This study reports the results of an experiment, testing the aflatoxin B1 (AfB1) carry-over from naturally contaminated feed to the dairy cows milk in the absence and in the presence of a mycotoxin deactivating product-Mycofix® Plus (MPL). The study was carried out using 18 healthy animals divided into 3 homogeneous groups of 6 animals each. The experimental design was a 3x3 Latin square with three periods of 7 days each without washout periods. The treatments were: (1) control diet without MPL (CTR); (2) control diet with 20 g/cow/day of MPL (T1) and (3) control diet with 50 g/cow/day of MPL (T2). The diet was a Total Mixed Ration (TMR) and 1 kg of a naturally contaminated maize meal (AfB1 = 91.7 ± 4.4 μg kg-1) was included in the diet of each cow. Each animal ingested daily 97.3 μg kg-1 AfB1 since the analysis of the TMR before inclusion of the contaminated maize revealed however the presence of 0.24 μg kg-1 DM of AfB1, corresponding to 5.6 μg per cow per day. In T1 and T2 diets, MPL was mixed with the contaminated maize meal. Feed intake and individual daily milk production were recorded during the study. Morning and evening milk samples from each cow were collected on day seven of each week. Samples derived from individual cows were mixed in proportion to the morning and evening milk production and then again combined in proportion to the daily milk production of each cow to constitute a representative bulk milk sample of each group; these samples were analyzed to determine the aflatoxin M1 (AfM1) content. The addition of MPL did not influence feed intake and milk production. The addition of MPL to the diet reduced significantly (p<0.01) the milk AfM1 content from 0.120 μg kg-1 (CTR group) to 0.083 μg kg-1 (-31%, T1 group) and 0.072 μg kg-1 (-41%, T2 group).
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Aflatoxins (B1, B2, G1, G2) are mycotoxins produced principally by Aspergillus flavus and A. parasiticus fungi (DMello and Macdonald, 1997), mainly in tropical and subtropical regions where the warm and humid weather provides optimum conditions for the growth of the molds (Rustom, 1997). They are considered as the mycotoxins that represent the greatest risk for human health. They occur in several products for human consumption, such as maize, almonds, peanuts and pistachio nuts. Nevertheless, their introduction in the food chain is greatly determined by the ingestion of contaminated feed such as maize and cotton seeds by livestock animals and their subsequent carry over into animal products for human consumption, particularly into milk and dairy products.
Aflatoxins (Afs) have been shown to negatively affect production, immune system function and rumen metabolism in cattle (Hussein and Brasel, 2001). However, their carry over into milk in the form of the hydroxylated metabolites aflatoxin M1 (AfM1) and aflatoxin M2 (AfM2) and consequent carcinogenic risks in humans, are one of the most worrying aspects of this group of mycotoxins. Aflatoxins and AfM1 have been included by the International Agency for Research on Cancer (IARC) in group 1 and 2B, as human and possible human carcinogens, respectively (IARC, 2002); their maximum levels both in animal feed and milk are regulated by European Commissions Directive (European Commission, 2003) and regulation (European Commission, 2006).
Although ruminants are globally more resistant to mycotoxins than most monogastric animals due to the partial detoxification capacity of ruminal microorganisms, aflatoxins ruminal degradation is less than 10% for contaminations between 1 and 10 μg mL-1 (Westlake et al., 1989). AfM1 is the main AfB1 metabolite, which secretion depends on the animals feed intake and lactation stage (Veldman et al., 1992). The liver is the main site of AfB1 biotransformation with the mitochondrial cytochrome P450 oxidative system (CYP) converting the AfB1 into AfM1 and other metabolites (Sudakin, 2003). A recent research demonstrated that Afs are quickly absorbed through the gastro-intestinal tract of cows and hypothesized that an early absorption might also take place in mouth or esophageal mucous membranes, before the rumen compartment (Gallo et al., 2008). Numerous strategies are described in the literature to avoid fungal growth and toxin production both in the field and during storage (Council for Agricultural Science and Technology, 2003). However, due to the existence of many factors that influence these processes (Ominsi et al., 1994) and in order to counteract the negative impacts of Afs and their metabolites in animal and human health, the development of practical, cost-effective and safe detoxifying processes is of great importance.
Many physical, chemical and biological techniques to neutralize mycotoxins have been developed and were reported in the literature throughout the years (Doyle et al., 1982; Samarajeewa et al., 1990). The adsorption of the aflatoxin molecule is possible due to its polarity. Deactivating agents used as feed additives are supposed to adsorb mycotoxins under the conditions within the digestive tract, whereby an absorption by the body is avoided (Dänicke, 2002). Chemisorbents have shown their capacity to tightly bind and immobilize Afs in the gastrointestinal tract of animals, resulting in a major reduction of the toxin bio-availability. Activated carbons (ACs) and Hydrated Sodium Calcium Alumino-Silicates (HSCAS) have shown a reduction of AfB1 carry-over into milk of respectively 50 and 36% (Galvano et al., 1996) in dairy cows with an inclusion rate of 2% in the pelleted contaminated feed. Although, in vitro experiments have shown promising results as regards to the use of HSCAS as effective AfB1 adsorbing compounds (Ramos and Hernandez, 1997) which have been corroborated by in vivo experiments (Harvey et al., 1991; Smith et al., 1994), special attention is recommended by several researchers regarding the use of ACs and HSCAS as feed additives (Galvano et al., 1996; Ramos and Hernandez, 1997) as the long-term effects of these materials on the utilization of essential nutrients are not well known yet and their effectiveness against other mycotoxins of importance in the animal production have not been thoroughly studied. If for other mycotoxins such as zearalenone, ochratoxin and trichothecenes the efficacy of adsorbing materials is very limited or even null, for AfB1 adsorption these substances seem to be very efficient (Huwig et al., 2001). Based on this knowledge, Vekiru et al. (2007) performed adsorption experiments in buffer solutions in order to evaluate the ability to bind AfB1 at various pH-values. The chemisorption index was calculated, in order to understand if the adsorption was mainly physical adsorption/physisorption (relatively weak bonding involving Van der Waals interactions and hydrogen bonding) or chemical adsorption/chemisorption (stronger interaction which involves ionic or covalent bonding). Isothermal analysis was used to determine the values of maximum binding capacity (qmax, [mol kg-1]) and the distribution constant (Kd), which describes the affinity of the binder to AfB1 (Grant et al., 1998). On the course of the same study, adsorption experiments in simulated gastrointestinal fluid and real gastric juice were also carried out. Furthermore, binding capability of the materials regarding selected vitamins was examined. Based on the obtained in vitro results, highly promising sorbent materials were ranked for further in vivo studies.
However, due to the multi-factorial nature of AfB1 carry-over into the milk, in vivo studies regarding the efficacy of the detoxifying product to avoid this phenomenon are always fundamental. Therefore, the main objective of this study was to determine whether the inclusion of Mycofix® Plus at different rates in the contaminated feed would reduce the carry-over of aflatoxin from feed into the milk of dairy cows.
MATERIALS AND METHODS
Mycofix® Plus (MPL) is a mycotoxin deactivator product, manufactured by BIOMIN GmbH, Herzogenburg, Austria, that presents as a beige greyish fine powder destined to be incorporated into the animal feed from 0.5 to 2.5% TMR, depending on the mycotoxin contamination level of feedstuffs. It has proven efficiency in the counteraction of the negative impacts of mycotoxins in animal health and performance (Cheng et al., 2006; Diaz et al., 2005; Politis et al., 2005). Polar mycotoxins (e.g., aflatoxins) are adsorbed by the inorganic components: a blend of bentonites selected for inclusion in the product after thorough scientific tests ascertaining AfB1-adsorption-properties (Vekiru et al., 2007) (solo constituent of Mycofix® Secure). Non-adsorbable mycotoxins (e.g., trichothecenes, zearalenone) are degraded by biological constituents, namely a Eubacterium strain (BBSH 797) and a yeast strain affiliated to the Trichosporon genus (MTV) (Schatzmayr et al., 2006). Finally, phycophytic substances and plant extracts act to compensate the adverse conditions caused by mycotoxins.
Experiment on Dairy Cows
The experiment was performed at the CERZOO research and experimental center (San Bonico, Piacenza, Italy) from April to May 2006.
A total of 18 healthy Italian Friesian dairy cows were used in this experiment. The average age of the animals given by the number of lactations was 1.9 ± 0.9. Their average days in milk was 130 ± 44 and the average daily milk production was 33.6 ± 6.3 kg.
The treatment groups were identified by different codes: CTR, T1 and T2. The animals were housed in a free-stall barn randomly divided in three pens of 6 cows each.
Animals were fed once daily with free access to water. The composition and chemical analysis of the experimental diet are presented in Table 1 and 2.
The experimental design was a 3x3 Latin square with periods of seven days each without washout periods.
|Table 1:||Composition (% dry matter) of the TMR used in the trial|
|1The premix did not contain any clays|
|Table 2:||Chemical analysis of DM for TMR used in the trial|
|*Limit of detection (LOD)|
The treatments were:
|•||Control diet containing 1 kg of contaminated maize meal, without MPL (CTR)|
|•||Control diet containing 1 kg of contaminated maize meal, with 20 g/cow/day of MPL (T1)|
|•||Control diet containing 1 kg of contaminated maize meal, with 50 g/cow/day of MPL (T2)|
MPL was always mixed with the aflatoxin contaminated maize meal, before inclusion in the Total Mixed Ration (TMR).
Evaluation of Parameters
During the experiment the collection and recording of data were made as follows:
|•||Feed intake: The amount of TMR fed to the animals was measured with an electronic scale on a mixer-feeder wagon|
|•||Milk production: The individual amount of milk produced (in kg) in each milking was recorded daily|
|•||Milk samples: Morning and evening milk samples from each cow were collected on day 7 of each week. The samples of each cow were mixed proportionately to the morning and evening milk production and then again combined in proportion in the daily milk production of each cow to constitute a representative bulk milk sample of each group|
|•||Feed samples: Samples of contaminated maize meal and of TMR were collected to determine composition and mycotoxin content|
|•||Health status: Daily inspections were carried out by qualified personnel and by the veterinary responsible for the animal welfare throughout the whole period of the study. The consistency of the feces was also examined|
Data were analyzed by ANOVA using the GLM procedure of SAS (SAS Institute, 1999-2001) in a Latin square design (Steel and Torrie, 1980). Linear and quadratic contrasts were used to determine the nature of the response to the feeding of Mycofix® Plus. Differences were considered statistically significant at p<0.05.
A TMR sample (approximately 1.0 kg) was collected and analyzed for proximate composition, Neutral Detergent Fibre (NDF), Acid Detergent Fibre (ADF), starch and mycotoxins content.
The proximate analysis of TMR was determined in compliance with the analytical methods of the Italian Ministero dellAgricoltura e Foreste (supplement n. 2 of 1975) for moisture, ash and starch in animal feed. The crude protein content was determined according to the Gazzetta Ufficiale Serie Generale n. 92 of April 21, 1996; the crude fat according to the Directive EEC n. 84/4/EEC of December 12, 1983 (Gazzetta Ufficiale EC n. L 15 of January 1, 1984) and crude fibre according to the EEC Directive n. 92/89 of November 3, 1992.
NDF and ADF contents were determined according to the methods reported in the literature (Martillotti et al., 1987; Mertens, 1992; Van Soest et al., 1991).
Aflatoxin B1, after extraction and purification by an immunoaffinity column, was determined by reversed-phase HPLC with fluorescence detection after post column derivatization involving bromination with pyridinium hydrobromide perbromide (Stroka et al., 2003).
Deoxynivalenol, after extraction and purification by a commercial (MycoSep®) clean-up column (Radova et al., 1998), was determined by GLC-MS after derivatization to trimethylsilylether (Eskola and Rizzo, 2001). Zearalenone, after extraction purification by an immunoaffinity column, was determined by reversed-phase HPLC with fluorescence detection (Kruger et al., 1999). These analysis were performed to certify that no mycotoxins other than aflatoxins were present in the rations fed to the animals.
AfM1 in milk, after extraction/purification by an immunoaffinity column, was determined by reversed-phase HPLC with fluorescence detection (Dragacci and Grosso, 2001).
RESULTS AND DISCUSSION
Effect of the Treatments on the Health Condition of the Animals
Daily inspections carried out by qualified personnel and by the veterinary responsible for animal welfare and examination of feces consistency reflected a good animal health condition throughout the experimental period. The fact that the animals were in contact with a low level of the mycotoxin for a short period might be an explanation for this fact, since a field experiment with natural contamination of feedstuffs and a longer period of aflatoxins intake, reported problems with the health condition of the herd, as animals had a higher incidence of lameness (subclinical laminitis) and impaired fertility (cystic ovaries) (Ozsoy and Altunatmaz, 2005). On the other hand, in controlled experiments, animals generally show a high tolerance to contaminated feeds (Fink-Gremmels, 2008).
Effect of the Treatments on Feed Intake, Milk Yield and Milk AfM1 Content
Addition of MPL did not influence dry matter feed intake (Table 3) or milk production, which evidences the non-interference of the feed additive with the animals performance in a negative way, demonstrating that it may be added to feed in practical conditions.
Aflatoxin B1 in Feed
The mean content of AfB1 in contaminated maize meal fed to the animals was 91.7 ± 4.4 μg kg-1. The analysis of the TMR before inclusion of the contaminated maize revealed however the presence of 0.24 μg kg-1 DM of AfB1, corresponding to 5.6 μg per cow per day. As each animal ingested one kg per day of contaminated maize meal, the sum of these two values represents the mean individual daily ingestion (μg) of AfB1, meaning that each animal ingested daily 97.3 μg kg-1 AfB1.
|Table 3:||Feed intake (kg DM/cow/day)|
AfB1 Intake and Effect of Treatments on Milk Yield and Aflatoxin M1 in Milk
Aflatoxin B1 carry-over into the milk as AfM1 is known to occur quickly, as this metabolite is detected in milk from the first milking after the animal has ingested AfB1-contaminated feed (Diaz et al., 2005; Masoeroa et al., 2007). Based on this information and since the main objective of this study was to determine whether the inclusion of MPL would reduce the carry-over of aflatoxin from feed into the milk of dairy cows, the occurrence of AfM1 in milk was recorded every 7th day of the experiment.
Raw data are shown in Table 4 and calculations are presented in Table 5. The continuous administration of aflatoxin contaminated maize meal to dairy cows has shown to negatively affect the milk quality regarding the content of the AfM1, resulting in higher contents of this toxic metabolite into the milk. The addition of MPL to the diet reduced significantly (p<0.01) the milk AfM1 content from 0.120 μg kg-1 (CTR group) to 0.083 μg kg-1 (-31%; T1 group = 20 g MPL day-1) and 0.072 μg kg-1 (-41%; T2 group = 50 g MPL day-1).
The inclusion of different quantities of MPL to the contaminated feed had positive effects, significantly reducing (p<0.01) the concentration of AfM1 in the treated groups (T1 and T2 groups). This reflects the efficacy of the product in the reduction of aflatoxin carry-over from feed to milk. A 31 and 41% reduction on the carry-over of AfB1 from feed to milk was achieved with the inclusion of respectively, 20 and 50 g MPL/cow/day. In percentage of TMR, these numbers represent a 0.047 and a 0.12% inclusion rate, respectively.
In comparison with earlier studies where other binding agents were tested, the results of this trial are very positive in a milk producers point of view as the inclusion rate of the product is much lower. A 24% decrease on the secretion of AfM1 into the milk was obtained by the addition of 1% HSCAS (Harvey et al., 1991) to the feed. Veldman (1992) reported no effects on the AfM1 content of milk when animals were fed 1% HSCAS. Galvano et al. (1996) achieved a 36% of carry-over reduction with the addition of 2% HSCAS. The results of this trial were positive as regards to the products ability to bind aflatoxins; however, the high initial contamination of the TMR impeded the fulfilment of the European Commission requirement of 0.050 μg kg-1 of AfM1 in the milk. Nevertheless, it should be considered that the AfB1 daily intake of each dairy cow was 97.3 μg; this amount represents a level of 8.3 μg kg-1, if calculated on the concentrate part of the TMR and therefore exceeds the 5 μg kg-1 level permitted by the EU Directive for dairy complete feeding stuffs. It should be also considered that there is a linear correlation between the AfM1 content of milk and the AfB1 intake, expressed by the regression equation (Veldman et al., 1992):
AfM1 (ng kg-1 of milk) = 1.19 AfB1 intake (μg/cow/day) + 1.9
|Table 4:||Aflatoxin M1 in milk (raw data in μg kg-1)|
|Table 5:||Reduction of AfB1 carry-over as AfM1 into milk over a 3 week period in three groups of cows with different treatments|
|a, bValues within the same column with different superscripts differ significantly (p<0.01), *na: Not applicable|
From this equation it can be calculated that the average daily AfB1 intake in a herd has to be below 40 μg per cow in order to produce milk with less than 0.050 μg kg-1 of AfM1. In this trial, the AfB1 amount ingested by each cow was 2.43 times higher (97.3 μg), but the T2 group (50 g MPL/cow/day) produced milk exceeding only 1.44 times the AfM1 limit fixed by the EC.
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