1. 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

2. 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

3. 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.

4. 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

5. 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 B 1 considered the most toxic and is produced by both Aspergillus flavus and Aspergillus parasiticus.  Aflatoxin G 1 and G 2 are produced exclusively by Aspergillus parasiticus  Aflatoxins M 1, M 2 discovered in the milk of cows which fed on moldy grain

6. 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

7. 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.

8. Formation of some Metabolites of Aflatoxin

9. Aflatoxins  The Joint Expert Committee on Food Additives (JECFA) has established an acceptable level of 0.5 mg/kg for aflatoxin M 1 in fluid milk.  Safety level for human subject- less than 0.5mg/kg

10. 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.

11. 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.

12. 4.MATERIALS AND METHODS Two Phases: 1.Field Survey At Ambagamuwa vs range in Nuwara Eliya District 2.Sample collection and Laboratory Analysis

13. 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

14. Selected Farms 10 farms-112 cows Feeding MethodNo. of FramsNo. of Animals 1.Coconut Poonac112 2.Concentrates116 3.Beer Pulp120 4.Concentrates+Coco nut Poonac 121 5.Concentrates +Beer Pulp 111 6.Coconut poonac +Beer Pulp 114 7.Concentrates+Beer Pulp+Coconut Poonac 117 8.Grasses311

15. 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.

16. 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

17. 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

18. Methods AFM 1 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).

19. HPLC

20.  HPLC(High Performance Liquid Chromatography)

21. Results and Discussion 1 Coconut poonac+concentrates 2 Concentrates+Coconut Poonac+Beer pulp 3 Concentrates+Beer pulp 4 Beer pulp 5 Grass mixtures 6 7 8 Coconut Poonac+Beer Pulp 9 concentrates 10 Coconut poonac

22. Number of Samples vs aFM1 Contamination Level

23. Relationship between concentrates (kg/day) and AFM 1 Concentration(ng L -1 ) Y=1.542x+5.240 R2=0.271 r=0.52

24. Relationship between coconut poonac (kg/day) and AFM 1 Concentration(ng L -1 ) R=0.65

25. Relationship between beer pulp(kg/day) and AFM 1 Concentration(ng L -1 ) r=0.405

26. Relationship between concentrates+coconut poonac (kg/day) and AFM 1 Concentration(ng L -1 ) r=0.655

27. Relationship between concentrates+beer pulp(kg/day) and AFM 1 Concentration(ng L -1 ) r=0.521

28. Relationship between coconut poonac+beer pulp(kg/day) and AFM 1 Concentration(ng L -1 ) r=0.767

29. Relationship between concentrates+coconut poonac+beer pulp(kg/day) and AFM 1 Concentration(ng L -1 ) Y=1.5X+- 1.39 R2=0.423 r=0.65

30. Relationship between grasses(kg/day) and AFM 1 Concentration(ng L -1 ) Y=0.031X+9.7 7 R2=0.002 r=0.053

31. N o. Feed Type Givenrelationship between feed type (kg/day) and AFM 1 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

32. 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.

33. 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.

34. 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

35. 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

36. 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.

37. 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.www.med.monash.edu.au/ascend  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.

38. Thank You!