Strategies for improving Strategies for improving CLA in milkCLA in milk

1 1 Ph.D. Student, Department of Animal Science, Ph.D. Student, Department of Animal Science, 2 2 Tropical Feed Resources Tropical Feed Resources Research and DevelopmentResearch and Development Center (TROFREC), Faculty of AgricultureCenter (TROFREC), Faculty of Agriculture

KhonKhon KaenKaen University, University, KhonKhon KaenKaen, Thailand, Thailand

R. R. LunsinLunsin1,2 1,2 and and MM..

WanapatWanapat22

CLA

CLA

CLA

IntroductionIntroduction……....

• Food products from animals are also known to contain microcomponents that have positive effects on human health and disease prevention (Allen,1993; Parodi, 1997; Molkentin, 1999)

• Conjugated linoleic acids (CLA) represent one of these microcomponents in animal products (Bauman et al., 1999)

• Food products from ruminants are the major dietary source of CLA in human diets (Chin et al., 1992)

CLA have been reported to have a wide range of beneficial effects on human health including (Bauman et al., 2001)

• anticarcinogenic

• antiatherogenic

• antidiabetic

• immune stimulatory

Thus, CLA are fascinating compounds of interest for animal scientists, human nutritionists, and the medical community

IntroductionIntroduction……....

Conjugated Conjugated linoleiclinoleic acids (acids (CLAsCLAs))……..

• CLA is a mixture of positional and geometric isomers of linoleic acid (cis-9, cis-12 C18:2 LA) with two conjugated unsaturated double bonds atvarious carbon positions in the fatty acid chain

• Each double bond can be cis or trans, but those with one trans double bond are bioactive (Jensen, 2002)

• Of all isomers of CLA that have been two importance isomers (Paroodi, 1977; 2003)

• cis-9, trans-11 is the major isomer comprising 80-90%

• trans-10, cis-12 isomer is present in amounts of 3-5% of total CLA

Figure 1. The structure of LinoleicLinoleic acidacid and two isomers of CLA

Source : https:/…/mcoov/web/GeneralInfo/cla.html

trans–10, cis–12 CLA

ciscis––9, 9, transtrans––1111 CLACLA

LinoleicLinoleic aciacid (d (ccisis––9,9, ciscis––12)12)

Biosynthesis of CLA in the ruminantBiosynthesis of CLA in the ruminant

CLA found in milk and meat of ruminant originate from two sources……

1. Synthesis in the rumen• Biohydrogenation of linoleic acid to steric

acid in the rumen

2. Endogenouse synthesis• Synthesis by animal’s tissues (adipose

tissue and mammary gland) from trans-11 C 18:1 (vaccenic acid)

Rumen biohydrogenation

• CLA is formed in the rumen during the biohydrogenation of linoleic acid and linolenic acid by the rumen bacteria such as (Tandon, 2006)…….

• Butyrivibrio fibrisovents (most active in CLA production)

• Micrococcus

• Treponema sp.

• Clostridium

• Bacteriodes

• Megasphera elsdenii (produce trans-10, cis-12 isomers of CLA) (Kim et al., 2000)

cis-9, cis-12 C18:2Linoleic Acid

cis-9, trans-11 C18:2Linoleic Acid (CLA)

cis-9, cis-12, cis-15 C18:3Linolenic Acid

cis-9, trans-11, cis-15 C18:3Linolenic Acid

trans-11, cis-15 C18:2

trans-11 C18:1Vaccenic Acid

trans-11, cis-15 C18:2

C18:0Stearic Acid

(Group A)

(Group A & B)

(Group A)

(Group A) (Group B)

(Group B)

(Group A & B)

Figure 2. Biochemical pathway for biohydrogenation of linoleic and linolenic acid in the rumen

Source : Harfoot and Hazlewood (1997)

Linoleic Acidcis-9, cis-12 C18:2

Linolenic Acidcis-9, cis-12, cis-15 C18:3

cis-9, trans-11, cis-15 C18:3 cis-9, trans-11 C18:2(CLA)

trans-11, cis-15 C18:2 trans-11 C18:1 (Vaccenic Acid)

C18:0(Stearic Acid)

cis-15 or trans-15 C18:3

trans-10, cis-12 C18:2(CLA)

trans-10 C18:1

High pHHigh pH Low pHLow pH

Figure 3. Biohydrogenation of linoleic and linolenic acid in the rumen

Source : Griinari and Bauman (2003)

Endogenouse synthesis

Dietary fat e.g. Linoleic acid

cis-9, cis-12 C18:2

cis-9, trans-11 C18:2 (CLA)

trans-11 C18:1 (vaccenic acid)

C18:0 (Steric acid)

cis-9, trans-11 C18:2 (CLA)

trans-11 C18:1

∆9-desaturase

Figure 4. Role of rumen biohydrogenation and tissue 9-desaturase in the production of cis-9, trans-11 CLA in milk fat. Adapted from Bauman et al. (2000)

∆

RumenRumen Mammary Mammary TissueTissue

The CLA content in milk and meat is affected by several factors, such as…….

• animal’s breed

• age

• diet and management factors related to feed supplements affecting the diet

Dietary factor are play an importance role on CLA content in milk

Manipulating the diet of dairy cow is one way to alter the fatty acid profile, especially CLA of milk fat

DietaryDietary manipulationmanipulation ofof CLA CLA inin ruminantruminant productsproducts

1. Dietary supply of plant and/or fish oils, that provide PUFA substrates for rumen production of CLA and trans-11 C18:1

2. Dietary supplements of CLA or trans-11 C18:1 fatty acids protected from rumen biohydrogentation

3. Dietary that affect rumen bacteria involved in biohydrogenation, either directly or via changes in rumen environment, such as modifying the forage : concentrate ratio of the diet or supplementation plant secondary compound (condensed tannins (CT), saponin)

ReferancesReferances TreatmentsTreatments Item (% of fatty acid)Item (% of fatty acid)

C18:1 tC18:1 t--1111Vaccenic acidVaccenic acid

C18:2 C18:2 LinoleicLinoleic acidacid

CC--9,t9,t--11 11 CLACLA

tt--10,c10,c--12 12 CLACLA

Total Total CLACLA

ChantaraprasarnChantaraprasarn and and WanapatWanapat (2008)(2008)

ConcentrateConcentrateCH+SFO 0%CH+SFO 0%CH+SFO 2.5%CH+SFO 2.5%CH+SFO 5%CH+SFO 5%

--------

--------

0.210.21aa

0.240.24aa

0.430.43bb

0.590.59cc

--------

0.260.26aa

0.280.28aa

0.520.52bb

0.730.73cc

ShingfieldShingfield et al. et al. (2006)(2006)

ControlControl4.5% FSO4.5% FSO

----

2.052.05dd

2.192.19ee

----

----

0.500.50dd

3.473.47ee

LeonardiLeonardi et al. et al. (2005)(2005)

ControlControl1.5% CO1.5% CO

0.860.86dd

1.771.77ee5.045.04dd

6.176.17ee0.450.45dd

0.890.89ee

0.010.010.020.02

----

LoorLoor et al. et al. (2005)(2005)

2.5% FO2.5% FO5% LO5% LO5% SFO5% SFO

6.586.585.435.434.224.22

------

2.292.292.362.361.831.83

------

2.732.732.842.842.402.40

ZhengZheng et al. et al. (2005)(2005)

ControlControl500 g of CSO500 g of CSO500 g of SBO500 g of SBO500 g of CO500 g of CO

1,181,18dd

1.991.99ee

2.392.39ff

2.032.03ee

2.602.60aa

3.433.43bb

3.873.87bb

2.952.95aa

0.350.35aa

0.600.60bb

1.021.02ee

0.690.69bb

0.010.010.020.020.010.010.010.01

--------

Table 1. Contents of fatty acids in milk fat from dairy cows influenced by supplementation of different plant oils

CH = Cassava hay in concentrate, SFO = sunflower oil, FSO = 45 g of a mixture (1:2, wt/wt) of fish oil 15 g and sunflower oil 30 g/kg of DM (FSO), CO = corn oil, FO = fish oil, LO = linseed oil, CSO = cottonseed oil, SBO = soybean oil

ReferancesReferances TreatmentsTreatments Item (% of fatty acid)Item (% of fatty acid)

C18:1 tC18:1 t--1111Vaccenic Vaccenic

acidacid

C18:2 C18:2 LinoleicLinoleic

acidacid

CC--9,t9,t--11 11 CLACLA

tt--10,c10,c--12 12 CLACLA

Total Total CLACLA

PiamphonPiamphon et et al. (2009)al. (2009)

0 g CLA0 g CLA--20 (RP20 (RP--CLA)CLA)20 g CLA 20 (RP20 g CLA 20 (RP--CLA)CLA)40 g CLA40 g CLA--20 (RP20 (RP--CLA)CLA)80 g CLA80 g CLA--20 (RP20 (RP--CLA)CLA)160 g CLA160 g CLA--20 (RP20 (RP--CLA)CLA)

----------

----------

0.620.62ee

1.171.17bb

1.941.94aa

1.871.87aa

1.821.82aa

0.010.01bb

0.630.63aa

0.670.67aa

0.930.93aa

0.950.95aa

0.800.80cc

2.252.25bb

3.163.16aa

3.973.97aa

3.943.94aa

CastanedaCastaneda-- CutierrezCutierrez et et al. (2005)al. (2005)

ControlControl31.6 g CLA (Ca31.6 g CLA (Ca--CLA)CLA)63.2 g CLA (Ca63.2 g CLA (Ca--CLA)CLA)

1.531.53dd

1.731.73ee

2.072.07ff

3.803.80aa

4.014.01abab

4.254.25bb

0.040.04dd

0.520.52dede

0.610.61ff

<0.01<0.01dd

0.020.02ee

0.040.04ff

------

PerfiledPerfiled et et al. (2004)al. (2004)

ControlControl54 g CLA (AP54 g CLA (AP--CLA)CLA)138 g CLA (LE138 g CLA (LE--CLA)CLA)

1.751.75dd

2.062.06ee

2.122.12ee

3.613.61dd

3.933.93ee

4.414.41ff

0.570.57dd

0.830.83ee

0.800.80ee

<0.01<0.01dd

0.080.08ee

0.090.09ee

------

PiperovaPiperova et et al. (2004)al. (2004)

ControlControl13 g CLA13 g CLA (Ca(Ca--CLA)CLA)

----

----

76.8276.82ee

67.9467.94dd1.321.32dd

3.483.48ee0.520.52aa

0.830.83bb

Table 2. Effects of rumen protected conjugated linoleic acid (RP-CLA) supplementation on milk fatty acids composition in lactating cows

RP-CLA = rumen protected conjugated linoleic acid, Ca-CLA = Calcium salts of conjugated linoleic acid, AP-CLA = Amide-protected of conjugated linoleic acid, LE-CLA = Lipid encapsulated of conjugated linoleic acid

Plant secondary compound effects on rumen biohydrogenation

Figure 5. Effect of dietary treatments on the share of linolenic acid as well as intermediates and end products of biohydrogenation made up from totally recovered C18 fatty acids. CH = control, TF = tannin containing forage (sainfoin), TH = tannin extract and SH = saponin extract. Source : Khiaosa-Ard et al. (2009)

∆

ImplicationsImplications……....

• The CLA content of ruminant fat (milk and meat) originate from two sources, including…..

• Rumen biohydrogenation of linoleic and linolenic aicd

• Synthesis in tissue (mammary gland and adipose tissue) from the desaturation of trans- 11 C 18:1 vaccenic acid by 9-desaturase

• The substantial variation in content of CLA in milk fat suggests that diet has a major influence

• Ruminant nutritionists have attempted to increase the naturally occurring CLA content of cow’s milk via diet changes and alterations of ruminal fermentation

Effects of protein and plant oil Effects of protein and plant oil sources on rumen fermentation, sources on rumen fermentation,

milk yield and qualitymilk yield and quality

Now……On going work???????

Hypothesis : 1. Supplementation of cassava hay (CH) as a protein source and rice bran oil (RBO) can enhance rumen fermentation and microbial population involved in rumen biohydrogenation of CLA

2. Supplementation CH and RBO can improve milk production and milk composition through increasing CLA production in milk of dairy cow

• To determine the effects of protein (cassava hay, protein (cassava hay, CH) and plant oil (rice bran oil, RBO) sources on DM CH) and plant oil (rice bran oil, RBO) sources on DM intake intake and feed digestibility in dairy cowsand feed digestibility in dairy cows

•• To determine the effects of CH and RBO onCH and RBO on rumen rumen fermentation, microbial population and diversityfermentation, microbial population and diversity

•• To determine the effects of CH and RBO onCH and RBO on milk milk yield and quality, especially accumulation of CLA in yield and quality, especially accumulation of CLA in milkmilk

Objective :

T2 : CHT2 : CH

T4 : T4 : CH+RiceCH+Rice bran oilbran oil

T1 : ControlT1 : Control

T3 : Rice bran oilT3 : Rice bran oil

Dietary treatment

2% Urea + 2% Lime (Ca(OH)2% Urea + 2% Lime (Ca(OH) 22 ))

Concentrate RoughageRoughage

2x2 factorial arrangement 2x2 factorial arrangement in a 4x4 LSin a 4x4 LS

Anticipated outcome :

1. To obtain the information of supplementation of CH and RBO on feed intake, nutrients digestibility, rumen fermentation and microbial population diversity, particularly microorganisms that involve biohydrogenation in the rumen of lactating dairy cows

2. To obtain the information of supplementation of CH and RBO on milk yield and milk quality, particularly CLA accumulation in milk of lactating dairy cows

Acknowledgements

Tropical Feed Resources Research and Development Center (TROFREC), KKU.

Dairy Production on Section, Department of Animal Science Faculty of Agriculture, KKU.

Strategic Scholarships Fellowships Frontier Research Networks, Commission on Higher Education

All participants