The effect of forbs on rumen biohydrogenation of fatty acids and bovine milk fatty acid composition

129 Pages·2014·3.05 MB·Danish

by  Majbritt Bonefeld Petersen

The effect of forbs on rumen biohydrogenation of
fatty acids and bovine milk fatty acid composition

Majbritt Bonefeld Petersen

PhD thesis Science and Technology 2014
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Preface
The present PhD thesis was carried out from June 2011 to June 2014, and was as an
industrial PhD project carried out as a cooperation between AgroTech and The
Department of Animal Science, Faculty of Science and Technology, Aarhus University.
External cooperation partners also contributed to the completion of the PhD project. The
study on farm level in the present PhD project was only possible because of the
participation of Frøsalget, Thise Dairy and three of their organic dairy farmers.
Senior Researcher Søren Krogh Larsen, The Department of Animal Science, Faculty of
Science and Technology, Aarhus University, was the main supervisor, Senior Researcher
Karen Søgaard, The Department of Agroecology, Faculty of Science and Technology,
Aarhus University, was co-supervisor and Finn Strudsholm, AgroTech, was the industrial
supervisor.
The PhD project was founded by the Danish Ministry of Higher Education and Science,
AgroTech, The Department of Animal Science, Faculty of Science and Technology, Aarhus
University, Thise Dairy, and the innovation environment Future Food Innovation.
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Table of contents
Preface
Acknowledgements……………………………………………………………………………………………………. 3
Summary…………………………………………………………………………………………………………………..4
Summary in Danish…………………………………………………………………………………………………… 5
List of original manuscripts……………………………………………………………………………………….. 6
List of abbreviations………………………………………………………………………………………………….. 7
1 General introduction………………………………………………………………………………………………. 8
1.1 Dairy fat and the human diet .......................................................................................... 8
1.2 Lipids .............................................................................................................................. 9
1.2.1 Fatty acids……………………………………………………………………………………………………. 9
1.3 Rumen pathway of lipolysis and biohydrogenation ...................................................... 11
1.4 Manipulation of milk fatty acid composition ................................................................ 13
1.5 Objectives and hypotheses of the Ph.D thesis ............................................................... 18
2 Materials and methods………………………………………………………………………………………….. 19
2.1 Fatty acid analysis .......................................................................................................... 19
2.2 Extraction of lipid fractions .......................................................................................... 21
2.3 In vitro biohydrogenation ............................................................................................ 23
2.4 Feeding forbs to dairy cows at farm level ..................................................................... 25
3 Main results and discussion…………………………………………………………………………………… 27
3.1 Fatty acid content and lipid fractions in herbs ............................................................. 27
3.1.1 Fatty acid content in forbs……………………………………………………………………………. 27
3.1.2 Fatty acids in lipid fractions………………………………………………………………………… 28
3.1.3 Hydrolysis versus no hydrolysis…………………………………………………………………… 29
3.2 In vitro biohydrogenation studies ................................................................................ 31
3.2.1 In vitro biohydrogenation and feeding regimen……………………………………………… 31
3.2.2 In vitro biohydrogenation and single plant species………………………………………… 34
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3.2.2 Differences in in vitro biohydrogenation of fatty acids between lipid fractions 39
3.3 Feeding forbs to dairy cows at farm level .................................................................... 42
3.3.1 Botanical composition of the swards on the three farms…………………………………. 43
3.3.2 Milk production in response to feeding forb-rich silage…………………………………. 45
3.3.3 The effect of forb-rich silage on milk fatty acid composition…………………………… 47
4 Conclusion and future perspectives………………………………………………………………………… 54
4.1. Conclusions ................................................................................................................... 54
4.2 Future perspectives ....................................................................................................... 55
5 References…………………………………………………………………………………………………………… 57
6 Appendix……………………………………………………………………………………………………………… 69
6.1 Paper I ........................................................................................................................... 69
6.2 Paper II……………………………………………………………………………………………………….…..73
6.3 Paper III………………………………………………………………………………………………………….80
6.4 Paper IV………………………………………………………………………………………………………….94

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Acknowledgements
A great number of people have contributed to the completion of the present PhD project
and the different studies which form the basis of the thesis. First of all I would like to thank
my supervisor team, Søren K. Jensen, Karen Søegaard and Finn Strudsholm for their
support and feedback throughout the three years of the project, and their trust in me to be
able to stand on my two feet, and for always being there, when support was needed.
Secondly, I would like to extend my gratitude to the lab technicians Mette Würtz Reeh and
Elsebeth Lyng Pedersen, who have been of great help and support during the three years of
the PhD project, both in regards to technical assistance and personal encouragement. I
would especially like to thank Mette Würtz Reeh for her patience and never fading
enthusiasm in teaching an almost complete novice the techniques of extraction and
analysis of fatty acids. I also send my thanks to technician Orla Nielsen for his much
appreciated help on the collection of data from the farms – most of all his willingness to
take the grave-yard shift when morning milk samples were to be collected.
I would also like to express my gratitude to my other colleagues at The Department of
Animals Science for their assistance and willingness to lend a hand – and an ear – in small
and large matters. A warm thank you to all of my colleagues at AgroTech, who never made
me feel alienated, although I most likely could be referred to as the guest of the week
during the three years of the PhD project.
I send my warmest thanks to Thise Dairy for their contribution to the on-farm study, and
especially the never wavering enthusiastic participation of the three organic farmers
through all the ups and downs – without them the study would not have been possible.
Finally, I send my sincere thanks to my family and friends for their understanding and
support. I extend an especially heartfelt thank you to my mother for her time and not least
patience in correcting my sometime lacking English skills.

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Summary
Milk and dairy products are major sources for the intake of dietary fatty acids in many
Western countries, and ways to increase milk content of beneficial poly unsaturated fatty
acids (PUFA) is of great interest in regards to human health. The increase of PUFA in milk
is highly dependent on rumen biohydrogenation, and forb-rich herbage has repeatedly
been shown to cause alterations in the rumen which maximizes the effect on milk content
of omega-3 fatty acids compared to clover-grass herbage. The aim of the present PhD
thesis is to explore, if single forb species and the embedding of fatty acids in lipid fractions
in the forb species can help clarify the mechanism for the higher milk content of omega-3
fatty acids in forb fed dairy cows.
Two in vitro studies were performed to investigate the effect of feeding regimen, single
forb species or individual lipid fractions on rumen biohydrogenation of PUFA, and one
large scale in vivo study was performed to study the effect on milk fatty acid composition
when clover-grass silage was substituted with a forb silage. The in vitro studies revealed
reduced biohydrogenation of C18:3n-3 and C18:2n-6 when cows where fed a forb silage
compared to feeding a total mixed ration containing concentrate and corn silage. For single
forb species biohydrogenation was reduced in salad burnet (Poterium sangusisorba),
alfalfa (Meticago sativa) and ribwort plantain (Plantago lanceolata) while birdsfoot trefoil
(Lotus corniculatus) had the fastest biohydrogenation. Regardless of the fatty acid studied,
biohydrogenation of lipid fractions occurred in the following order: Free fatty acid >
triglycerides > phospholipids > cholesterol ester. On three Danish organic farms the milk
fatty acid proportions of c18:3n-3 and C18:2n-6 were increased when the cows were fed a
forb silage. Milk yield only decreased when the forb-rich silage contributed with >40 % of
dry matter intake. The changes in milk fatty acid composition can probably be linked to the
contribution of red clover (Trifolium pretense), caraway (Carum carvi), plantain and
alfalfa to the botanical composition of the forb silage on the individual farms.
In conclusion the results from the present study revealed that biohydrogenation of PUFA
was dependent on feeding regimen and single forb species, and these differences can
possibly be linked to which lipid fraction the PUFA are esterified to in the individual forb
species. The in vitro results were confirmed in the large scale in vivo study, as feeding the
forb silage significantly increased the milk fatty acid proportion of PUFA compared to
feeding clover-grass silage on all three farms.
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Summary in Danish
Forskere har i mange år været interesserede i at øge indholdet af de gavnlige polyumættede
fedtsyrer (PUFA) i mælken, da mejeriprodukter udgør en stor del af det daglige indtag af
fedtsyrer i mange vestlige lande. Biohydrogenering (BH) af PUFA har stor betydning for
mælkens fedtsyresammensætning, da en reduceret BH vil øge indholdet af de gavnlige
PUFA i mælken. Flere studier har vist, at urter reducerer BH af PUFA, hvilket øger
indholdet af PUFA i mælken. Mekanismerne for denne urteeffekt er ikke fuldt afklaret, og
formålet med denne PhD afhandling er at undersøge, om enkelte urtearter og fedtsyrernes
indlejring i planternes forskellige lipid fraktioner kan afklare den specifikke effekt af urter
på BH af PUFA og mælkens fedtsyreindhold.
Der er gennemført to in vitro forsøg for at undersøge betydningen af fodring, de enkelte
urtearter og enkelte lipid fraktioner på BH af PUFA. Derudover blev et større in vivo forsøg
hos tre danske økologiske mælkeproducenter gennemført for at undersøge effekten på
mælkens fedtsyresammensætning, når kløvergræs ensilage blev udskiftet med
urteensilage. Resultaterne viste, at en større andel af fedtsyrerne i urter er indlejret i sterol
fraktioner sammenlignet med rajgræs (Lolium perenne). Uafhængigt af fedtsyrer var
rækkefølgen for BH af de enkelte fedtfraktioner: Frie fedtsyrer > triglycerider >
fosfolipider > kolesterolestre. Derudover viste in vitro forsøgene, at BH af C18:3n-3 og
C18:2n-6 var betydeligt reduceret i bibernelle (Poterium sangusisorba), lucerne(Meticago
sativa) og vejbred (Plantago lanceolata), men markant hurtigere i kællingetand (Lotus
corniculatus). Det ene in vitro forsøg viste, at BH af PUFA var betydeligt reduceret, når
køerne blev fodret med en urteensilage sammenlignet med fodring med kløvergræs
ensilage eller fodring baseret på majsensilage og kraftfoder. Dette blev bekræftet i in vivo
forsøget, hvor udskiftning af kløvergræs ensilage hos alle tre mælkeproducenter med
urteensilage medførte en øget andel af gavnlige PUFA i mælken. Mælkeydelsen kunne
opretholdes indtil 40 % af foderindtag var urteensilage. Ændringer i mælkens
fedtsammensætning skyldes formodentlig andelen af rødkløver (Trifolium pretense),
kommen (Carum carvi), vejbred og lucerne i den botaniske sammensætning af urte-
ensilagen.
Konklusionen af resultaterne fra den foreliggende PhD afhandling er, at BH af PUFA er
afhængig af koens fodring samt de enkelte urtearter, og disse forskelle kan muligvis
afhænge af, hvilke lipidfraktioner PUFA er esterificeret til. Disse in vitro resultater blev
bekræftet i in vivo-forsøget, hvor fodring med urteensilage øgede mælkens andel af
gavnlige PUFA i forhold til fodring med kløvergræs ensilage.
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List of original manuscripts
The present PhD thesis is based on the following original manuscripts referred to in the
text as follows:
Paper I: Petersen, M.B., Søegaard, K. and Jensen, S.K. 2011. Fatty acid content and lipid
fractions in herbs. Grassland Science in Europe. 17: 314-216.

Paper II: Petersen, M.B. and Jensen, S.K. 2014: Biohydrogenation of Fatty Acids Is
Dependent on Plant Species and Feeding Regimen of Dairy Cows. Journal of
Agricultural and Food Chemistry. 62: 3570-3576

Paper III: Petersen, M.B. and Jensen, S.K. Biohydrogenation of C18:2n-2 and C18:3n-3 is
reduced when esterified to complex lipid fractions. Draft intended for submission to
British Journal of Nutrition
Paper IV: Petersen, M.B, Søegaard, K., Strudsholm, F., Leiber, F. and Jensen, S.K. Forb-
rich silages to dairy cows increase milk n-3 and n-6 fatty acid content: Results from
three Danish organic farms. Draft intended for submission to Journal of Dairy
Research

Paper I and II are reproduced with kind permission from the publishers

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List of abbreviations
ALA α-linolenic acid
B&D Bligh and Dyer method
BH Biohydrogenation
CE Cholesterol ester
CLA Conjugated linoleic acid
CLOVER-GRASS Clover-grass silage
CT Condensed tannins
DM Dry matter
DMI Dry matter intake
EKM Energy corrected milk
FA Fatty acids
FFA Free fatty acids
FAME Fatty acids methyl esters
FORBS Forb-rich silage
GL Glycolipids
GRASS Ryegrass silage
LA Linoleic acid
LCFA Long chained fatty acids
MCFA Medium chained fatty acids
MFD Milk fat depression
ML Membrane lipids
NEFA Non-esterified fatty acids
OBCFA Odd and branched chained fatty acids
PL Phospholipids
PPO Poly phenol oxidase
PUFA Poly unsaturated fatty acids
SCFA Short chained fatty acids
SFA Saturated fatty acids
ST Sterols
SPE Solid phase extraction
TAG Triglycerides
TMR Total mixed ration
VFA Volatile fatty acids
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1 General introduction
1.1 Dairy fat and the human diet
In many western countries milk has a long tradition for providing humans with energy and
other essential nutritional components like vitamins and minerals. Depending on
continent and country, milk is also an important fat source, but the intake of milk varies
because of culture, history, and eating habits. In recent years the milk intake has
decreased, probably due to the debate of the negative impact of milk on human health
(German et al., 2009). Milk fat is normally around four percentages, and thus a main
contributor to the composition of milk, and the milk fat is estimated to contain > 400
different fatty acids (FA), which differ in both chain length and number of unsaturated
bonds. Approximately 50 % of the FA in milk are saturated FA (SFA), with C14:0 and
C16:0 as the main FA, while the polyunsaturated fatty acids (PUFA) only contribute with
around 0.5 % of the FA (Jensen, 2002).
Research has shown that a high intake of certain SFA is one possible factor in the
development of a number of the so-called modern western diseases like obesity,
cardiovascular diseases, and type II diabetis (Simopoulos, 1999), and therefore several
advisory bodies, including the 4th edition of the Nordic Recommendations (Nordic Council
of Ministers, 2004) have recommended to avoid high fat dairy products. In the recently
published New Nordic Nutrition Recommendation, the emphasis on fat quantity has
shifted to focus more on fat quality (Nordic Council of Ministers, 2013), as some FA are
considered beneficial for human health. The major FA that are considered to be beneficial
are the n-3 FA (Simopoulos, 1991; Simopoulos, 2008), which are also essential FA, and
conjugated linolenic acid (CLA) (Dilzer and Park, 2012), which is a FA particularly formed
in the ruminant lipid metabolism. As a consequence of the public awareness of specific FA,
animal scientists have over the last couple of decades investigated ways to increase the
content of these FA in ruminant derived products. The feedstuffs included in the cow´s
diet have major impact on milk FA composition, and if dairy farmers were to produce milk
with a high content of the beneficial PUFA, one prerequisite would be that the milk yield
was not compromised. Thus, a compromise which meets both the demand for a high
content of PUFA in milk and stabile milk yield would have to be found.
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