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
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
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].
A Thermo Scientific Exactive (San Jose, CA, USA) mass spectrometer was employed for the experiments. DESI-MS profiles were acquired in lab-built stage and the DESI spray was positioned ~2 mm from the surface at an incident angle of 50°.
The DESI spray had 5 kV applied to the stainless steel needle syringe and nitrogen gas pressure was set at 180 psi. Detailed instrumental conditions were described recently [18].
Molecular formula matching and error calculations were performed using the instrument software Xcalibur v.1.0.1.03 (Thermo Fisher Scientific San Jose, CA, USA) and online search of lipids containing the calculated molecular formulae was carried out in the LIPID MAPS and METLIN database [37, 38]
IGF1, non-esterified fatty acid and cholesterol analysis
Plasma and follicular fluid samples were analyzed for concentrations of IGF1 in duplicate by a commercially available immunoradiometric assay according to manufacturer’s instructions (IRMA IGF1; IMMUNOTECH SAS, Marseille, France).
Briefly, insulin-like growth factor 1 was separated from its binding proteins by incubating 25 μL of the samples with 500 μL dissociation buffer, followed by mixing with a vortex-type shaker. The assay was tested for bovine plasma and follicular fluid by determining the recovery and intra- and interassay coefficient of variation. The recovery was tested by adding 200 ng/mL of IGF1 (obtained from the National Hormone and Peptide Program, NHPP; National Institute of Diabetes and Digestive and Kidney Diseases, NIDDK and the A. F. Parlow) to 30 plasma and 30 follicular fluid samples. Afterwards, the acid-ethanol extraction and IRMA was performed according the manufacturer’s instructions. Recovery rate ranged from 91 % and 103
%. The intra-assay coefficient of variation was ≤ 6.3 % and the inter-assay coefficient of variation was ≤6.8 %.
Plasma NEFA and cholesterol concentrations were measured using an automatic
clinical chemistry analyzer based on photometric detection (Eurolyser CCA180,
Eurolab, Hallein, Austria).
Lipid extraction and transmethylation of erythrocytes and follicular fluid
One mL of either erythrocytes or follicular fluid was used, respectively, for total lipid extraction based on the method of Bligh and Dyer [39] as reported previously [40].
Lipid extracts were transmethylated by using a combination of 0.5 N methanolic sodium hydroxide (Merck) and 10 % (w/w, Supelco) boron trifluoride-methanol (100
°C for 5 min each). Subsequently, fatty acid methyl esters (FAME) were purified by thin layer chromatography and dissolved in n-hexane for analysis [41].
Fatty acid methyl ester (FAME) profiling by gas chromatography
A system of two GC/FID methods was employed to analyze the full fatty acid spectrum from C4 to C26 including CLA (GC-17 V3 Shimadzu; DB-225MS: 60 m, i.d.
0.25 mm, 0.25 μm film thickness; Agilent Technologies) as well as cis and trans isomers of C18:1, trans C18:2 and C18:3 (GC-2010, Shimadzu; CP-select 200 m x 0.25 mm i.d. with 0.25 μm film thickness; Varian) [42]. Briefly, injector and detector temperatures were maintained at 260 °C and 270 °C, respectively, with hydrogen as carrier gas [42]. Fatty acid concentrations were expressed as the percentage of the total area of all FA peaks (% of total FAME).
Determination of the relative abundance of mRNA transcripts for IGF1R, GJA1, FASN, SREBP1 and SCAP by RT-qPCR
Poly (A)+ RNA from single oocytes and blastocysts collected from animals fed with the supplements CLA and SA was isolated using the Dynabeads® mRNA DIRECT™
kit (Invitrogen, Carlsbad, USA) as reported previously [25, 43]. Two μL of the RT
reaction were used for quantitative PCR (RT-qPCR). Quantitative PCR was
performed in 96-Well Optical Reaction Plates (Applied Biosystems, Darmstadt,
Germany). The PCR mix, in each well, included 10 μL 2X Power SYBR Green PCR
Master Mix (Applied Biosystems, Darmstadt, Germany), 7.2 μL dH 2 O, 0.4 μL each of
the forward and reverse primers (5 μM) in a final reaction volume of 20 μL. Rabbit
globin (50 fg) was amplified in parallel with the target genes for normalization. Primer
sequences of the target genes are summarized in Table S2. The PCR reaction was
carried out in an ABI 7500 Fast Real-Time System (Applied Biosystems, Darmstadt,
Germany) using the following program: denaturation and activation of the Taq
Polymerase for 10 min at 95 °C, followed by 40 cycles at 95 °C for 15 sec and 60 °C for 1 min and a final slow heating cycle to obtain dissociation curves. A cDNA standard dilution from pooled immature oocytes was included on every plate to generate standard curves for each target gene. Data were processed using the Sequence Detection Software 1.3.1 (Applied Biosystems, Darmstadt, Germany).
Relative mRNA concentration of each gene was calculated by the standard curve method. Normalization of the results obtained for each gene was performed by calculating the ratio of the target gene to the level of globin mRNA.
In vivo oocyte maturation
In vivo matured oocytes were produced from the grass-silage fed heifers as controls in the gene expression analysis. The estrus cycle of heifers fed with a grass silage diet was synchronized by means of an intrauterine device (PRID ® Delta 1.55 g progesterone, Ceva Sante Animale, Libourne, France). Five days after insertion of the device, two daily doses of FSH (12 h intervals), were injected over 4 days with decreasing doses of 2 mL, 2 mL, 1.5 mL, 1.5 mL, 1 mL, 1 mL, 0.5 mL and 0.5 mL) for a total of 10 mL of FSH (Pluset ® , Calier, Barcelona, Spain). At the time of the seventh and eighth injection of FSH, 3 mL Cloprostenol-Natrium (Estrumate ® 250 ug/mL, Intervet, Unterschleissheim, Germany) were injected and 48 h later, oocytes were collected via ovum pick-up. Oocytes were separated from the cumulus cells by incubating in 1 % hyaluronidase for 10 min under a stereomicroscope. After visualization of the first polar body, matured oocytes were washed three times with PBS and frozen at -70 °C in PBS-PVA until further analysis.
Statistical analysis
For each sample submitted to DESI-MS lipid profiling, a list of m/z values and ion
abundances (after background subtraction) from average mass spectra was imported
into Matlab (The MathWorks, Inc., Natick, USA). Multivariate data processing was
performed by means of in-house Matlab routines in order to explore the entire
complex information contained in the DESI mass spectra [18, 32]. Briefly, for the
positive ion mode, the mass range of m/z 600-1200 was structured with 60001 m/z
variables, whereas in negative ion mode, each representative mass spectrum was
structured with 65001 m/z variables in the reduced mass range of m/z 250-900.
Principal component analysis (PCA) was applied to the two different data sets (51
rows and 60001 variables; 34 rows and 65001 variables) in order to separately
explore the information contents detected using the positive (n= 51) and negative ion
mode (n= 34) mass spectra for characterizing samples according to the maturation
stage and CLA vs. SA diet supplementation. PCA was performed separately in the
positive and negative ion mode datasets on column-centered data, after
normalization with respect to total ion current (TIC) to correct unwanted signal
intensity variation of instrumental variability. NEFA plasma concentration, cholesterol
plasma level, IGF1 plasma and follicular fluid concentrations, plasma and follicular
fluid lipid profiles were analyzed by one-way ANOVA using JMP 8.0 (SAS Institute
Inc., Cary, NC, USA), followed by multiple comparisons of means using the
Tukey-Kramer test. Gene expression data were also analyzed by univariate one-way
ANOVA and pairwise comparison of means. For all tests, a p-value ≤ 0.05 was
considered as statistically significant. For the maturation and blastocyst rates a
Chi-square test was performed using R 2.15.3 (R Development Core Team (2008). R: A
language and environment for statistical computing. R Foundation for Statistical
Computing, Vienna, Austria) for comparing absolute values derived from in vitro
