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 Farms10 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+Coconut Poonac

1 21

5.Concentrates +Beer Pulp

1 11

6.Coconut poonac +Beer Pulp

1 14

7.Concentrates+Beer Pulp+Coconut Poonac

1 17

8.Grasses 3 11

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

MethodsAFM1 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

1 2 3 4 5 6 7 8 9 100

5

10

15

20

25

30

35

40

AFM1 contamination levels in each sample

Feed Type

AFM

i concentr

ati

on(n

g L

-1)

1 Coconut poonac+concentrates

2 Concentrates+Coconut

Poonac+Beer pulp

3 Concentrates+Beer pulp

4 Beer pulp

5 Grass mixtures

6 Grass mixtures

7 Grass mixtures

8 Coconut Poonac+Beer Pulp

9 concentrates

10 Coconut poonac

Number of Samples vs aFM1 Contamination Level

0-10 11-21 22-32 32-430

1

2

3

4

5

6

7

Number of samples vs AFM1 contamination ranges

No of samples

Range of AFM1 levels (ng L-1)

Num

ber

of

sam

ple

s

Relationship between concentrates (kg/day) and AFM1

Concentration(ng L-1)

0 2 4 6 8 10 120

5

10

15

20

25

30

35

40

concentrates+coconut poonac+beer pulp kg/day

AFM

1 C

oncentr

ati

on (

ng/L

)

AFM1 Conc. vs Coconut poonac+Beer Pulp

Y=1.542x+5.240R2=0.271

r=0.52

Relationship between coconut poonac (kg/day) and AFM1

Concentration(ng L-1)

0 2 4 6 8 10 120

5

10

15

20

25

30

35

40

coconut poonac kg/day

AFM

1 c

oncentr

ati

on(n

g/L

)

Y=6.195x-11.20R2=0.422

R=0.65

Relationship between beer pulp(kg/day) and AFM1

Concentration(ng L-1)

0 2 4 6 8 10 120

5

10

15

20

25

30

35

40

Beer pulp Kg/day

AFM

1 c

oncentr

ati

on(n

g/L

)

Y=7.523x+7.714R2=0.164

r=0.405

Relationship between concentrates+coconut poonac (kg/day) and AFM1

Concentration(ng L-1)

0 2 4 6 8 10 120

5

10

15

20

25

30

35

40

coconut poonac kg/day

AFM

1 c

oncentr

ati

on(n

g/L

)

Y=1.76x+-1.99R2=0.429

r=0.655

Relationship between concentrates+beer pulp(kg/day) and AFM1

Concentration(ng L-1)

0 2 4 6 8 10 120

5

10

15

20

25

30

35

40

concentrates+beer pulp kg/day

AFM

1 C

oncentr

ati

on (

ng/L

)

Y=1.37x+5.26R2=0.272 r=0.521

Relationship between coconut poonac+beer pulp(kg/day) and AFM1

Concentration(ng L-1)

0 2 4 6 8 10 120

5

10

15

20

25

30

35

40

coconut poonac+beer pulp

AFM

1 C

oncentr

ati

on (

ng/L

)

AFM1 Conc. vs Coconut poonac+Beer Pulp

Y=7.38x+-19.9R2=0.588

r=0.767

Relationship between concentrates+coconut poonac+beer pulp(kg/day) and AFM1

Concentration(ng L-1)

0 2 4 6 8 10 120

5

10

15

20

25

30

35

40

concentrates+coconut poonac+beer pulp kg/day

AFM

1 C

oncentr

ati

on (

ng/L

)

AFM1 Conc. vs Coconut poonac+Beer Pulp

Y=1.5X+-1.39R2=0.423

r=0.65

Relationship between grasses(kg/day) and AFM1

Concentration(ng L-1)

14 16 18 20 22 24 26 28 30 320

5

10

15

20

25

grasses(Kg/Day)

AFM

1 c

onc

Y=0.031X+9.77R2=0.002

r=0.053

No.

Feed Type Given relationship between feed type (kg/day) and AFM1 concentration (ng L-1)

1 Coconut

poonac+concentrates

Significant relationship observed

2 Concentrates+Coconut

Poonac+Beer pulp

Significant relationship observed

3 Concentrates+Beer pulp No significant relationship

4 Beer pulp No significant relationship

5 Grass No significant relationship

6 Coconut Poonac+Beer

Pulp

Significant relationship observed

7 concentrates No significant relationship

8 Coconut poonac Significant relationship observed

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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