Effects of a rumen-protected conjugated linoleic acid supplementation on the quality of bovine oocytes and embryos

University of Veterinary Medicine Hannover

Institute of Farm Animal Genetics (Mariensee) Friedrich-Loeffler-Institut

Effects of a rumen-protected conjugated linoleic acid supplementation on the quality of bovine oocytes and

embryos

Thesis

Submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY

(PhD)

awarded by the University of Veterinary Medicine Hannover by

Andrés Felipe González Serrano (Caracas)

Hannover, Germany 2014

Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes

University of Veterinary Medicine Hannover

Institute of Farm Animal Genetics (Mariensee) Friedrich-Loeffler-Institut

Effects of a rumen-protected conjugated linoleic acid supplementation on the quality of bovine oocytes and

embryos

Thesis

Submitted in partial fulfilment of the requirements for the degree DOCTOR OF PHILOSOPHY

(PhD)

awarded by the University of Veterinary Medicine Hannover by

Andrés Felipe González Serrano (Caracas)

Hannover, Germany 2014

Supervisor: Prof. Dr. Heiner Niemann

Advisory Committee: Prof. Dr. Heiner Niemann Prof. Dr. Sven Dänicke Prof. Dr. Gerhard Breves 1 ^st Evaluation:

Prof. Dr. Heiner Niemann

Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut Mariensee, Germany

Prof. Dr. Sven Dänicke

Institute of Animal Nutrition, Friedrich-Loeffler-Institut Braunschweig, Germany

Prof. Dr. Gerhard Breves

Institute of Physiology, University of Veterinary Medicine Hannover Hannover, Germany

2 ^nd Evaluation

Prof. Dr. Markus Rodehutscord

Institute of Animal Nutrition, University of Hohenheim Stuttgart, Germany

Date of oral exam: 22.4.2014

This PhD Thesis was conducted at the Institute of Farm Animal Genetics, Friedrich-

Loeffler Institut (FLI) in Mariensee, Germany

To Mirian, Argenis, Ligia and Reyna. My inspiration.

Presentations of the thesis

Results of this doctoral project have been presented at national and international conferences:

AF González-Serrano, J Heinzmann, K-G Hadeler, D Herrmann, P Aldag, M Diedrich, U Meyer, C Rohrer, G Jahreis, M Piechotta, S Dänicke and H Niemann (2012). Effects of rumen-protected conjugated linoleic acids supplementation on the expression of developmentally important genes in bovine oocytes. Poster presentation at the 45 ^th Annual Conference of Physiology and Pathology of Reproduction in Berlin, Germany. Abstract published in Reproduction in Domestic Animals. Vol. 47 p. 24.

AF González-Serrano, J Heinzmann, K-G Hadeler, D Herrmann, P Aldag, M Diederich, U Meyer, C Rohrer, G Jahreis, M Piechotta, S Dänicke and H Niemann (2012). Supplementation of rumen-protected conjugated linoleic acids regulates gene expression of bovine oocytes after in vitro maturation. Poster presentation at the 17 ^th International Congress on Biotechnology in Animal Reproduction (ICBAR) in Leipzig, Germany. (Abstract #: 15). Poster prize winner.

AF González-Serrano, CR Ferreira, LS Eberlin, V Pirro, J Heinzmann, A Lucas- Hahn, RG Cooks and H Niemann (2013). Rapid, untargeted lipid determination in individual bovine oocytes and pre-implantation embryos by high resolution desorption electrospray ionization mass spectrometry (DESI-MS). Poster presentation at the 39 ^th Annual Meeting of the International Embryo Transfer Society (IETS) in Hannover, Germany (Abstract #: 229). Abstract published in Reproduction Fertility and Development t 25(1) 262.

AF González-Serrano, CR Ferreira, V Pirro, J Heinzmann, K-G Hadeler, D Herrmann, P Aldag, U Meyer, M Piechotta, S Dänicke, RG Cooks and H Niemann (2013). Transcript expression level and lipid determination in bovine oocytes after supplementation of rumen-protected conjugated linoleic acids and stearic acid.

Poster presentation at the 46 ^th Annual Conference of Physiology and Pathology of

Reproduction in Gdansk, Poland. Abstract published in Reproductive Biology 13 (1)

59.

AF González-Serrano, CR Ferreira, V Pirro, J Heinzmann, K-G Hadeler, D Herrmann, P Aldag, U Meyer, M Piechotta, C Rohrer, G Jahreis, S Dänicke, RG Cooks, H Niemann (2014). Specific fatty acid follow-up reveals rumen-protected fat supplementation effects on bovine oocyte quality and embryo development. Oral presentation as a finalist of the student competition at the 40 ^th Annual meeting of the International Embryo Transfer Society (IETS) in Reno, USA (Abstract #: 2). Abstract published in Reproduction Fertility and Development 26(1) 115-116. Runner up winner at the student competition.

AF González-Serrano, CR Ferreira, V Pirro, A Lucas-Hahn, U Baulain, J Heinzmann, K-G Hadeler, P Aldag, U Meyer, M Piechotta, G Jahreis, S Dänicke, RG Cooks and H Niemann (2014). Fatty acid profiling after rumen-protected fat supplementation reveals nutritional footprints on bovine oocyte quality and embryo development.

Poster presentation at the 47 ^th Annual Conference of Physiology and Pathology of

Reproduction in Gießen, Germany (Abstract #: 42). Abstract published in

Reproduction in Domestic Animals 49(1) p. 20.

Content

List of Abbreviations ... I Summary ... V Zusammenfassung ... VII

1 General Introduction ... 1

1.1 Fatty acid supplementation of dairy cows ... 1

1.2 Rumen-protected fatty acid supplements... 2

1.3 Conjugated linoleic acid (CLA) ... 3

1.3.1 Biosysnthesis of CLA ... 3

1.3.2 Rumen biohydrogenation... 3

1.3.3 Tissue synthesis of CLA ... 4

1.4 Rumen-protected CLA supplementation ... 5

1.5 CLA supplementation and cattle fertility ... 6

1.6 Fatty acid effects on bovine oocyte quality and embryo development ... 7

1.7 Determination of fatty acid profiles in oocytes and embryos ... 8

1.8 Motivation and goals of this study ... 10

2 Publication 1 ... 13

Abstract ... 14

3 Publication 2 ... 15

Abstract ... 16

3.1 Introduction ... 17

3.2 Material and methods ... 18

3.3 Results ... 26

3.4 Discussion ... 39

3.5 Acknowledgements ... 43

3.6 References ... 44

3.7 Supplementary information ... 51

4 General Discussion ... 61

4.1 Fatty acid profiling of single oocytes and embryos by DESI-MS ... 61

4.2 Lipid metabolism and gene expression ... 66

4.3 Importance of this PhD thesis ... 67

4.4 Conclusions and perspectives ... 67

5 References ... 69

Acknowledgments ... 79

List of Abbreviations

DMI- Dry matter intake

NEFA- Non-esterified fatty acid CLA- Conjugated linoleic acid FFA- Free fatty acids

PUFA- Polyunsaturated fatty acids ECE1- Endothelin converting ezyme 1

PTGS2- Prostaglandin-endoperoxide synthase 2 PTGFR- Prostaglandin F receptor

STAR- Steroidogenic acute regulatory protein

HSD3B1- Hydroxy-delta-5-steroid dehydrogenase, 3 beta- isomerase 1 TAG- Triacylglycerols

Chol- Cholesterol PL- Phospholipid

MAPK- Mitogen- activated protein kinase

MALDI-MS- Matrix-assisted laser desorption/ionization mass spectrometry DESI-MS- Desorption electrospray ionization mass spectrometry

GC- Gas chromatography

ACAT1- Cholesterol acyl transferase 1 CPT1b- Carnitine palmitoyltransferase FASN- Fatty acid synthase

SREBP1- Sterol regulatory element binding protein 1

SCAP- Sterol regulatory element binding protein-activating protein PCA- Principal component analysis

LDA- Linear discriminant analysis ART- Assisted reproductive techniques TLC- Thin-layer chromatography

CID- Collision-induced dissociation

CE- Cholesteryl ester

DBH- Dihydroxibenzoic acid PE- Phosphatidylethanolamine

PEp- alkenyl ether of Phosphatidylethanolamine (Plasmalogen) PC- Phosphatidylcholine

PCp- alkenyl ether of Phosphatidylcholine (Plasmalogen) PS- Phosphatidylserine

PG- Phosphatidylglycerol PI- Phosphatidylinositol

DF-PCA- Data fusion- Principal component Analysis SLC2A8- Solute carrier family 2, member 8

GDF9- Growth differentiation factor 9 PRDX1- Peroxiredoxin 1

ZAR1- Zygote arrest 1

NADPH- Nicotinamide adenine dinucleotide phosphate HSD17B7- Hydroxysteroid (17-beta) dehydrogenase 7

CYP11A1- Cytochrome P450, family 11, subfamily A, polypeptide 1

HSD3B1- Hydroxy-delta-5-steroid dehydrogenase, 3-beta- and steroid delta- isomerase 1

APOA1- Apolipoprotein A-1

MSMO1- Methylsterol monooxygenase 1 ANXA1- Annexin A1

ANXA2- Annexin A2

ABCC2- ATP-binding cassette, subfamily C, member 2 TCM- Tissue culture medium

COC- Cumulus-oocyte-complex TIC- Total ion current

CV- Cross validation SA- Stearic acid

BCS- Body condition score

FAME- Fatty acid methyl esters

EDTA- Ethylenediaminetetraacetic acid IGF1- Insulin-like growth factor 1

OPU- Ovum pick-up

hCG- human chorionic gonadotropin

GC/FID- Gas chromatographic/ flame ionization detector GJA1- Gap junction alpha 1

FSH- Follicle-stimulating hormone MUFA- Monounsaturated fatty acids SFA- Saturated fatty acids

ADF- Acid detergent fiber

NDF- Neutral detergent fiber

Summary

Andrés Felipe González Serrano:

Effects of a rumen-protected conjugated linoleic acid supplementation on the quality of bovine oocytes and embryos

Linoleic acid is an essential polyunsaturated fatty acid in the diet of ruminants.

Conjugated linoleic acid (CLA) is a general term used for different spatial isomers of linoleic acid. CLA is produced in the rumen as result of the incomplete biohydrogenation of the linoleic acid. Supplementation of lactating cows with CLA has been reported to reduce milk fat content and is thought to improve reproductive performance by increasing the probability of pregnancy and reducing the interval between parturition and first ovulation. Direct effects of conjugated linoleic acid diet supplementation on oocyte quality and the developmental capacity of embryos have not yet been investigated.

Here, Holstein-Friesian heifers (n=84), fed with a grass silage basis diet, received a supplement consisting of either rumen-protected CLA (10% of cis9,trans11-CLA and 10% of trans10,cis12-CLA) or stearic acid (C18:0; SA) on top of an isocaloric diet.

Two different supplementation doses were given depending on the experiment (100 g/d and 200 g/d). After an initial phase of supplementation (45 days), oocytes were collected by ultra-sound guided ovum pick-up (OPU) twice weekly for four weeks.

Oocytes from the CLA-supplemented (n=413) and the SA-supplemented (n=350) groups were in vitro matured, fertilized and cultured to the blastocyst stage. Blood and follicular fluid samples were collected at the start and end of the supplementation period in order to analyze cholesterol, IGF1, NEFA and for fatty acid profiling.

Maturation rates did not differ between experimental groups

(CLA=73% vs. SA=70%). A trend towards a lower blastocyst rate in the SA-

supplemented group was observed when compared to the CLA-supplemented group

(CLA=22.4% vs. SA=13.8%). While IGF and NEFA levels did not differ among

experimental groups, lipid profiles in blood and follicular fluid were affected in a dose-

dependent manner by both supplements. Cholesterol plasma concentration was

significantly increased after 200 g/d of CLA-supplementation. Relative mRNA

abundance of selected developmentally important genes (IGF1R, GJA1, FASN,

SREBP1 and SCAP) was analyzed in single immature and in vitro matured oocytes

by RT-qPCR (n=6/group). In vivo matured oocytes (collected by OPU 20h after GnRH injection) were used as control. Poly(A ⁺ ) mRNA abundance did not differ between treatments for the selected genes, however SCAP mRNA was significantly down-regulated in in vitro matured oocytes from supplemented heifers in comparison to their in vivo matured counterparts fed only the regular grass silage diet.

Lipid profiling of single oocytes from the CLA-supplemented (n=37) and the SA- supplemented (n=50) animals was performed by desorption electrospray ionization mass spectrometry (DESI-MS). Fatty acid analysis using DESI-MS in immature oocytes revealed that CLA supplementation led to an increase of triacylglycerol 52:3 [TAG (52:3)] and TAG (52:2), squalene, palmitic acid (C16:0) and oleic acid (C18:1), and decreased abundance of TAG (56:3), TAG (50:2) and TAG (48:1) compared to SA-supplemented animals. In vitro matured oocytes from CLA-supplemented animals showed a different lipid profile, with increased abundances of TAG (52:3), and TAG (52:2) as well as phosphatidylinositol 34:1 [Plo (34:1)], whereas plasmalogen species [PCp (38:4) and Pep (38:4)] and palmitic acid (C16:0) were more abundant in in vitro matured oocytes collected from SA-supplemented heifers.

In conclusion, it is shown here that supplementation of the diet of dairy heifers with rumen-protected fatty acids can be monitored and analyzed in different sample types (i.e., blood system, follicular fluid and intra-oocyte). Single oocyte lipid determination using state-of-the-art high resolution mass spectrometry was employed for the first time for monitoring the effects of fatty acid supplementation of the daily diet and revealed specific nutritional footprints on oocyte quality and embryo development.

These results demonstrate the close relationship between nutrition and herd fertility

and at the same time support the role of the bovine model for understanding

nutritional-dependent fertility impairments.

Zusammenfassung

Andrés Felipe González Serrano

Effekte der Fütterungssupplementierung mit pansengeschützten konjugierten Linolsäuren auf die Qualität boviner Eizellen und Embryonen

Die Linolsäure ist eine für die Ernährung von Wiederkäuern essentielle mehrfach ungesättigte Fettsäure. Als Folge einer unvollständigen Hydrierung der Linolsäure im Pansen werden konjugierte Linolsäuren (CLA / conjugated linoleic acid) gebildet.

CLA ist ein Überbegriff für verschiedene Positions-Isomere der Linolsäure, die im Darm adsorbiert werden. In laktierenden Kühen konnte eine Reduzierung des Milchfetts durch Supplementierung mit CLA erreicht werden, auch scheint sich die Supplementierung positiv auf die Reproduktion auszuwirken, da sich die Wahrscheinlichkeit einer Trächtigkeit erhöht und dem Intervall zwischen Abkalbung und erster Ovulation verkürzt wird. Direkte Effekte der Supplementierung mit konjugierten Linolsäuren auf Eizellqualität und Entwicklungspotential von Embryonen sind bisher noch nicht beschrieben worden.

Im Rahmen dieser Arbeit erhielten Holstein-Friesian Färsen (n=84) entweder

pansengeschützte CLA (10% cis9,trans11-CLA und 10% trans10,cis12-CLA) oder

Stearinsäure (C18:0; SA) als Supplementierung zu einer isokalorischen Grassilage-

Fütterung. Zwei verschiedene Mengen wurden dabei in zwei Experimenten

eingesetzt (100 g/d und 200 g/d). Nach einer initialen Supplementationsphase (45

Tage), wurden die Eizellen mittels Ultraschall-geleiteter Ovum-pick-up (OPU)

zweimal wöchentlich über einen Zeitraum von vier Wochen gewonnen. Oozyten der

CLA-supplementierten (n=413) und der SA-supplementierten (n=350) Gruppen

wurden in vitro gereift, befruchtet und kultiviert, um Reifungs- und Blastozystenraten

zu bestimmen. Blut und Follikelflüssigkeit wurden am Anfang und Ende der

Supplementationsphase gewonnen, um Cholesterin, IGF1, NEFA und das

Fettsäureprofil zu bestimmen. Die Reifungsraten haben sich zwischen den

Versuchsgruppen nicht unterschieden (CLA=73% vs. SA=70%). Die Blastozystenrate

in der SA-supplementierten Gruppe war tendenziell niedriger als in der CLA-

supplementierten Gruppe (CLA=22,4% vs. SA=13,8%). Während sich die IGF1- und

NEFA-Spiegel zwischen den Versuchgruppen nicht unterschieden, waren die

Lipidprofile in Blut und Follikelflüssigkeit in beiden Versuchsgruppen dosis-abhängig

verschieden. Die Cholesterinspiegel wurde durch die CLA-Supplementierung beeinflusst. Die relative mRNA Expression der Gene IGF1R, GJA1, FASN, SREBP1 und SCAP wurde in einzelnen unreiften und in vitro gereiften Eizellen mittels RT- qPCR analysiert (n=6/Gruppe). In vivo gereifte Oozyten, gewonnen durch OPU 20h nach GnRH Injektion, wurden als Kontrolle verwendet. Die relative poly(A ⁺ ) mRNA unterschied sich zwischen den verschiedenen Fütterungsgruppen für die untersuchten Gene nicht; SCAP war aber signifikant erniedrigt in in vitro gereiften Eizellen von supplementierten Färsen im Vergleich zu in vivo gereiften Eizellen, die von nur mit Grassilage gefütterten Tieren stammen.

Die Lipid-Profile wurden von Oozyten CLA- (n=37) und SA- (n=50) supplementierter Tiere mittels Desorptions-Elektrospray Ionisation Massenspektrometrie (DESI-MS) erstellt.

Die Fettsäureanalyse mittels DESI-MS ergaben, dass die CLA-Supplementation zu einem Anstieg der Triglyceride 52:3 [TAG (52:3)] und TAG (52:2), Squalen, Palmitinsäure (C16:0) und Ölsäure (C18:1)100, und einer Abnahme von TAG (56:3), TAG (50:2) und TAG (48:1) im Vergleich zu SA-supplementierten Tieren, führte. In vitro gereifte Eizellen CLA-supplementierter Tiere zeigten veränderte Lipidprofile, mit einer Anreicherung von TAG (52:3) und TAG (52:2) sowie Phosphatidylinositol 34:1 [Plo (34:1)], wohingegen Plasmalogene [PCp (38:4) und Pep (38:4)] und Palmitinsäure (C16:0) im Vergleich zu Oozyten von SA-supplemenierten Tieren deutlich erniedrigt waren.

In dieser Arbeit konnte erstmals gezeigt werden, dass sich eine Supplementierung

mit pansengeschützten Fettsäuren bei Färsen mit hoher Spezifität und Sensitivität in

verschiedenen Probenmaterialien (z.B. Blut, Follikelflüssigkeit und Oozyte)

nachverfolgen lässt. Die Supplementierung mit CLA hat einen signifikanten Einfluss

auf das Lipidmuster in Eizellen gezeigt. Diese Ergebnisse demonstrieren die enge

Beziehung zwischen Fütterung und Herdenfertilität und unterstützen die Bedeutung

des Rindermodells für das Verständnis von ernährungsabhängigen

Fertilitätsstörungen.

1 General Introduction

1.1 Fatty acid supplementation of dairy cows

Modern dairy cows have been selected over generations for high milk production, with many successful dairy herds yielding an excess of 13,500kg of milk per lactation/cow. This is associated with marked changes in metabolism during the transition from pregnancy to lactation (Drackley 1999). These fundamental physiological changes ensure provision of adequate nutrients for the calf, both prenatally and postnatally. After parturition, nutrient demand cannot be met through feed intake alone because the rate of the dry matter intake (DMI) increase is slower than the rate of milk energy output. These nutrient and energy deficits after parturition are physiologically met by mobilization of body fat and protein as well as by decreasing non-essential use of glucose in non-mammary tissue (Bauman et al.

1980). Moreover, as one of the hormonal shifts and changes in tissue responsiveness to the metabolic adaptations, somatotropin is increased around parturition and in early lactation, which in turn increases responsiveness of adipose tissue to lipolytic signals such as norepinephrine. The resulting non-esterified fatty acids (NEFA) mobilization from adipose tissue is used directly as fuel by muscle and other tissues and NEFA are converted in the liver to ketone bodies (acetoacetate, ß- hydroxybutyrate). The ketone bodies can serve as energy source by replacing glucose in some metabolic pathways, and thereby provide glucose for milk synthesis (Grum et al. 1996). However, in most of the cases this complex metabolic adaptation is not able to compensate for the high-energy demands and nutritional strategies need to be used. Since fats supplements of the diets can concentrate higher amounts of energy in a relatively small mass, they allow for a better relation of mass/energy, benefiting in this way the metabolic homeostasis and thus a healthy lactation, which is followed by the physiological reactivation of the reproductive function.

The influence of dietary fat supplements on reproductive performance is not clear.

Most of the published data comes from studies with nutritional rather than

reproductive objectives. These studies are often designed primarily to examine

supplemental fat effects on dry matter intake, digestibility, milk production and

composition, and do not focus on return to estrus, follicle growth and pregnancy rates

(Staples et al. 1998). As a result, many uncontrolled management factors have influenced reproductive parameters in these studies (Barton et al. 1996). A large number of cows with the same physiological, productive and reproductive characteristics is needed to ensure a reasonable chance of detecting small differences between treatments in important parameters such as number of inseminations prior to conception and pregnancy rates. Thus the acquisition of reliable and consistent conclusions about effects of fatty acid supplementation remains a challenge (Staples et al. 1998).

1.2 Rumen-protected fatty acid supplements

Fatty acid supplements defined as “rumen-protected” are designed to resist biohydrogenation by ruminal microorganisms and thus enhance the postruminal flow and reach higher concentration that facilitate intestinal absorption. Important requirements for a successful rumen protection of fatty acids include (i) consistent and predictable enhancement of unsaturated fatty acid flow to the duodenum, (ii) adequate release and absorption of the fatty acids in the intestines and (iii) minimal adverse effects on ruminal fermentation (Jenkins et al. 2007). The goal of ruminal fatty acid protection is to provide high amounts of the two essential fatty acids linoleic and linolenic acid. Recent rumen protection technologies involve either encapsulation of unsaturated fatty acids inside a formaldehyde-treated protein shell or alteration of the fatty acid structure to resist the action of microbial enzymes (Børsting et al. 1992;

Faichney et al. 1972; Cook et al. 1972; Hogan et al. 1976). Fatty acid amides were developed for the first time at Clemson University (South Carolina, USA) to avoid ruminal biohydrogenation and increase contents of unsaturated fatty acids in milk and red meat. The amides consist of an unsaturated fat source such as linoleic acid or soybean fatty acids, which are chemically linked through an amide bond to an amine source, such as butylamine, ethanolamine or ammonia (Steen et al. 1989).

Ruminal biohydrogenation is avoided because a free acid or carboxyl group is a

target for microbial hydrogenation (Harfoot et al. 1997). Another form of protection of

fatty acid against ruminal degradation is the formation of calcium salt of fatty acids

(Klusmeyer et al. 1991; Chouinard et al. 1998). Calcium salts of fatty acids were

developed at Ohio University (Ohio, USA) for the first time as a form of rumen-inert

fat, even though they provide only a partial ruminal protection (Wu et al. 1991).

1.3 Conjugated linoleic acid (CLA)

Conjugated linoleic acid (CLA) is a general term given to a mixture of positional and geometric isomers of the linoleic acid (C18:2, n6). Linoleic acid is an essential fatty acid and must be provided by the diet since desaturation of fatty acid does not occur at positions greater than Δ ⁹ . Linoleic acid is an essential fatty acid required for the synthesis of arachidonic acid (C20:4, n6) and eicosanoids, which are important factors in many biological processes.

Food products derived from ruminants are the major source of CLA in human diets.

More than three decades ago, the role of CLA as “functional food” was identified when it was found that ground beef contained an anticarcinogenic factor that consisted of a series of conjugated dienoic isomers of linoleic acid (Pariza et al.

1979; Ha et al. 1987; Pariza et al. 1985). Subsequent studies observed that dietary CLA reduce the incidence of tumors in animal models for mammary, forestomach, colon and skin tumorgenesis (Banni et al. 1998; Belury 1995; Scimeca et al. 1994).

Indeed, the National Academy of Science of the United States of America published the document “ Carcinogens, and Anticarcinogens in the Human Diet” in 1996 and concluded that conjugated linoleic acid (CLA) is the only fatty acid that unequivocally inhibits carcinogenesis in experimental animals. Moreover, positive effects associated with CLA in experimental models include reduction in body fat content, antidiabetic effects, reduction in the development of atherosclerosis, enhanced bone mineralization and modulation of the immune system (Belury 1995; Banni et al. 1998;

Houseknecht et al. 1998).

1.3.1 Biosysnthesis of CLA

In ruminants, CLA originate from two sources. The first one is CLA as product of the partial biohydrogenation of linoleic acid in the rumen. The second source is CLA synthesized by animal tissues from trans-11 C18:1, which is another intermediate in the biohydrogenation of unsaturated fatty acids (Griinari et al. 1999).

1.3.2 Rumen biohydrogenation

The lipid composition of forages consists largely of glycolipids and phospholipids,

and the major fatty acids are the unsaturated fatty acids linolenic (C18:3) and linoleic

(C18:2) acid. When consumed by ruminants, dietary lipids undergo two important transformations in the rumen compartment (Dawson et al. 1977; Dawson et al. 1970;

Keeney 1970; Harfoot et al. 1997). The initial transformation is hydrolysis of ester linkages catalyzed by microbial lipases. This step is a prerequisite for the second transformation, which is the biohydrogenation of the unsaturated fatty acids. For several years, Butybivibrio fibrisolvens was the only bacterium known to be responsible of biohydrogeation (Kepler et al. 1966). However, as research efforts expanded, other rumen bacteria have been identified to be capable to biohydrogenate unsaturated fatty acids (Harfoot et al. 1988).

The first step of the biohydrogenation of linoleic acid is the isomerization of the cis-12 double bond. The isomerase reaction is unusual, since it has no cofactor requirement and occurs in the middle of a long hydrocarbon chain, remote from any activating functional group. Linoleate isomerase is the enzyme responsible for forming conjugated double bonds from the cis-9, cis-12 double bond structure of linoleic as well as α- and γ-linolenic acid.

The second reaction is a reduction in which it is converted to trans-11 C18:1. This hydrogenation occurs less rapidly and therefore cis9,trans11-CLA concentration increases in the rumen. The subsequent reduction of trans-11-C18:1 seems to be rate-limiting in the biohydrogenation of C18 fatty acids. The complete biohydrogenation process of both linoleic and linolenic fatty acid terminates with the formation of stearic acid (C18:0) (Griinari et al. 1999). Rumen biohydrogenation of linoleic acid is depicted in Figure 1.

1.3.3 Tissue synthesis of CLA

Studies performed to evaluate milk fat contents of dairy cows demonstrated that the trans11-C18:1 isomer is related to cis9,trans11-CLA concentrations in milk fat in a linear manner, and this relationship was observed in a wide range of diets (Jahreis et al. 1997; Jiang et al. 1996). This is generally attributed to the fact that these molecules act as intermediates in ruminal biohydrogenation. It has been hypothesized that non-ruminal cis9,trans11-CLA would originate from the desaturation of trans11-C18:1 by Δ ⁹ -desaturase (Corl et al. 1998; Corl et al. 2001).

Moreover, adipose tissue seems to be a major site of endogenous synthesis of

cis9,trans11-CLA in growing ruminants (Cameron et al. 1994; Chang et al. 1992;

Kinsella 1972; Page et al. 1997), while the mammary gland is the apparent site for the major endogenous synthesis of cis9,trans11-CLA for lactating ruminants, based on the activity of Δ ⁹ -desaturase (Bickerstaffe et al. 1970).

Linolenic acid C18:3 cis9,cis12, cis15

C18:3 cis9,trans11,cis15

C18:2 trans11,cis15

Linoleic acid C18:2 cis9,cis12

Rumenic acid C18:2 cis9,trans11 CLA

Vaccenic acid C18:1 trans11

Stearic acid C18:0

Rumen

Mammary gland/ adipose tissue

Rumenic acid C18:2 cis9, trans11 CLA

Vaccenic acid C18:1 trans11

Δ

-desaturase

Figure 1. Pathways for ruminal and endogenous synthesis of cis9,trans11-CLA (Rumenic acid).

Pathways for biohydrogenation of linoleic and linolenic acids yielding trans11-C18:1 (Vaccenic acid) are shown in the rumen cavity and endogenous synthesis by Δ ⁹ -desaturase is shown in the mammary gland/adipose tissue box. Modified from Bauman et al (2003).

1.4 Rumen-protected CLA supplementation

Conjugated linoleic acid supplementation of ruminant diets has become a common strategy to improve the energy status of high-yielding dairy cows during lactation.

One reason for this is the isomer-specific effects of CLA. In the field of animal and

human nutrition the most investigated CLA isomers, are cis9,trans11-CLA and

trans10,cis12-CLA (Figure 2). To increase the availability of CLA for absorption in

the abomasum and the intestine, a number of processes have been used to produce

rumen-protected supplements, and their efficacy is characterized by the extent of

their protection from rumen bacteria (Wu and Papas, 1997). The majority of studies

using rumen-protected CLA employed supplements consisting of calcium salts of free

fatty acids (FFA) (Giesy et al. 2002; Perfield et al. 2002; Bernal-Santos et al. 2003).

However, other technologies to protect CLA against ruminal degradation have been developed, including amide bonds and lipid-encapsulated supplements that contain methyl esters of CLA.

cis9 cis12

trans11 cis9

trans10

cis12

(a)

(b)

(c)

Figure 2. Chemical structure of conjugated linoleic acid (CLA) isomers and linoleic acid. (a) cis9,cis12-octadecadienoic acid (linoleic acid), (b) cis9,trans11-CLA, (c) trans10,cis12-CLA.

1.5 CLA supplementation and cattle fertility

The majority of studies using CLA supplementation of the daily diet has been

performed in the high-yielding dairy cows. The reason for this could be the enormous

energy demand of lactating cows during the transition period, in which energy intake

is unable to meet the requirements for milk synthesis and the other physiological requirements. To compensate for energy requirements, the animals mobilize lipids from the adipose depots. Dietary fat supplements in early lactation may benefit reproductive outcome by improving energy intake and reducing the extent of the negative energy balance, as well as by increasing size of the ovulatory follicle and lifespan of the corpus luteum (Mattos et al. 2000). The trans10,cis12-CLA isomer causes reduction of milk fat synthesis, and studies in dairy cows have established that CLA-induced milk fat depression is an important approach to prevent the negative energy balance during early lactation. A decrease of the interval between postpartum and first ovulation and increased pregnancy rates have been shown after supplementation of the diet with a mixture of CLA providing less than 10g/d of trans10,cis12-CLA (De Veth et al. 2009). Dietary polyunsaturated fatty acids (PUFA) may mediate suppression of uterine PGF 2α synthesis induced by the developing conceptus, having the potential to decrease embryo losses (Mattos et al. 2000). CLA has been shown to inhibit the prostaglandin synthesis in in vitro models (Belury 2002), but in vivo uterine PGF 2α responses still need to be studied. Recently, high-yielding dairy cows and heifers supplemented with a rumen-protected CLA daily ration showed a significantly increased blood progesterone concentration and down- regulation of genes critically involved in luteal function such ECE1, PTGS2, PTGFR, STAR and HSD3B1 (Stinshoff et al. 2011) Although there are positive effects of CLA supplementation in reproductive performance of dairy cows, the mechanisms of action and the possible isomer-specific effects of CLA need to be clarified.

1.6 Fatty acid effects on bovine oocyte quality and embryo development

Free fatty acids (FFA) are stored as triacylglycerols (TAG), which constitute uncharged esters of glycerol arranged as virtually anhydrous cytoplasmic droplets (McKeegan et al. 2011) and represent a compact energy reserve (McEvoy et al.

2000). The esterification of fatty acids and storage in lipid droplets may also protect the oocyte against fatty acid-induced lipotoxicity (Listenberger et al. 2003).

Cholesterol (Chol) and phospholipids (PL) are essential for the formation of cellular

membranes and are critically important for cell division after fertilization. However,

the lipid abundance in oocytes and embryos seems to be species-specific (Prates et

al. 2013). Interestingly, oocytes and embryos of mammalian species, including

porcine, bovine and ovine species contain high quantities of lipids in their cytoplasm

(Coull et al. 1998; Ferguson and Leese 1999; McEvoy et al. 2000). Cat oocytes are also high in lipid concentration (Guraya 1965). On the other hand, oocytes and embryos from mice are pale, and contain a low level of endogenous lipids (Loewenstein and Cohen 1964). Human oocytes have been shown to possess an intermediate level of endogenous lipids (Matorras et al. 1998).

Oocyte development is a multistep process, including nuclear and cytoplasmic maturation. During progression from the primordial follicle to the mature oocyte in metaphase II, there are several periods when the oocyte accumulates lipids. Based on in vitro oocyte maturation experiments, the final stages of maturation are the most susceptible to cytoplasmic modifications of the lipid contents. The vast majority of studies that investigated effects of specific fatty acids on oocyte quality and embryo development have been performed under in vitro conditions. Indeed, the supplementation of linolenic acid into bovine maturation medium in vitro increased the number of oocytes that reached the metaphase II stage and more oocytes exhibited active mitogen-activated protein kinase (MAPK) signaling pathways (Marei et al. 2010). In another in vitro experiment, the three predominant fatty acids in follicular fluid (palmitic, stearic and oleic acids) were added to in vitro maturation medium of bovine oocytes. Palmitic and stearic acids had a dose-dependent inhibitory effect on the amount of fat stored in lipid droplets, associated with detrimental effects on oocyte developmental competence. In contrast, oleic acid showed a positive effect, which increased lipid storage in droplets, and thus enhancing oocyte development capacity. Even more, adverse effects of palmitic and stearic acid were counteracted by oleic acid (Aardema et al. 2011). Moreover, CLA supplementation of in vitro culture medium of bovine embryos decreased blastocyst rates after 50uM trans10,cis12-CLA and 50uM cis9,trans11-CLA supplementation. In addition, genes involved in oocyte lipid metabolism were down-regulated showing that CLA affects bovine embryonic fat metabolism (Stinshoff et al. 2013).

1.7 Determination of fatty acid profiles in oocytes and embryos

Oocytes and preimplantation embryos of mammals, including mice, cattle and

humans are of microscopic dimensions (100-200μm in diameter) with a total lipid

content of few nanograms. Non-fertilized mouse oocytes contain approximately 3.8

ng total lipids (Loewenstein et al. 1964), and bovine oocytes contain around 63 ng

total lipids, thereby phospholipids comprising a quarter of the total (McEvoy et al.

2000). Structural studies on lipids and lipid complexes in single oocytes and embryos clearly represent an analytical challenge for improving assisted reproductive techniques such as in vitro embryo production and cryopreservation. Knowledge on the biological function of lipids within the cell has been derived from the development of increasingly sensitive and selective analytical techniques, particularly those based on mass spectrometry (Roberts et al. 2008; Ejsing et al. 2009). Attempts to characterize lipid composition of bovine oocytes/embryos have been performed primarily because lipid composition affects cryosensivity and thus post-thaw survival of bovine embryos. These experiments used pools of hundreds of embryos to obtain sufficient amounts of lipid for gas chromatographic analysis (McEvoy et al. 2000; Kim et al. 2001). Employing approximately 1000 oocytes/assay, palmitic, stearic and oleic acids were found to be the predominant fatty acids and their proportion varied in a species-specific manner in cattle, pig and sheep oocytes (McEvoy et al. 2000).

Another drawback of the traditional methods like gas chromatography and radioactive labeling used for analysis of lipid content in oocytes and embryos is the demand of lipid extraction with organic solvents, followed by chemical manipulation (hydrolysis/ derivatization), separation and characterization by chromatographic techniques (Matorras et al. 1998; Kim et al. 2001; Haggarty et al. 2006). A novel technique, matrix-assisted laser desorption/ionization mass spectrometry (MALDI- MS) has been performed to analyze complex lipid profiles (i.e., phosphatidylcholines, sphingomyelins and triacylglycerols) in single human, sheep and fish oocytes and bovine in vitro produced embryos and oocytes.

In previous studies, MALDI-MS analysis has provided lipid profiles of single gametes

from different mammalian species and bovine embryos (Ferreira et al. 2010). Another

analytical tool, desorption electrospray ionization high-resolution mass spectrometry

(DESI-MS) has been used for elucidating lipid profiles from single oocytes and

embryos. DESI-MS reduces sample preparation steps needed for lipid analysis and

has originally been used for mass spectrometric analysis and imaging of drugs,

metabolites and lipids directly from biological samples (Harris et al. 2011; Eberlin et

al. 2011; Huang et al. 2011). Important differences between DESI-MS lipid profiling

and GC analysis include the following aspects. First, in the GC analysis, structural

information on complex lipids is lost because the esterification process involves

disassembling the fatty acyl residues from complex lipid structures. In contrast, by DESI-MS the intact components of complex lipids are ionized so that most fatty acyl residues are detected as subunits of complex lipid structures. Second, sample preparation is minimized by DESI-MS, so that no lipid extraction or methylation is needed. Third, intact single samples are used in DESI-MS procedures making individual profiling possible with a high repeatability and a little variation (less than 20%). DESI-MS and MALDI-MS are considered to be complementary to each other (Ferreira et al. 2012).

1.8 Motivation and goals of this study

Conjugated linoleic acid supplementation of the diet of high-yielding dairy cows is a strategy commonly used to manage energetic demands during the lactation period.

Reduction of milk fat synthesis affects indirectly reproductive performance by saving

energy originally destined for milk production and redirecting this energy to other

physiological functions, such as reactivation of ovarian follicular growth thus

preparing the cow for the start of a new reproductive cycle. Nearly all experimental

animals used for such CLA-supplementation experiments were lactating cows, which

makes it difficult to separate the effects of CLA as lipid molecule, able to concentrate

important amounts of energy, and their isomer-specific effects on female gametes. At

the start of this project (October 2010) no data had been reported regarding direct

effects of diet supplementation with conjugated linoleic acid on the quality of oocytes

and embryo developmental capacity. The young heifer animal model was chosen in

this study in order to address the main question regarding specific effects of lipid

molecules, in this case conjugated linoleic acids, on the quality of oocytes collected

from diet supplemented donors. The host laboratory at the Institute of Farm Animal

Genetics, FLI, Mariensee, has extensive experience in embryology and reproductive

assisted techniques of farm animals including in vitro production of embryos and

follicular-guided oocyte retrieval from living donors by ovum pick-up (OPU) (Oropeza

et al. 2004; Zaraza et al. 2010; Diederich et al. 2012; Heinzmann et al. 2011). The

cooperation with the Institute of Animal Nutrition FLI in Braunschweig, Germany,

provided the technical platform for preparation of the rumen-protected fatty acid

supplements used in these experiments. Lipid profiles of bovine oocyte and embryos

were analyzed at the laboratory of Professor Robert Graham Cooks at the Purdue

University (Indiana, USA). This research group is a world leader in developing high-

resolution mass spectrometry protocols. As a result of the joint efforts, expertise of

the two groups made possible the improvement and adaptation of desorption

electrospray ionization mass spectrometry (DESI-MS) technique for a fatty acid

profiling of single bovine oocytes and embryos. This technique combined with gene

expression analysis and follow-up of fatty acid profiles throughout different body

compartments (i.e., blood circulation and follicular fluid) allowed for monitoring of

physiological responses during and after the CLA supplementation of heifers.

2 Publication 1

Desorption electrospray ionization mass spectrometry reveals lipid metabolism of individual oocytes and

embryos

Andrés Felipe González-Serrano ¹ , Valentina Pirro ² , Christina R. Ferreira ³ , Paolo Oliveri ⁴ , Livia S. Eberlin ³ , Julia Heinzmann ¹ , Andrea Lucas-Hahn ¹ , Heiner Niemann ¹ , Robert Graham Cooks ³

1 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, Hoeltystrasse 10, 31535 Neustadt, Mariensee, Germany. Fax: +49 5034 871 143; Tel: +49 5034 871 136/148. E-mail:

heiner.niemann@fli.bund.de

2 Department of Chemistry,University of Turin, Via Pietro Giuria 7, Turin 10125, Italy

3 Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, IN 47907, USA. Fax:

765-494-9421; Tel: 765-494-5263; E-mail: cooks@purdue.edu

4 Department of Pharmacy, University of Genova, Via Brigata Salermo 13, Genova 16147, Italy

Published in PloS ONE. DOI: 10.1371/journal.pone.0074981 (September 20 ^th , 2013)

Contribution of Andrés Felipe González Serrano to this publication: 80%

Corresponding authors:

Robert Graham Cooks Purdue University 560 Oval Drive

West Lafayette, IN 47907, USA Email: cooks@purdue.edu Fax: 765-494-9421

Tel: 765-494-5263

Heiner Niemann

Institute of Farm Animal Genetics (FLI) Mariensee, Höltystr. 10

31535 Neustadt

Email: heiner.niemann@fli.bund.de Fax: +49 5034 871 143

Tel: +49 5034 871 136

Abstract

Alteration of maternal lipid metabolism early in development has been shown to trigger obesity, insulin resistance, type 2 diabetes and cardiovascular diseases later in life in humans and animal models. Here, we set out to determine (i) lipid composition dynamics in single oocytes and preimplantation embryos by high mass resolution desorption electrospray ionization mass spectrometry (DESI-MS), using the bovine species as biological model, (ii) the metabolically most relevant lipid compounds by multivariate data analysis and (iii) lipid upstream metabolism by quantitative real-time PCR (RT-qPCR) analysis of several target genes (ACAT1, CPT1b, FASN, SREBP1 and SCAP). Bovine oocytes and blastocysts were individually analyzed by DESI-MS in both positive and negative ion modes, without lipid extraction and under ambient conditions, and were profiled for free fatty acids (FFA), phospholipids (PL), cholesterol-related molecules, and triacylglycerols (TAG).

Principal component analysis (PCA) and linear discriminant analysis (LDA),

performed for the first time on DESI-MS fused data, allowed unequivocal

discrimination between oocytes and blastocysts based on specific lipid profiles. This

analytical approach resulted in broad and detailed lipid annotation of single oocytes

and blastocysts. Results of DESI-MS and transcript regulation analysis demonstrate

that blastocysts produced in vitro and their in vivo counterparts differed significantly

in the homeostasis of cholesterol and FFA metabolism. These results should assist in

the production of viable and healthy embryos by elucidating in vivo embryonic lipid

metabolism.

3 Publication 2

Nutritional footprints on bovine oocyte quality and embryo development after long-term feeding rumen-protected fatty acid supplements

AF González-Serrano ¹ , CR Ferreira ² , V Pirro ³ , A Lucas-Hahn ¹ , J Heinzmann ¹ , K-G Hadeler ¹ , U Baulain ¹ , P Aldag ¹ , U Meyer ⁴ , M Piechotta ⁵ , G Jahreis ⁶ , S Dänicke ⁴ , RG Cooks ² and H Niemann ¹

1 Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut (FLI), 31535 Neustadt, Germany; ² Department of Chemistry and Center for Analytical Instrumentation Development, Purdue University, West Lafayette, 47907 IN, USA; ³ Department of Chemistry, University of Turin, 10125 Turin, Italy; ⁴ Institute of Animal Nutrition, Friedrich-Loeffler-Institut (FLI), 38116 Braunschweig, Germany; ⁵ Clinic for Cattle, University of Veterinary Medicine, 30173 Hannover, Germany, ⁶ Institute of Nutrition, Friedrich Schiller University of Jena, 07743 Jena, Germany

Submitted/ under review Corresponding authors:

Contribution of Andrés Felipe González Serrano to this publication: 90%

Running title: Effects of diet on oocyte lipid composition

Summary sentence: Plasmalogens were significantly more abundant in mass spectra of in vitro matured bovine oocytes after rumen-protected stearic acid supplementation when compared to those collected from animals supplemented with rumen-protected conjugated linoleic acid.

Key words: Desorption electrospray ionization mass spectrometry (DESI-MS), bovine oocytes, blastocysts, rumen-protected fat supplements, plasmalogens, ultrasound-guided follicular aspiration, gene expression, lipid metabolism.

Heiner Niemann

Institute of Farm Animal Genetics (FLI) Mariensee, Höltystr. 10

31535 Neustadt

Email: heiner.niemann@fli.bund.de Fax: +49 5034 871 143

Tel: +49 5034 871 136

Robert Graham Cooks Purdue University 560 Oval Drive

West Lafayette, IN 47907, USA Email: cooks@purdue.edu Fax: 765-494-9421

Tel: 765-494-5263

Abstract

It is now well established that nutritional and environmental conditions around

conception and during early embryonic development may have significant effects on

health and well-being in adult life. Here, we used the bovine model to investigate the

effects of rumen-protected fat supplements on oocytes and early embryos. Holstein-

Friesian heifers (n=84) received a diet supplement consisting of rumen-protected

conjugated linoleic acid (CLA) or stearic acid (SA), each on top of a grass silage

basic diet. Oocytes were collected via ultrasound guided follicular aspiration and

subjected to in vitro maturation (IVM) followed by either desorption electrospray

ionization mass spectrometry (DESI-MS) for lipid profiling of individual oocytes or in

vitro fertilization and embryo culture. The type of supplement significantly affected

lipid profiles of IVM oocytes. Plasmalogens were more abundant in the mass spectra

of in vitro matured oocytes after rumen-protected stearic acid supplementation when

compared to those collected from animals supplemented with CLA. Plasmalogens

are phospholipids commonly associated with human diseases like cardiac failure,

obesity and type II diabetes, and are herein reported for the first time in mammalian

oocytes. Lipid concentrations in blood and follicular fluid were significantly affected in

a dose-dependent manner by both supplements. CLA supplementation increased

cholesterol plasma levels. Results show that nutrition and fertility are closely linked

with each other and pave the way for the establishment of a large animal model for

studies towards a better understanding of metabolic disorders associated with human

infertility.

3.1 Introduction

Rumen-protected fat supplementation of the daily diet is commonly used for improving the energy status and concomitantly fertility in high yielding dairy cows.

Different types of fatty acids and combinations thereof from vegetal and animal sources have been successfully used for this purpose [1, 2]. Protection of lipids against degradation in the rumen can be achieved by formaldehyde treatment, high melting point saturated fatty acid encapsulation and blocking the carboxyl group of fatty acids by forming an ionic bound with calcium or by formation of fatty acid amides [3]. This protection of lipids is necessary to increase intestinal absorption by preventing their ruminal degradation.

Conjugated linoleic acids (CLA) are fatty acids composed of an 18-carbon chain with two double bonds separated by one single bound between them as a typical feature.

CLA diet supplementation of dairy cows increased IGF1 plasma concentration, which in turn improved fertility of dairy cows [4]. In addition, plasma concentrations of NEFA, which are critically involved in the negative energy balance during the early lactation period were significantly reduced after diet supplementation of dairy cows with rumen-protected CLA [5]. Importantly, reproductive parameters, including the probability to get pregnant, the interval from parturition to successful insemination and the onset of first ovulation postpartum were improved after feeding rumen- protected CLA supplements to dairy cows [6]. However, the underlying mechanisms through which CLA bovine diet supplementation improves bovine female fertility remain an enigma. It has also been shown that CLA supplementation of human diets is associated with several health benefits, including anti-mutagenic properties [7, 8]

and anti-carcinogenic effects [9-12]. Indeed, the CLA constitute a mixture of positional and geometric isomers of linoleic acid (cis9,cis12-octadecadienoic acid) the exact nature of which is critical since CLA biological activities can be related to specific isomers. The two best investigated CLA isomers with a high metabolic activity in animal and human diet supplements are cis9,trans11-CLA and trans10,cis12-CLA. Ruminants produce high amounts of endogenous CLA as intermediates in the biohydrogenation of exogenous linoleic acid [13] and these CLA can be observed in dairy products mainly as the isomer cis9,trans11-CLA [14].

Moreover, milk fat synthesis was decreased by the trans10,cis12-CLA [15] by

specifically inhibiting key lipogenic enzymes involved in the de novo synthesis of milk fatty acids [16, 17].

Recently, we reported the lipid profile of bovine individual oocytes and embryos by DESI-MS and identified several genes that are critically involved in lipid metabolism of bovine oocytes and in vitro produced preimplantation embryos [18]. In vitro produced embryos presented a distinct lipid profile compared to their in vivo derived counterparts. The direct effects of lipid feed supplements on lipid contents in oocytes and early embryos raises the question on how fatty acids are incorporated in female gametes and/or early embryos. Moreover, nutritional and environmental conditions around conception and during early embryonic development are well known to exert long-term effects on health and well-being in adult life [19-21]. However, the mechanisms and bioactive lipids involved are poorly understood. To further understand the relationship between nutrition and fertility, we have investigated rumen-protected CLA supplementation impact in different body compartments (oocyte, blood, follicular fluid) using lipid and gene expression analysis as well as characterized CLA effects on oocyte quality and in vitro embryo development in a bovine heifer model.

3.2 Material and methods

Experimental groups, supplementation diets and experimental design

The experiments were conducted according to the German animal welfare regulations and the guidelines of LAVES (Lower Saxony State Office for Consumer Protection and Food Safety). Holstein-Friesian heifers (16-18 months old) were fed individually in tie-stalls with an isocaloric grass silage diet and water ad libitum. The experiments were performed between April 2011 and March 2013. Heifers (n=84) were divided in groups of 6-10 animals each for the dietary treatments.

Subsequently, groups were separated in subgroups so that one received a rumen-

protected CLA mixture in which trans10,cis12-CLA and cis9,trans11-CLA were

prevalent isomers while the other group received a rumen-protected fat preparation

in which the CLA content was substituted by stearic acid (C18:0) in order to obtain an

isocaloric diet. Two different doses of the CLA and stearic acid containing fat

supplements (100 g/d [CLA100 and SA100] and 200 g/d [CLA200 and SA200]) were

used during the experiments. The fat supplements were mixed into a concentrate feed (Table S1) which was fed individually at an amount of 2 kg/d in two equal portions. Each individual feed supplementation period lasted 3 months and consisted of an adaptation period of 15 days, followed by the main supplementation period of 45 days, followed by oocyte retrieval performed twice per week over 30 days at 3 or 4 days intervals. In parallel, another subgroup of heifers received only grass silage for collecting immature and in vivo matured oocytes to be used for gene expression analysis. The animals were weighed at the beginning and at the end of the supplementation period and body condition (BCS) was determined weekly using a 1- 5 scale [22] and just heifers with approximately 300 kg of body weight and a BCS between 3-3.5 points were selected for the experimental groups.

Grass silage and concentrate samples were collected weekly for chemical analysis (dry matter, crude ash, crude protein, ether extract, NDF and ADF) following the established methods of the Association of German Agricultural Analysis and Research Center (VDLUFA, 1993). The collected samples were pooled prior to analysis. The chemical composition of feedstuff is presented in the supplementary information (Table S1).

Table1. Fatty acid profiles of fat supplements.

CLA containing in fat supplement were mixed in the concentrates as a rumen-protected preparation. In the SA supplement CLA were substituted by stearic acid (C 18:0 ).

Fatty Acid Methyl Ester.

Fatty Acid (% of total FAME

) CLA SA

C 16:0 10.9 10.9

C 18:0 50.3 87.3

C 18:1 cis-9 10.7 <0.01

Conjugated Linoleic Acid

C 18:2 cis-9, trans-11 12.0 0.1

C 18:2 trans-10, cis-12 11.9 0.02

Other CLAs 0.9 0.1

Other fatty acids 3.3 1.6

Silage dry matter was monitored twice a week using the forced-air oven technique (114 °C for 24 h). Fatty acid supplements were analyzed for verification of lipid contents by established methods [23]. The fatty acid profile of concentrates is given in Table 1.

Blood sample collection

Blood samples (~8 mL) were collected from the Vena jugularis externa at the beginning and the end of the supplementation period by means of a 10 mL syringe and an 18 G sterile needle (Terumo Europe, Leuven, Belgium) and immediately placed in sample tubes with EDTA. Subsequently, samples were centrifuged (1000xg for 10 min) with a centrifuge pre-cooled to 8 °C (Megafuge 1.0 R) for separating erythrocytes and plasma. At least 3 mL plasma were pipetted and frozen in two different cryotubes; one was used for determination of cholesterol and NEFA and the second one was used for determination of IGF1 concentrations. A total of 5 mL PBS (Applichem, Darmstadt, Germany) was added to the erythrocytes samples followed for centrifugation (1000xg for 5 min). The supernatant was removed and erythrocytes were washed again with 5 mL of PBS and centrifuged for 10 min at 1000xg.

Erythrocytes and plasma samples were frozen at -20 °C until further analysis.

Oocyte and follicular fluid retrieval by ovum pick-up

Cumulus-complex-oocytes (COCs) were collected by ultrasound-guided follicular

aspiration (OPU) as reported [24, 25]. Briefly, an epidural anesthesia was performed

in the experimental animals by injection 3.5 mL of Procasel-2 %

(Procainhydrochlorid; Selectavet, Weyarn-Holzolling, Germany). Ovaries were

visualized using a ProsoundSSD-4000SV ultrasound device (Aloka, Tokyo, Japan)

connected to a UST-987 7.5 MHz ultrasound transducer (Aloka, Tokyo, Japan). The

ultrasound transducer was covered with a hygienic protection cover (Servoprax ^® ,

GmbH, Wesel, Germany). Ovarian follicles (3-10 mm) were aspirated after ten weeks

of fatty acid supplementation (i.e., CLA and SA) and from the non-supplemented

heifers using a 20 G disposable needle (Terumo Europe, Leuven, Belgium) fitted to a

vacuum pump (IVF Ultra quiet; Cook Veterinary Products, Moenchengladbach,

Germany) connected to a flexible plastic tube. The aspiration pressure was adjusted to 40 mmHg. After aspiration of a maximum of four follicles, the system was rinsed with PBS (Applichem, Darmstadt, Germany) supplemented with 1 % heat-inactivated newborn calf serum (NBCS; PPA Laboratories, Coelbe, Germany), 36 μg/mL sodium pyruvate (Applichem, Darmstadt, Germany), 1 mg/mL glucose (Roth, Karlsruhe, Germany), 133 μg/mL calcium chloride dehydrate (Fluka, Munich, Germany), 50 μg/mL streptomycin (Applichem, Darmstadt, Germany), 6 μg/mL penicillin G (Applichem, Darmstadt, Germany), and 2.2 IU/mL sodium heparin (Applichem, Darmstadt, Germany). At the beginning and the end of the fatty acid supplementation period, one ovarian follicle (approx. 6-8 mm) was aspirated to collect follicular fluid that was placed in a 2 mL cryovial. The follicular fluid was immediately transported to the lab and centrifuged for 5min at 1000xg at 10 C°, for separating fluid from tissue material. The supernatant was pipetted, collected in two cryovials (one for IGF1 analysis and the other one for fatty acid profiling) and frozen at -20 °C until further analysis.

In vitro oocyte maturation and embryo production

COCs collected by OPU were selected under a stereomicroscope in TCM-air (TCM199, Sigma-Aldrich, Munich, Germany) supplemented with 50 μg/mL gentamycin sulphate (Sigma-Aldrich), 0.2 mM sodium pyruvate (Sigma-Aldrich), 4.2 mM NaHCO 3 (Roth, Karlsruhe, Germany), and 1 mg/mL BSA (Sigma-Aldrich), and only oocytes with at least three layers of compact cumulus cells with an homogeneous granulated cytoplasm were used for the experiments [26].

Maturation of oocytes and in vitro embryo production were carried out as described recently [18]. Briefly, the maturation medium consisted of TCM199 at pH 7.4, supplemented with 0.2 mM sodium pyruvate, 25 mM NaHCO 3 , 50 μg/mL gentamycin, 10 IU/mL eCG, 5 IU/mL of hCG (Suigonan ^® , Intervet, Tönisvorst, Germany), and 0.1

% fatty acid free BSA (Sigma-Aldrich, Munich, Germany). Oocytes were matured in a

humidified atmosphere composed of 5 % CO 2 at 39 °C for 24 h under silicone oil. For

in vitro fertilization, COCs were placed in Fert-TALP medium containing HHE (10 μM

hypotaurine (Sigma-Aldrich), 1 μM epinephrine (Sigma-Aldrich), and 0.1 IU/mL

heparin (Serva, Heidelberg, Germany)), and 6 mg/mL BSA [27, 28]. Frozen semen

from one bull of proven fertility was thawed at 30 °C for 25 sec and layered carefully

on 1 mL of BoviPure™ 90 % (Labotec, Goettingen, Germany). The semen was centrifuged at 400 g for 10 min, followed by removal of the supernatant and re- suspension in 750 μL of fertilization medium (Fert-TALP) containing 6 mg/mL BSA (fraction V) and centrifuged at 400xg for 3 min. This washing step was repeated once using Fert-TALP containing HHE. Finally, the supernatant was completely removed.

The final sperm concentration added per 100 μL/fertilization drop was 1 ×10 ⁶ spermcells/mL. COCs and sperm cells were co-incubated for 19 h under silicone oil at 5 % CO 2 in air at 39 °C. Modified synthetic oviduct fluid (mSOF) medium supplemented with BSA-FAF was employed for in vitro culture [29]. Presumptive zygotes were transferred into drops containing 30 μL of mSOF after complete removal of the adhering cumulus cells by repeated pipetting. Embryos were cultured under silicone oil (Serva, Heidelberg, Germany) at 39 °C in a humidified atmosphere composed of 5 % CO 2 and 5 % O 2 to the expanded blastocyst stage (day 8) [30, 31].

The respective in vitro maturation and embryo development (day 8) rates were recorded.

DESI-MS analysis and attribution of lipid species

Lipid profiles were determined as reported recently [18]. Briefly, oocytes were stored in minimal volume (2-5 μL) of PBS supplemented with 0.1 % polyvinyl alcohol (PVA) and shipped on dry ice from the Institute of Farm Animal Genetics (Mariensee, Germany) to Purdue University (West Lafayette, IN, USA; USDA permit 118624 Research). Mouse brain tissue sections and some of the samples were used for DESI system optimization (Purdue University Animal Care and Use Committee approved protocol No. 1111000314, see [32]).

Individual oocytes from the 100 g/d CLA-supplemented animals (in vitro matured

n=15; immature n=15) and from the 100 g/d SA-supplemented animals (in vitro

matured n=11; immature n=10) were submitted to DESI-MS analysis in the positive

ion mode by doping the spray solvent with silver nitrate in order to detect cholesteryl

esters and triacylglycerols (TAG) [18, 33-35]. Also by DESI-MS, oocytes from CLA-

supplemented animals (in vitro matured n=6; immature n=11) and from SA-

supplemented animals (in vitro matured n=10; immature n=7) were profiled in the

negative ion mode for FFA and PL using experimental conditions previously reported

[18, 32, 36].