra-Ben 20A Ò group 20.2 ± ± ± 5.23 concentrations in milk from lactating Holstein cows. However, the present results for MTB-100 Ò are consistent with Stroud (2006), but in contrast to Diaz et al. (2004). Data from this study indicate that under the conditions of these experiments, MTB- 100 Ò was not effective at reducing milk AFM 1 concentrations. This trial included a 7-day feeding period with the different EFAs tested. This protocol has been used several times previously and reductions in AFM 1 concentrations usually occur immediately after an effective binder is added to the diet (Harvey et al., 1991; Diaz et al., 2004; Kutz et al., 2009). However, we cannot exclude the possibility Journal of Animal Physiology and Animal Nutrition ª 2012 Blackwell Verlag GmbH 5

6 Efficacy of feed additives to bind aflatoxin L. Kissell et al. that this product might have had some positive effects if fed for a greater length of time. Several studies in swine and poultry have found products containing non-digestible oligosaccharides derived from yeast to be effective at preventing toxicity associated with feeding aflatoxin. An experiment conducted using chickens showed that yeast culture residue enhanced the performance of broiler breeder hens fed aflatoxin-contaminated diets (Stanley et al., 2004). Basmacioglu et al. (2005) reported that the addition of an esterified glucan polymer, extracted from yeast cell wall reduced the deleterious effects caused by aflatoxin in broiler chickens. The differences in the effectiveness of the tested EFAs to reduce milk AFM 1 concentrations may be due to the feed additives composition and mechanism of action. Sodium bentonite, which is a phyllosilicate, has a layered crystalline microstructure that enables the adsorption of aflatoxin. Bentonites are comprised mainly of montmorillonite and have interchangeable cations. Cell wall derived products typically contain glucan polymers and/or mannan oligosaccharides. The proposed mechanism of action for this category of EFA is an interaction between the glucan portion of the yeast cell wall and the mycotoxin (Yiannikouris et al., 2003, 2004). However, this mechanism of action was only evaluated using the yeast cell wall and zearalenone, not aflatoxin. In conclusion, this study demonstrated that sodium bentonite (AB-20 Ò ) fed at 227 g per cow daily, reduced AFM 1 concentrations by 60.4% when cows were fed diets containing 86.0 ppb of AFB 1 ; however, AB-20 Ò did not reduce AFM 1 concentrations below the action level set by the FDA of 0.5 ppb. Experimental feed additives partially consisting of non-digestible yeast oligosaccharides used in this study did not affect AFM 1 concentrations when cows were fed diets containing 80 ppb AFB 1. References Basmacioglu, H.; Oguz, H.; Ergul, M.; Col, R.; Birdane, Y. O., 2005: Effect of dietary esterified glucomannan on performance, serum biochemistry and haematology in broilers exposed to aflatoxin. Czech Journal of Animal Science 50, Diaz, D. E.; Hagler, W. M. Jr; Hopkins, B. A.; Whitlow, L. W., 2002: Aflatoxin binders I: in vitro binding assay for aflatoxin B1 by several potential sequestering agents. Mycopathologia 156, Diaz, D. E.; Hagler, W. M. Jr; Blackwelder, J. T.; Eve, J. A.; Hopkins, B. A.; Anderson, K. L.; Jones, F. T.; Whitlow, L. W., 2004: Aflatoxin binders II: reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed. Mycopathologia 157, Dwyer, M. R.; Kubena, L. F.; Harvey, R. B.; Mayura, K.; Sarr, A. B.; Buckley, S.; Bailey, R. H.; Phillips, T. D., 1997: Effects of inorganic adsorbents and cyclopiazonic acid in broiler chicks. Poultry Science 76, Harvey, R. B.; Phillips, T. D.; Ellis, J. A.; Kubena, L. F.; Huff, W. E.; Petersen, H. D., 1991: Effects on aflatoxin M 1 residues in milk by addition of hydrated sodium calcium aluminosilicate to aflatoxin-contaminated diets of dairy cows. American Journal of Veterinary Research 52, Hsieh, D. P.; Cullen, J. M.; Reubner, B. H., 2004: Comparative hepatocarcinogenicity of aflatoxins B1 and M1 in the rat. Food and Chemical Toxicology 22, Kutz, R. E.; Sampson, J. D.; Pompeu, L. B.; Leduox, D. R.; Spain, J. N.; Vazquez-Anon, M.; Rottinghaus, G. E., 2009: Efficacy of Solis, NovasilPlus, and MTB- 100 to reduce aflatoxin M 1 levels in milk of early to mid lactation dairy cows fed aflatoxin B 1. Journal of Dairy Science 92, Maryamma, K. I.; Rajan, A.; Gangadharan, B.; Manomohan, C. B., 1991: In vitro and in vivo studies on aflatoxin B1 neutralization. Indian Journal of Animal Sciences 61, National Research Council, 2001: Nutrient Requirements of Dairy Cattle, 7th edn. National Academies Press, Washington, DC. Phillips, T. D.; Kubena, L. F.; Harvey, R. B.; Taylor, D. R.; Heidelbaugh, N. D., 1988: Hydrated sodium calcium aluminosilicate: a high affinity sorbent for aflatoxin. Poultry Science 67, Ramos, A. J.; Fink, G. J.; Hernandez, J., 1996: Prevention of toxic effects of mycotoxins by means of nonnutritive adsorbent compounds. Journal of Food Protection 59, Sinnhuber, R. O.; Lee, D. J.; Wales, J. H.; Landers, M. K.; Keyl, A. C., 1974: Hepatic carcinogenesis of aflatoxin M1 in rainbow trout (Salmo gairdneri) and its enhancement by cyclopropene fatty acids. Journal of the National Cancer Institute 53, Stanley, V. G.; Winsman, M.; Dunkley, C.; Ogunleye, T.; Daley, M.; Krueger, W. F.; Sefton, A. E.; Hinton, A. Jr, 2004: The impact of yeast culture residue on the suppression of dietary aflatoxin on the performance of broiler breeder hens. Journal of Applied Poultry Research 13, Stroud, J. S., 2006: The effect of feed additives on aflatoxin in milk of dairy cows fed aflatoxin-contaminated diets. Disseration, North Carolina State University. Van Egmond, H. P.; Paulsch, W. E., 1986: Mycotoxins in milk and milk products. Netherlands Milk and Dairy Journal 40, Journal of Animal Physiology and Animal Nutrition ª 2012 Blackwell Verlag GmbH

7 L. Kissell et al. Efficacy of feed additives to bind aflatoxin Yiannikouris, A.; Poughon, L.; Cameleyre, X.; Dussap, C.; Francois, J.; Berting, G.; Jouany, J. P., 2003: A novel technique to evaluate interactions between Saccharomyces cerevisiae cell wall and mycotoxins: application to zearalenone. Biotechnology Letters 25, Yiannikouris, A.; Andre, G.; Buleon, A.; Jeminet, G.; Canet, I.; Francois, J.; Bertin, G.; Jouany, J. P., 2004: Comprehensive conformational study of key interactions involved in zearalenone complexation with b-d-glucan. Biomacromolecules 5, Journal of Animal Physiology and Animal Nutrition ª 2012 Blackwell Verlag GmbH 7