Effect of feeding type on the
occurrence of Aflatoxin M1 in
cow milk of high producing Dairy
cattle in Sri Lanka.
G.S.SUMANASEKARA
ASCEND RESEARCH NETWORK TRAINEE
SRI LANKA
Introduction

Dairy Sector in Sri Lanka

One of the priority sectors

Currently in the process of achieving self sufficiency
in milk production by 2020

Lack of the lands for roughage production

Promote Feeding with more concentrated feeds

Major problem associated with concentrated feeds
are contamination with aflatoxins
Introduction

Aflatoxins are naturally occurring mycotoxins

Metabolites produced by certain fungi in/on foods
and feeds.

The best known and most intensively researched
mycotoxins in the world.

The toxin producing fungi -Aspergillus flavus and
Aspergillus parasiticus .
Aflatoxins

Heat stable

Produced on many grains and legumes intended for
human and animal consumption.

Present in grains and legumes used for animal feed
production

Introduce to the digestive system of the animals
through the animal feed.

With food processing, aflatoxins get into the general
food supply

Found in both pet and human foods as well as in feed
stocks for agricultural animals
Aflatoxins

About 14 different types of aflatoxins produced in nature

B1,B2,G1,G2 plus two additional metabolic products, M1 and M2, that
are of significance as direct contaminants of foods and feeds.

Aflatoxin B1 considered the most toxic and is produced by both
Aspergillus flavus and Aspergillus parasiticus.

Aflatoxin G1 and G2 are produced exclusively by Aspergillus parasiticus

Aflatoxins M1, M2 discovered in the milk of cows which fed on moldy
grain
Aflatoxins

Aflatoxin B1 present in cattle feed will be metabolized
in the animals liver and produce Aflatoxin M1

subsequently excreted in milk

Higher levels of Aflatoxin M1 in milk is hazardous to
human health

Most harmful if present in infant formulae
Aflatoxins
-Aflatoxins associated with Aflatoxicosis, primarily a
hepatic disease, in livestock, domestic animals and
humans throughout the world
-Carcinogenic in long term exposure
-Natural habitat - soil,
-decaying vegetation, hay, and grains undergoing
microbiological deterioration are more suitable for
their growth.
Formation of some Metabolites of
Aflatoxin
Aflatoxins

The Joint Expert Committee on Food Additives
(JECFA) has established an acceptable level of 0.5
mg/kg for aflatoxin M1 in fluid milk.

Safety level for human subject- less than 0.5mg/kg
Aflatoxins

No regulations for accepted levels of AFM1 in food
in Sri Lanka

No special testing methods practice to detect
Aflatoxin M1 in milk and food for human
consumption in Sri Lanka

Therefore, it is important to study on Aflatoxins in
milk of high yielding cows fed with concentrate
feeds.
Objectives
Main Objective
To identify the relationship between feed type and
presence of Aflatoxin M1 in milk of the high producing
Dairy Cattle
Specific Objectives
1. Detection of presence of Aflatoxin M1 in the high
yielding dairy cattle in Sri Lanka
2. Detection of levels of Aflatoxin M1 present in the milk
3. Identify the risk of Aflatoxin M1 in milk for humans.
4.MATERIALS AND METHODS
Two Phases:
1.Field Survey
At Ambagamuwa vs range in Nuwara Eliya District
2.Sample collection and Laboratory Analysis
MATERIALS AND METHODS

Visited 32 farms and selected 10 farms based on;
Feeding type(fed with concentrates and with
grasses(Cut & Fed- Mixed with nutrients)
Management system(Intensive Management)
Number of Animals > 10 cows
Milk yield>6L

Data Collection – based on Questionnaire
Selected Farms
10 farms-112 cows
Feeding Method
No. of Frams
No. of Animals
1.Coconut Poonac
1
12
2.Concentrates
1
16
3.Beer Pulp
1
20
4.Concentrates+Coco
nut Poonac
1
21
5.Concentrates +Beer
Pulp
1
11
6.Coconut poonac
+Beer Pulp
1
14
7.Concentrates+Beer
1
Pulp+Coconut Poonac
17
8.Grasses
11
3
2.Sample Collection
1.
10 Milk samples of 60 ml in volume collected from
each farm according to the type of feed
combination given. Morning milk from all milking
cows in the farm was bulked and representative
sample taken
2. For the main study, two animals from each farm was
selected to test the AFM1 levels with different
quantities
3. Other animals were fed same amount of
concentrate feeds to explore the appearance of
AFM1 under different feeding regimes.
METHODS
TLC(Thin Layer Chromatography) Analysis

Before HPLC analysis, TLC was done to confirm the
presence of M1

Used Official Methods of Analysis of Official
Analytical Chemists(AOAC) official
chromatographic method
METHODS
HPLC (High Performance Liquid Chromatography)
Analysis :

Used Official Methods of Analysis of Official Analytical
Chemists(AOAC) official chromatographic method 986.16- For
Aflatoxins M1 and M2 in fluid milk
Methods
AFM1 idenetified on the retention time with respect to the standard.
Aflatoxin M1 concentrations calculated as follows:
FM1 concentration (ppb) = (H × C’ × VI’ × V) / (H’ × VI × W)
where H and H’ = peak height of sample and standard, respectively;
C’ = concentration of standard (ng µL-1);
VI’ and VI = volume injected of standard and sample, respectively;
V = final total sample volume (µL);
W = volume of milk represented by final extract (typically 20 mL).
HPLC
HPLC

HPLC(High Performance Liquid Chromatography)
Results and Discussion
AFM1 contamination levels in each sample
Coconut poonac+concentrates
2
Concentrates+Coconut
Poonac+Beer pulp
40
AFMi concentration(ng L-1)
1
3
Concentrates+Beer pulp
35
4
Beer pulp
30
5
Grass mixtures
6
Grass mixtures
7
Grass mixtures
8
Coconut Poonac+Beer Pulp
15
9
concentrates
10
10
Coconut poonac
25
20
5
0
1
2
3
4
5
6
7
8
9 Feed 10
Type
Number of Samples vs aFM1
Contamination Level
Number of samples vs AFM1 contamination ranges
7
Number of samples
6
5
4
3
No of samples
2
1
0
0-10
11-21
22-32
Range of AFM1 levels (ng L-1)
32-43
Relationship between concentrates (kg/day) and AFM1
Concentration(ng L-1)
40
AFM1 Conc. vs Coconut poonac+Beer Pulp
AFM1 Concentration (ng/L)
35
Y=1.542x+5.240
R2=0.271
30
25
20
15
r=0.52
10
5
0
0
2
4
6
8
concentrates+coconut poonac+beer pulp kg/day
10
12
Relationship between coconut poonac (kg/day) and
AFM1
Concentration(ng L-1)
40
AFM1 concentration(ng/L)
35
30
25
Y=6.195x-11.20
R2=0.422
20
15
10
R=0.65
5
0
0
2
4
6
coconut poonac
kg/day
8
10
12
Relationship between beer pulp(kg/day) and AFM1
Concentration(ng L-1)
40
AFM1 concentration(ng/L)
35
30
25
20
Y=7.523x+7.714
R2=0.164
15
r=0.405
10
5
0
0
2
4
6
Beer pulp Kg/day
8
10
12
Relationship between concentrates+coconut poonac
(kg/day) and AFM1
Concentration(ng L-1)
40
35
AFM1 concentration(ng/L)
30
25
20
Y=1.76x+-1.99
R2=0.429
15
10
r=0.655
5
0
0
2
4
6
coconut poonac kg/day
8
10
12
Relationship between concentrates+beer pulp(kg/day)
and AFM1
Concentration(ng L-1)
40
AFM1 Concentration (ng/L)
35
30
25
20
15
Y=1.37x+5.26
R2=0.272
10
r=0.521
5
0
0
2
4
6
8
concentrates+beer pulp kg/day
10
12
Relationship between coconut poonac+beer
pulp(kg/day) and AFM1
Concentration(ng L-1)
40
AFM1 Conc. vs Coconut poonac+Beer Pulp
AFM1 Concentration (ng/L)
35
30
25
20
r=0.767
Y=7.38x+-19.9
R2=0.588
15
10
5
0
0
2
4
6
coconut poonac+beer pulp
8
10
12
Relationship between concentrates+coconut
poonac+beer pulp(kg/day) and AFM1
Concentration(ng L-1)
40
AFM1 Conc. vs Coconut poonac+Beer Pulp
AFM1 Concentration (ng/L)
35
30
25
Y=1.5X+1.39
R2=0.423
20
15
r=0.65
10
5
0
0
2
4
6
8
concentrates+coconut poonac+beer pulp kg/day
10
12
Relationship between grasses(kg/day)
and AFM1
Concentration(ng L-1)
25
AFM1 conc
20
15
Y=0.031X+9.7
7
R2=0.002
10
r=0.053
5
0
0
5
10
15
20
grasses(Kg/Day)
25
30
35
N
o.
Feed Type Given
relationship between feed type (kg/day) and AFM1
concentration (ng L-1)
Coconut
Significant relationship observed
poonac+concentrates
Concentrates+Coconut
Significant relationship observed
Poonac+Beer pulp
Concentrates+Beer pulp
No significant relationship
Beer pulp
No significant relationship
Grass
No significant relationship
Coconut Poonac+Beer
Significant relationship observed
Pulp
concentrates
No significant relationship
Discussion
Majority of the milk samples showed AFM1 levels
between 0-10 ng L-1.Only one sample lied between
AFM1 levels between 32-43 ng L-1.
Two samples had AFM1 concentrations between
22-32 ng L-1. None of the samples exceeded above
the EU recommended level of 50 ng L-1.
Discussion

Study results indicated that AFM1 was present in the
50% of milk samples which showed evidence of
association between feed type and AFM1
contamination.

Compared individual feed types, coconut poonac was
considered to have the most significant relationship
with the AFM1 occurrence having a correlation of 0.65.

Among the feed type combinations, coconut poonac
and beer pulp combination had showed the highest
correlation of 0.77.

Grasses had shown a very poor relationship with the
AFM1 occurrence in milk.
Discussion

Within combinations concentrates+coconut
poonac,concentrates+coconut poonac+beer pulp,
coconut poonac +beer pulp, and beer
pulp+concentrates showed increased levels of AFM1 in
milk

When consider the relationship between overall
concentrate feeds present in the study and AFM1
contamination in milk, it was clear that they had a
significant relationship.

Compared to grass only fed animals concentrates fed
animals either fed concentrate alone or combination
has increased M1 levels in cow milk
Discussion

Within the feeding with concentrates alone
,coconut poonac and beer pulp showed increased
AFM1 levels

Compared to concentrates alone, combinations of
concentrates showed higher levels of AFM1

But non of the detected levels in milk did not
exceed the risk level for humans.<0.5mg/kg
Conclusion

The study revealed that concentrated formulae
feeding has caused increased AFM1 levels in milk of
high yielding dairy cattle fed with concentrated
feeds indicated that proper care should be taken in
feeding practices for dairy cows.
Acknowledgements

Supported
by
the
ASCEND
Program
(www.med.monash.edu.au/ascend) funded by the
Fogarty International Centre, National Institutes of
Health, under Award Number: D43TW008332.

The
contents
of
this
[poster/booklet/publication/presentation] is solely
the responsibility of the author(s) and does not
necessarily represent the official views of the
National Institutes of Health or the ASCEND Program.
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