Aflatoxin M 1 in Milk of Cow in Relation to Lactation Period and Yields

Aflatoxin produced from few molds as Aspergillus flavus, A. parasiticus and A. nomius is highly carcinogenic, mutagenic to the animals and humans. These molds are present in the dietary intake of cattle feed. Present investigation was performed to observe the carryover of AFB1 to the milk toxin of lactating cows. Cows during early lactation stage yields more milk compared to late lactating stage, hence, the same cows were used to keep the variable of physiology of cows as nearly constant. AFM1 concentration was found independent to the intake of AFB1 with the compound foods ingested by cows. Carryover of aflatoxin was observed comparatively less in case of cows of late lactation stage. With the yield of milk per day (milking twice), the carryover rate also varied. AFM1 concentration was observed within the limit as set by EU. Frequent evaluation and testing of milk is necessary.


Introduction
Mycotoxins are secondary metabolites produced by some toxigenic fungi under conducive conditions of temperature and humidity [1]. Broad range of food and feeds are contaminated by this toxin. Aflatoxins produced by Aspergillus flavus, A. parasiticus and A. nomius is highly toxic and hazardous to animals and human beings [2]. Aflatoxin M1, also known as milk toxin, is a hydroxyl metabolite of aflatoxin B1, metabolized in the liver of cattle and is being secreted in bile, urine, dung and also in milk of lactating cattle [3], [4]. These toxins can cause carcinogenic, mutagenic, teratogenic and immunosuppressive effect on many animal species including human. Humans may be exposed to aflatoxin M1 through the consumption of contaminated milk. The toxin might cause unfavourable effects in the human body [1], [3]. However, aflatoxin M1 is less poisonous than aflatoxin B1, and according to the International Agency for Research on Cancer (IARC) classification, aflatoxin B1 and M1 were classified as group 1 and 2 of the human carcinogens, respectively [2], [4]. Milk in almost every part of world is the main dietary intake of infants, old and weak/ sick people. Hence, consumption of AFM1 contaminated milk (exceeding the regulatory limit of USFDA, EU and CAC) can adversely affect the health of children, old and weak and susceptible people.
The complications related to aflatoxin M1 contaminated milk and the significance of healthy milk and its dairy products have rendered various researchers to monitor the factors affecting the level of AFM1 contamination in milk and its products [5]- [7]. Impact of various factors such as geographical regions, climatic factors, types of feeds etc. have been evidenced to influence the incidence and prevalence of aflatoxin M1 in milk [1], [4], [5], [6]. However, in this specified geographical region, there is no previous recorded study with regard to aflatoxin M1 level in milk in relation to the milk yields and lactation period of cows. The aim of this study was to determine the carryover rate of aflatoxin M1 in the milk of early lactating period (3-4 weeks after calving) and late lactating period (34-36 weeks after calving) of cows.
The present study was undertaken in specified geographical regions of Bhagalpur and its nearby district. This maintained the type and quality feed, the hygienic practices performed by the breeders and the similar breed of cows for the comparative experimental study. During the early lactation stage, cows produce comparatively higher amount of milk (kg/day) than those of late lactation, however, the diet intake remains the same on an average. This point towards the scenario of metabolism of production of milk and level of aflatoxin M1 in milk yield though with the same dietary intake. The study was done with the same cow in early lactation stage (2 -4 weeks) and late lactation stage (34 -36 weeks) after calving.

A. AFB1 analysis in feed of cows
The qualitative and quantitative test for AFB1 was done employing the methods previously discussed [7].

B. Milk sample collection
Raw milk was collected twice (morning and evening) from each cow and was stored in sterile plastic container. The samples of milk obtained during morning and evening time from each cow were mixed in the ratio of the quantity it was produced. The resultant quantity was labelled as Day 1 and sample from the mixture was considered for AFM1 testing.

C. Extraction
For the determination of AFM1, the method used was the AOAC, 2000. 50 ml of different milk samples was added to the 10 ml saturated salt solution (40gm NaCl in 100ml of water) and was shaken gently. Final preparation was further added to chloroform (120 ml) at 30 ºC in a 250 ml separating funnel and allowed filtrate to settle for 2-3 minutes. Filtrate was then taken for column chromatography.

D. Column chromatography
Silica gel slurry made with 2gm silica gel and CHCl3 was added to the column containing half-filled CHCl3. Sodium sulphate (2 gm) was further added in the column. Excess CHCl3 was drained out and sides of column was rinsed with CHCl3. The milk sample was added to the column and drained entire solution through column simultaneously Sodium sulphate was stirred gently. The cylinder was rinsed with CHCl3 and it was again added to the column. Column was then washed off with 25 ml toluene and acetic acid (9+1). This removed any coloured compounds in the column. Further, column was washed with 25 ml of hexane, ether and acetonitrile (5+3+2) for fat removal. Finally, aflatoxin M1 eluted with 40 ml CHCl3 and acetone (4+1). The elute was evaporated to dry and was used for TLC technique.

E. Thin Layer chromatography
The residue of sample was dissolved in 100 μl of benzene and acetonitrile (9+1) solution by mixing them and further, spots on TLC plate were marked. Spot 20μl of test solutions and 2, 4, 6, 8 and 10μl aflatoxin M1 standard (0.25 μg/ml). The plate was developed in chloroform -actoneisopropanol (87+10+3) and Aflatoxin M1 was calculated in μg/ kg or ppb.

Results and Discussion
Carry-over rate of low milking cows (<20 kg milk yield/ day) was observed (milked twice daily) as 1-2% of AFB1 and high milking cows showed upto 6%. Multiple factors affecting the carry over rates were differences in species/ breed, physiology/ health of the cattle, biotransformation capacity of liver, types of feed etc. [8], [9]. C1, C2.....C10 indicate the cow numbered for experimental purposes. Table 1 shows the summarized data of milk yield during early lactation stage of ten cows and their respective carryover rates of AFB1. During early lactation, the cows were in 2-4 weeks after calving and same cows while in late lactation period (34 to 36 weeks after calving) were employed for our present investigation. Consumption of AFB1 cattle feed was analysed for each cow and accordingly it was found that the mean intake of aflatoxin B1 during early lactation period was 40.7 ± 3.6µg/ day. The mean concentration of AFM1 toxin during early lactating cows was 0.05 ± 0.01µg/kg, which was 2.1± 0.027µg/day. The average carry over rate was 0.050. The carryover rate varied from 0.041 ± 0.007 to 0.058 ± 0.011. Various researchers have studied the metabolism of AFB1 in cattle feed to AFM1 in the milk yield [10]- [13]. Table 2 shows the AFB1 intake and AFM1 excretion by the selected cows during late lactation (34.36 weeks after calving). An average the daily intake of AFB1 during late lactating period was 34.4 ± 1.08 µg/day. During late lactation period the yield of milk from same cow, however, decreased (9.4 ± 1.45 kg/day). Mean production of milk from the cows were 10.1 ± 1.39 kg/day during this period of milking. Mean AFM1 concentration in milk was 0.04 ± 0.02µg/kg, which was 0.5 ± 0.21 µg/day. The mean carry-over rate observed in this period was recorded to be 0.016 ± 0.005 (ranging from 0.009 ± 0.008 to 0.024 ± 0.009).
There was no significant correlation among AFM1 concentration in milk, carryover rate by individual cow and the amount of AFB1 consumed by respective cow. Our observations are in conformity with the previous work [14]. They fed the cattle with AFB1 contaminated feed (57 to 311µg/day per cow) and the resultant carry-over rate was found independent of AFB1 intake ingested.
Remarkable differences were found in the carry over rate of aflatoxin between early lactation and late lactat/ion period of milking. While comparing two different lactating periods, the difference of carryover rate was found in a factor of 3.1. Variation among the AFM1 excretion has been observed among the cows of same stage fed with similar AFB1 concentration. This has been supported by the fact that conversion of AFB1 to AFM1 in liver is partially excreted by the urine and by way of bile to faeces (apart from milk) [12]. The variation in the carryover rate of each cow can be supported with the fact that the metabolism/ conversion of AFB1 to AFM1 can be due to variation in activities of enzyme of the MFO (mixed function oxidase) system. Moreover, the conversion of AFB1 is not only converted to AFM1 but also to AFP1 and AFQ1 [10].  Figure 1 shows relation between the milk yield and carryover rate of aflatoxin from AFB1 to AFM1. The data reflects the facts that with the variation in the milk yield, the carry-over rate can be predicted (if other variables such as lactation period, age of the cows etc. remain same). This is in conformity with the results obtained by earlier researchers [15], [16]. The metabolism of AFB1 to AFM1 is performed in the liver of lactating cows and are disposed out of the body through bile, urine, faeces and milk [3], [4]. Partial conversion of AFB1 occurs to AFM1 occurs and rest to other forms of toxins. It is evident that more milk production by the cow, there are the chances of more AFM1 contamination in milk. Previous workers [11], [17] observed that high milk yield could intensify the effect on AFM1.

Conclusion
The presence of AFM1 in the milk of the cows is a result of carryover by the ingestion of AFB1 contaminated feed. AFB1 is metabolized and hydroxylated to form AFM1 in the cows which are excreted through the milk. Mean percent AFM1 contamination was comparatively high in the mik produced by early lactating cows than late lactating cows. The food intake by early lactating cows and late lactating cows are nearly similar. The intake of diet/feed were metabolized to excrete bile, urine, faeces and milk. Hence, the toxin gets excreted out through various discharges. Thus it relates to the finding that amount of toxins will vary with the amount of excretions. The observation leads to a finding that amount of milk yielded by the cows is in correlation with the AFM1 contamination of the milk.