Bovine embryo elongation is altered due to maternal fatty acid supplementation

Katrin Giller; Barbara Drews; Joel Berard; Hermine Kienberger; Marion Schmicke; Jan Frank; Britta Spanier; Hannelore Daniel; Gerd Geisslinger; Susanne E Ulbrich

doi:10.1093/biolre/ioy084

Bovine embryo elongation is altered due to maternal fatty acid supplementation

Giller, Katrin;Drews, Barbara;Berard, Joel;Kienberger, Hermine;Schmicke, Marion;Frank, Jan;Spanier, Britta;Daniel, Hannelore;Geisslinger, Gerd;Ulbrich, Susanne E 2018-04-12 00:00:00 Abstract The pre-implantation period is prone to embryonic losses in bovine. Embryo–maternal communication is crucial to support embryo development. Thereby, factors of the uterine fluid (UF) are of specific importance. The maternal diet can affect the UF composition. Since omega 3 fatty acids (omega 3 FA) are considered to be beneficial for reproduction, we investigated if dietary omega 3 FA affected factors in the UF related to embryo elongation. Angus heifers (n = 37) were supplemented with either 450 g of rumen-protected fish oil (omega 3 FA) or sunflower oil (omega 6 FA) for a period of 8 weeks. Following cycle synchronization and artificial insemination, the uteri were flushed post mortem to recover the embryos on day 15 of pregnancy. The UF and tissue samples of endometrium and corpus luteum (CL) were collected. Strikingly, the embryo elongation in the omega 3 group was enhanced compared to the omega 6 group. No differences were observed in uterine prostaglandins, even though the endometrial concentration of their precursor arachidonic acid was reduced in omega 3 compared to omega 6 heifers. The dietary FA neither led to differential expression of target genes in endometrium nor CL nor to a differential abundance of low-density lipoprotein cholesterol, cortisol or amino acids in the UF. Interestingly, the omega 3 group displayed a higher plasma progesterone concentration during luteal growth than the omega 6 group, possibly promoting embryo elongation. Further research should include an ovarian perspective to understand the functional link between dietary omega 3 FA and reproductive outcome. Introduction Early embryonic losses in the pre-implantation phase are commonly observed in cattle. Even though the fertilization rate averages 90%, the actual calving rate reaches only 55% [1]. In high-producing dairy cows, the rate of early embryonic losses is even more pronounced [2, 3]. The most critical period during which embryo losses occur in bovines is prior to implantation between day 8 and 16 of pregnancy. As reason for this, a failure of embryo elongation and maternal recognition of pregnancy is considered [4]. For successful establishment and maintenance of pregnancy, an intense embryo–maternal communication via embryonic signals and maternal factors is crucial. In bovines, direct contact of mother and embryo is established through implantation only from day 18 on. Until then, the communication is crucially dependent on the uterine fluid (UF), a complex mixture of nutritive and regulatory factors such as amino acids, sugars, fatty acids (FA), enzymes and other proteins, growth factors and hormones (reviewed by Filant and Spencer [5]). The UF comprises secretions by both the endometrium and the embryo as well as molecules that are transudated from the blood. It may be affected by the maternal diet [6]. Dietary supplementation with fat in general and specifically with ω3 FA has been discussed to improve bovine fertility [7]. However, the study outcomes are inconsistent. Some studies showed an increased conception or pregnancy rate or reduced pregnancy losses following dietary polyunsaturated FA (PUFA) supplementation compared to supplementation with saturated FA (SFA) [8, 9]. In contrast, other authors [10–12] found no effect of dietary FA on the pregnancy rate. In a study by Burke et al. [13], dietary ω3 FA increased the pregnancy rate in only one of two investigated farms where overall fertility was initially poor. Supplementing beef cows with linoleic acid (LA) reduced pregnancy rates [14]. Before implantation, the bovine trophoblast undergoes rapid elongation, starting around day 15 of pregnancy. At this developmental stage, the trophoblast mononuclear cells synthesize large amounts of interferon tau (IFNT), the signal for maternal recognition of pregnancy [15, 16]. IFNT induces the endometrial gene expression of interferon-stimulated genes (ISGs), which are affecting the endometrium and the trophoblast to support embryo elongation and implantation [17]. IFNT also prevents the pulsatile release of endometrium-derived prostaglandin (PG) F2α and thus luteolysis, whereby the progesterone (P4) production of the corpus luteum (CL) is maintained [15]. Starting shortly after ovulation, luteal P4 affects the endometrium by alterating gene expression and secretion of embryotropic factors [18–20]. The administration of P4 during days 1–4 or days 3–16 of pregnancy caused an increased elongation of the embryo on days 13–16 of pregnancy in beef heifers [18, 21] and was associated with the induction of gene expression changes in the endometrium [22, 23]. Factors secreted by both the embryo (e.g. IFNT, PG, and cortisol) and the endometrium (e.g. PG and cortisol) are then assumed to further modulate the endometrial gene expression [24, 25] and lead to an ongoing modification of the UF [24–27]. Prostaglandins are local signaling molecules that exhibit a broad spectrum of functions. The main series-2 PG precursor is the ω6 FA arachidonic acid (AA; C20:4) that is incorporated into the phospholipid bilayer of cell membranes. After liberation from the cell membrane by phospholipase A2 (PLA2), prostaglandin-endoperoxide synthase 2 (PTGS2; previously known as prostaglandin G/H synthase and cyclooxygenase), the key enzyme of PG synthesis, converts AA to PGH2 which is the common precursor for all proinflammatory downstream series-2 PG, namely PGF2α, PGE2, PGD2, PGI2, and thromboxane A2 (TXA2). Increasing endometrial PTGS2 expression during diestrus induces the synthesis and release of luteolytic PGF2α pulses [28–30]. In addition, PG in the uterine lumen promote embryonic development and elongation [29, 31, 32]. As both the endometrium and the embryo synthesize PG [28, 33–35], the concentration of uterine luminal PG is higher during pregnancy than during the estrous cycle [31, 36]. Even though luteolytic PGF2α pulses are absent during pregnancy, constant levels of PGE2 have been detected in the bovine caudal vena cava [37]. An increased PGE2 production during early pregnancy is induced by IFNT through the upregulation of endometrial PTGS2 [38–40]. PGE2 has furthermore been shown to promote luteal maintenance and subsequent embryo elongation and implantation [38]. A modification of the PG synthesis may be at least in part involved in the improved fertility observed following a dietary FA supply. The utilization of the ω3 FA eicosapentaenoic acid (EPA; C20:5) as a substrate for PTGS2 results in the formation of series-3 PG. Series-3 PG are much less bioactive than the AA-derived series-2 PG and do not seem to be involved in luteolysis in bovines [41, 42]. The ω3 FA docosahexaenoic acid (DHA; C22:6) does not serve as a precursor for PG synthesis, but has been shown to inhibit PTGS2 activity [43]. Both ω6 and ω3 FA compete for the incorporation into cell membrane phospholipids as well as for an utilization by PTGS2. Therefore, dietary ω3 FA may reduce the formation of AA-derived series-2 PG in favor of increasing the synthesis of series-3 PG. Discussed pathways for ω3 FA-induced reduction of series-2 PG include an inhibiting effect on the synthesis and activity of Δ-6-desaturase involved in AA synthesis. Another potential pathway is the ω3 FA-induced inhibition of PTGS2 involved in PGH2 synthesis and the replacement of cell membrane AA by ω3 FA, thereby reducing the precursor for series-2 PG production [14, 41]. PG as well as ω3 and ω6 FA are ligands for nuclear receptors such as the transcription factor family of peroxisome proliferator activated receptors (PPARs), which impact on gene expression [44–47]. An involvement of PPARs in development and reproduction has already been demonstrated [44, 48] and a supplementation with ω3 FA has been reported to alter the expression of genes involved in reproduction in the bovine endometrium [49]. Trophectodermal peroxisome proliferator activated receptor gamma (PPARG) signaling involved in glucose and FA uptake and metabolism has been shown to be essential for early embryo elongation and survival in sheep [50]. A number of studies have investigated the effects of dietary ω3 and ω6 FA on PG concentrations in plasma. At parturition, ω3 FA from fish oil have been shown to decrease the concentration of plasma PGF metabolites (PGFM) compared to olive oil rich in ω6 FA [51]. Contrarily, feeding diets rich in ω3 FA significantly increased PGFM plasma concentrations post partum in beef cattle [52] as well as in primiparous, but not in multiparous dairy cattle [9]. Several studies have also determined the effect of ω3 FA on oxytocin-stimulated PGFM concentrations. A supplementation of fish oil and feeding fish meal or linseed decreased the PGFM response to oxytocin stimulation in beef heifers and dairy cows compared to control groups which lacked additional dietary fat or which were supplemented with palmitic acid [41, 53–55]. However, feeding linseed in comparison to soybean meal did not affect the oxytocin-induced PGFM response [56]. We hypothesize an effect of dietary ω3 FA on embryo elongation. As the cell membrane composition and thus the content of the PG precursors ω6 and ω3 FA can vary depending on dietary available FA, we supplemented growing Angus heifers with rumen-protected fish oil rich in ω3 FA (EPA and DHA) to ensure the incorporation of FA into the body tissues. To maximize the differences in the ω6/ω3 ratio between the experimental and the control group, we supplemented the latter with rumen-protected sunflower oil rich in the ω6 FA LA, a precursor for AA. We studied embryo elongation and development and further associated parameters in the endometrium and the CL. We specifically aimed at focusing whether the composition of the UF was affected by the diet. Materials and methods Animals and study design In total, 37 Angus heifers (Bos taurus) aged between 10 and 27 months (mean ± SEM: 19 ± 0.7) with an average body weight of 348.1 ± 6.4 kg were randomly assigned to two age- and weight-matched study groups. The animals were housed in a freestall barn with ad libitum access to water at the former ETH research station Chamau (Canton Zug, Switzerland) and fed a ration (7.54 kg/day; containing barley straw [3.98 kg], hay [0.57 kg], soy [01.02 kg], wheat [0.69 kg], molasses [0.68 kg], beta-carotene [0.09 kg], commercial mineral mix [0.04 kg] and sodium chloride [0.02 kg]) that included either 450 g of a rumen-protected sunflower oil (“Sonnenblumenöl 100528” from NUTRISWISS AG, Lyss, Switzerland) supplement rich in ω6 FA (ω6 group, n = 15 animals) or 450 g of a rumen-protected fish oil (“Marineöl Omega-3-Konzentrat 33/22” from Henry Lamotte Oils GmbH, Bremen, Germany) supplement rich in in ω3 FA (ω3 group, n = 22 animals). The detailed diet composition is presented in Supplementary Table S1 according to Wolf et al. [57]. The feeding experiment was performed in two separate runs, with 18 animals (n = 9 for both ω3 and ω6 groups) in 2014 and 19 animals (n = 13 for ω3 and n = 6 for ω6) in 2015. Management reasons prevented equal group sizes in the second run. The feeding gate in the free-stall barn was equipped with a gridlock where animals were fixed during feeding. The troughs between animals were separated by wooden barriers during the last two weeks before slaughter. Rumen protection of the supplement oils was established by mixing of the respective oil with hydrogenated rapeseed oil and subsequent spray chilling (Erbo Spraytec AG, Bützberg, Switzerland). The ω6/ω3 ratio of the diet in the ω6 group was 4:1 and in the ω3 group 1:2. The selected fish oil contained 33% EPA and 22% DHA, leading to a combined intake of EPA (37g) and DHA (25 g) of 62 g. Approximately 30 g EPA plus DHA were provided to the animals’ intestinal metabolism, assuming a protection of ruminal biohydrogenation of 50%. The animals were maintained on the respective diets for 8 weeks, leading to a similar average weight gain during the supplementation period of 70 kg. After 5 weeks on the diet, the animals were cycle synchronized by using a controlled internal drug release (Eazi-Breed CIDR 1380®, Zoetis, Zurich, Switzerland) that was removed after 8 days. During the first run, for a few animals in both groups, synchronization by two injections of PGF2α (Estrumate®, MSD Animal Health/Intervet International GmbH, Unterschleissheim, Germany) in an interval of 11 days was applied instead of the CIDR due to management reasons. One day before CIDR removal, animals were injected intramuscularly with 2 ml Estrumate (MSD Animal Health GmbH, Luzern, Switzerland). Three days after Estrumate injection, independently from the synchronization protocol used, the animals were artificially inseminated with sperm from the same Angus bull. The day of artificial insemination (AI) was defined as day 0 of pregnancy. Blood samples were collected on days 0, 3, 6, 9, 12, and 15 from a randomly chosen subset of animals of both groups (ω3: n = 11–17; ω6: n = 6–13). Immediately after slaughter on day 15, the reproductive tract was removed and the uterus was flushed ex vivo with 10 ml PBS to obtain the embryo. The length of the embryos was determined using a stereo microscope prior to snap freezing in liquid nitrogen. Endometrial and CL tissue samples were carefully collected and likewise snap frozen. The recovered UF was centrifuged to remove cellular debris at 800 × g for 10 min. Tissues, plasma, and UF were stored at −80°C until analysis. Animals were only included in the study if an embryo was recovered (ω3: n = 18; ω6: n = 13). With the presence of an embryo on day 15 the respective animal was defined pregnant. During the collection, one of the embryos from the ω3 group was destroyed and thus its length could not be determined. However, this animal was included in the study as the presence of an embryo was confirmed. Because of the high prevalence of pre-implantation embryo mortality in bovine, the embryos’ genomic DNA was controlled for apoptotic laddering on an agarose gel to ensure the inclusion of animals with viable embryos only. Apoptotic DNA fragmentation was not observed for any of the embryos. The experimental protocol was approved by the Veterinary office of the Canton Zug (Switzerland) in accordance with the Swiss legislation on animal rights and welfare (Permit numbers: ZG 64/14 and ZG 71/15). Quantification of fatty acids by gas chromatography with flame ionization detector The FA composition of plasma and endometrial tissue was determined using FA methyl esters (FAME) prepared by transesterification with trimethylsulfonium hydroxide (TMSH). Briefly, for both plasma and endometrial tissue 1 g was extracted two times with 7 ml of chloroform/methanol. The chloroform layer was drained and evaporated. The residues were resuspended in tert-butylmethyl-ether with TMSH added. The analysis of FAMEs was performed by gas chromatography with a flame ionization detector (GC 6890, Agilent Technologies, Waldbronn, Germany). The quantification of FA was performed using Chromeleon® 6.8 Chromatography Software (Dionex, Sunnyvale, USA) [58]. Quantification of prostanoids by liquid chromatography-tandem mass spectrometry Prostaglandins (PGF2α, PGD2, PGE2, 6-keto-PGF1α) and thromboxane B2 (TXB2) were determined in the UF by solid-phase sample extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extraction and analysis were performed as previously described [31]. Briefly, a stock solution with 100 μg/ml PGE2, PGD2, TXB2, PGF2α, and 6-keto PGF1α was prepared in methanol and then further diluted to obtain working standards. Bovine UF samples were prepared with 200 μl sample and 20 μl methanol were added. The prepared samples were extracted, the organic phase was removed and the residues were reconstituted with 50 μl acetonitrile/water/formic acid (20:80:0.0025, v/v, pH 4.0), centrifuged and then transferred to glass vials (Macherey-Nagel, Düren, Germany) prior to injection into the LC-MS/MS system. A Synergi Hydro-RP column (150 × 2 mm I.D., 4 μm particle size, and 80 Å pore size from Phenomenex, Aschaffenburg, Germany) was used to separate PG and TXB2 before being determined with an API 4000 tandem mass spectrometer (Applied Biosystems, Darmstadt, Germany). The quantification was performed with Analyst Software V1.4.2 (Applied Biosystems). Analysis of cortisol in the uterine fluid The cortisol content was determined in UF using a commercial immunoassay (Cortisol Free Saliva Kit, Demeditec, Kiel, Germany) according to the manufacturer's instructions. Analysis of amino acids and total protein content The analysis of amino acids was performed as described earlier [59]. Briefly, the aTRAQ Reagent Kit was used for 40 μl of UF according to the manufacturer's instructions (Applied Biosystems) by subsequent analysis via targeted LC-MS/MS (AB SCIEX QTrap 3200 LC-MS/MS System, AB SCIEX, Framingham, MA, USA). The data were analyzed using the Analyst® 1.5 Software, and quantitative measurements of the amino acids were obtained. The total protein content in UF and plasma was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA). Because total protein content and total amino acid concentration (sum of all measured amino acids) were well correlated (r = 0.82, P < 0.001), the data are presented as mean nmol/mg total protein ± SEM. Total RNA isolation Total RNA was extracted from the ipsilateral intercaruncular endometrial and CL tissue using the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Tissues were homogenized using the MagNA Lyser and MagNA Lyser Green Beads (Roche, Rotkreuz, Switzerland). RNA concentrations were quantified with the NanoDrop 2000 (peqLab, Erlangen, Germany). RNA integrity was monitored using the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) with the Agilent RNA 6000 Nano Kit. RNA integrity numbers ranged between 8.9 and 10 for all samples. RNA aliquots were stored at −80°C until cDNA synthesis. Reverse transcription and qPCR Total RNA (1 μg from endometrium and 500 ng from CL, respectively) was used for cDNA synthesis with the GoScript Reverse Transcription System (Promega, Madison, USA). The reaction mix was composed as follows: 10 μl RNA in H2O, 0.5 μl Oligo(dT)15 primer, 0.5 μl random primer, 4 μl reaction buffer, 2.5 μl MgCl2, 1 μl dNTPs, 0.5 μl RNasin, 1 μl reverse transcriptase. Incubation of the reaction mix was performed in a PCR cycler (25°C for 5 min, 42°C for 60 min, 70°C for 15 min). Quantitative real-time PCR (qPCR) was carried out using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, USA) on a CFX384 Real-Time PCR Detection System (Bio-Rad, Munich, Germany). The cycle of quantification (Cq) values were obtained using a single threshold. The relative expression level (ΔCq) of each gene was generated by scaling the target gene Cq of each individual sample to the geometric mean of the Cqs of three reference genes (ubiquitin B (UBB), H3 histone family member 3A (H3F3A), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), according to the bestkeeper method [60]. Fold-changes were calculated according to the ΔΔCt method. The sequences of commercially synthesized primers (Microsynth, Balgach, Switzerland) used are listed in Supplementary Table S2. Plasma progesterone concentration Plasma P4 was determined using the Immunotech RIA (Beckman Coulter, CA, USA), previously validated for bovine plasma [61]. The analytical sensitivity for P4 was 0.03 ng/ml, intraassay CV was 7.6%, and interassay CV was 9.7%. Quantification of LDL cholesterol A commercial assay kit (high-density lipoprotein (HDL) and low-density lipoprotein (LDL)/very low-density lipoprotein (VLDL) Cho-lesterol Assay kit, abcam, Cambridge, UK) was used to measure cholesterol concentration in the LDL fractions of plasma samples collected at slaughter according to the manufacturer's instructions. Quantification of tocopherols by high-performance liquid chromatography Tocopherols were quantified in plasma (collected at slaughter), liver, and endometrium by a validated method described earlier [62]. Briefly, tissue samples were saponified for 30 min at 70°C in a shaking water bath. To both tissue and plasma samples butylated hydroxytoluene (BHT; 25 μL of a 1 mg/ml ethanolic solution) was added. Then all samples were extracted twice with n-Hexane. The supernatants were pooled and evaporated (Christ SpeedDry; Christ, Osterode Germany). The dried residues were resuspended and injected into a Jasco HPLC system (AS-950 Plus autosampler, PU-980 Plus pump, FP-950 Plus fluorescence detector, LG-980-02 gradient unit, and a 3-line degasser; Jasco, Groß-Umstadt, Germany). The tocopherols were separated on a Kinetex PFP column (2.6 μm, 150 × 4.6 mm; Phenomenex, Aschaffenburg, Germany) using a methanol: water (85:15, vol/vol) mobile phase. The fluorescence detector was set to an excitation wavelength of 296 nm and emission wavelength of 325 nm. Peaks were recorded and integrated using ChromPass version 1.8.6.1 (Jasco). The concentrations of tocopherols were quantified against external standard curves with authentic compounds (Sigma Aldrich, St. Louis, MO). Statistical analysis The statistical analysis was performed using SPSS version 22 (SPSS GmbH Software, Munich, Germany). The Shapiro–Wilk test was used to test for normal distribution of data and residuals. For comparisons between diet groups, data following a Gaussian distribution were analyzed by Student t test. For data not following a Gaussian distribution (as observed for embryo length), a Mann–Whitney U test was performed. In case of a significant effect of embryo length on dependent variables (endometrial gene expression, uterine prostaglandins, and amino acids), the “least-square ANOVA general linear models procedure” was used with diet group (ω3 or ω6) as a fixed factor and embryo length (in cm) as a covariable. Data analysis for qPCR results was performed on ΔCq values. Results are presented as means ± SEM and P values ≤ 0.05 were considered statistically significant. Results Rumen protected ω3 FA lowered the ω6/ω3 ratio and changed the FA pattern in plasma and endometrium The amounts of single ω3 and ω6 FA in both plasma and endometrium differed significantly between animals supplemented with fish oil (ω3 FA) and animals supplemented with sunflower oil (ω6 FA) as presented in Table 1. In both diet groups, the percentage of AA, docosapentaenoic acid, and DHA of total ω6 and ω3 FA was higher in the endometrium compared to plasma whereas the percentages of LA, alpha-linolenic acid (ALA), and EPA were decreased in endometrium compared to plasma (all P < 0.001). Table 1. Concentration and percentage of selected ω3 and ω6 FA to total ω3 and ω6 FA in plasma and endometrium of Angus heifers. Plasma Endometrium μg/g P value % of total ω6 and ω3 FA μg/g P value % of total ω6 and ω3 FA ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet SFA 596 ± 37 675 ± 33 0.1 1616 ± 117 1489 ± 105 0.4 MUFA 304 ± 15 264 ± 12 0.06 1266 ± 106 1030 ± 65 0.1 PUFA 749 ± 42 916 ± 47 0.02 1449 ± 105 1352 ± 103 0.5 Linoleic acid 513 ± 30 458 ± 22 0.2 68 50 338 ± 25 315 ± 24 0.3 23 23 gamma-Linolenic acid 19.8 ± 1.5 5.3 ± 0.5 <0.001 3 1 17.0 ± 1.8 10.2 ± 1.1 0.002 1 1 Eicosadienoic acid ω6 0.8 ± 0.1 1.7 ± 0.1 0.005 - - 18.8 ± 3.0 15.1 ± 1.6 0.1 1 1 Eicosatrienoic 8c,11c,14c acid 29.6 ± 1.9 13.3 ± 0.7 <0.001 4 2 117 ± 10 80.7 ± 7.4 0.006 8 6 Arachidonic acid 51.6 ± 2.7 59.3 ± 3.6 0.4 7 6 584 ± 45 388 ± 22 <0.001 40 29 ω6 FA (total) 614 ± 35 536 ± 26 0.09 82 59 1075 ± 80 810 ± 53 0.009 73* 60 Linolenic acid 69.0 ± 4.7 117 ± 6 <0.001 9 13 12.2 ± 4.9 17.8 ± 1.9 0.1 1 1 Eicosapentaenoic acid 34.7 ± 3.2 187 ± 18 <0.001 5 20 24.6 ± 3.1 101 ± 13 <0.001 2 7 Docosapentaenoic acid ω3 20.8 ± 1.2 30.8 ± 1.4 <0.001 3 3 116 ± 8 158 ± 14 0.2 8 12 Docosahexaenoic acid 10.7 ± 0.9 43.7 ± 2.6 <0.001 1 5 221 ± 19 266 ± 23 0.2 15 20 ω3 FA (total) 135 ± 9 378 ± 26 <0.001 18 41 376 ± 29 545 ± 50 0.045 26 40 ω6/ω3 4.7 ± 0.2 1.5 ± 0.1 <0.001 2.8 ± 0.1 1.6 ± 0.1 <0.001 Plasma Endometrium μg/g P value % of total ω6 and ω3 FA μg/g P value % of total ω6 and ω3 FA ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet SFA 596 ± 37 675 ± 33 0.1 1616 ± 117 1489 ± 105 0.4 MUFA 304 ± 15 264 ± 12 0.06 1266 ± 106 1030 ± 65 0.1 PUFA 749 ± 42 916 ± 47 0.02 1449 ± 105 1352 ± 103 0.5 Linoleic acid 513 ± 30 458 ± 22 0.2 68 50 338 ± 25 315 ± 24 0.3 23 23 gamma-Linolenic acid 19.8 ± 1.5 5.3 ± 0.5 <0.001 3 1 17.0 ± 1.8 10.2 ± 1.1 0.002 1 1 Eicosadienoic acid ω6 0.8 ± 0.1 1.7 ± 0.1 0.005 - - 18.8 ± 3.0 15.1 ± 1.6 0.1 1 1 Eicosatrienoic 8c,11c,14c acid 29.6 ± 1.9 13.3 ± 0.7 <0.001 4 2 117 ± 10 80.7 ± 7.4 0.006 8 6 Arachidonic acid 51.6 ± 2.7 59.3 ± 3.6 0.4 7 6 584 ± 45 388 ± 22 <0.001 40 29 ω6 FA (total) 614 ± 35 536 ± 26 0.09 82 59 1075 ± 80 810 ± 53 0.009 73* 60 Linolenic acid 69.0 ± 4.7 117 ± 6 <0.001 9 13 12.2 ± 4.9 17.8 ± 1.9 0.1 1 1 Eicosapentaenoic acid 34.7 ± 3.2 187 ± 18 <0.001 5 20 24.6 ± 3.1 101 ± 13 <0.001 2 7 Docosapentaenoic acid ω3 20.8 ± 1.2 30.8 ± 1.4 <0.001 3 3 116 ± 8 158 ± 14 0.2 8 12 Docosahexaenoic acid 10.7 ± 0.9 43.7 ± 2.6 <0.001 1 5 221 ± 19 266 ± 23 0.2 15 20 ω3 FA (total) 135 ± 9 378 ± 26 <0.001 18 41 376 ± 29 545 ± 50 0.045 26 40 ω6/ω3 4.7 ± 0.2 1.5 ± 0.1 <0.001 2.8 ± 0.1 1.6 ± 0.1 <0.001 Heifers in the ω3 group have significantly more ω3 FA in plasma and endometrium than ω6 heifers. This was caused by an increase in all ω3 FA in plasma whereas in endometrium it caused by solely a higher EPA concentration. Conversely, heifers in the ω6 group display a higher concentration of AA in endometrium. Values are given as means ± sem. *Rounding error. View Large Table 1. Concentration and percentage of selected ω3 and ω6 FA to total ω3 and ω6 FA in plasma and endometrium of Angus heifers. Plasma Endometrium μg/g P value % of total ω6 and ω3 FA μg/g P value % of total ω6 and ω3 FA ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet SFA 596 ± 37 675 ± 33 0.1 1616 ± 117 1489 ± 105 0.4 MUFA 304 ± 15 264 ± 12 0.06 1266 ± 106 1030 ± 65 0.1 PUFA 749 ± 42 916 ± 47 0.02 1449 ± 105 1352 ± 103 0.5 Linoleic acid 513 ± 30 458 ± 22 0.2 68 50 338 ± 25 315 ± 24 0.3 23 23 gamma-Linolenic acid 19.8 ± 1.5 5.3 ± 0.5 <0.001 3 1 17.0 ± 1.8 10.2 ± 1.1 0.002 1 1 Eicosadienoic acid ω6 0.8 ± 0.1 1.7 ± 0.1 0.005 - - 18.8 ± 3.0 15.1 ± 1.6 0.1 1 1 Eicosatrienoic 8c,11c,14c acid 29.6 ± 1.9 13.3 ± 0.7 <0.001 4 2 117 ± 10 80.7 ± 7.4 0.006 8 6 Arachidonic acid 51.6 ± 2.7 59.3 ± 3.6 0.4 7 6 584 ± 45 388 ± 22 <0.001 40 29 ω6 FA (total) 614 ± 35 536 ± 26 0.09 82 59 1075 ± 80 810 ± 53 0.009 73* 60 Linolenic acid 69.0 ± 4.7 117 ± 6 <0.001 9 13 12.2 ± 4.9 17.8 ± 1.9 0.1 1 1 Eicosapentaenoic acid 34.7 ± 3.2 187 ± 18 <0.001 5 20 24.6 ± 3.1 101 ± 13 <0.001 2 7 Docosapentaenoic acid ω3 20.8 ± 1.2 30.8 ± 1.4 <0.001 3 3 116 ± 8 158 ± 14 0.2 8 12 Docosahexaenoic acid 10.7 ± 0.9 43.7 ± 2.6 <0.001 1 5 221 ± 19 266 ± 23 0.2 15 20 ω3 FA (total) 135 ± 9 378 ± 26 <0.001 18 41 376 ± 29 545 ± 50 0.045 26 40 ω6/ω3 4.7 ± 0.2 1.5 ± 0.1 <0.001 2.8 ± 0.1 1.6 ± 0.1 <0.001 Plasma Endometrium μg/g P value % of total ω6 and ω3 FA μg/g P value % of total ω6 and ω3 FA ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet SFA 596 ± 37 675 ± 33 0.1 1616 ± 117 1489 ± 105 0.4 MUFA 304 ± 15 264 ± 12 0.06 1266 ± 106 1030 ± 65 0.1 PUFA 749 ± 42 916 ± 47 0.02 1449 ± 105 1352 ± 103 0.5 Linoleic acid 513 ± 30 458 ± 22 0.2 68 50 338 ± 25 315 ± 24 0.3 23 23 gamma-Linolenic acid 19.8 ± 1.5 5.3 ± 0.5 <0.001 3 1 17.0 ± 1.8 10.2 ± 1.1 0.002 1 1 Eicosadienoic acid ω6 0.8 ± 0.1 1.7 ± 0.1 0.005 - - 18.8 ± 3.0 15.1 ± 1.6 0.1 1 1 Eicosatrienoic 8c,11c,14c acid 29.6 ± 1.9 13.3 ± 0.7 <0.001 4 2 117 ± 10 80.7 ± 7.4 0.006 8 6 Arachidonic acid 51.6 ± 2.7 59.3 ± 3.6 0.4 7 6 584 ± 45 388 ± 22 <0.001 40 29 ω6 FA (total) 614 ± 35 536 ± 26 0.09 82 59 1075 ± 80 810 ± 53 0.009 73* 60 Linolenic acid 69.0 ± 4.7 117 ± 6 <0.001 9 13 12.2 ± 4.9 17.8 ± 1.9 0.1 1 1 Eicosapentaenoic acid 34.7 ± 3.2 187 ± 18 <0.001 5 20 24.6 ± 3.1 101 ± 13 <0.001 2 7 Docosapentaenoic acid ω3 20.8 ± 1.2 30.8 ± 1.4 <0.001 3 3 116 ± 8 158 ± 14 0.2 8 12 Docosahexaenoic acid 10.7 ± 0.9 43.7 ± 2.6 <0.001 1 5 221 ± 19 266 ± 23 0.2 15 20 ω3 FA (total) 135 ± 9 378 ± 26 <0.001 18 41 376 ± 29 545 ± 50 0.045 26 40 ω6/ω3 4.7 ± 0.2 1.5 ± 0.1 <0.001 2.8 ± 0.1 1.6 ± 0.1 <0.001 Heifers in the ω3 group have significantly more ω3 FA in plasma and endometrium than ω6 heifers. This was caused by an increase in all ω3 FA in plasma whereas in endometrium it caused by solely a higher EPA concentration. Conversely, heifers in the ω6 group display a higher concentration of AA in endometrium. Values are given as means ± sem. *Rounding error. View Large The total PUFA concentration in plasma was significantly increased in the ω3 group compared to the ω6 group (Table 1). This change was due to increased concentrations of total ω3 FA, whereas total ω6 FA concentration was unaffected in plasma. However, in the endometrium, the total PUFA concentration did not differ between diet groups. This was caused by a significant increase in total ω3 FA and a simultaneous significant decrease in total ω6 FA. Saturated FA and monounsaturated FA (MUFA) were not affected by diet neither in plasma nor in endometrium. The supplementation with rumen-protected ω3 FA significantly decreased the ω6/ω3 ratio in plasma as well as in endometrium compared to supplementation with rumen-protected ω6 FA (Table 1). This change in the ω6/ω3 ratio in plasma was caused by significant increases of all detectable ω3 FA including EPA and DHA in the ω3 group compared to the ω6 group. The amount of AA in plasma did not significantly differ between the two groups. In the endometrium, dietary ω3 FA significantly decreased the amount of AA and significantly increased the amount of EPA compared to dietary ω6 FA. Endometrial DHA did not differ between groups. Day 15 preimplantation embryos differed in length due to supplementation with rumen protected ω3 and ω6 FA The embryo length varied greatly within and between the experimental groups (Figure 1), namely from 0.2 to 20.3 cm in the ω3 group, but only from 1.4 to 7.7 cm in the ω6 group. On average, embryos from ω3 FA supplemented animals were significantly longer than embryos in the ω6 group (median ± SEM: ω6 group: 2.5 ± 0.5 cm; ω3 group: 6.4 ± 1.3 cm; P = 0.043). The pregnancy rate did not differ between treatment groups and was 82% in the ω3 and 87% in ω6 group, respectively. Figure 1. View largeDownload slide Supplementation with ω3 FA compared to ω6 FA led to significantly longer embryos on day 15 of pregnancy in Angus heifers (*P ≤ 0.05). Figure 1. View largeDownload slide Supplementation with ω3 FA compared to ω6 FA led to significantly longer embryos on day 15 of pregnancy in Angus heifers (*P ≤ 0.05). Different concentrations of AA and EPA in the endometrium caused by FA supplementation did not affect uterine prostaglandin concentrations The concentrations of different series-2 PG and their metabolites (PGE2, PGD2, TXB2, PGF2α, PGFM, and 6-keto-PGF1α) in the UF did not differ between groups (Table 2). PGF2α and the PGI2 metabolite 6-keto-PGF1α were the most abundant prostaglandines followed by PGFM and PGE2. PGD2 and TXB2 were present in similar concentrations and represented the least prominent PG in the UF. Table 2. Uterine concentrations of prostaglandins (ng/ml uterine fluid; means ± SEM). ω6 FA diet ω3 FA diet P-value PGF2α 17.5 ± 7.0 31.5 ± 5.8 0.9 PGE2 0.6 ± 0.2 1.0 ± 0.2 0.4 6-keto-PGF1α 12.6 ± 3.4 15.2 ± 2.8 0.6 PGD2 0.2 ± 0.1 0.2 ± 0.03 0.5 TXB2 0.2 ± 0.1 0.1 ± 0.04 0.4 PGFM 1.5 ± 0.2 1.3 ± 0.2 0.4 ω6 FA diet ω3 FA diet P-value PGF2α 17.5 ± 7.0 31.5 ± 5.8 0.9 PGE2 0.6 ± 0.2 1.0 ± 0.2 0.4 6-keto-PGF1α 12.6 ± 3.4 15.2 ± 2.8 0.6 PGD2 0.2 ± 0.1 0.2 ± 0.03 0.5 TXB2 0.2 ± 0.1 0.1 ± 0.04 0.4 PGFM 1.5 ± 0.2 1.3 ± 0.2 0.4 View Large Table 2. Uterine concentrations of prostaglandins (ng/ml uterine fluid; means ± SEM). ω6 FA diet ω3 FA diet P-value PGF2α 17.5 ± 7.0 31.5 ± 5.8 0.9 PGE2 0.6 ± 0.2 1.0 ± 0.2 0.4 6-keto-PGF1α 12.6 ± 3.4 15.2 ± 2.8 0.6 PGD2 0.2 ± 0.1 0.2 ± 0.03 0.5 TXB2 0.2 ± 0.1 0.1 ± 0.04 0.4 PGFM 1.5 ± 0.2 1.3 ± 0.2 0.4 ω6 FA diet ω3 FA diet P-value PGF2α 17.5 ± 7.0 31.5 ± 5.8 0.9 PGE2 0.6 ± 0.2 1.0 ± 0.2 0.4 6-keto-PGF1α 12.6 ± 3.4 15.2 ± 2.8 0.6 PGD2 0.2 ± 0.1 0.2 ± 0.03 0.5 TXB2 0.2 ± 0.1 0.1 ± 0.04 0.4 PGFM 1.5 ± 0.2 1.3 ± 0.2 0.4 View Large Dietary FA affected intrauterine alanine concentration but did neither impact on other amino acids nor intrauterine cortisol With the exception of a significantly higher concentration of alanine in the ω6 compared to the ω3 group, no differences were observed in amino acid concentrations in the UF of day 15 pregnant heifers (Supplementary Table S3). The cortisol concentration in the UF did not significantly differ between groups (ω6 group: 0.59 ± 0.1 ng/ml; ω3 group: 0.58 ± 0.1 ng/ml). The different concentrations of AA and EPA in the endometrium had only a minor impact on endometrial gene expression The endometrial expression of a broad range of genes, which were selected due to their respective involvement in embryo elongation and metabolism as well embryo–maternal communication, was widely unaffected by the dietary FA supplementation. Out of the genes under investigation, only perilipin 2 (PLIN2; previously known as adipose differentiation-related protein (ADFP)), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG), and sperm associated antigen 9 (SPAG9) were differently regulated in the endometrium of pregnant ω3 supplemented heifers compared to pregnant ω6 supplemented heifers (Table 3). The insulin receptor (INSR) transcript abundance showed a trend (P = 0.058). Table 3. Endometrial relative gene expression (means ± SEM) and mean fold-changes of pregnant heifers as determined by qPCR. ΔCt ω6 FA diet (n = 13) ΔCt ω3 FA diet (n = 18) Mean fold-change (ω3 vs ω6) P-value IFNt-stimulated genes ISG15 $$-$$1.19 ± 0.13 $$-$$1.05 ± 0.27 1.1 0.4 OAS1 $$-$$0.39 ± 0.13 $$-$$0.30 ± 0.17 1.1 0.7 MX1 0.25 ± 0.10 0.34 ± 0.18 1.1 0.9 Antioxidant defense GPX1 $$-$$2.09 ± 0.18 $$-$$2.15 ± 0.09 1.0 0.6 GPX4 $$-$$3.72 ± 0.14 $$-$$3.71 ± 0.09 1.0 0.6 SOD1 $$-$$3.00 ± 0.48 $$-$$3.66 ± 0.75 0.6 0.8 SOD2 $$-$$5.39 ± 0.94 $$-$$5.35 ± 0.86 1.0 0.8 NQO1 $$-$$9.85 ± 0.31 $$-$$10.25 ± 0.21 0.8 0.4 GSTA2 $$-$$7.65 ± 0.86 $$-$$7.02 ± 0.17 1.6 0.7 GSR $$-$$4.44 ± 0.10 $$-$$4.42 ± 0.10 1.0 0.6 GSS $$-$$4.44 ± 0.62 $$-$$3.67 ± 0.19 1.7 0.7 CAT $$-$$4.35 ± 0.13 $$-$$4.52 ± 0.23 0.9 0.6 MT1 $$-$$8.33 ± 1.32 $$-$$7.44 ± 0.77 1.9 0.7 MT2 $$-$$7.86 ± 0.68 $$-$$7.18 ± 0.28 1.6 0.7 NFE2L2 $$-$$2.85 ± 0.12 $$-$$4.14 ± 0.87 0.4 0.3 Amino acid transport and metabolism SLC1A1 $$-$$1.08 ± 0.15 $$-$$1.19 ± 0.13 0.9 0.7 SLC1A5 $$-$$5.94 ± 0.21 $$-$$5.81 ± 0.23 1.1 0.3 SLC6A6 $$-$$5.86 ± 0.14 $$-$$5.98 ± 0.12 0.9 0.2 SLC7A1 $$-$$4.95 ± 0.10 $$-$$4.93 ± 0.11 1.0 0.2 SLC7A5 $$-$$8.33 ± 0.15 $$-$$8.29 ± 0.19 1.0 0.8 SLC7A8 $$-$$5.81 ± 0.30 $$-$$5.67 ± 0.27 1.1 0.9 SLC15A1 $$-$$5.99 ± 0.19 $$-$$6.00 ± 0.16 1.0 0.4 SLC15A2 $$-$$6.20 ± 0.11 $$-$$6.05 ± 0.13 1.1 0.09 SLC15A3 $$-$$6.10 ± 0.17 $$-$$5.98 ± 0.23 1.1 0.5 GTP $$-$$6.78 ± 0.13 $$-$$6.90 ± 0.15 0.9 0.5 HDC $$-$$10.37 ± 0.50 $$-$$10.29 ± 0.52 1.1 0.8 IGF signaling IGF1 $$-$$5.59 ± 0.39 $$-$$5.32 ± 0.30 1.2 0.8 IGF1R $$-$$3.53 ± 0.14 $$-$$3.39 ± 0.16 1.1 0.3 IGF2 $$-$$4.82 ± 0.27 $$-$$4.10 ± 0.21 1.7 0.2 IGF2R $$-$$5.66 ± 0.23 $$-$$5.40 ± 0.24 1.2 0.7 INSR $$-$$5.00 ± 0.06 $$-$$4.89 ± 0.07 1.1 0.06 PPAR signaling PPARA $$-$$4.10 ± 0.09 $$-$$4.18 ± 0.07 0.9 0.2 PPARD $$-$$5.47 ± 0.15 $$-$$5.38 ± 0.11 1.1 0.8 PPARG $$-$$9.97 ± 0.29 $$-$$9.72 ± 0.20 1.2 0.6 Prostaglandin signaling PLA2 $$-$$5.80 ± 0.12 $$-$$5.80 ± 0.07 1.0 0.5 PTGS2 $$-$$1.81 ± 0.20 $$-$$1.99 ± 0.15 0.9 0.4 PTGER2 $$-$$5.38 ± 0.11 $$-$$5.74 ± 0.41 0.8 0.6 PTGER4 $$-$$7.21 ± 0.19 $$-$$7.11 ± 0.14 1.1 0.9 PTGIS $$-$$7.76 ± 1.04 $$-$$7.33 ± 0.72 1.4 0.7 PTGIR $$-$$9.62 ± 0.21 $$-$$9.48 ± 0.29 1.1 0.8 HPGD $$-$$2.04 ± 0.38 $$-$$1.66 ± 0.15 1.3 0.8 SLCO2A1 $$-$$7.65 ± 0.13 $$-$$7.89 ± 0.24 0.9 0.1 EDN1 $$-$$7.44 ± 0.26 $$-$$7.54 ± 0.24 0.9 0.8 Lipid and cholesterol metabolism and transport LPL $$-$$9.37 ± 0.15 $$-$$9.26 ± 0.16 1.1 0.9 FABP3 $$-$$2.84 ± 0.27 $$-$$2.55 ± 0.31 1.2 0.1 STAR $$-$$7.95 ± 0.19 $$-$$8.11 ± 0.21 0.9 0.3 PLIN2 $$-$$3.67 ± 0.17 $$-$$3.97 ± 0.14 0.8 0.04 Immunoregulation CPN1 $$-$$5.55 ± 0.23 $$-$$5.90 ± 0.18 0.8 0.9 TCF7 $$-$$5.44 ± 0.11 $$-$$5.47 ± 0.07 1.0 0.4 Wnt signaling SFRP2 $$-$$4.57 ± 0.35 $$-$$4.54 ± 0.26 1.0 0.9 WNT11 $$-$$6.00 ± 0.16 $$-$$5.97 ± 0.12 1.0 0.5 Signal transduction YWHAG $$-$$1.44 ± 0.07 $$-$$1.50 ± 0.06 1.0 0.04 SPAG9 $$-$$2.61 ± 0.06 $$-$$2.72 ± 0.05 0.9 0.03 ΔCt ω6 FA diet (n = 13) ΔCt ω3 FA diet (n = 18) Mean fold-change (ω3 vs ω6) P-value IFNt-stimulated genes ISG15 $$-$$1.19 ± 0.13 $$-$$1.05 ± 0.27 1.1 0.4 OAS1 $$-$$0.39 ± 0.13 $$-$$0.30 ± 0.17 1.1 0.7 MX1 0.25 ± 0.10 0.34 ± 0.18 1.1 0.9 Antioxidant defense GPX1 $$-$$2.09 ± 0.18 $$-$$2.15 ± 0.09 1.0 0.6 GPX4 $$-$$3.72 ± 0.14 $$-$$3.71 ± 0.09 1.0 0.6 SOD1 $$-$$3.00 ± 0.48 $$-$$3.66 ± 0.75 0.6 0.8 SOD2 $$-$$5.39 ± 0.94 $$-$$5.35 ± 0.86 1.0 0.8 NQO1 $$-$$9.85 ± 0.31 $$-$$10.25 ± 0.21 0.8 0.4 GSTA2 $$-$$7.65 ± 0.86 $$-$$7.02 ± 0.17 1.6 0.7 GSR $$-$$4.44 ± 0.10 $$-$$4.42 ± 0.10 1.0 0.6 GSS $$-$$4.44 ± 0.62 $$-$$3.67 ± 0.19 1.7 0.7 CAT $$-$$4.35 ± 0.13 $$-$$4.52 ± 0.23 0.9 0.6 MT1 $$-$$8.33 ± 1.32 $$-$$7.44 ± 0.77 1.9 0.7 MT2 $$-$$7.86 ± 0.68 $$-$$7.18 ± 0.28 1.6 0.7 NFE2L2 $$-$$2.85 ± 0.12 $$-$$4.14 ± 0.87 0.4 0.3 Amino acid transport and metabolism SLC1A1 $$-$$1.08 ± 0.15 $$-$$1.19 ± 0.13 0.9 0.7 SLC1A5 $$-$$5.94 ± 0.21 $$-$$5.81 ± 0.23 1.1 0.3 SLC6A6 $$-$$5.86 ± 0.14 $$-$$5.98 ± 0.12 0.9 0.2 SLC7A1 $$-$$4.95 ± 0.10 $$-$$4.93 ± 0.11 1.0 0.2 SLC7A5 $$-$$8.33 ± 0.15 $$-$$8.29 ± 0.19 1.0 0.8 SLC7A8 $$-$$5.81 ± 0.30 $$-$$5.67 ± 0.27 1.1 0.9 SLC15A1 $$-$$5.99 ± 0.19 $$-$$6.00 ± 0.16 1.0 0.4 SLC15A2 $$-$$6.20 ± 0.11 $$-$$6.05 ± 0.13 1.1 0.09 SLC15A3 $$-$$6.10 ± 0.17 $$-$$5.98 ± 0.23 1.1 0.5 GTP $$-$$6.78 ± 0.13 $$-$$6.90 ± 0.15 0.9 0.5 HDC $$-$$10.37 ± 0.50 $$-$$10.29 ± 0.52 1.1 0.8 IGF signaling IGF1 $$-$$5.59 ± 0.39 $$-$$5.32 ± 0.30 1.2 0.8 IGF1R $$-$$3.53 ± 0.14 $$-$$3.39 ± 0.16 1.1 0.3 IGF2 $$-$$4.82 ± 0.27 $$-$$4.10 ± 0.21 1.7 0.2 IGF2R $$-$$5.66 ± 0.23 $$-$$5.40 ± 0.24 1.2 0.7 INSR $$-$$5.00 ± 0.06 $$-$$4.89 ± 0.07 1.1 0.06 PPAR signaling PPARA $$-$$4.10 ± 0.09 $$-$$4.18 ± 0.07 0.9 0.2 PPARD $$-$$5.47 ± 0.15 $$-$$5.38 ± 0.11 1.1 0.8 PPARG $$-$$9.97 ± 0.29 $$-$$9.72 ± 0.20 1.2 0.6 Prostaglandin signaling PLA2 $$-$$5.80 ± 0.12 $$-$$5.80 ± 0.07 1.0 0.5 PTGS2 $$-$$1.81 ± 0.20 $$-$$1.99 ± 0.15 0.9 0.4 PTGER2 $$-$$5.38 ± 0.11 $$-$$5.74 ± 0.41 0.8 0.6 PTGER4 $$-$$7.21 ± 0.19 $$-$$7.11 ± 0.14 1.1 0.9 PTGIS $$-$$7.76 ± 1.04 $$-$$7.33 ± 0.72 1.4 0.7 PTGIR $$-$$9.62 ± 0.21 $$-$$9.48 ± 0.29 1.1 0.8 HPGD $$-$$2.04 ± 0.38 $$-$$1.66 ± 0.15 1.3 0.8 SLCO2A1 $$-$$7.65 ± 0.13 $$-$$7.89 ± 0.24 0.9 0.1 EDN1 $$-$$7.44 ± 0.26 $$-$$7.54 ± 0.24 0.9 0.8 Lipid and cholesterol metabolism and transport LPL $$-$$9.37 ± 0.15 $$-$$9.26 ± 0.16 1.1 0.9 FABP3 $$-$$2.84 ± 0.27 $$-$$2.55 ± 0.31 1.2 0.1 STAR $$-$$7.95 ± 0.19 $$-$$8.11 ± 0.21 0.9 0.3 PLIN2 $$-$$3.67 ± 0.17 $$-$$3.97 ± 0.14 0.8 0.04 Immunoregulation CPN1 $$-$$5.55 ± 0.23 $$-$$5.90 ± 0.18 0.8 0.9 TCF7 $$-$$5.44 ± 0.11 $$-$$5.47 ± 0.07 1.0 0.4 Wnt signaling SFRP2 $$-$$4.57 ± 0.35 $$-$$4.54 ± 0.26 1.0 0.9 WNT11 $$-$$6.00 ± 0.16 $$-$$5.97 ± 0.12 1.0 0.5 Signal transduction YWHAG $$-$$1.44 ± 0.07 $$-$$1.50 ± 0.06 1.0 0.04 SPAG9 $$-$$2.61 ± 0.06 $$-$$2.72 ± 0.05 0.9 0.03 View Large Table 3. Endometrial relative gene expression (means ± SEM) and mean fold-changes of pregnant heifers as determined by qPCR. ΔCt ω6 FA diet (n = 13) ΔCt ω3 FA diet (n = 18) Mean fold-change (ω3 vs ω6) P-value IFNt-stimulated genes ISG15 $$-$$1.19 ± 0.13 $$-$$1.05 ± 0.27 1.1 0.4 OAS1 $$-$$0.39 ± 0.13 $$-$$0.30 ± 0.17 1.1 0.7 MX1 0.25 ± 0.10 0.34 ± 0.18 1.1 0.9 Antioxidant defense GPX1 $$-$$2.09 ± 0.18 $$-$$2.15 ± 0.09 1.0 0.6 GPX4 $$-$$3.72 ± 0.14 $$-$$3.71 ± 0.09 1.0 0.6 SOD1 $$-$$3.00 ± 0.48 $$-$$3.66 ± 0.75 0.6 0.8 SOD2 $$-$$5.39 ± 0.94 $$-$$5.35 ± 0.86 1.0 0.8 NQO1 $$-$$9.85 ± 0.31 $$-$$10.25 ± 0.21 0.8 0.4 GSTA2 $$-$$7.65 ± 0.86 $$-$$7.02 ± 0.17 1.6 0.7 GSR $$-$$4.44 ± 0.10 $$-$$4.42 ± 0.10 1.0 0.6 GSS $$-$$4.44 ± 0.62 $$-$$3.67 ± 0.19 1.7 0.7 CAT $$-$$4.35 ± 0.13 $$-$$4.52 ± 0.23 0.9 0.6 MT1 $$-$$8.33 ± 1.32 $$-$$7.44 ± 0.77 1.9 0.7 MT2 $$-$$7.86 ± 0.68 $$-$$7.18 ± 0.28 1.6 0.7 NFE2L2 $$-$$2.85 ± 0.12 $$-$$4.14 ± 0.87 0.4 0.3 Amino acid transport and metabolism SLC1A1 $$-$$1.08 ± 0.15 $$-$$1.19 ± 0.13 0.9 0.7 SLC1A5 $$-$$5.94 ± 0.21 $$-$$5.81 ± 0.23 1.1 0.3 SLC6A6 $$-$$5.86 ± 0.14 $$-$$5.98 ± 0.12 0.9 0.2 SLC7A1 $$-$$4.95 ± 0.10 $$-$$4.93 ± 0.11 1.0 0.2 SLC7A5 $$-$$8.33 ± 0.15 $$-$$8.29 ± 0.19 1.0 0.8 SLC7A8 $$-$$5.81 ± 0.30 $$-$$5.67 ± 0.27 1.1 0.9 SLC15A1 $$-$$5.99 ± 0.19 $$-$$6.00 ± 0.16 1.0 0.4 SLC15A2 $$-$$6.20 ± 0.11 $$-$$6.05 ± 0.13 1.1 0.09 SLC15A3 $$-$$6.10 ± 0.17 $$-$$5.98 ± 0.23 1.1 0.5 GTP $$-$$6.78 ± 0.13 $$-$$6.90 ± 0.15 0.9 0.5 HDC $$-$$10.37 ± 0.50 $$-$$10.29 ± 0.52 1.1 0.8 IGF signaling IGF1 $$-$$5.59 ± 0.39 $$-$$5.32 ± 0.30 1.2 0.8 IGF1R $$-$$3.53 ± 0.14 $$-$$3.39 ± 0.16 1.1 0.3 IGF2 $$-$$4.82 ± 0.27 $$-$$4.10 ± 0.21 1.7 0.2 IGF2R $$-$$5.66 ± 0.23 $$-$$5.40 ± 0.24 1.2 0.7 INSR $$-$$5.00 ± 0.06 $$-$$4.89 ± 0.07 1.1 0.06 PPAR signaling PPARA $$-$$4.10 ± 0.09 $$-$$4.18 ± 0.07 0.9 0.2 PPARD $$-$$5.47 ± 0.15 $$-$$5.38 ± 0.11 1.1 0.8 PPARG $$-$$9.97 ± 0.29 $$-$$9.72 ± 0.20 1.2 0.6 Prostaglandin signaling PLA2 $$-$$5.80 ± 0.12 $$-$$5.80 ± 0.07 1.0 0.5 PTGS2 $$-$$1.81 ± 0.20 $$-$$1.99 ± 0.15 0.9 0.4 PTGER2 $$-$$5.38 ± 0.11 $$-$$5.74 ± 0.41 0.8 0.6 PTGER4 $$-$$7.21 ± 0.19 $$-$$7.11 ± 0.14 1.1 0.9 PTGIS $$-$$7.76 ± 1.04 $$-$$7.33 ± 0.72 1.4 0.7 PTGIR $$-$$9.62 ± 0.21 $$-$$9.48 ± 0.29 1.1 0.8 HPGD $$-$$2.04 ± 0.38 $$-$$1.66 ± 0.15 1.3 0.8 SLCO2A1 $$-$$7.65 ± 0.13 $$-$$7.89 ± 0.24 0.9 0.1 EDN1 $$-$$7.44 ± 0.26 $$-$$7.54 ± 0.24 0.9 0.8 Lipid and cholesterol metabolism and transport LPL $$-$$9.37 ± 0.15 $$-$$9.26 ± 0.16 1.1 0.9 FABP3 $$-$$2.84 ± 0.27 $$-$$2.55 ± 0.31 1.2 0.1 STAR $$-$$7.95 ± 0.19 $$-$$8.11 ± 0.21 0.9 0.3 PLIN2 $$-$$3.67 ± 0.17 $$-$$3.97 ± 0.14 0.8 0.04 Immunoregulation CPN1 $$-$$5.55 ± 0.23 $$-$$5.90 ± 0.18 0.8 0.9 TCF7 $$-$$5.44 ± 0.11 $$-$$5.47 ± 0.07 1.0 0.4 Wnt signaling SFRP2 $$-$$4.57 ± 0.35 $$-$$4.54 ± 0.26 1.0 0.9 WNT11 $$-$$6.00 ± 0.16 $$-$$5.97 ± 0.12 1.0 0.5 Signal transduction YWHAG $$-$$1.44 ± 0.07 $$-$$1.50 ± 0.06 1.0 0.04 SPAG9 $$-$$2.61 ± 0.06 $$-$$2.72 ± 0.05 0.9 0.03 ΔCt ω6 FA diet (n = 13) ΔCt ω3 FA diet (n = 18) Mean fold-change (ω3 vs ω6) P-value IFNt-stimulated genes ISG15 $$-$$1.19 ± 0.13 $$-$$1.05 ± 0.27 1.1 0.4 OAS1 $$-$$0.39 ± 0.13 $$-$$0.30 ± 0.17 1.1 0.7 MX1 0.25 ± 0.10 0.34 ± 0.18 1.1 0.9 Antioxidant defense GPX1 $$-$$2.09 ± 0.18 $$-$$2.15 ± 0.09 1.0 0.6 GPX4 $$-$$3.72 ± 0.14 $$-$$3.71 ± 0.09 1.0 0.6 SOD1 $$-$$3.00 ± 0.48 $$-$$3.66 ± 0.75 0.6 0.8 SOD2 $$-$$5.39 ± 0.94 $$-$$5.35 ± 0.86 1.0 0.8 NQO1 $$-$$9.85 ± 0.31 $$-$$10.25 ± 0.21 0.8 0.4 GSTA2 $$-$$7.65 ± 0.86 $$-$$7.02 ± 0.17 1.6 0.7 GSR $$-$$4.44 ± 0.10 $$-$$4.42 ± 0.10 1.0 0.6 GSS $$-$$4.44 ± 0.62 $$-$$3.67 ± 0.19 1.7 0.7 CAT $$-$$4.35 ± 0.13 $$-$$4.52 ± 0.23 0.9 0.6 MT1 $$-$$8.33 ± 1.32 $$-$$7.44 ± 0.77 1.9 0.7 MT2 $$-$$7.86 ± 0.68 $$-$$7.18 ± 0.28 1.6 0.7 NFE2L2 $$-$$2.85 ± 0.12 $$-$$4.14 ± 0.87 0.4 0.3 Amino acid transport and metabolism SLC1A1 $$-$$1.08 ± 0.15 $$-$$1.19 ± 0.13 0.9 0.7 SLC1A5 $$-$$5.94 ± 0.21 $$-$$5.81 ± 0.23 1.1 0.3 SLC6A6 $$-$$5.86 ± 0.14 $$-$$5.98 ± 0.12 0.9 0.2 SLC7A1 $$-$$4.95 ± 0.10 $$-$$4.93 ± 0.11 1.0 0.2 SLC7A5 $$-$$8.33 ± 0.15 $$-$$8.29 ± 0.19 1.0 0.8 SLC7A8 $$-$$5.81 ± 0.30 $$-$$5.67 ± 0.27 1.1 0.9 SLC15A1 $$-$$5.99 ± 0.19 $$-$$6.00 ± 0.16 1.0 0.4 SLC15A2 $$-$$6.20 ± 0.11 $$-$$6.05 ± 0.13 1.1 0.09 SLC15A3 $$-$$6.10 ± 0.17 $$-$$5.98 ± 0.23 1.1 0.5 GTP $$-$$6.78 ± 0.13 $$-$$6.90 ± 0.15 0.9 0.5 HDC $$-$$10.37 ± 0.50 $$-$$10.29 ± 0.52 1.1 0.8 IGF signaling IGF1 $$-$$5.59 ± 0.39 $$-$$5.32 ± 0.30 1.2 0.8 IGF1R $$-$$3.53 ± 0.14 $$-$$3.39 ± 0.16 1.1 0.3 IGF2 $$-$$4.82 ± 0.27 $$-$$4.10 ± 0.21 1.7 0.2 IGF2R $$-$$5.66 ± 0.23 $$-$$5.40 ± 0.24 1.2 0.7 INSR $$-$$5.00 ± 0.06 $$-$$4.89 ± 0.07 1.1 0.06 PPAR signaling PPARA $$-$$4.10 ± 0.09 $$-$$4.18 ± 0.07 0.9 0.2 PPARD $$-$$5.47 ± 0.15 $$-$$5.38 ± 0.11 1.1 0.8 PPARG $$-$$9.97 ± 0.29 $$-$$9.72 ± 0.20 1.2 0.6 Prostaglandin signaling PLA2 $$-$$5.80 ± 0.12 $$-$$5.80 ± 0.07 1.0 0.5 PTGS2 $$-$$1.81 ± 0.20 $$-$$1.99 ± 0.15 0.9 0.4 PTGER2 $$-$$5.38 ± 0.11 $$-$$5.74 ± 0.41 0.8 0.6 PTGER4 $$-$$7.21 ± 0.19 $$-$$7.11 ± 0.14 1.1 0.9 PTGIS $$-$$7.76 ± 1.04 $$-$$7.33 ± 0.72 1.4 0.7 PTGIR $$-$$9.62 ± 0.21 $$-$$9.48 ± 0.29 1.1 0.8 HPGD $$-$$2.04 ± 0.38 $$-$$1.66 ± 0.15 1.3 0.8 SLCO2A1 $$-$$7.65 ± 0.13 $$-$$7.89 ± 0.24 0.9 0.1 EDN1 $$-$$7.44 ± 0.26 $$-$$7.54 ± 0.24 0.9 0.8 Lipid and cholesterol metabolism and transport LPL $$-$$9.37 ± 0.15 $$-$$9.26 ± 0.16 1.1 0.9 FABP3 $$-$$2.84 ± 0.27 $$-$$2.55 ± 0.31 1.2 0.1 STAR $$-$$7.95 ± 0.19 $$-$$8.11 ± 0.21 0.9 0.3 PLIN2 $$-$$3.67 ± 0.17 $$-$$3.97 ± 0.14 0.8 0.04 Immunoregulation CPN1 $$-$$5.55 ± 0.23 $$-$$5.90 ± 0.18 0.8 0.9 TCF7 $$-$$5.44 ± 0.11 $$-$$5.47 ± 0.07 1.0 0.4 Wnt signaling SFRP2 $$-$$4.57 ± 0.35 $$-$$4.54 ± 0.26 1.0 0.9 WNT11 $$-$$6.00 ± 0.16 $$-$$5.97 ± 0.12 1.0 0.5 Signal transduction YWHAG $$-$$1.44 ± 0.07 $$-$$1.50 ± 0.06 1.0 0.04 SPAG9 $$-$$2.61 ± 0.06 $$-$$2.72 ± 0.05 0.9 0.03 View Large Table 4. Luteal relative gene expression (mean ± SEM) and mean fold-changes of pregnant heifers as measured via qPCR. ΔCt ω6 FA diet (n = 6) ΔCt ω3 FA diet (n = 12) Mean fold- change (ω3 vs ω6) P-value HSD3B1 2.80 ± 0.16 2.73 ± 0.13 0.95 0.8 STAR 2.43 ± 0.28 2.58 ± 0.14 1.11 0.6 PTGFR 0.57 ± 0.11 0.73 ± 0.13 1.12 0.4 AKR1C3 $$-$$4.13 ± 0.08 $$-$$4.07 ± 0.05 1.04 0.5 CASP8 $$-$$5.45 ± 0.10 $$-$$5.39 ± 0.08 1.04 0.7 CASP3 $$-$$11.88 ± 0.24 $$-$$11.97 ± 0.06 0.94 0.6 PPARG $$-$$8.22 ± 0.28 $$-$$8.50 ± 0.11 0.82 0.3 ΔCt ω6 FA diet (n = 6) ΔCt ω3 FA diet (n = 12) Mean fold- change (ω3 vs ω6) P-value HSD3B1 2.80 ± 0.16 2.73 ± 0.13 0.95 0.8 STAR 2.43 ± 0.28 2.58 ± 0.14 1.11 0.6 PTGFR 0.57 ± 0.11 0.73 ± 0.13 1.12 0.4 AKR1C3 $$-$$4.13 ± 0.08 $$-$$4.07 ± 0.05 1.04 0.5 CASP8 $$-$$5.45 ± 0.10 $$-$$5.39 ± 0.08 1.04 0.7 CASP3 $$-$$11.88 ± 0.24 $$-$$11.97 ± 0.06 0.94 0.6 PPARG $$-$$8.22 ± 0.28 $$-$$8.50 ± 0.11 0.82 0.3 View Large Table 4. Luteal relative gene expression (mean ± SEM) and mean fold-changes of pregnant heifers as measured via qPCR. ΔCt ω6 FA diet (n = 6) ΔCt ω3 FA diet (n = 12) Mean fold- change (ω3 vs ω6) P-value HSD3B1 2.80 ± 0.16 2.73 ± 0.13 0.95 0.8 STAR 2.43 ± 0.28 2.58 ± 0.14 1.11 0.6 PTGFR 0.57 ± 0.11 0.73 ± 0.13 1.12 0.4 AKR1C3 $$-$$4.13 ± 0.08 $$-$$4.07 ± 0.05 1.04 0.5 CASP8 $$-$$5.45 ± 0.10 $$-$$5.39 ± 0.08 1.04 0.7 CASP3 $$-$$11.88 ± 0.24 $$-$$11.97 ± 0.06 0.94 0.6 PPARG $$-$$8.22 ± 0.28 $$-$$8.50 ± 0.11 0.82 0.3 ΔCt ω6 FA diet (n = 6) ΔCt ω3 FA diet (n = 12) Mean fold- change (ω3 vs ω6) P-value HSD3B1 2.80 ± 0.16 2.73 ± 0.13 0.95 0.8 STAR 2.43 ± 0.28 2.58 ± 0.14 1.11 0.6 PTGFR 0.57 ± 0.11 0.73 ± 0.13 1.12 0.4 AKR1C3 $$-$$4.13 ± 0.08 $$-$$4.07 ± 0.05 1.04 0.5 CASP8 $$-$$5.45 ± 0.10 $$-$$5.39 ± 0.08 1.04 0.7 CASP3 $$-$$11.88 ± 0.24 $$-$$11.97 ± 0.06 0.94 0.6 PPARG $$-$$8.22 ± 0.28 $$-$$8.50 ± 0.11 0.82 0.3 View Large Plasma progesterone was affected by dietary FA In both groups, the concentration of plasma P4 increased from day 3 to day 15 (Figure 2). Animals from the ω3 group had higher P4 concentrations compared to animals from the ω6 group. The average higher concentration of P4 in the ω3 group was due to the animals from which embryos >8 cm in length were recovered. Figure 2. View largeDownload slide Plasma progesterone concentrations were higher in ω3 FA supplemented heifers than in ω6 FA supplemented heifers. Values are given as means ± SEM. Figure 2. View largeDownload slide Plasma progesterone concentrations were higher in ω3 FA supplemented heifers than in ω6 FA supplemented heifers. Values are given as means ± SEM. Luteal gene expression of enzymes involved in P4 synthesis did not differ on day 15 between diet groups The luteal gene expression of neither hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), steroidogenic acute regulatory protein (STAR), prostaglandin F receptor (PTGFR), aldo-keto reductase family 1 member C3 (AKR1C3; previously known as hydroxysteroid (17-beta) dehydrogenase 5 (HSD17B5)), caspase 3 (CASP3), caspase 8 (CASP8) or PPARG differed between diet groups (Table 4). Dietary FA neither impacted on plasma LDL nor on plasma nor endometrial α- and γ-tocopherol concentrations The LDL cholesterol concentration in plasma did not differ between the two diet groups (ω6 group: 0.24 ± 0.03 mmol/l; ω3 group: 0.23 ± 0.03 mmol/l) on day 15 of pregnancy. Despite liver concentrations of α- and γ-tocopherol being significantly lower in the ω3 group compared to the ω6 group, neither plasma nor endometrial concentrations of α- and γ-tocopherol differed between groups, respectively (Supplementary Table S4). Discussion The present study is the first to report differences in embryo elongation in heifers supplemented with ω3 FA-rich fish oil and ω6 FA-rich sunflower oil, respectively. The main findings were that (1) day 15 embryos were significantly longer in ω3 compared to ω6 fed animals; (2) the concentration of AA was significantly lower, while the concentration of EPA was significantly higher and DHA was unaffected in the endometrium of ω3 compared to ω6 fed animals; (3) the concentrations of uterine prostaglandins did not differ between diet groups despite significantly different endometrial amounts of their precursor AA; and (4) increased plasma P4 concentration in ω3 compared to ω6 fed animals during luteal growth was associated with filamentous embryos present on day 15 of pregnancy. There is no general agreement on how to categorize embryos according to their length, specifically at the beginning of elongation. The classification of embryos differs between published studies [63–67]. Bovine embryos are suggested to have an ovoid (day 12), a tubular (day 14), or a filamentous (day 16–18) shape, and the term “elongation” is generally applied to embryos with an ovoid to slightly tubular shape [63, 68]. The day 15 embryos of the present study showed a great variance in length from the ovoid to the filamentous forms. The embryos recovered from ω3 FA supplemented animals were not only significantly longer than those recovered from ω6 FA supplemented animals, but also the variance in embryo length was greater in the ω3 group than in the ω6 group. Staples et al. [69] reported bovine embryo lengths on day 15 of pregnancy ranging from 0.6 to 17 cm, whereas Betteridge et al. [70] found day 15 embryo lengths ranging between 0.3 and 4 cm. Following superovulation, an enormous range of variation in embryo length (e.g. 0.4–4 cm on day 14) was observed even within a single donor [70]. Therefore, it remains difficult to state with certainty whether ω3 FA promoted or ω6 FA delayed embryo elongation in comparison to the respective other supplementation, or both. Dietary PUFA normally undergo partial biohydrogenation by ruminal microorganisms. Our FA supplements were coated with hydrogenated rapeseed oil for rumen protection. We chose heifers during their postpubertal growing phase to maximize the incorporation of dietary PUFA into the body lipid pools. The plasma and the endometrial FA profiles of our heifers confirm the successful rumen protection of ω3 FA. The higher EPA and DHA concentration in plasma of ω3 compared to ω6 supplemented animals is in accordance with the literature, where a stronger increase in EPA compared to DHA concentration as well as an increase in total ω3 PUFA lacking a difference in total ω6 PUFA has been shown [71, 72]. Increased plasma levels of supplemented ω3 FA led to an increased endometrial EPA concentration compared to the supplementation with ω6 FA. We observed an incorporation of dietary ω3 FA into endometrial tissue. This is in line with previously published studies [55, 71, 72], leading to a decreased ω6/ω3 ratio [55, 72, 73]. The latter has also been observed for luteal tissue [72]. During early embryonic development, PUFA play an important role by affecting membrane fluidity and permeability. They also serve as essential precursors for signaling molecules such as PG. Childs et al. [55] did not find any difference in plasma AA concentrations after 50 days of ω3 FA supplementation compared to an unsupplemented control group. This is in accordance with our findings, though we compared to a supplementation with ω6 FA. The supplementation with ω3 FA increased EPA almost threefold and decreased AA concentration in the UF of beef heifers compared to an ω6 supplementation, leading to significantly different ω6/ω3 ratios in the UF at day 7 of pregnancy [54]. Even though we did not analyze the FA in the UF, we have observed similar changes in EPA and AA in the endometrium. Consequently, we assume that our rumen-protected ω3 and ω6 FA supplementation has most likely resulted in a similar FA profile in the UF. Interestingly, Childs et al. [54] did not detect differences in DHA concentrations in the UF when comparing ω3 and ω6 supplemented animals. Accordingly, despite significant differences in DHA concentrations in plasma, we neither observed differing DHA concentrations in the endometrium of the two diet groups. Furthermore, the concentration of EPA was lower in endometrium compared to plasma, although endometrial AA and DHA concentrations were higher than those in plasma. Thus, we assume that AA and DHA have preferentially been incorporated into the endometrium. These results point toward a selective uptake of specific FA into the endometrium and the UF, respectively. We hypothesize that this regulatory mechanism might be due to specific needs of the developing embryo. It has been hypothesized that ω3 FA may act synergistically with IFNT to inhibit endometrial production of PGF2α and thus luteolysis [74]. In our study, we could not observe any diet-induced differences in endometrial gene expression of enzymes involved in PG synthesis. Moreover, with regard to the significantly decreased endometrial availability of AA in ω3 compared to ω6 FA supplemented animals, we hypothesized to find decreased series-2 PG in the UF. Surprisingly, this was not case. In several animal cell culture systems including bovine endometrial cells, incubation with ω3 FA decreased PGF2α production and secretion [74–77]. In addition, the PG-reducing effect of ω3 FA in the bovine reproductive tract has repeatedly been reported, especially in cyclic animals [78]. However, this was assessed by determining circulating PGFM. Our data show that irrespective of decreased AA, the local intrauterine series-2 PG concentrations during preimplantation development remained unaffected. We doubt whether the AA shift induced by a maternal diet can be pronounced enough at all to impact on the intrauterine hormonal signaling of series-2 PG. Most studies that report an effect of PUFA supplementation on PG concentrations were performed with cyclic animals. Therefore, we cannot rule out an effect of dietary ω3 and ω6 PUFA on PG concentrations in nonpregnant animals. Increasing plasma P4 concentrations might present a mechanism by which ω3 FA increase pregnancy rates in cows [79]. Intervention studies showed that higher P4 concentrations during the early stage of pregnancy were associated with a greater embryo elongation [18, 20–22, 80–82]. The importance of the P4 regulatory effect has been demonstrated in synchronous versus asynchronous embryo transfer [83, 84]. However, dietary ω3 and ω6 FA effects on plasma P4 concentrations led to contradictory results. In lactating Holstein cows, the supplementation of ω3 FA slightly increased mean P4 concentrations in some studies [85, 86], but lacked to show an effect in others [85, 87, 88]. Robinson et al. [89] observed lower plasma P4 concentrations when dairy cows were supplemented with FA. However, the effect was irrespective of a ω3 or ω6 FA supplementation. The incubation of bovine luteal cells with ω3 FA decreased P4 synthesis [90], whereas ω6 FA increased P4 in bovine CL cells [91]. Dietary ω3 FA are suggested to prevent the regression of the CL by decreased endometrial PGF2α released into the circulation (indicated by reduced PGFM), which may result in a prolonged P4 secretion [13, 92]. Indeed, a fish meal supplementation to lactating animals resulted in P4 concentrations >1 ng/ml 2 days after PGF2α injection. This led to the assumption of an inhibitory effect of ω3 FA on PGF2α-induced CL regression [13]. Dietary supplementation with flaxseed containing ALA led to higher P4 values on the day of AI in lactating Holstein cows compared to sunflower seed, also suggesting a delay in luteolysis [87]. In our experiment, we did not determine the timing of luteolysis and ovulation. If ω3 FA supplementation had caused a delay in luteolysis in our animals as described in the literature [13, 87], a delayed ovulation would be expected to result in less elongated embryos because of a relative younger embryonic age. However, we observed the opposite, thereby rather strengthening the hypothesis of promoted embryo elongation via ω3 FA supplementation. In our data, the heterogeneity in P4 was higher in the ω3 than in the ω6 group. Those heifers carrying an elongated embryo on day 15 had higher P4 from day 6 onwards. The supplementation with dietary ω3 FA has been associated with reduced plasma cholesterol compared to dietary ω6 FA [89]. While our data indicate the lack of change in plasma cholesterol, we found higher plasma P4 concentrations in the ω3 FA supplemented animals compared to the ω6 FA supplemented animals during luteal growth. However, this appeared due to those animals in the ω3 group where a filamentous embryo was present. Clearly, the rise in P4 of these latter ones could have caused the enhanced embryo elongation. However, it is unclear why only some animals responded to the ω3 treatment with an increase in P4 while others of the same treatment group did not. A direct effect of the dietary ω3 FA on P4 synthesis and luteal function will thus need further substantiation. We further analyzed whether the supplementation with ω3 compared to ω6 FA altered the response of the endometrium to P4. Specifically, gene expression changes via FA-sensitive transcription factors were of interest. In general, ω3 FA seem to be more effective in modifying gene expression than ω6 FA or SFA and increased EPA plasma levels are associated with increased PPAR gene expression [93, 94]. Interestingly, the endometrial gene expression of PPARs did not differ in the present study. Waters et al. [49] demonstrated that feeding ω3 FA led to differential gene expression of a variety of genes in the endometrium. We could confirm only some of these reported gene expression changes in our study. However, the control group in Waters et al. [49] received palmitic acid and not ω6 FA as reference. With the exception of alanine, neither the concentration of amino acids in the UF, the expression of amino acid transporters, the presence of endometrial α- and gamma-tocopherol, or uterine cortisol was affected by the diet. Surprisingly, even the gene expression of ISGs did not significantly differ between animals with embryos of different lengths. Because day 7 embryos already induce endometrial ISG15 ubiquitin-like modifier (ISG15; previously known as interferon, alpha-inducible protein (clone IFI-15K) (G1P2)), 2΄-5΄-oligoadenylate synthetase 1 (OAS1), and MX dynamin like GTPase 1 (MX1) gene expression [95], we assume that embryos may have induced the respective gene expression to a maximum already at an earlier stage of elongation. This would explain the lack of an observable difference. Taken together, endometrial gene expression differences in the present study in general were very scarce, even though the endometrial concentration of ω3 and ω6 FA was significantly altered. It remains to be analyzed to which extent gene expression and PG concentration differed before day 15 of pregnancy and may hereby had affected embryo elongation. Despite a significant difference in embryo length between the ω3 and ω6 FA groups, we cannot identify with certainty a mechanism by which ω3 and ω6 FA differentially affected embryo elongation. Our study rules out (1) a direct effect of ω3 FA altering endometrial embryotropic series-2 PG as well as (2) a direct or indirect effect of ω3 FA altering uterine amino acids, α- and γ-tocopherol, or cortisol furthering embryo elongation. In the present study, we neither investigated possible effects of dietary FA on uterine series-3 PG nor FA effects on the ovaries and thus follicles and oocytes. The latter remains a specifically interesting target because dietary ω3 and ω6 FA have been shown to affect follicular development and oocyte competency [96–98]. Therefore, further research is needed to understand the functional link between dietary ω3 FA and reproductive outcome. Supplementary data Supplementary data are available at BIOLRE online. Supplementary Table S1 Proximate (mean of pooled diet samples) and fatty acid composition of diets (n = 3) adapted from [57]. Values are given as means ± standard deviation. Supplementary Table S2 Sequences of primers. Supplementary Table S3 Amino acid concentrations in the uterine fluid. Values are given as means ± SEM. Supplementary Table S4 Plasma, liver and endometrial concentrations of alpha- and gamma-tocopherol. Values are given as means ± SEM. Acknowledgments We are thankful for the kind support of Marcus Wysshaar (ErboSpraytech), Michael Kreuzer for expert consulting, as well as the staff of the former ETH research station Chamau. The authors are active members of the European Union COST action CA16119 CELLFIT. References 1. Diskin MG , Murphy JJ , Sreenan JM . Embryo survival in dairy cows managed under pastoral conditions . Anim Reprod Sci 2006 ; 96 ( 3-4 ): 297 – 311 . Google Scholar CrossRef Search ADS PubMed 2. Moore K , Thatcher WW . Major advances associated with reproduction in dairy cattle . J Dairy Sci 2006 ; 89 : 1254 – 1266 . Google Scholar CrossRef Search ADS PubMed 3. Dobson H , Smith R , Royal M , Knight C , Sheldon I . The high-producing dairy cow and its reproductive performance . Reprod Domest Anim 2007 ; 42 ( s2 ): 17 – 23 . Google Scholar CrossRef Search ADS PubMed 4. Diskin MG , Parr MH , Morris DG . Embryo death in cattle: an update . Reprod Fertil Dev 2012 ; 24 : 244 – 251 . Google Scholar CrossRef Search ADS 5. Filant J , Spencer TE . Uterine glands: biological roles in conceptus implantation, uterine receptivity and decidualization . Int J Dev Biol 2014 ; 58 : 107 – 116 . Google Scholar CrossRef Search ADS PubMed 6. Ashworth CJ , Toma LM , Hunter MG . Nutritional effects on oocyte and embryo development in mammals: implications for reproductive efficiency and environmental sustainability . Philos T Roy Soc B Biol Sci 2009 ; 364 : 3351 – 3361 . Google Scholar CrossRef Search ADS 7. Mattos R , Staples CR , Thatcher WW . Effects of dietary fatty acids on reproduction in ruminants . Rev Reprod 2000 ; 5 : 38 – 45 . Google Scholar CrossRef Search ADS PubMed 8. Lopes CN , Scarpa AB , Cappellozza BI , Cooke RF , Vasconcelos JL . Effects of rumen-protected polyunsaturated fatty acid supplementation on reproductive performance of Bos indicus beef cows . J Anim Sci 2009 ; 87 : 3935 – 3943 . Google Scholar CrossRef Search ADS PubMed 9. Juchem SO , Cerri RLA , Villasenor M , Galvao KN , Bruno RGS , Rutigliano HM , DePeters EJ , Silvestre FT , Thatcher WW , Santos JEP . Supplementation with calcium salts of linoleic and trans -octadecenoic acids improves fertility of lactating dairy cows . Reprod Dom Anim 2010 ; 45 : 55 – 62 . Google Scholar CrossRef Search ADS 10. Colazo MG , Kastelic JP , Martinez MF , Whittaker PR , Wilde R , Ambrose JD , Corbett R , Mapletoft RJ . Fertility following fixed-time AI in CIDR-treated beef heifers given GnRH or estradiol cypionate and fed diets supplemented with flax seed or sunflower seed . Theriogenology 2004 ; 61 : 1115 – 1124 . Google Scholar CrossRef Search ADS PubMed 11. Bork NR , Schroeder JW , Lardy GP , Vonnahme KA , Bauer ML , Buchanan DS , Shaver RD , Fricke PM . Effect of feeding rolled flaxseed on milk fatty acid profiles and reproductive performance of dairy cows . J Anim Sci 2010 ; 88 : 3739 – 3748 . Google Scholar CrossRef Search ADS PubMed 12. Richardson GF , McNiven MA , Petit HV , Duynisveld JL . The effects of dietary omega fatty acids on pregnancy rate, plasma prostaglandin metabolite levels, serum progesterone levels, and milk fatty-acid profile in beef cows . Can J Vet Res 2013 ; 77 : 314 – 318 . Google Scholar PubMed 13. Burke JM , Staples CR , Risco CA , de la Sota RL , Thatcher WW . Effect of ruminant grade Menhaden fish meal on reproductive and productive performance of lactating dairy cows . J Dairy Sci 1997 ; 80 : 3386 – 3398 . Google Scholar CrossRef Search ADS PubMed 14. Gulliver CE , Friend MA , King BJ , Clayton EH . The role of omega-3 polyunsaturated fatty acids in reproduction of sheep and cattle . Anim Reprod Sci 2012 ; 131 : 9 – 22 . Google Scholar CrossRef Search ADS PubMed 15. Spencer TE , Ott TL , Bazer FW . tau-Interferon: pregnancy recognition signal in ruminants . Exp Biol Med 1996 ; 213 : 215 – 229 . Google Scholar CrossRef Search ADS 16. Roberts RM , Ealy AD , Alexenko AP , Han CS , Ezashi T . Trophoblast interferons . Placenta 1999 ; 20 : 259 – 264 . Google Scholar CrossRef Search ADS PubMed 17. Hansen TR , Austin KJ , Perry DJ , Pru JK , Teixeira MG , Johnson GA . Mechanism of action of interferon-tau in the uterus during early pregnancy . J Reprod Fertil Suppl 1999 ; 54 : 329 – 339 . Google Scholar PubMed 18. Garrett JE , Geisert RD , Zavy MT , Morgan GL . Evidence for maternal regulation of early conceptus growth and development in beef cattle . Reproduction 1988 ; 84 : 437 – 446 . Google Scholar CrossRef Search ADS 19. Mann GE , Lamming GE . Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows . Reproduction 2001 ; 121 : 175 – 180 . Google Scholar CrossRef Search ADS PubMed 20. Satterfield MC , Bazer FW , Spencer TE . Progesterone regulation of preimplantation conceptus growth and galectin 15 (LGALS15) in the ovine uterus . Biol Reprod 2006 ; 75 : 289 – 296 . Google Scholar CrossRef Search ADS PubMed 21. Carter F , Forde N , Duffy P , Wade M , Fair T , Crowe MA , Evans AC , Kenny DA , Roche JF , Lonergan P . Effect of increasing progesterone concentration from Day 3 of pregnancy on subsequent embryo survival and development in beef heifers . Reprod Fertil Dev 2008 ; 20 : 368 – 375 . Google Scholar CrossRef Search ADS PubMed 22. Forde N , Carter F , Fair T , Crowe MA , Evans AC , Spencer TE , Bazer FW , McBride R , Boland MP , O’Gaora P , Lonergan P , Roche JF . Progesterone-regulated changes in endometrial gene expression contribute to advanced conceptus development in cattle . Biol Reprod 2009 ; 81 : 784 – 794 . Google Scholar CrossRef Search ADS PubMed 23. Mesquita FS , Pugliesi G , Scolari SC , Franca MR , Ramos RS , Oliveira M , Papa PC , Bressan FF , Meirelles FV , Silva LA , Nogueira GP , Membrive CM et al. Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows . Theriogenology 2014 ; 81 : 861 – 869 . Google Scholar CrossRef Search ADS PubMed 24. Dorniak P , Welsh TH Jr , Bazer FW , Spencer TE . Cortisol and interferon tau regulation of endometrial function and conceptus development in female sheep . Endocrinology 2013 ; 154 : 931 – 941 . Google Scholar CrossRef Search ADS PubMed 25. Brooks K , Burns G , Spencer TE . Conceptus elongation in ruminants: roles of progesterone, prostaglandin, interferon tau and cortisol . J Anim Sci Biotechnol 2014 ; 5 : 53 . Google Scholar CrossRef Search ADS PubMed 26. Bazer FW , Wu G , Spencer TE , Johnson GA , Burghardt RC , Bayless K . Novel pathways for implantation and establishment and maintenance of pregnancy in mammals . Mol Hum Reprod 2010 ; 16 : 135 – 152 . Google Scholar CrossRef Search ADS PubMed 27. Forde N , Lonergan P . Transcriptomic Analysis of the bovine endometrium: what is required to establish uterine receptivity to implantation in cattle? J Reprod Dev 2012 ; 58 : 189 – 195 . Google Scholar CrossRef Search ADS PubMed 28. Charpigny G , Reinaud P , Tamby JP , Creminon C , Martal J , Maclouf J , Guillomot M . Expression of cyclooxygenase-1 and -2 in ovine endometrium during the estrous cycle and early pregnancy . Endocrinology 1997 ; 138 : 2163 – 2171 . Google Scholar CrossRef Search ADS PubMed 29. Simmons RM , Satterfield MC , Welsh TH Jr , Bazer FW , Spencer TE . HSD11B1, HSD11B2, PTGS2, and NR3C1 expression in the peri-implantation ovine uterus: effects of pregnancy, progesterone, and interferon tau . Biol Reprod 2010 ; 82 : 35 – 43 . Google Scholar CrossRef Search ADS PubMed 30. Wathes DC , Lamming GE . The oxytocin receptor, luteolysis and the maintenance of pregnancy . J Reprod Fertil Suppl 1995 ; 49 : 53 – 67 . Google Scholar PubMed 31. Ulbrich SE , Schulke K , Groebner AE , Reichenbach HD , Angioni C , Geisslinger G , Meyer HH . Quantitative characterization of prostaglandins in the uterus of early pregnant cattle . Reproduction 2009 ; 138 : 371 – 382 . Google Scholar CrossRef Search ADS PubMed 32. Dorniak P , Bazer FW , Spencer TE . Prostaglandins regulate conceptus elongation and mediate effects of interferon tau on the ovine uterine endometrium . Biol Reprod 2011 ; 84 : 1119 – 1127 . Google Scholar CrossRef Search ADS PubMed 33. Charpigny G , Reinaud P , Tamby JP , Creminon C , Guillomot M . Cyclooxygenase-2 unlike cyclooxygenase-1 is highly expressed in ovine embryos during the implantation period . Biol Reprod 1997 ; 57 : 1032 – 1040 . Google Scholar CrossRef Search ADS PubMed 34. Lewis GS , Waterman RA . Effects of endometrium on metabolismof arachidonic acid by bovine blastocysts . Prostaglandins 1983 ; 25 : 881 – 889 . Google Scholar CrossRef Search ADS PubMed 35. Lewis GS , Waterman RA . Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy . Prostaglandins 1985 ; 30 : 263 – 283 . Google Scholar CrossRef Search ADS PubMed 36. Bartol FF , Thatcher WW , Bazer FW , Kimball FA , Chenault JR , Wilcox CJ , Roberts RM . Effects of the estrous cycle and early pregnancy on bovine uterine, luteal, and follicular responses . Biol Reprod 1981 ; 25 : 759 – 776 . Google Scholar CrossRef Search ADS PubMed 37. Schallenberger E , Schams D , Meyer HH . Sequences of pituitary, ovarian and uterine hormone secretion during the first 5 weeks of pregnancy in dairy cattle . J Reprod Fertil Suppl 1989 ; 37 : 277 – 286 . Google Scholar PubMed 38. Arosh JA , Banu SK , Kimmins S , Chapdelaine P , Maclaren LA , Fortier MA . Effect of interferon-tau on prostaglandin biosynthesis, transport, and signaling at the time of maternal recognition of pregnancy in cattle: evidence of polycrine actions of prostaglandin E2 . Endocrinology 2004 ; 145 : 5280 – 5293 . Google Scholar CrossRef Search ADS PubMed 39. Emond V , MacLaren LA , Kimmins S , Arosh JA , Fortier MA , Lambert RD . Expression of cyclooxygenase-2 and granulocyte-macrophage colony-stimulating factor in the endometrial epithelium of the cow is up-regulated during early pregnancy and in response to intrauterine infusions of interferon-tau . Biol Reprod 2004 ; 70 : 54 – 64 . Google Scholar CrossRef Search ADS PubMed 40. Krishnaswamy N , Chapdelaine P , Tremblay JP , Fortier MA . Development and characterization of a simian virus 40 immortalized bovine endometrial stromal cell line . Endocrinology 2009 ; 150 : 485 – 491 . Google Scholar CrossRef Search ADS PubMed 41. Mattos R , Staples CR , Williams J , Amorocho A , McGuire MA , Thatcher WW . Uterine, ovarian, and production responses of lactating dairy cows to increasing dietary concentrations of menhaden fish meal . J Dairy Sci 2002 ; 85 : 755 – 764 . Google Scholar CrossRef Search ADS PubMed 42. Needleman P , Raz A , Minkes MS , Ferrendelli JA , Sprecher H . Triene prostaglandins: Prostacyclin and thromboxane biosynthesis and unique biological properties . Proc Natl Acad Sci USA 1979 ; 76 : 944 – 948 . Google Scholar CrossRef Search ADS PubMed 43. Ringbom T , Huss U , Stenholm A , Flock S , Skattebol L , Perera P , Bohlin L . COX-2 inhibitory effects of naturally occurring and modified fatty acids . J Nat Prod 2001 ; 64 : 745 – 749 . Google Scholar CrossRef Search ADS PubMed 44. Desvergne B , Wahli W . Peroxisome proliferator-activated receptors: nuclear control of metabolism . Endocr Rev 1999 ; 20 : 649 – 688 . Google Scholar PubMed 45. Calder PC , Yaqoob P . Understanding omega-3 polyunsaturated fatty acids . Postgrad Med 2009 ; 121 : 148 – 157 . Google Scholar CrossRef Search ADS PubMed 46. Lim H , Gupta RA , Ma WG , Paria BC , Moller DE , Morrow JD , DuBois RN , Trzaskos JM , Dey SK . Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta . Genes Dev 1999 ; 13 : 1561 – 1574 . Google Scholar CrossRef Search ADS PubMed 47. Waters SM , Kelly JP , O’Boyle P , Moloney AP , Kenny DA . Effect of level and duration of dietary n-3 polyunsaturated fatty acid supplementation on the transcriptional regulation of Delta9-desaturase in muscle of beef cattle . J Anim Sci 2009 ; 87 : 244 – 252 . Google Scholar CrossRef Search ADS PubMed 48. Berger J , Moller DE . The mechanisms of action of PPARs . Annu Rev Med 2002 ; 53 : 409 – 435 . Google Scholar CrossRef Search ADS PubMed 49. Waters SM , Coyne GS , Kenny DA , MacHugh DE , Morris DG . Dietary n-3 polyunsaturated fatty acid supplementation alters the expression of genes involved in the control of fertility in the bovine uterine endometrium . Physiol Genomics 2012 ; 44 : 878 – 888 . Google Scholar CrossRef Search ADS PubMed 50. Brooks K , Spencer TE . Biological roles of interferon tau (IFNT) and type I IFN receptors in elongation of the ovine conceptus . Biol Reprod 2015 ; 92 : 47 . Google Scholar PubMed 51. Mattos R , Staples CR , Arteche A , Wiltbank MC , Diaz FJ , Jenkins TC , Thatcher WW . The effects of feeding fish oil on uterine secretion of PGF2alpha, milk composition, and metabolic status of periparturient holstein cows . J Dairy Sci 2004 ; 87 : 921 – 932 . Google Scholar CrossRef Search ADS PubMed 52. Filley SJ , Turner HA , Stormshak F . Plasma fatty acids, prostaglandin F-2alpha metabolite, and reproductive response in postpartum heifers fed rumen bypass fat . J Anim Sci 2000 ; 78 : 139 – 144 . Google Scholar CrossRef Search ADS PubMed 53. Petit HV , Germiquet C , Lebel D . Effect of feeding whole, unprocessed sunflower seeds and flaxseed on milk production, milk composition, and prostaglandin secretion in dairy cows . J Dairy Sci 2004 ; 87 : 3889 – 3898 . Google Scholar CrossRef Search ADS PubMed 54. Childs S , Carter F , Lynch CO , Sreenan JM , Lonergan P , Hennessy AA , Kenny DA . Embryo yield and quality following dietary supplementation of beef heifers with n-3 polyunsaturated fatty acids (PUFA) . Theriogenology 2008 ; 70 : 992 – 1003 . Google Scholar CrossRef Search ADS PubMed 55. Childs S , Hennessy AA , Sreenan JM , Wathes DC , Cheng Z , Stanton C , Diskin MG , Kenny DA . Effect of level of dietary n-3 polyunsaturated fatty acid supplementation on systemic and tissue fatty acid concentrations and on selected reproductive variables in cattle . Theriogenology 2008 ; 70 : 595 – 611 . Google Scholar CrossRef Search ADS PubMed 56. Fuentes MC , Calsamiglia S , Sanchez C , Gonzalez A , Newbold JR , Santos JEP , Rodriguez-Alcala LM , Fontecha J . Effect of extruded linseed on productive and reproductive performance of lactating dairy cows . Livestock Sci 2008 ; 113 : 144 – 154 . Google Scholar CrossRef Search ADS 57. Wolf C , Ulbrich SE , Kreuzer M , Berard J , Giller K . Differential partitioning of rumen-protected n –3 and n –6 fatty acids into muscles with different metabolism . Meat Sci 2018 ; 137 : 106 – 113 . Google Scholar CrossRef Search ADS PubMed 58. Firl N , Kienberger H , Hauser T , Rychlik M . Determination of the fatty acid profile of neutral lipids, free fatty acids and phospholipids in human plasma . Clin Chem Lab Med 2013 ; 51 : 799 – 810 . Google Scholar CrossRef Search ADS PubMed 59. Giesbertz P , Padberg I , Rein D , Ecker J , Hofle A , Spanier B , Daniel H . Metabolite profiling in plasma and tissues of ob/ob and db/db mice identifies novel markers of obesity and type 2 diabetes . Diabetologia 2015 ; 58 : 2133 – 2143 . Google Scholar CrossRef Search ADS PubMed 60. Pfaffl MW , Tichopad A , Prgomet C , Neuvians TP . Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations . Biotechnol Lett 2004 ; 26 : 509 – 515 . Google Scholar CrossRef Search ADS PubMed 61. Ruhmann B , Giller K , Hankele A , Ulbrich SE , Schmicke M . Interferon-tau induced gene expression in hepatocytes during early pregnancy in Angus heifers . Reprod Domest Anim 2017 ; 52 : 45 – 45 . Google Scholar CrossRef Search ADS PubMed 62. Grebenstein N , Frank J . Rapid baseline-separation of all eight tocopherols and tocotrienols by reversed-phase liquid-chromatography with a solid-core pentafluorophenyl column and their sensitive quantification in plasma and liver . J Chromatogr A 2012 ; 1243 : 39 – 46 . Google Scholar CrossRef Search ADS PubMed 63. Hue I , Degrelle SA , Campion E , Renard J-P . Gene expression in elongating and gastrulating embryos from ruminants . Soc Reprod Fertil Suppl 2007 ; 64 : 13 . Google Scholar PubMed 64. Degrelle SA , Jaffrezic F , Campion E , Le Cao KA , Le Bourhis D , Richard C , Rodde N , Fleurot R , Everts RE , Lecardonnel J , Heyman Y , Vignon X et al. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer . PLoS One 2012 ; 7 : e38309 . Google Scholar CrossRef Search ADS PubMed 65. Degrelle SA , Campion E , Cabau C , Piumi F , Reinaud P , Richard C , Renard JP , Hue I . Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts . Dev Biol 2005 ; 288 : 448 – 460 . Google Scholar CrossRef Search ADS PubMed 66. Barnwell CV , Farin PW , Ashwell CM , Farmer WT , Galphin SP , Farin CE . Differences in mRNA populations of short and long bovine conceptuses on Day 15 of gestation . Mol Reprod Dev 2016 ; 83 : 424 – 441 . Google Scholar CrossRef Search ADS PubMed 67. Bazer FW , Song GH , Thatcher WW . Roles of conceptus secretory proteins in establishment and maintenance of pregnancy in ruminants . Asian Australas J Anim Sci 2012 ; 25 : 1 – 16 . Google Scholar CrossRef Search ADS PubMed 68. Hue I . Determinant molecular markers for peri-gastrulating bovine embryo development . Reprod Fertil Dev 2016 ; 28 : 51 – 65 . Google Scholar CrossRef Search ADS PubMed 69. Staples RE , Hansel W , Mcentee K . Luteal function as related to pituitary and ovarian cytology and embryo development in the bovine . J Dairy Sci 1961 ; 44 : 2049 – 2057 . Google Scholar CrossRef Search ADS 70. Betteridge KJ , Eaglesome MD , Randall GC , Mitchell D . Collection, description and transfer of embryos from cattle 10-16 days after oestrus . Reproduction 1980 ; 59 : 205 – 216 . Google Scholar CrossRef Search ADS 71. Burns PD , Engle TE , Harris MA , Enns RM , Whittier JC . Effect of fish meal supplementation on plasma and endometrial fatty acid composition in nonlactating beef cows . J Anim Sci 2003 ; 81 : 2840 – 2846 . Google Scholar CrossRef Search ADS PubMed 72. White NR , Burns PD , Cheatham RD , Romero RM , Nozykowski JP , Bruemmer JE , Engle TE . Fish meal supplementation increases bovine plasma and luteal tissue omega-3 fatty acid composition . J Anim Sci 2012 ; 90 : 771 – 778 . Google Scholar CrossRef Search ADS PubMed 73. Moussavi ARH , Gilbert RO , Overton TR , Bauman DE , Butler WR . Effects of feeding fish meal and n-3 fatty acids on ovarian and uterine responses in early lactating dairy cows . J Dairy Sci 2007 ; 90 : 145 – 154 . Google Scholar CrossRef Search ADS PubMed 74. Mattos R , Guzeloglu A , Badinga L , Staples CR , Thatcher WW . Polyunsaturated fatty acids and bovine interferon-tau modify phorbol ester-induced secretion of prostaglandin f2 alpha and expression of prostaglandin endoperoxide synthase-2 and phospholipase-a2 in bovine endometrial cells . Biol Reprod 2003 ; 69 : 780 – 787 . Google Scholar CrossRef Search ADS PubMed 75. Levine L , Worth N . Eicosapentaenoic acid: Its effects on arachidonic acid metabolism by cells in culture . J Allergy Clin Immunol 1984 ; 74 : 430 – 436 . Google Scholar CrossRef Search ADS PubMed 76. Achard F , Gilbert M , Benistant C , Ben Slama S , DeWitt DL , Smith WL , Lagarde M . Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1 expression in bovine aortic endothelial cells . Biochem Biophys Res Commun 1997 ; 241 : 513 – 518 . Google Scholar CrossRef Search ADS PubMed 77. Caldari-Torres C , Rodriguez-Sallaberry C , Greene ES , Badinga L . Differential effects of n-3 and n-6 fatty acids on prostaglandin F2 alpha production by bovine endometrial cells . J Dairy Sci 2006 ; 89 : 971 – 977 . Google Scholar CrossRef Search ADS PubMed 78. Wathes DC , Abayasekara DR , Aitken RJ . Polyunsaturated fatty acids in male and female reproduction . Biol Reprod 2007 ; 77 : 190 – 201 . Google Scholar CrossRef Search ADS PubMed 79. Mann GE , Lamming GE . The influence of progesterone during early pregnancy in cattle . Reprod Domest Anim 1999 ; 34 : 269 – 274 . Google Scholar CrossRef Search ADS 80. Rizos D , Carter F , Besenfelder U , Havlicek V , Lonergan P . Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows . J Dairy Sci 2010 ; 93 : 1022 – 1029 . Google Scholar CrossRef Search ADS PubMed 81. O’Hara L , Forde N , Kelly AK , Lonergan P . Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: Can supplementary progesterone rescue small embryos? Theriogenology 2014 ; 81 : 1123 – 1128 . Google Scholar CrossRef Search ADS PubMed 82. Silva JCE , da Costa LL , Silva JR . Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers . Anim Reprod Sci 2002 ; 69 : 1 – 8 . Google Scholar CrossRef Search ADS PubMed 83. Ledgard AM , Berg MC , McMillan WH , Smolenski G , Peterson AJ . Effect of asynchronous transfer on bovine embryonic development and relationship with early cycle uterine proteome profiles . Reprod Fertil Dev 2012 ; 24 : 962 – 972 . Google Scholar CrossRef Search ADS PubMed 84. Randi F , Kenny DA , Lonergan P . The use of fixed time artificial insemination in beef suckler herds . Cattle Practice 2015 ; 23 : 235 – 236 . 85. Petit HV , Twagiramungu H . Conception rate and reproductive function of dairy cows fed different fat sources . Theriogenology 2006 ; 66 : 1316 – 1324 . Google Scholar CrossRef Search ADS PubMed 86. Thangavelu G , Colazo MG , Ambrose DJ , Oba M , Okine EK , Dyck MK . Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows . Theriogenology 2007 ; 68 : 949 – 957 . Google Scholar CrossRef Search ADS PubMed 87. Ambrose DJ , Kastelic JP , Corbett R , Pitney PA , Petit HV , Small JA , Zalkovic P . Lower pregnancy losses in lactating dairy cows fed a diet enriched in alpha-linolenic acid . J Dairy Sci 2006 ; 89 : 3066 – 3074 . Google Scholar CrossRef Search ADS PubMed 88. Bilby TR , Sozzi A , Lopez MM , Silvestre FT , Ealy AD , Staples CR , Thatcher WW . Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows: i. ovarian, conceptus, and growth hormone–insulin-like growth factor system responses . J Dairy Sci 2006 ; 89 : 3360 – 3374 . Google Scholar CrossRef Search ADS PubMed 89. Robinson RS , Pushpakumara PGA , Cheng Z , Peters AR , Abayasekara DRE , Wathes DC . Effects of dietary polyunsaturated fatty acids on ovarian and uterine function in lactating dairy cows . Reproduction 2002 ; 124 : 119 – 131 . Google Scholar CrossRef Search ADS PubMed 90. Hinckley T , Clark RM , Bushmich SL , Milvae RA . Long chain polyunsaturated fatty acids and bovine luteal cell function . Biol Reprod 1996 ; 55 : 445 – 449 . Google Scholar CrossRef Search ADS PubMed 91. Weems YS , Kim L , Humphreys V , Tsuda V , Blankfein R , Wong A , Weems CW . Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE1 and PGE2) and F2 alpha (PGF2 alpha) by ovine corpora lutea of the estrous cycle or pregnancy in vitro . Prostaglandins Other Lipid Mediat 2007 ; 84 : 163 – 173 . Google Scholar CrossRef Search ADS PubMed 92. McCracken JA , Samuelsson B , Goding JR , Glew ME , Green K , Carlson JC , Baird DT . Prostaglandin F2-Alpha identified as a Luteolytic Hormone in Sheep . Nat New Biol 1972 ; 238 : 129 – 134 . Google Scholar CrossRef Search ADS PubMed 93. MacLaren LA , Guzeloglu A , Michel F , Thatcher WW . Peroxisome proliferator-activated receptor (PPAR) expression in cultured bovine endometrial cells and response to omega-3 fatty acid, growth hormone and agonist stimulation in relation to series 2 prostaglandin production . Domest Anim Endocrinol 2006 ; 30 : 155 – 169 . Google Scholar CrossRef Search ADS PubMed 94. Clarke SD , Jump DB . Dietary polyunsaturated fatty acid regulation of gene transcription . Annu Rev Nutr 1994 ; 14 : 83 – 98 . Google Scholar CrossRef Search ADS PubMed 95. Sponchiado M , Gomes NS , Fontes PK , Martins T , del Collado M , Pastore AD , Pugliesi G , Nogueira MFG , Binelli M . Pre-hatching embryo-dependent and -independent programming of endometrial function in cattle . PLoS One 2017 ; 12 : e0175954 . Google Scholar CrossRef Search ADS PubMed 96. Marei WFA , De Bie J , Mohey-Elsaeed O , Wydooghe E , Bols PEJ , Leroy JLMR . Alpha-linolenic acid protects the developmental capacity of bovine cumulus–oocyte complexes matured under lipotoxic conditions in vitro . Biol Reprod 2017 ; 96 : 1181 – 1196 . Google Scholar CrossRef Search ADS PubMed 97. Oseikria M , Elis S , Maillard V , Corbin E , Uzbekova S . N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle . Theriogenology 2016 ; 85 : 1625 – 1634.e2 . Google Scholar CrossRef Search ADS PubMed 98. Hughes J , Kwong WY , Li DF , Salter AM , Lea RG , Sinclair KD . Effects of omega-3 and -6 polyunsaturated fatty acids on ovine follicular cell steroidogenesis, embryo development and molecular markers of fatty acid metabolism . Reproduction 2011 ; 141 : 105 – 118 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biology of Reproduction Oxford University Press http://www.deepdyve.com/lp/oxford-university-press/bovine-embryo-elongation-is-altered-due-to-maternal-fatty-acid-0Qt1knVvpK

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References (108)

M. Dehnhard, S. Naidenko, K. Jewgenow (2017)
Metabolism of prostaglandin F2alpha in Eurasian lynx (Lynx lynx) and Asian leopard cat (Prionailurus bengalensis euptilura).
Reproduction in domestic animals = Zuchthygiene, 52 Suppl 2
F. Bazer, G. Song, W. Thatcher (2011)
Roles of Conceptus Secretory Proteins in Establishment and Maintenance of Pregnancy in Ruminants
Asian-Australasian Journal of Animal Sciences, 25
I. Hue (2016)
Determinant molecular markers for peri-gastrulating bovine embryo development.
Reproduction, fertility, and development, 28 1-2
M. Satterfield, F. Bazer, T. Spencer (2006)
Progesterone Regulation of Preimplantation Conceptus Growth and Galectin 15 (LGALS15) in the Ovine Uterus1
, 75
T. Ringbom, Ulrika Huss, Å. Stenholm, S. Flock, L. Skattebol, P. Perera, L. Bohlin (2001)
Cox-2 inhibitory effects of naturally occurring and modified fatty acids.
Journal of natural products, 64 6
Nadine Grebenstein, J. Frank (2012)
Rapid baseline-separation of all eight tocopherols and tocotrienols by reversed-phase liquid-chromatography with a solid-core pentafluorophenyl column and their sensitive quantification in plasma and liver.
Journal of chromatography. A, 1243
R. Roberts, A. Ealy, A. Alexenko, C. Han, T. Ezashi (1999)
Current TopicTrophoblast Interferons
Placenta, 20
N. Krishnaswamy, P. Chapdelaine, J. Tremblay, M. Fortier (2009)
Development and characterization of a simian virus 40 immortalized bovine endometrial stromal cell line.
Endocrinology, 150 1
Nina Firl, H. Kienberger, T. Hauser, M. Rychlik (2012)
Determination of the fatty acid profile of neutral lipids, free fatty acids and phospholipids in human plasma
Clinical Chemistry and Laboratory Medicine (CCLM), 51
A. Ledgard, M. Berg, W. Mcmillan, G. Smolenski, A. Peterson (2012)
Effect of asynchronous transfer on bovine embryonic development and relationship with early cycle uterine proteome profiles.
Reproduction, fertility, and development, 24 7
Ruhmann (2017)
Interferon-tau induced gene expression in hepatocytes during early pregnancy in Angus heifers
Reprod Domest Anim, 52
P. Giesbertz, I. Padberg, D. Rein, J. Ecker, Anja Hoefle, Britta Spanier, H. Daniel (2015)
Metabolite profiling in plasma and tissues of ob/ob and db/db mice identifies novel markers of obesity and type 2 diabetes
Diabetologia, 58
T. Bilby, A. Sozzi, M. López, F. Silvestre, A. Ealy, C. Staples, W. Thatcher (2006)
Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows: I. Ovarian, conceptus, and growth hormone-insulin-like growth factor system responses.
Journal of dairy science, 89 9
R. Simmons, M. Satterfield, Thomas Welsh, F. Bazer, T. Spencer (2010)
HSD11B1, HSD11B2, PTGS2, and NR3C1 Expression in the Peri-Implantation Ovine Uterus: Effects of Pregnancy, Progesterone, and Interferon Tau1
, 82
S. Waters, J. Kelly, P. O’Boyle, A. Moloney, D. Kenny (2009)
Effect of level and duration of dietary n-3 polyunsaturated fatty acid supplementation on the transcriptional regulation of Delta9-desaturase in muscle of beef cattle.
Journal of animal science, 87 1
R. Mattos, C. Staples, W. Thatcher (2000)
EFFECTS OF DIETARY FATTY ACIDS ON REPRODUCTION IN RUMINANTS
Reviews of Reproduction, 5
B. Desvergne, W. Wahli (1999)
Peroxisome proliferator-activated receptors: nuclear control of metabolism.
Endocrine reviews, 20 5
J. Mccracken, J. Carlson, M. Glew, J. Goding, D. Baird, K. Gréen, B. Samuelsson (1972)
Prostaglandin F 2 identified as a luteolytic hormone in sheep.
Nature: New biology, 238 83
S. Waters, Gerard Coyne, D. Kenny, D. MacHugh, D. Morris (2012)
Dietary n-3 polyunsaturated fatty acid supplementation alters the expression of genes involved in the control of fertility in the bovine uterine endometrium.
Physiological genomics, 44 18
M. Diskin, J. Murphy, J. Sreenan (2006)
Embryo survival in dairy cows managed under pastoral conditions.
Animal reproduction science, 96 3-4
P. Burns, T. Engle, M. Harris, R. Enns, J. Whittier (2003)
Effect of fish meal supplementation on plasma and endometrial fatty acid composition in nonlactating beef cows.
Journal of animal science, 81 11
Y. Weems, L. Kim, V. Humphreys, V. Tsuda, R. Blankfein, A. Wong, C. Weems (2007)
Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE(1) and PGE(2)) and F(2alpha) (PGF(2alpha)) by ovine corpora lutea of the estrous cycle or pregnancy in vitro.
Prostaglandins & other lipid mediators, 84 3-4
J. Garrett, R. Geisert, M. Zavy, G. Morgan (1988)
Evidence for maternal regulation of early conceptus growth and development in beef cattle.
Journal of reproduction and fertility, 84 2
G. Charpigny, P. Reinaud, J. Tamby, C. Créminon, M. Guillomot (1997)
Cyclooxygenase-2 unlike cyclooxygenase-1 is highly expressed in ovine embryos during the implantation period.
Biology of reproduction, 57 5
S. Juchem, R. Cerri, M. Villaseñor, K. Galvão, R. Bruno, H. Rutigliano, E. Depeters, F. Silvestre, W. Thatcher, J. Santos (2010)
Supplementation with calcium salts of linoleic and trans-octadecenoic acids improves fertility of lactating dairy cows.
Reproduction in domestic animals = Zuchthygiene, 45 1
E. Schallenberger, D. Schams, Meyer Hh (1989)
Sequences of pituitary, ovarian and uterine hormone secretion during the first 5 weeks of pregnancy in dairy cattle.
Journal of reproduction and fertility. Supplement, 37
N. Forde, F. Carter, T. Fair, M. Crowe, A. Evans, T. Spencer, F. Bazer, R. McBride, M. Boland, P. O'Gaora, P. Lonergan, J. Roche (2009)
Progesterone-Regulated Changes in Endometrial Gene Expression Contribute to Advanced Conceptus Development in Cattle1
, 81
H. Lim, Rajnish Gupta, Wen-ge Ma, B. Paria, D. Moller, J. Morrow, R. Dubois, J. Trzăskos, S. Dey (1999)
Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta.
Genes & development, 13 12
Roberts (1999)
Trophoblast interferons
Placenta, 20
K. Moore, W. Thatcher (2006)
Major advances associated with reproduction in dairy cattle.
Journal of dairy science, 89 4
P. Dorniak, T. Welsh, F. Bazer, T. Spencer (2013)
Cortisol and interferon tau regulation of endometrial function and conceptus development in female sheep.
Endocrinology, 154 2
J. Silva, L. Costa, J. Silva (2002)
Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers.
Animal Reproduction Science, 69
Catherine Gulliver, Michael Friend, B. King, Edward Clayton (2012)
The role of omega-3 polyunsaturated fatty acids in reproduction of sheep and cattle.
Animal reproduction science, 131 1-2
(2004)
Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations.
Nicole White, Patrick Burns, Robert Cheatham, R. Romero, J. Nozykowski, J. Bruemmer, Terry Engle (2012)
Fish meal supplementation increases bovine plasma and luteal tissue omega-3 fatty acid composition.
Journal of animal science, 90 3
M. Fuentes, S. Calsamiglia, C. Sánchez, Antonio Gonzalez, J. Newbold, J. Santos, Luis Rodríguez-Alcalá, J. Fontecha (2008)
Effect of extruded linseed on productive and reproductive performance of lactating dairy cows
Livestock Science, 113
S. Ulbrich, Katy Schulke, Anna Groebner, H. Reichenbach, C. Angioni, G. Geisslinger, H. Meyer (2009)
Quantitative characterization of prostaglandins in the uterus of early pregnant cattle.
Reproduction, 138 2
H. Petit, C. Germiquet, D. Lebel (2004)
Effect of feeding whole, unprocessed sunflower seeds and flaxseed on milk production, milk composition, and prostaglandin secretion in dairy cows.
Journal of dairy science, 87 11
G. Lewis, R. Waterman (1985)
Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy.
Prostaglandins, 30 2
Isabelle Hue, S. Degrelle, É. Campion, Jean-Paul Renard (2019)
Gene expression in elongating and gastrulating embryos from ruminants
Bioscientifica Proceedings
K. Kokawa, T. Shikone, R. Nakano (1998)
Apoptosis in human chorionic villi and decidua in normal pregnancy, spontaneous abortion and ectopic pregnancy
Placenta, 20
G. Richardson, M. Mcniven, H. Petit, J. Duynisveld (2013)
The effects of dietary omega fatty acids on pregnancy rate, plasma prostaglandin metabolite levels, serum progesterone levels, and milk fatty-acid profile in beef cows.
Canadian journal of veterinary research = Revue canadienne de recherche veterinaire, 77 4
F. Carter, N. Forde, Patrick Duffy, M. Wade, T. Fair, M. Crowe, A. Evans, David Kenny, J. Roche, P. Lonergan (2008)
Effect of increasing progesterone concentration from Day 3 of pregnancy on subsequent embryo survival and development in beef heifers.
Reproduction, fertility, and development, 20 3
P. Needleman, A. Raz, M. Minkes, J. Ferrendelli, H. Sprecher (1979)
Triene prostaglandins: prostacyclin and thromboxane biosynthesis and unique biological properties.
Proceedings of the National Academy of Sciences of the United States of America, 76 2
D. Wathes, G. Lamming (2019)
The oxytocin receptor, luteolysis and the maintenance of pregnancy.
Journal of reproduction and fertility. Supplement, 49
S. Degrelle, F. Jaffrézic, É. Campion, K. Cao, D. Bourhis, C. Richard, N. Rodde, Renaud Fleurot, R. Everts, J. Lecardonnel, Y. Heyman, X. Vignon, X. Yang, X. Tian, H. Lewin, J. Renard, I. Hue (2012)
Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer
PLoS ONE, 7
McCracken (1972)
Prostaglandin F2-Alpha identified as a Luteolytic Hormone in Sheep
Nat New Biol, 238
L. Maclaren, A. Guzeloglu, F. Michel, W. Thatcher (2006)
Peroxisome proliferator-activated receptor (PPAR) expression in cultured bovine endometrial cells and response to omega-3 fatty acid, growth hormone and agonist stimulation in relation to series 2 prostaglandin production.
Domestic animal endocrinology, 30 3
D. Rizos, F. Carter, U. Besenfelder, Vitezslav Havlicek, Patrick Lonergan (2010)
Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows.
Journal of dairy science, 93 3
L. O'Hara, N. Forde, A. Kelly, P. Lonergan (2014)
Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: can supplementary progesterone rescue small embryos?
Theriogenology, 81 8
T. Hinckley, R. Clark, S. Bushmich, R. Milvae (1996)
Long chain polyunsaturated fatty acids and bovine luteal cell function.
Biology of reproduction, 55 2
L. Levine, Nancy Worth (1984)
Eicosapentaenoic acid: its effects on arachidonic acid metabolism by cells in culture.
The Journal of allergy and clinical immunology, 74 3 Pt 2
G. Lewis, R. Waterman (1983)
Effects of endometrium on metabolism of arachidonic acid by bovine blastocysts in vitro.
Prostaglandins, 25 6
Betteridge (1980)
Collection, description and transfer of embryos from cattle 10-16 days after oestrus
Reproduction, 59
R. Mattos, C. Staples, Janet Williams, A. Amorocho, Mark McGuire, W. Thatcher (2002)
Uterine, ovarian, and production responses of lactating dairy cows to increasing dietary concentrations of menhaden fish meal.
Journal of dairy science, 85 4
C. Caldari-Torres, C. Rodriguez-Sallaberry, E. Greene, L. Badinga (2006)
Differential effects of n-3 and n-6 fatty acids on prostaglandin F2alpha production by bovine endometrial cells.
Journal of dairy science, 89 3
Randi (2015)
The use of fixed time artificial insemination in beef suckler herds
Cattle Practice, 23
G. Charpigny, P. Reinaud, J. Tamby, Christophe Minon, J. Martal, J. Maclouf, M. Guillomot (1997)
Expression of Cyclooxygenase-1 and -2 in Ovine Endometrium During the Estrous Cycle and Early Pregnancy.
Endocrinology, 138 5
P. Dorniak, F. Bazer, T. Spencer (2011)
Prostaglandins Regulate Conceptus Elongation and Mediate Effects of Interferon Tau on the Ovine Uterine Endometrium1
, 84
J. Hughes, W. Kwong, Dongfang Li, A. Salter, R. Lea, K. Sinclair (2011)
Effects of omega-3 and -6 polyunsaturated fatty acids on ovine follicular cell steroidogenesis, embryo development and molecular markers of fatty acid metabolism.
Reproduction, 141 1
C. Ashworth, L. Toma, M. Hunter (2009)
Nutritional effects on oocyte and embryo development in mammals: implications for reproductive efficiency and environmental sustainability
Philosophical Transactions of the Royal Society B: Biological Sciences, 364
S. Childs, F. Carter, C. Lynch, J. Sreenan, P. Lonergan, A. Hennessy, D. Kenny (2008)
Embryo yield and quality following dietary supplementation of beef heifers with n-3 polyunsaturated fatty acids (PUFA).
Theriogenology, 70 6
S. Childs, S. Childs, A. Hennessy, A. Hennessy, J. Sreenan, D. Wathes, Zhangrui Cheng, C. Stanton, M. Diskin, David Kenny (2008)
Effect of level of dietary n-3 polyunsaturated fatty acid supplementation on systemic and tissue fatty acid concentrations and on selected reproductive variables in cattle.
Theriogenology, 70 4
C. Lopes, A. Scarpa, B. Cappellozza, R. Cooke, J. Vasconcelos (2009)
Effects of rumen-protected polyunsaturated fatty acid supplementation on reproductive performance of Bos indicus beef cows.
Journal of animal science, 87 12
K. Betteridge, M. Eaglesome, G. Randall, D. Mitchell (1980)
Collection, description and transfer of embryos from cattle 10--16 days after oestrus.
Journal of reproduction and fertility, 59 1
J. Filant, T. Spencer (2014)
Uterine glands: biological roles in conceptus implantation, uterine receptivity and decidualization.
The International journal of developmental biology, 58 2-4
M. Diskin, M. Parr, M. Parr, D. Morris (2011)
Embryo death in cattle: an update.
Reproduction, fertility, and development, 24 1
F. Achard, M. Gilbert, C. Benistant, S. Slama, D. Dewitt, W. Smith, M. Lagarde (1997)
Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1 expression in bovine aortic endothelial cells.
Biochemical and biophysical research communications, 241 2
S. Degrelle, É. Campion, C. Cabau, F. Piumi, P. Reinaud, C. Richard, J. Renard, I. Hue (2005)
Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts.
Developmental biology, 288 2
J. Burke, C. Staples, C. Risco, R. Sota, W. Thatcher (1997)
Effect of ruminant grade Menhaden fish meal on reproductive and productive performance of lactating dairy cows.
Journal of dairy science, 80 12
R. Staples, K. Mcentee, W. Hansel (1961)
Luteal Function as Related to Pituitary and Ovarian Cytology and Embryo Development in the Bovine
Journal of Dairy Science, 44
Needleman (1979)
Triene prostaglandins: Prostacyclin and thromboxane biosynthesis and unique biological properties
Proc Natl Acad Sci USA, 76
K. Brooks, T. Spencer (2015)
Biological Roles of Interferon Tau (IFNT) and Type I IFN Receptors in Elongation of the Ovine Conceptus1
, 92
J. Berger, D. Moller (2002)
The mechanisms of action of PPARs.
Annual review of medicine, 53
S. Filley, H. Turner, F. Stormshak (2000)
Plasma fatty acids, prostaglandin F2alpha metabolite, and reproductive response in postpartum heifers fed rumen bypass fat.
Journal of animal science, 78 1
F. Bazer, Guoyao Wu, T. Spencer, G. Johnson, R. Burghardt, K. Bayless (2010)
Novel pathways for implantation and establishment and maintenance of pregnancy in mammals.
Molecular human reproduction, 16 3
F. Bartol, W. Thatcher, F. Bazer, F. Kimball, J. Chenault, C. Wilcox, R. Roberts (1981)
Effects of the estrous cycle and early pregnancy on bovine uterine, luteal, and follicular responses.
Biology of reproduction, 25 4
R. Robinson, PG Pushpakumara, Z. Cheng, AR Peters, DR Abayasekara, D. Wathes (2002)
Effects of dietary polyunsaturated fatty acids on ovarian and uterine function in lactating dairy cows.
Reproduction, 124 1
D. Wathes, D. Abayasekara, R. Aitken (2007)
Polyunsaturated Fatty Acids in Male and Female Reproduction1
, 77
R. Mattos, C. Staples, A. Arteche, M. Wiltbank, Francisco Diaz, T. Jenkins, W. Thatcher (2004)
The effects of feeding fish oil on uterine secretion of PGF2alpha, milk composition, and metabolic status of periparturient Holstein cows.
Journal of dairy science, 87 4
H. Petit, H. Twagiramungu (2006)
Conception rate and reproductive function of dairy cows fed different fat sources.
Theriogenology, 66 5
T. Spencer, T. Ott, F. Bazer (1996)
τ-Interferon: Pregnancy Recognition Signal in Ruminants
Proceedings of the Society for Experimental Biology and Medicine, 213
T. Hansen, KJ Austin, D. Perry, JK Pru, MG Teixeira, G. Johnson (2019)
Mechanism of action of interferon-tau in the uterus during early pregnancy.
Journal of reproduction and fertility. Supplement, 54
D. Ambrose, J. Kastelic, R. Corbett, P. Pitney, H. Petit, J. Small, P. Zalkovic (2006)
Lower Pregnancy Losses in Lactating Dairy Cows Fed a Diet Enriched in α-Linolenic Acid
Journal of Dairy Science, 89
V. Émond, L. Maclaren, S. Kimmins, J. Arosh, M. Fortier, R. Lambert (2004)
Expression of Cyclooxygenase-2 and Granulocyte-Macrophage Colony-Stimulating Factor in the Endometrial Epithelium of the Cow Is Up-Regulated During Early Pregnancy and in Response to Intrauterine Infusions of Interferon-τ1
, 70
G. Mann, G. Lamming (1999)
The influence of progesterone during early pregnancy in cattle
Reproduction in Domestic Animals, 34
N. Forde, P. Lonergan (2012)
Transcriptomic analysis of the bovine endometrium: What is required to establish uterine receptivity to implantation in cattle?
The Journal of reproduction and development, 58 2
A. Moussavi, R. Gilbert, T. Overton, D. Bauman, W. Butler (2007)
Effects of feeding fish meal and n-3 fatty acids on ovarian and uterine responses in early lactating dairy cows.
Journal of dairy science, 90 1
W. Marei, J. Bie, O. Mohey-Elsaeed, E. Wydooghe, P. Bols, J. Leroy (2017)
Alpha-linolenic acid protects the developmental capacity of bovine cumulus–oocyte complexes matured under lipotoxic conditions in vitro†
Biology of Reproduction, 96
F. Mesquita, G. Pugliesi, S. Scolari, M. França, R. Ramos, M. Oliveira, P. Papa, F. Bressan, F. Meirelles, L. Silva, G. Nogueira, C. Membrive, M. Binelli (2014)
Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows.
Theriogenology, 81 6
Spencer (1996)
tau-Interferon: pregnancy recognition signal in ruminants
Exp Biol Med, 213
P. Calder, P. Yaqoob (2009)
Understanding Omega-3 Polyunsaturated Fatty Acids
Postgraduate Medicine, 121
K. Brooks, G. Burns, T. Spencer (2014)
Conceptus elongation in ruminants: roles of progesterone, prostaglandin, interferon tau and cortisol
Journal of Animal Science and Biotechnology, 5
S. Clarke, D. Jump (1994)
Dietary polyunsaturated fatty acid regulation of gene transcription.
Annual review of nutrition, 14
J. Arosh, S. Banu, S. Kimmins, P. Chapdelaine, L. Maclaren, M. Fortier (2004)
Effect of interferon-tau on prostaglandin biosynthesis, transport, and signaling at the time of maternal recognition of pregnancy in cattle: evidence of polycrine actions of prostaglandin E2.
Endocrinology, 145 11
N. Bork, J. Schroeder, G. Lardy, K. Vonnahme, M. Bauer, D. Buchanan, R. Shaver, P. Fricke (2010)
Effect of feeding rolled flaxseed on milk fatty acid profiles and reproductive performance of dairy cows.
Journal of animal science, 88 11
M. Colazo, J. Kastelic, M.F Martı́nez, P. Whittaker, R. Wilde, J.D Ambrose, R. Corbett, R. Mapletoft (2004)
Fertility following fixed-time AI in CIDR-treated beef heifers given GnRH or estradiol cypionate and fed diets supplemented with flax seed or sunflower seed.
Theriogenology, 61 6
M. Sponchiado, N. Gomes, P. Fontes, T. Martins, Maite Collado, A. Pastore, G. Pugliesi, M. Nogueira, M. Binelli (2017)
Pre-hatching embryo-dependent and -independent programming of endometrial function in cattle
PLoS ONE, 12
Garrett (1988)
Evidence for maternal regulation of early conceptus growth and development in beef cattle
Reproduction, 84
M. Oseikria, S. Elis, V. Maillard, E. Corbin, S. Uzbekova (2016)
N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle.
Theriogenology, 85 9
C. Wolf, S. Ulbrich, M. Kreuzer, J. Berard, K. Giller (2018)
Differential partitioning of rumen-protected n-3 and n-6 fatty acids into muscles with different metabolism.
Meat science, 137
C. Barnwell, P. Farin, C. Ashwell, W. Farmer, S. Galphin, C. Farin (2016)
Differences in mRNA populations of short and long bovine conceptuses on Day 15 of gestation
Molecular Reproduction and Development, 83
Ashworth (2009)
Nutritional effects on oocyte and embryo development in mammals: implications for reproductive efficiency and environmental sustainability
Philos T Roy Soc B Biol Sci, 364
G. Thangavelu, M. Colazo, D. Ambrose, M. Oba, E. Okine, M. Dyck (2007)
Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows.
Theriogenology, 68 7
H. Dobson, R.F. Smith, M. Royal, C. Knight, I. Sheldon (2007)
The high-producing dairy cow and its reproductive performance.
Reproduction in domestic animals = Zuchthygiene, 42 Suppl 2
G. Mann, G. Lamming (2001)
Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows.
Reproduction, 121 1
R. Mattos, A. Guzeloglu, L. Badinga, C. Staples, W. Thatcher (2003)
Polyunsaturated Fatty Acids and Bovine Interferon-τ Modify Phorbol Ester-Induced Secretion of Prostaglandin F2α and Expression of Prostaglandin Endoperoxide Synthase-2 and Phospholipase-A2 in Bovine Endometrial Cells1
, 69
Hue (2007)
Gene expression in elongating and gastrulating embryos from ruminants
Soc Reprod Fertil Suppl, 64

Publisher: Oxford University Press
Copyright: © The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction.
ISSN: 0006-3363
eISSN: 1529-7268
DOI: 10.1093/biolre/ioy084
Publisher site: See Article on Publisher Site

Abstract

Abstract The pre-implantation period is prone to embryonic losses in bovine. Embryo–maternal communication is crucial to support embryo development. Thereby, factors of the uterine fluid (UF) are of specific importance. The maternal diet can affect the UF composition. Since omega 3 fatty acids (omega 3 FA) are considered to be beneficial for reproduction, we investigated if dietary omega 3 FA affected factors in the UF related to embryo elongation. Angus heifers (n = 37) were supplemented with either 450 g of rumen-protected fish oil (omega 3 FA) or sunflower oil (omega 6 FA) for a period of 8 weeks. Following cycle synchronization and artificial insemination, the uteri were flushed post mortem to recover the embryos on day 15 of pregnancy. The UF and tissue samples of endometrium and corpus luteum (CL) were collected. Strikingly, the embryo elongation in the omega 3 group was enhanced compared to the omega 6 group. No differences were observed in uterine prostaglandins, even though the endometrial concentration of their precursor arachidonic acid was reduced in omega 3 compared to omega 6 heifers. The dietary FA neither led to differential expression of target genes in endometrium nor CL nor to a differential abundance of low-density lipoprotein cholesterol, cortisol or amino acids in the UF. Interestingly, the omega 3 group displayed a higher plasma progesterone concentration during luteal growth than the omega 6 group, possibly promoting embryo elongation. Further research should include an ovarian perspective to understand the functional link between dietary omega 3 FA and reproductive outcome. Introduction Early embryonic losses in the pre-implantation phase are commonly observed in cattle. Even though the fertilization rate averages 90%, the actual calving rate reaches only 55% [1]. In high-producing dairy cows, the rate of early embryonic losses is even more pronounced [2, 3]. The most critical period during which embryo losses occur in bovines is prior to implantation between day 8 and 16 of pregnancy. As reason for this, a failure of embryo elongation and maternal recognition of pregnancy is considered [4]. For successful establishment and maintenance of pregnancy, an intense embryo–maternal communication via embryonic signals and maternal factors is crucial. In bovines, direct contact of mother and embryo is established through implantation only from day 18 on. Until then, the communication is crucially dependent on the uterine fluid (UF), a complex mixture of nutritive and regulatory factors such as amino acids, sugars, fatty acids (FA), enzymes and other proteins, growth factors and hormones (reviewed by Filant and Spencer [5]). The UF comprises secretions by both the endometrium and the embryo as well as molecules that are transudated from the blood. It may be affected by the maternal diet [6]. Dietary supplementation with fat in general and specifically with ω3 FA has been discussed to improve bovine fertility [7]. However, the study outcomes are inconsistent. Some studies showed an increased conception or pregnancy rate or reduced pregnancy losses following dietary polyunsaturated FA (PUFA) supplementation compared to supplementation with saturated FA (SFA) [8, 9]. In contrast, other authors [10–12] found no effect of dietary FA on the pregnancy rate. In a study by Burke et al. [13], dietary ω3 FA increased the pregnancy rate in only one of two investigated farms where overall fertility was initially poor. Supplementing beef cows with linoleic acid (LA) reduced pregnancy rates [14]. Before implantation, the bovine trophoblast undergoes rapid elongation, starting around day 15 of pregnancy. At this developmental stage, the trophoblast mononuclear cells synthesize large amounts of interferon tau (IFNT), the signal for maternal recognition of pregnancy [15, 16]. IFNT induces the endometrial gene expression of interferon-stimulated genes (ISGs), which are affecting the endometrium and the trophoblast to support embryo elongation and implantation [17]. IFNT also prevents the pulsatile release of endometrium-derived prostaglandin (PG) F2α and thus luteolysis, whereby the progesterone (P4) production of the corpus luteum (CL) is maintained [15]. Starting shortly after ovulation, luteal P4 affects the endometrium by alterating gene expression and secretion of embryotropic factors [18–20]. The administration of P4 during days 1–4 or days 3–16 of pregnancy caused an increased elongation of the embryo on days 13–16 of pregnancy in beef heifers [18, 21] and was associated with the induction of gene expression changes in the endometrium [22, 23]. Factors secreted by both the embryo (e.g. IFNT, PG, and cortisol) and the endometrium (e.g. PG and cortisol) are then assumed to further modulate the endometrial gene expression [24, 25] and lead to an ongoing modification of the UF [24–27]. Prostaglandins are local signaling molecules that exhibit a broad spectrum of functions. The main series-2 PG precursor is the ω6 FA arachidonic acid (AA; C20:4) that is incorporated into the phospholipid bilayer of cell membranes. After liberation from the cell membrane by phospholipase A2 (PLA2), prostaglandin-endoperoxide synthase 2 (PTGS2; previously known as prostaglandin G/H synthase and cyclooxygenase), the key enzyme of PG synthesis, converts AA to PGH2 which is the common precursor for all proinflammatory downstream series-2 PG, namely PGF2α, PGE2, PGD2, PGI2, and thromboxane A2 (TXA2). Increasing endometrial PTGS2 expression during diestrus induces the synthesis and release of luteolytic PGF2α pulses [28–30]. In addition, PG in the uterine lumen promote embryonic development and elongation [29, 31, 32]. As both the endometrium and the embryo synthesize PG [28, 33–35], the concentration of uterine luminal PG is higher during pregnancy than during the estrous cycle [31, 36]. Even though luteolytic PGF2α pulses are absent during pregnancy, constant levels of PGE2 have been detected in the bovine caudal vena cava [37]. An increased PGE2 production during early pregnancy is induced by IFNT through the upregulation of endometrial PTGS2 [38–40]. PGE2 has furthermore been shown to promote luteal maintenance and subsequent embryo elongation and implantation [38]. A modification of the PG synthesis may be at least in part involved in the improved fertility observed following a dietary FA supply. The utilization of the ω3 FA eicosapentaenoic acid (EPA; C20:5) as a substrate for PTGS2 results in the formation of series-3 PG. Series-3 PG are much less bioactive than the AA-derived series-2 PG and do not seem to be involved in luteolysis in bovines [41, 42]. The ω3 FA docosahexaenoic acid (DHA; C22:6) does not serve as a precursor for PG synthesis, but has been shown to inhibit PTGS2 activity [43]. Both ω6 and ω3 FA compete for the incorporation into cell membrane phospholipids as well as for an utilization by PTGS2. Therefore, dietary ω3 FA may reduce the formation of AA-derived series-2 PG in favor of increasing the synthesis of series-3 PG. Discussed pathways for ω3 FA-induced reduction of series-2 PG include an inhibiting effect on the synthesis and activity of Δ-6-desaturase involved in AA synthesis. Another potential pathway is the ω3 FA-induced inhibition of PTGS2 involved in PGH2 synthesis and the replacement of cell membrane AA by ω3 FA, thereby reducing the precursor for series-2 PG production [14, 41]. PG as well as ω3 and ω6 FA are ligands for nuclear receptors such as the transcription factor family of peroxisome proliferator activated receptors (PPARs), which impact on gene expression [44–47]. An involvement of PPARs in development and reproduction has already been demonstrated [44, 48] and a supplementation with ω3 FA has been reported to alter the expression of genes involved in reproduction in the bovine endometrium [49]. Trophectodermal peroxisome proliferator activated receptor gamma (PPARG) signaling involved in glucose and FA uptake and metabolism has been shown to be essential for early embryo elongation and survival in sheep [50]. A number of studies have investigated the effects of dietary ω3 and ω6 FA on PG concentrations in plasma. At parturition, ω3 FA from fish oil have been shown to decrease the concentration of plasma PGF metabolites (PGFM) compared to olive oil rich in ω6 FA [51]. Contrarily, feeding diets rich in ω3 FA significantly increased PGFM plasma concentrations post partum in beef cattle [52] as well as in primiparous, but not in multiparous dairy cattle [9]. Several studies have also determined the effect of ω3 FA on oxytocin-stimulated PGFM concentrations. A supplementation of fish oil and feeding fish meal or linseed decreased the PGFM response to oxytocin stimulation in beef heifers and dairy cows compared to control groups which lacked additional dietary fat or which were supplemented with palmitic acid [41, 53–55]. However, feeding linseed in comparison to soybean meal did not affect the oxytocin-induced PGFM response [56]. We hypothesize an effect of dietary ω3 FA on embryo elongation. As the cell membrane composition and thus the content of the PG precursors ω6 and ω3 FA can vary depending on dietary available FA, we supplemented growing Angus heifers with rumen-protected fish oil rich in ω3 FA (EPA and DHA) to ensure the incorporation of FA into the body tissues. To maximize the differences in the ω6/ω3 ratio between the experimental and the control group, we supplemented the latter with rumen-protected sunflower oil rich in the ω6 FA LA, a precursor for AA. We studied embryo elongation and development and further associated parameters in the endometrium and the CL. We specifically aimed at focusing whether the composition of the UF was affected by the diet. Materials and methods Animals and study design In total, 37 Angus heifers (Bos taurus) aged between 10 and 27 months (mean ± SEM: 19 ± 0.7) with an average body weight of 348.1 ± 6.4 kg were randomly assigned to two age- and weight-matched study groups. The animals were housed in a freestall barn with ad libitum access to water at the former ETH research station Chamau (Canton Zug, Switzerland) and fed a ration (7.54 kg/day; containing barley straw [3.98 kg], hay [0.57 kg], soy [01.02 kg], wheat [0.69 kg], molasses [0.68 kg], beta-carotene [0.09 kg], commercial mineral mix [0.04 kg] and sodium chloride [0.02 kg]) that included either 450 g of a rumen-protected sunflower oil (“Sonnenblumenöl 100528” from NUTRISWISS AG, Lyss, Switzerland) supplement rich in ω6 FA (ω6 group, n = 15 animals) or 450 g of a rumen-protected fish oil (“Marineöl Omega-3-Konzentrat 33/22” from Henry Lamotte Oils GmbH, Bremen, Germany) supplement rich in in ω3 FA (ω3 group, n = 22 animals). The detailed diet composition is presented in Supplementary Table S1 according to Wolf et al. [57]. The feeding experiment was performed in two separate runs, with 18 animals (n = 9 for both ω3 and ω6 groups) in 2014 and 19 animals (n = 13 for ω3 and n = 6 for ω6) in 2015. Management reasons prevented equal group sizes in the second run. The feeding gate in the free-stall barn was equipped with a gridlock where animals were fixed during feeding. The troughs between animals were separated by wooden barriers during the last two weeks before slaughter. Rumen protection of the supplement oils was established by mixing of the respective oil with hydrogenated rapeseed oil and subsequent spray chilling (Erbo Spraytec AG, Bützberg, Switzerland). The ω6/ω3 ratio of the diet in the ω6 group was 4:1 and in the ω3 group 1:2. The selected fish oil contained 33% EPA and 22% DHA, leading to a combined intake of EPA (37g) and DHA (25 g) of 62 g. Approximately 30 g EPA plus DHA were provided to the animals’ intestinal metabolism, assuming a protection of ruminal biohydrogenation of 50%. The animals were maintained on the respective diets for 8 weeks, leading to a similar average weight gain during the supplementation period of 70 kg. After 5 weeks on the diet, the animals were cycle synchronized by using a controlled internal drug release (Eazi-Breed CIDR 1380®, Zoetis, Zurich, Switzerland) that was removed after 8 days. During the first run, for a few animals in both groups, synchronization by two injections of PGF2α (Estrumate®, MSD Animal Health/Intervet International GmbH, Unterschleissheim, Germany) in an interval of 11 days was applied instead of the CIDR due to management reasons. One day before CIDR removal, animals were injected intramuscularly with 2 ml Estrumate (MSD Animal Health GmbH, Luzern, Switzerland). Three days after Estrumate injection, independently from the synchronization protocol used, the animals were artificially inseminated with sperm from the same Angus bull. The day of artificial insemination (AI) was defined as day 0 of pregnancy. Blood samples were collected on days 0, 3, 6, 9, 12, and 15 from a randomly chosen subset of animals of both groups (ω3: n = 11–17; ω6: n = 6–13). Immediately after slaughter on day 15, the reproductive tract was removed and the uterus was flushed ex vivo with 10 ml PBS to obtain the embryo. The length of the embryos was determined using a stereo microscope prior to snap freezing in liquid nitrogen. Endometrial and CL tissue samples were carefully collected and likewise snap frozen. The recovered UF was centrifuged to remove cellular debris at 800 × g for 10 min. Tissues, plasma, and UF were stored at −80°C until analysis. Animals were only included in the study if an embryo was recovered (ω3: n = 18; ω6: n = 13). With the presence of an embryo on day 15 the respective animal was defined pregnant. During the collection, one of the embryos from the ω3 group was destroyed and thus its length could not be determined. However, this animal was included in the study as the presence of an embryo was confirmed. Because of the high prevalence of pre-implantation embryo mortality in bovine, the embryos’ genomic DNA was controlled for apoptotic laddering on an agarose gel to ensure the inclusion of animals with viable embryos only. Apoptotic DNA fragmentation was not observed for any of the embryos. The experimental protocol was approved by the Veterinary office of the Canton Zug (Switzerland) in accordance with the Swiss legislation on animal rights and welfare (Permit numbers: ZG 64/14 and ZG 71/15). Quantification of fatty acids by gas chromatography with flame ionization detector The FA composition of plasma and endometrial tissue was determined using FA methyl esters (FAME) prepared by transesterification with trimethylsulfonium hydroxide (TMSH). Briefly, for both plasma and endometrial tissue 1 g was extracted two times with 7 ml of chloroform/methanol. The chloroform layer was drained and evaporated. The residues were resuspended in tert-butylmethyl-ether with TMSH added. The analysis of FAMEs was performed by gas chromatography with a flame ionization detector (GC 6890, Agilent Technologies, Waldbronn, Germany). The quantification of FA was performed using Chromeleon® 6.8 Chromatography Software (Dionex, Sunnyvale, USA) [58]. Quantification of prostanoids by liquid chromatography-tandem mass spectrometry Prostaglandins (PGF2α, PGD2, PGE2, 6-keto-PGF1α) and thromboxane B2 (TXB2) were determined in the UF by solid-phase sample extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extraction and analysis were performed as previously described [31]. Briefly, a stock solution with 100 μg/ml PGE2, PGD2, TXB2, PGF2α, and 6-keto PGF1α was prepared in methanol and then further diluted to obtain working standards. Bovine UF samples were prepared with 200 μl sample and 20 μl methanol were added. The prepared samples were extracted, the organic phase was removed and the residues were reconstituted with 50 μl acetonitrile/water/formic acid (20:80:0.0025, v/v, pH 4.0), centrifuged and then transferred to glass vials (Macherey-Nagel, Düren, Germany) prior to injection into the LC-MS/MS system. A Synergi Hydro-RP column (150 × 2 mm I.D., 4 μm particle size, and 80 Å pore size from Phenomenex, Aschaffenburg, Germany) was used to separate PG and TXB2 before being determined with an API 4000 tandem mass spectrometer (Applied Biosystems, Darmstadt, Germany). The quantification was performed with Analyst Software V1.4.2 (Applied Biosystems). Analysis of cortisol in the uterine fluid The cortisol content was determined in UF using a commercial immunoassay (Cortisol Free Saliva Kit, Demeditec, Kiel, Germany) according to the manufacturer's instructions. Analysis of amino acids and total protein content The analysis of amino acids was performed as described earlier [59]. Briefly, the aTRAQ Reagent Kit was used for 40 μl of UF according to the manufacturer's instructions (Applied Biosystems) by subsequent analysis via targeted LC-MS/MS (AB SCIEX QTrap 3200 LC-MS/MS System, AB SCIEX, Framingham, MA, USA). The data were analyzed using the Analyst® 1.5 Software, and quantitative measurements of the amino acids were obtained. The total protein content in UF and plasma was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Massachusetts, USA). Because total protein content and total amino acid concentration (sum of all measured amino acids) were well correlated (r = 0.82, P < 0.001), the data are presented as mean nmol/mg total protein ± SEM. Total RNA isolation Total RNA was extracted from the ipsilateral intercaruncular endometrial and CL tissue using the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Tissues were homogenized using the MagNA Lyser and MagNA Lyser Green Beads (Roche, Rotkreuz, Switzerland). RNA concentrations were quantified with the NanoDrop 2000 (peqLab, Erlangen, Germany). RNA integrity was monitored using the Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) with the Agilent RNA 6000 Nano Kit. RNA integrity numbers ranged between 8.9 and 10 for all samples. RNA aliquots were stored at −80°C until cDNA synthesis. Reverse transcription and qPCR Total RNA (1 μg from endometrium and 500 ng from CL, respectively) was used for cDNA synthesis with the GoScript Reverse Transcription System (Promega, Madison, USA). The reaction mix was composed as follows: 10 μl RNA in H2O, 0.5 μl Oligo(dT)15 primer, 0.5 μl random primer, 4 μl reaction buffer, 2.5 μl MgCl2, 1 μl dNTPs, 0.5 μl RNasin, 1 μl reverse transcriptase. Incubation of the reaction mix was performed in a PCR cycler (25°C for 5 min, 42°C for 60 min, 70°C for 15 min). Quantitative real-time PCR (qPCR) was carried out using the KAPA SYBR FAST qPCR Kit (Kapa Biosystems, Wilmington, USA) on a CFX384 Real-Time PCR Detection System (Bio-Rad, Munich, Germany). The cycle of quantification (Cq) values were obtained using a single threshold. The relative expression level (ΔCq) of each gene was generated by scaling the target gene Cq of each individual sample to the geometric mean of the Cqs of three reference genes (ubiquitin B (UBB), H3 histone family member 3A (H3F3A), and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), according to the bestkeeper method [60]. Fold-changes were calculated according to the ΔΔCt method. The sequences of commercially synthesized primers (Microsynth, Balgach, Switzerland) used are listed in Supplementary Table S2. Plasma progesterone concentration Plasma P4 was determined using the Immunotech RIA (Beckman Coulter, CA, USA), previously validated for bovine plasma [61]. The analytical sensitivity for P4 was 0.03 ng/ml, intraassay CV was 7.6%, and interassay CV was 9.7%. Quantification of LDL cholesterol A commercial assay kit (high-density lipoprotein (HDL) and low-density lipoprotein (LDL)/very low-density lipoprotein (VLDL) Cho-lesterol Assay kit, abcam, Cambridge, UK) was used to measure cholesterol concentration in the LDL fractions of plasma samples collected at slaughter according to the manufacturer's instructions. Quantification of tocopherols by high-performance liquid chromatography Tocopherols were quantified in plasma (collected at slaughter), liver, and endometrium by a validated method described earlier [62]. Briefly, tissue samples were saponified for 30 min at 70°C in a shaking water bath. To both tissue and plasma samples butylated hydroxytoluene (BHT; 25 μL of a 1 mg/ml ethanolic solution) was added. Then all samples were extracted twice with n-Hexane. The supernatants were pooled and evaporated (Christ SpeedDry; Christ, Osterode Germany). The dried residues were resuspended and injected into a Jasco HPLC system (AS-950 Plus autosampler, PU-980 Plus pump, FP-950 Plus fluorescence detector, LG-980-02 gradient unit, and a 3-line degasser; Jasco, Groß-Umstadt, Germany). The tocopherols were separated on a Kinetex PFP column (2.6 μm, 150 × 4.6 mm; Phenomenex, Aschaffenburg, Germany) using a methanol: water (85:15, vol/vol) mobile phase. The fluorescence detector was set to an excitation wavelength of 296 nm and emission wavelength of 325 nm. Peaks were recorded and integrated using ChromPass version 1.8.6.1 (Jasco). The concentrations of tocopherols were quantified against external standard curves with authentic compounds (Sigma Aldrich, St. Louis, MO). Statistical analysis The statistical analysis was performed using SPSS version 22 (SPSS GmbH Software, Munich, Germany). The Shapiro–Wilk test was used to test for normal distribution of data and residuals. For comparisons between diet groups, data following a Gaussian distribution were analyzed by Student t test. For data not following a Gaussian distribution (as observed for embryo length), a Mann–Whitney U test was performed. In case of a significant effect of embryo length on dependent variables (endometrial gene expression, uterine prostaglandins, and amino acids), the “least-square ANOVA general linear models procedure” was used with diet group (ω3 or ω6) as a fixed factor and embryo length (in cm) as a covariable. Data analysis for qPCR results was performed on ΔCq values. Results are presented as means ± SEM and P values ≤ 0.05 were considered statistically significant. Results Rumen protected ω3 FA lowered the ω6/ω3 ratio and changed the FA pattern in plasma and endometrium The amounts of single ω3 and ω6 FA in both plasma and endometrium differed significantly between animals supplemented with fish oil (ω3 FA) and animals supplemented with sunflower oil (ω6 FA) as presented in Table 1. In both diet groups, the percentage of AA, docosapentaenoic acid, and DHA of total ω6 and ω3 FA was higher in the endometrium compared to plasma whereas the percentages of LA, alpha-linolenic acid (ALA), and EPA were decreased in endometrium compared to plasma (all P < 0.001). Table 1. Concentration and percentage of selected ω3 and ω6 FA to total ω3 and ω6 FA in plasma and endometrium of Angus heifers. Plasma Endometrium μg/g P value % of total ω6 and ω3 FA μg/g P value % of total ω6 and ω3 FA ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet SFA 596 ± 37 675 ± 33 0.1 1616 ± 117 1489 ± 105 0.4 MUFA 304 ± 15 264 ± 12 0.06 1266 ± 106 1030 ± 65 0.1 PUFA 749 ± 42 916 ± 47 0.02 1449 ± 105 1352 ± 103 0.5 Linoleic acid 513 ± 30 458 ± 22 0.2 68 50 338 ± 25 315 ± 24 0.3 23 23 gamma-Linolenic acid 19.8 ± 1.5 5.3 ± 0.5 <0.001 3 1 17.0 ± 1.8 10.2 ± 1.1 0.002 1 1 Eicosadienoic acid ω6 0.8 ± 0.1 1.7 ± 0.1 0.005 - - 18.8 ± 3.0 15.1 ± 1.6 0.1 1 1 Eicosatrienoic 8c,11c,14c acid 29.6 ± 1.9 13.3 ± 0.7 <0.001 4 2 117 ± 10 80.7 ± 7.4 0.006 8 6 Arachidonic acid 51.6 ± 2.7 59.3 ± 3.6 0.4 7 6 584 ± 45 388 ± 22 <0.001 40 29 ω6 FA (total) 614 ± 35 536 ± 26 0.09 82 59 1075 ± 80 810 ± 53 0.009 73* 60 Linolenic acid 69.0 ± 4.7 117 ± 6 <0.001 9 13 12.2 ± 4.9 17.8 ± 1.9 0.1 1 1 Eicosapentaenoic acid 34.7 ± 3.2 187 ± 18 <0.001 5 20 24.6 ± 3.1 101 ± 13 <0.001 2 7 Docosapentaenoic acid ω3 20.8 ± 1.2 30.8 ± 1.4 <0.001 3 3 116 ± 8 158 ± 14 0.2 8 12 Docosahexaenoic acid 10.7 ± 0.9 43.7 ± 2.6 <0.001 1 5 221 ± 19 266 ± 23 0.2 15 20 ω3 FA (total) 135 ± 9 378 ± 26 <0.001 18 41 376 ± 29 545 ± 50 0.045 26 40 ω6/ω3 4.7 ± 0.2 1.5 ± 0.1 <0.001 2.8 ± 0.1 1.6 ± 0.1 <0.001 Plasma Endometrium μg/g P value % of total ω6 and ω3 FA μg/g P value % of total ω6 and ω3 FA ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet SFA 596 ± 37 675 ± 33 0.1 1616 ± 117 1489 ± 105 0.4 MUFA 304 ± 15 264 ± 12 0.06 1266 ± 106 1030 ± 65 0.1 PUFA 749 ± 42 916 ± 47 0.02 1449 ± 105 1352 ± 103 0.5 Linoleic acid 513 ± 30 458 ± 22 0.2 68 50 338 ± 25 315 ± 24 0.3 23 23 gamma-Linolenic acid 19.8 ± 1.5 5.3 ± 0.5 <0.001 3 1 17.0 ± 1.8 10.2 ± 1.1 0.002 1 1 Eicosadienoic acid ω6 0.8 ± 0.1 1.7 ± 0.1 0.005 - - 18.8 ± 3.0 15.1 ± 1.6 0.1 1 1 Eicosatrienoic 8c,11c,14c acid 29.6 ± 1.9 13.3 ± 0.7 <0.001 4 2 117 ± 10 80.7 ± 7.4 0.006 8 6 Arachidonic acid 51.6 ± 2.7 59.3 ± 3.6 0.4 7 6 584 ± 45 388 ± 22 <0.001 40 29 ω6 FA (total) 614 ± 35 536 ± 26 0.09 82 59 1075 ± 80 810 ± 53 0.009 73* 60 Linolenic acid 69.0 ± 4.7 117 ± 6 <0.001 9 13 12.2 ± 4.9 17.8 ± 1.9 0.1 1 1 Eicosapentaenoic acid 34.7 ± 3.2 187 ± 18 <0.001 5 20 24.6 ± 3.1 101 ± 13 <0.001 2 7 Docosapentaenoic acid ω3 20.8 ± 1.2 30.8 ± 1.4 <0.001 3 3 116 ± 8 158 ± 14 0.2 8 12 Docosahexaenoic acid 10.7 ± 0.9 43.7 ± 2.6 <0.001 1 5 221 ± 19 266 ± 23 0.2 15 20 ω3 FA (total) 135 ± 9 378 ± 26 <0.001 18 41 376 ± 29 545 ± 50 0.045 26 40 ω6/ω3 4.7 ± 0.2 1.5 ± 0.1 <0.001 2.8 ± 0.1 1.6 ± 0.1 <0.001 Heifers in the ω3 group have significantly more ω3 FA in plasma and endometrium than ω6 heifers. This was caused by an increase in all ω3 FA in plasma whereas in endometrium it caused by solely a higher EPA concentration. Conversely, heifers in the ω6 group display a higher concentration of AA in endometrium. Values are given as means ± sem. *Rounding error. View Large Table 1. Concentration and percentage of selected ω3 and ω6 FA to total ω3 and ω6 FA in plasma and endometrium of Angus heifers. Plasma Endometrium μg/g P value % of total ω6 and ω3 FA μg/g P value % of total ω6 and ω3 FA ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet SFA 596 ± 37 675 ± 33 0.1 1616 ± 117 1489 ± 105 0.4 MUFA 304 ± 15 264 ± 12 0.06 1266 ± 106 1030 ± 65 0.1 PUFA 749 ± 42 916 ± 47 0.02 1449 ± 105 1352 ± 103 0.5 Linoleic acid 513 ± 30 458 ± 22 0.2 68 50 338 ± 25 315 ± 24 0.3 23 23 gamma-Linolenic acid 19.8 ± 1.5 5.3 ± 0.5 <0.001 3 1 17.0 ± 1.8 10.2 ± 1.1 0.002 1 1 Eicosadienoic acid ω6 0.8 ± 0.1 1.7 ± 0.1 0.005 - - 18.8 ± 3.0 15.1 ± 1.6 0.1 1 1 Eicosatrienoic 8c,11c,14c acid 29.6 ± 1.9 13.3 ± 0.7 <0.001 4 2 117 ± 10 80.7 ± 7.4 0.006 8 6 Arachidonic acid 51.6 ± 2.7 59.3 ± 3.6 0.4 7 6 584 ± 45 388 ± 22 <0.001 40 29 ω6 FA (total) 614 ± 35 536 ± 26 0.09 82 59 1075 ± 80 810 ± 53 0.009 73* 60 Linolenic acid 69.0 ± 4.7 117 ± 6 <0.001 9 13 12.2 ± 4.9 17.8 ± 1.9 0.1 1 1 Eicosapentaenoic acid 34.7 ± 3.2 187 ± 18 <0.001 5 20 24.6 ± 3.1 101 ± 13 <0.001 2 7 Docosapentaenoic acid ω3 20.8 ± 1.2 30.8 ± 1.4 <0.001 3 3 116 ± 8 158 ± 14 0.2 8 12 Docosahexaenoic acid 10.7 ± 0.9 43.7 ± 2.6 <0.001 1 5 221 ± 19 266 ± 23 0.2 15 20 ω3 FA (total) 135 ± 9 378 ± 26 <0.001 18 41 376 ± 29 545 ± 50 0.045 26 40 ω6/ω3 4.7 ± 0.2 1.5 ± 0.1 <0.001 2.8 ± 0.1 1.6 ± 0.1 <0.001 Plasma Endometrium μg/g P value % of total ω6 and ω3 FA μg/g P value % of total ω6 and ω3 FA ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet ω6 FA diet ω3 FA diet SFA 596 ± 37 675 ± 33 0.1 1616 ± 117 1489 ± 105 0.4 MUFA 304 ± 15 264 ± 12 0.06 1266 ± 106 1030 ± 65 0.1 PUFA 749 ± 42 916 ± 47 0.02 1449 ± 105 1352 ± 103 0.5 Linoleic acid 513 ± 30 458 ± 22 0.2 68 50 338 ± 25 315 ± 24 0.3 23 23 gamma-Linolenic acid 19.8 ± 1.5 5.3 ± 0.5 <0.001 3 1 17.0 ± 1.8 10.2 ± 1.1 0.002 1 1 Eicosadienoic acid ω6 0.8 ± 0.1 1.7 ± 0.1 0.005 - - 18.8 ± 3.0 15.1 ± 1.6 0.1 1 1 Eicosatrienoic 8c,11c,14c acid 29.6 ± 1.9 13.3 ± 0.7 <0.001 4 2 117 ± 10 80.7 ± 7.4 0.006 8 6 Arachidonic acid 51.6 ± 2.7 59.3 ± 3.6 0.4 7 6 584 ± 45 388 ± 22 <0.001 40 29 ω6 FA (total) 614 ± 35 536 ± 26 0.09 82 59 1075 ± 80 810 ± 53 0.009 73* 60 Linolenic acid 69.0 ± 4.7 117 ± 6 <0.001 9 13 12.2 ± 4.9 17.8 ± 1.9 0.1 1 1 Eicosapentaenoic acid 34.7 ± 3.2 187 ± 18 <0.001 5 20 24.6 ± 3.1 101 ± 13 <0.001 2 7 Docosapentaenoic acid ω3 20.8 ± 1.2 30.8 ± 1.4 <0.001 3 3 116 ± 8 158 ± 14 0.2 8 12 Docosahexaenoic acid 10.7 ± 0.9 43.7 ± 2.6 <0.001 1 5 221 ± 19 266 ± 23 0.2 15 20 ω3 FA (total) 135 ± 9 378 ± 26 <0.001 18 41 376 ± 29 545 ± 50 0.045 26 40 ω6/ω3 4.7 ± 0.2 1.5 ± 0.1 <0.001 2.8 ± 0.1 1.6 ± 0.1 <0.001 Heifers in the ω3 group have significantly more ω3 FA in plasma and endometrium than ω6 heifers. This was caused by an increase in all ω3 FA in plasma whereas in endometrium it caused by solely a higher EPA concentration. Conversely, heifers in the ω6 group display a higher concentration of AA in endometrium. Values are given as means ± sem. *Rounding error. View Large The total PUFA concentration in plasma was significantly increased in the ω3 group compared to the ω6 group (Table 1). This change was due to increased concentrations of total ω3 FA, whereas total ω6 FA concentration was unaffected in plasma. However, in the endometrium, the total PUFA concentration did not differ between diet groups. This was caused by a significant increase in total ω3 FA and a simultaneous significant decrease in total ω6 FA. Saturated FA and monounsaturated FA (MUFA) were not affected by diet neither in plasma nor in endometrium. The supplementation with rumen-protected ω3 FA significantly decreased the ω6/ω3 ratio in plasma as well as in endometrium compared to supplementation with rumen-protected ω6 FA (Table 1). This change in the ω6/ω3 ratio in plasma was caused by significant increases of all detectable ω3 FA including EPA and DHA in the ω3 group compared to the ω6 group. The amount of AA in plasma did not significantly differ between the two groups. In the endometrium, dietary ω3 FA significantly decreased the amount of AA and significantly increased the amount of EPA compared to dietary ω6 FA. Endometrial DHA did not differ between groups. Day 15 preimplantation embryos differed in length due to supplementation with rumen protected ω3 and ω6 FA The embryo length varied greatly within and between the experimental groups (Figure 1), namely from 0.2 to 20.3 cm in the ω3 group, but only from 1.4 to 7.7 cm in the ω6 group. On average, embryos from ω3 FA supplemented animals were significantly longer than embryos in the ω6 group (median ± SEM: ω6 group: 2.5 ± 0.5 cm; ω3 group: 6.4 ± 1.3 cm; P = 0.043). The pregnancy rate did not differ between treatment groups and was 82% in the ω3 and 87% in ω6 group, respectively. Figure 1. View largeDownload slide Supplementation with ω3 FA compared to ω6 FA led to significantly longer embryos on day 15 of pregnancy in Angus heifers (*P ≤ 0.05). Figure 1. View largeDownload slide Supplementation with ω3 FA compared to ω6 FA led to significantly longer embryos on day 15 of pregnancy in Angus heifers (*P ≤ 0.05). Different concentrations of AA and EPA in the endometrium caused by FA supplementation did not affect uterine prostaglandin concentrations The concentrations of different series-2 PG and their metabolites (PGE2, PGD2, TXB2, PGF2α, PGFM, and 6-keto-PGF1α) in the UF did not differ between groups (Table 2). PGF2α and the PGI2 metabolite 6-keto-PGF1α were the most abundant prostaglandines followed by PGFM and PGE2. PGD2 and TXB2 were present in similar concentrations and represented the least prominent PG in the UF. Table 2. Uterine concentrations of prostaglandins (ng/ml uterine fluid; means ± SEM). ω6 FA diet ω3 FA diet P-value PGF2α 17.5 ± 7.0 31.5 ± 5.8 0.9 PGE2 0.6 ± 0.2 1.0 ± 0.2 0.4 6-keto-PGF1α 12.6 ± 3.4 15.2 ± 2.8 0.6 PGD2 0.2 ± 0.1 0.2 ± 0.03 0.5 TXB2 0.2 ± 0.1 0.1 ± 0.04 0.4 PGFM 1.5 ± 0.2 1.3 ± 0.2 0.4 ω6 FA diet ω3 FA diet P-value PGF2α 17.5 ± 7.0 31.5 ± 5.8 0.9 PGE2 0.6 ± 0.2 1.0 ± 0.2 0.4 6-keto-PGF1α 12.6 ± 3.4 15.2 ± 2.8 0.6 PGD2 0.2 ± 0.1 0.2 ± 0.03 0.5 TXB2 0.2 ± 0.1 0.1 ± 0.04 0.4 PGFM 1.5 ± 0.2 1.3 ± 0.2 0.4 View Large Table 2. Uterine concentrations of prostaglandins (ng/ml uterine fluid; means ± SEM). ω6 FA diet ω3 FA diet P-value PGF2α 17.5 ± 7.0 31.5 ± 5.8 0.9 PGE2 0.6 ± 0.2 1.0 ± 0.2 0.4 6-keto-PGF1α 12.6 ± 3.4 15.2 ± 2.8 0.6 PGD2 0.2 ± 0.1 0.2 ± 0.03 0.5 TXB2 0.2 ± 0.1 0.1 ± 0.04 0.4 PGFM 1.5 ± 0.2 1.3 ± 0.2 0.4 ω6 FA diet ω3 FA diet P-value PGF2α 17.5 ± 7.0 31.5 ± 5.8 0.9 PGE2 0.6 ± 0.2 1.0 ± 0.2 0.4 6-keto-PGF1α 12.6 ± 3.4 15.2 ± 2.8 0.6 PGD2 0.2 ± 0.1 0.2 ± 0.03 0.5 TXB2 0.2 ± 0.1 0.1 ± 0.04 0.4 PGFM 1.5 ± 0.2 1.3 ± 0.2 0.4 View Large Dietary FA affected intrauterine alanine concentration but did neither impact on other amino acids nor intrauterine cortisol With the exception of a significantly higher concentration of alanine in the ω6 compared to the ω3 group, no differences were observed in amino acid concentrations in the UF of day 15 pregnant heifers (Supplementary Table S3). The cortisol concentration in the UF did not significantly differ between groups (ω6 group: 0.59 ± 0.1 ng/ml; ω3 group: 0.58 ± 0.1 ng/ml). The different concentrations of AA and EPA in the endometrium had only a minor impact on endometrial gene expression The endometrial expression of a broad range of genes, which were selected due to their respective involvement in embryo elongation and metabolism as well embryo–maternal communication, was widely unaffected by the dietary FA supplementation. Out of the genes under investigation, only perilipin 2 (PLIN2; previously known as adipose differentiation-related protein (ADFP)), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG), and sperm associated antigen 9 (SPAG9) were differently regulated in the endometrium of pregnant ω3 supplemented heifers compared to pregnant ω6 supplemented heifers (Table 3). The insulin receptor (INSR) transcript abundance showed a trend (P = 0.058). Table 3. Endometrial relative gene expression (means ± SEM) and mean fold-changes of pregnant heifers as determined by qPCR. ΔCt ω6 FA diet (n = 13) ΔCt ω3 FA diet (n = 18) Mean fold-change (ω3 vs ω6) P-value IFNt-stimulated genes ISG15 $$-$$1.19 ± 0.13 $$-$$1.05 ± 0.27 1.1 0.4 OAS1 $$-$$0.39 ± 0.13 $$-$$0.30 ± 0.17 1.1 0.7 MX1 0.25 ± 0.10 0.34 ± 0.18 1.1 0.9 Antioxidant defense GPX1 $$-$$2.09 ± 0.18 $$-$$2.15 ± 0.09 1.0 0.6 GPX4 $$-$$3.72 ± 0.14 $$-$$3.71 ± 0.09 1.0 0.6 SOD1 $$-$$3.00 ± 0.48 $$-$$3.66 ± 0.75 0.6 0.8 SOD2 $$-$$5.39 ± 0.94 $$-$$5.35 ± 0.86 1.0 0.8 NQO1 $$-$$9.85 ± 0.31 $$-$$10.25 ± 0.21 0.8 0.4 GSTA2 $$-$$7.65 ± 0.86 $$-$$7.02 ± 0.17 1.6 0.7 GSR $$-$$4.44 ± 0.10 $$-$$4.42 ± 0.10 1.0 0.6 GSS $$-$$4.44 ± 0.62 $$-$$3.67 ± 0.19 1.7 0.7 CAT $$-$$4.35 ± 0.13 $$-$$4.52 ± 0.23 0.9 0.6 MT1 $$-$$8.33 ± 1.32 $$-$$7.44 ± 0.77 1.9 0.7 MT2 $$-$$7.86 ± 0.68 $$-$$7.18 ± 0.28 1.6 0.7 NFE2L2 $$-$$2.85 ± 0.12 $$-$$4.14 ± 0.87 0.4 0.3 Amino acid transport and metabolism SLC1A1 $$-$$1.08 ± 0.15 $$-$$1.19 ± 0.13 0.9 0.7 SLC1A5 $$-$$5.94 ± 0.21 $$-$$5.81 ± 0.23 1.1 0.3 SLC6A6 $$-$$5.86 ± 0.14 $$-$$5.98 ± 0.12 0.9 0.2 SLC7A1 $$-$$4.95 ± 0.10 $$-$$4.93 ± 0.11 1.0 0.2 SLC7A5 $$-$$8.33 ± 0.15 $$-$$8.29 ± 0.19 1.0 0.8 SLC7A8 $$-$$5.81 ± 0.30 $$-$$5.67 ± 0.27 1.1 0.9 SLC15A1 $$-$$5.99 ± 0.19 $$-$$6.00 ± 0.16 1.0 0.4 SLC15A2 $$-$$6.20 ± 0.11 $$-$$6.05 ± 0.13 1.1 0.09 SLC15A3 $$-$$6.10 ± 0.17 $$-$$5.98 ± 0.23 1.1 0.5 GTP $$-$$6.78 ± 0.13 $$-$$6.90 ± 0.15 0.9 0.5 HDC $$-$$10.37 ± 0.50 $$-$$10.29 ± 0.52 1.1 0.8 IGF signaling IGF1 $$-$$5.59 ± 0.39 $$-$$5.32 ± 0.30 1.2 0.8 IGF1R $$-$$3.53 ± 0.14 $$-$$3.39 ± 0.16 1.1 0.3 IGF2 $$-$$4.82 ± 0.27 $$-$$4.10 ± 0.21 1.7 0.2 IGF2R $$-$$5.66 ± 0.23 $$-$$5.40 ± 0.24 1.2 0.7 INSR $$-$$5.00 ± 0.06 $$-$$4.89 ± 0.07 1.1 0.06 PPAR signaling PPARA $$-$$4.10 ± 0.09 $$-$$4.18 ± 0.07 0.9 0.2 PPARD $$-$$5.47 ± 0.15 $$-$$5.38 ± 0.11 1.1 0.8 PPARG $$-$$9.97 ± 0.29 $$-$$9.72 ± 0.20 1.2 0.6 Prostaglandin signaling PLA2 $$-$$5.80 ± 0.12 $$-$$5.80 ± 0.07 1.0 0.5 PTGS2 $$-$$1.81 ± 0.20 $$-$$1.99 ± 0.15 0.9 0.4 PTGER2 $$-$$5.38 ± 0.11 $$-$$5.74 ± 0.41 0.8 0.6 PTGER4 $$-$$7.21 ± 0.19 $$-$$7.11 ± 0.14 1.1 0.9 PTGIS $$-$$7.76 ± 1.04 $$-$$7.33 ± 0.72 1.4 0.7 PTGIR $$-$$9.62 ± 0.21 $$-$$9.48 ± 0.29 1.1 0.8 HPGD $$-$$2.04 ± 0.38 $$-$$1.66 ± 0.15 1.3 0.8 SLCO2A1 $$-$$7.65 ± 0.13 $$-$$7.89 ± 0.24 0.9 0.1 EDN1 $$-$$7.44 ± 0.26 $$-$$7.54 ± 0.24 0.9 0.8 Lipid and cholesterol metabolism and transport LPL $$-$$9.37 ± 0.15 $$-$$9.26 ± 0.16 1.1 0.9 FABP3 $$-$$2.84 ± 0.27 $$-$$2.55 ± 0.31 1.2 0.1 STAR $$-$$7.95 ± 0.19 $$-$$8.11 ± 0.21 0.9 0.3 PLIN2 $$-$$3.67 ± 0.17 $$-$$3.97 ± 0.14 0.8 0.04 Immunoregulation CPN1 $$-$$5.55 ± 0.23 $$-$$5.90 ± 0.18 0.8 0.9 TCF7 $$-$$5.44 ± 0.11 $$-$$5.47 ± 0.07 1.0 0.4 Wnt signaling SFRP2 $$-$$4.57 ± 0.35 $$-$$4.54 ± 0.26 1.0 0.9 WNT11 $$-$$6.00 ± 0.16 $$-$$5.97 ± 0.12 1.0 0.5 Signal transduction YWHAG $$-$$1.44 ± 0.07 $$-$$1.50 ± 0.06 1.0 0.04 SPAG9 $$-$$2.61 ± 0.06 $$-$$2.72 ± 0.05 0.9 0.03 ΔCt ω6 FA diet (n = 13) ΔCt ω3 FA diet (n = 18) Mean fold-change (ω3 vs ω6) P-value IFNt-stimulated genes ISG15 $$-$$1.19 ± 0.13 $$-$$1.05 ± 0.27 1.1 0.4 OAS1 $$-$$0.39 ± 0.13 $$-$$0.30 ± 0.17 1.1 0.7 MX1 0.25 ± 0.10 0.34 ± 0.18 1.1 0.9 Antioxidant defense GPX1 $$-$$2.09 ± 0.18 $$-$$2.15 ± 0.09 1.0 0.6 GPX4 $$-$$3.72 ± 0.14 $$-$$3.71 ± 0.09 1.0 0.6 SOD1 $$-$$3.00 ± 0.48 $$-$$3.66 ± 0.75 0.6 0.8 SOD2 $$-$$5.39 ± 0.94 $$-$$5.35 ± 0.86 1.0 0.8 NQO1 $$-$$9.85 ± 0.31 $$-$$10.25 ± 0.21 0.8 0.4 GSTA2 $$-$$7.65 ± 0.86 $$-$$7.02 ± 0.17 1.6 0.7 GSR $$-$$4.44 ± 0.10 $$-$$4.42 ± 0.10 1.0 0.6 GSS $$-$$4.44 ± 0.62 $$-$$3.67 ± 0.19 1.7 0.7 CAT $$-$$4.35 ± 0.13 $$-$$4.52 ± 0.23 0.9 0.6 MT1 $$-$$8.33 ± 1.32 $$-$$7.44 ± 0.77 1.9 0.7 MT2 $$-$$7.86 ± 0.68 $$-$$7.18 ± 0.28 1.6 0.7 NFE2L2 $$-$$2.85 ± 0.12 $$-$$4.14 ± 0.87 0.4 0.3 Amino acid transport and metabolism SLC1A1 $$-$$1.08 ± 0.15 $$-$$1.19 ± 0.13 0.9 0.7 SLC1A5 $$-$$5.94 ± 0.21 $$-$$5.81 ± 0.23 1.1 0.3 SLC6A6 $$-$$5.86 ± 0.14 $$-$$5.98 ± 0.12 0.9 0.2 SLC7A1 $$-$$4.95 ± 0.10 $$-$$4.93 ± 0.11 1.0 0.2 SLC7A5 $$-$$8.33 ± 0.15 $$-$$8.29 ± 0.19 1.0 0.8 SLC7A8 $$-$$5.81 ± 0.30 $$-$$5.67 ± 0.27 1.1 0.9 SLC15A1 $$-$$5.99 ± 0.19 $$-$$6.00 ± 0.16 1.0 0.4 SLC15A2 $$-$$6.20 ± 0.11 $$-$$6.05 ± 0.13 1.1 0.09 SLC15A3 $$-$$6.10 ± 0.17 $$-$$5.98 ± 0.23 1.1 0.5 GTP $$-$$6.78 ± 0.13 $$-$$6.90 ± 0.15 0.9 0.5 HDC $$-$$10.37 ± 0.50 $$-$$10.29 ± 0.52 1.1 0.8 IGF signaling IGF1 $$-$$5.59 ± 0.39 $$-$$5.32 ± 0.30 1.2 0.8 IGF1R $$-$$3.53 ± 0.14 $$-$$3.39 ± 0.16 1.1 0.3 IGF2 $$-$$4.82 ± 0.27 $$-$$4.10 ± 0.21 1.7 0.2 IGF2R $$-$$5.66 ± 0.23 $$-$$5.40 ± 0.24 1.2 0.7 INSR $$-$$5.00 ± 0.06 $$-$$4.89 ± 0.07 1.1 0.06 PPAR signaling PPARA $$-$$4.10 ± 0.09 $$-$$4.18 ± 0.07 0.9 0.2 PPARD $$-$$5.47 ± 0.15 $$-$$5.38 ± 0.11 1.1 0.8 PPARG $$-$$9.97 ± 0.29 $$-$$9.72 ± 0.20 1.2 0.6 Prostaglandin signaling PLA2 $$-$$5.80 ± 0.12 $$-$$5.80 ± 0.07 1.0 0.5 PTGS2 $$-$$1.81 ± 0.20 $$-$$1.99 ± 0.15 0.9 0.4 PTGER2 $$-$$5.38 ± 0.11 $$-$$5.74 ± 0.41 0.8 0.6 PTGER4 $$-$$7.21 ± 0.19 $$-$$7.11 ± 0.14 1.1 0.9 PTGIS $$-$$7.76 ± 1.04 $$-$$7.33 ± 0.72 1.4 0.7 PTGIR $$-$$9.62 ± 0.21 $$-$$9.48 ± 0.29 1.1 0.8 HPGD $$-$$2.04 ± 0.38 $$-$$1.66 ± 0.15 1.3 0.8 SLCO2A1 $$-$$7.65 ± 0.13 $$-$$7.89 ± 0.24 0.9 0.1 EDN1 $$-$$7.44 ± 0.26 $$-$$7.54 ± 0.24 0.9 0.8 Lipid and cholesterol metabolism and transport LPL $$-$$9.37 ± 0.15 $$-$$9.26 ± 0.16 1.1 0.9 FABP3 $$-$$2.84 ± 0.27 $$-$$2.55 ± 0.31 1.2 0.1 STAR $$-$$7.95 ± 0.19 $$-$$8.11 ± 0.21 0.9 0.3 PLIN2 $$-$$3.67 ± 0.17 $$-$$3.97 ± 0.14 0.8 0.04 Immunoregulation CPN1 $$-$$5.55 ± 0.23 $$-$$5.90 ± 0.18 0.8 0.9 TCF7 $$-$$5.44 ± 0.11 $$-$$5.47 ± 0.07 1.0 0.4 Wnt signaling SFRP2 $$-$$4.57 ± 0.35 $$-$$4.54 ± 0.26 1.0 0.9 WNT11 $$-$$6.00 ± 0.16 $$-$$5.97 ± 0.12 1.0 0.5 Signal transduction YWHAG $$-$$1.44 ± 0.07 $$-$$1.50 ± 0.06 1.0 0.04 SPAG9 $$-$$2.61 ± 0.06 $$-$$2.72 ± 0.05 0.9 0.03 View Large Table 3. Endometrial relative gene expression (means ± SEM) and mean fold-changes of pregnant heifers as determined by qPCR. ΔCt ω6 FA diet (n = 13) ΔCt ω3 FA diet (n = 18) Mean fold-change (ω3 vs ω6) P-value IFNt-stimulated genes ISG15 $$-$$1.19 ± 0.13 $$-$$1.05 ± 0.27 1.1 0.4 OAS1 $$-$$0.39 ± 0.13 $$-$$0.30 ± 0.17 1.1 0.7 MX1 0.25 ± 0.10 0.34 ± 0.18 1.1 0.9 Antioxidant defense GPX1 $$-$$2.09 ± 0.18 $$-$$2.15 ± 0.09 1.0 0.6 GPX4 $$-$$3.72 ± 0.14 $$-$$3.71 ± 0.09 1.0 0.6 SOD1 $$-$$3.00 ± 0.48 $$-$$3.66 ± 0.75 0.6 0.8 SOD2 $$-$$5.39 ± 0.94 $$-$$5.35 ± 0.86 1.0 0.8 NQO1 $$-$$9.85 ± 0.31 $$-$$10.25 ± 0.21 0.8 0.4 GSTA2 $$-$$7.65 ± 0.86 $$-$$7.02 ± 0.17 1.6 0.7 GSR $$-$$4.44 ± 0.10 $$-$$4.42 ± 0.10 1.0 0.6 GSS $$-$$4.44 ± 0.62 $$-$$3.67 ± 0.19 1.7 0.7 CAT $$-$$4.35 ± 0.13 $$-$$4.52 ± 0.23 0.9 0.6 MT1 $$-$$8.33 ± 1.32 $$-$$7.44 ± 0.77 1.9 0.7 MT2 $$-$$7.86 ± 0.68 $$-$$7.18 ± 0.28 1.6 0.7 NFE2L2 $$-$$2.85 ± 0.12 $$-$$4.14 ± 0.87 0.4 0.3 Amino acid transport and metabolism SLC1A1 $$-$$1.08 ± 0.15 $$-$$1.19 ± 0.13 0.9 0.7 SLC1A5 $$-$$5.94 ± 0.21 $$-$$5.81 ± 0.23 1.1 0.3 SLC6A6 $$-$$5.86 ± 0.14 $$-$$5.98 ± 0.12 0.9 0.2 SLC7A1 $$-$$4.95 ± 0.10 $$-$$4.93 ± 0.11 1.0 0.2 SLC7A5 $$-$$8.33 ± 0.15 $$-$$8.29 ± 0.19 1.0 0.8 SLC7A8 $$-$$5.81 ± 0.30 $$-$$5.67 ± 0.27 1.1 0.9 SLC15A1 $$-$$5.99 ± 0.19 $$-$$6.00 ± 0.16 1.0 0.4 SLC15A2 $$-$$6.20 ± 0.11 $$-$$6.05 ± 0.13 1.1 0.09 SLC15A3 $$-$$6.10 ± 0.17 $$-$$5.98 ± 0.23 1.1 0.5 GTP $$-$$6.78 ± 0.13 $$-$$6.90 ± 0.15 0.9 0.5 HDC $$-$$10.37 ± 0.50 $$-$$10.29 ± 0.52 1.1 0.8 IGF signaling IGF1 $$-$$5.59 ± 0.39 $$-$$5.32 ± 0.30 1.2 0.8 IGF1R $$-$$3.53 ± 0.14 $$-$$3.39 ± 0.16 1.1 0.3 IGF2 $$-$$4.82 ± 0.27 $$-$$4.10 ± 0.21 1.7 0.2 IGF2R $$-$$5.66 ± 0.23 $$-$$5.40 ± 0.24 1.2 0.7 INSR $$-$$5.00 ± 0.06 $$-$$4.89 ± 0.07 1.1 0.06 PPAR signaling PPARA $$-$$4.10 ± 0.09 $$-$$4.18 ± 0.07 0.9 0.2 PPARD $$-$$5.47 ± 0.15 $$-$$5.38 ± 0.11 1.1 0.8 PPARG $$-$$9.97 ± 0.29 $$-$$9.72 ± 0.20 1.2 0.6 Prostaglandin signaling PLA2 $$-$$5.80 ± 0.12 $$-$$5.80 ± 0.07 1.0 0.5 PTGS2 $$-$$1.81 ± 0.20 $$-$$1.99 ± 0.15 0.9 0.4 PTGER2 $$-$$5.38 ± 0.11 $$-$$5.74 ± 0.41 0.8 0.6 PTGER4 $$-$$7.21 ± 0.19 $$-$$7.11 ± 0.14 1.1 0.9 PTGIS $$-$$7.76 ± 1.04 $$-$$7.33 ± 0.72 1.4 0.7 PTGIR $$-$$9.62 ± 0.21 $$-$$9.48 ± 0.29 1.1 0.8 HPGD $$-$$2.04 ± 0.38 $$-$$1.66 ± 0.15 1.3 0.8 SLCO2A1 $$-$$7.65 ± 0.13 $$-$$7.89 ± 0.24 0.9 0.1 EDN1 $$-$$7.44 ± 0.26 $$-$$7.54 ± 0.24 0.9 0.8 Lipid and cholesterol metabolism and transport LPL $$-$$9.37 ± 0.15 $$-$$9.26 ± 0.16 1.1 0.9 FABP3 $$-$$2.84 ± 0.27 $$-$$2.55 ± 0.31 1.2 0.1 STAR $$-$$7.95 ± 0.19 $$-$$8.11 ± 0.21 0.9 0.3 PLIN2 $$-$$3.67 ± 0.17 $$-$$3.97 ± 0.14 0.8 0.04 Immunoregulation CPN1 $$-$$5.55 ± 0.23 $$-$$5.90 ± 0.18 0.8 0.9 TCF7 $$-$$5.44 ± 0.11 $$-$$5.47 ± 0.07 1.0 0.4 Wnt signaling SFRP2 $$-$$4.57 ± 0.35 $$-$$4.54 ± 0.26 1.0 0.9 WNT11 $$-$$6.00 ± 0.16 $$-$$5.97 ± 0.12 1.0 0.5 Signal transduction YWHAG $$-$$1.44 ± 0.07 $$-$$1.50 ± 0.06 1.0 0.04 SPAG9 $$-$$2.61 ± 0.06 $$-$$2.72 ± 0.05 0.9 0.03 ΔCt ω6 FA diet (n = 13) ΔCt ω3 FA diet (n = 18) Mean fold-change (ω3 vs ω6) P-value IFNt-stimulated genes ISG15 $$-$$1.19 ± 0.13 $$-$$1.05 ± 0.27 1.1 0.4 OAS1 $$-$$0.39 ± 0.13 $$-$$0.30 ± 0.17 1.1 0.7 MX1 0.25 ± 0.10 0.34 ± 0.18 1.1 0.9 Antioxidant defense GPX1 $$-$$2.09 ± 0.18 $$-$$2.15 ± 0.09 1.0 0.6 GPX4 $$-$$3.72 ± 0.14 $$-$$3.71 ± 0.09 1.0 0.6 SOD1 $$-$$3.00 ± 0.48 $$-$$3.66 ± 0.75 0.6 0.8 SOD2 $$-$$5.39 ± 0.94 $$-$$5.35 ± 0.86 1.0 0.8 NQO1 $$-$$9.85 ± 0.31 $$-$$10.25 ± 0.21 0.8 0.4 GSTA2 $$-$$7.65 ± 0.86 $$-$$7.02 ± 0.17 1.6 0.7 GSR $$-$$4.44 ± 0.10 $$-$$4.42 ± 0.10 1.0 0.6 GSS $$-$$4.44 ± 0.62 $$-$$3.67 ± 0.19 1.7 0.7 CAT $$-$$4.35 ± 0.13 $$-$$4.52 ± 0.23 0.9 0.6 MT1 $$-$$8.33 ± 1.32 $$-$$7.44 ± 0.77 1.9 0.7 MT2 $$-$$7.86 ± 0.68 $$-$$7.18 ± 0.28 1.6 0.7 NFE2L2 $$-$$2.85 ± 0.12 $$-$$4.14 ± 0.87 0.4 0.3 Amino acid transport and metabolism SLC1A1 $$-$$1.08 ± 0.15 $$-$$1.19 ± 0.13 0.9 0.7 SLC1A5 $$-$$5.94 ± 0.21 $$-$$5.81 ± 0.23 1.1 0.3 SLC6A6 $$-$$5.86 ± 0.14 $$-$$5.98 ± 0.12 0.9 0.2 SLC7A1 $$-$$4.95 ± 0.10 $$-$$4.93 ± 0.11 1.0 0.2 SLC7A5 $$-$$8.33 ± 0.15 $$-$$8.29 ± 0.19 1.0 0.8 SLC7A8 $$-$$5.81 ± 0.30 $$-$$5.67 ± 0.27 1.1 0.9 SLC15A1 $$-$$5.99 ± 0.19 $$-$$6.00 ± 0.16 1.0 0.4 SLC15A2 $$-$$6.20 ± 0.11 $$-$$6.05 ± 0.13 1.1 0.09 SLC15A3 $$-$$6.10 ± 0.17 $$-$$5.98 ± 0.23 1.1 0.5 GTP $$-$$6.78 ± 0.13 $$-$$6.90 ± 0.15 0.9 0.5 HDC $$-$$10.37 ± 0.50 $$-$$10.29 ± 0.52 1.1 0.8 IGF signaling IGF1 $$-$$5.59 ± 0.39 $$-$$5.32 ± 0.30 1.2 0.8 IGF1R $$-$$3.53 ± 0.14 $$-$$3.39 ± 0.16 1.1 0.3 IGF2 $$-$$4.82 ± 0.27 $$-$$4.10 ± 0.21 1.7 0.2 IGF2R $$-$$5.66 ± 0.23 $$-$$5.40 ± 0.24 1.2 0.7 INSR $$-$$5.00 ± 0.06 $$-$$4.89 ± 0.07 1.1 0.06 PPAR signaling PPARA $$-$$4.10 ± 0.09 $$-$$4.18 ± 0.07 0.9 0.2 PPARD $$-$$5.47 ± 0.15 $$-$$5.38 ± 0.11 1.1 0.8 PPARG $$-$$9.97 ± 0.29 $$-$$9.72 ± 0.20 1.2 0.6 Prostaglandin signaling PLA2 $$-$$5.80 ± 0.12 $$-$$5.80 ± 0.07 1.0 0.5 PTGS2 $$-$$1.81 ± 0.20 $$-$$1.99 ± 0.15 0.9 0.4 PTGER2 $$-$$5.38 ± 0.11 $$-$$5.74 ± 0.41 0.8 0.6 PTGER4 $$-$$7.21 ± 0.19 $$-$$7.11 ± 0.14 1.1 0.9 PTGIS $$-$$7.76 ± 1.04 $$-$$7.33 ± 0.72 1.4 0.7 PTGIR $$-$$9.62 ± 0.21 $$-$$9.48 ± 0.29 1.1 0.8 HPGD $$-$$2.04 ± 0.38 $$-$$1.66 ± 0.15 1.3 0.8 SLCO2A1 $$-$$7.65 ± 0.13 $$-$$7.89 ± 0.24 0.9 0.1 EDN1 $$-$$7.44 ± 0.26 $$-$$7.54 ± 0.24 0.9 0.8 Lipid and cholesterol metabolism and transport LPL $$-$$9.37 ± 0.15 $$-$$9.26 ± 0.16 1.1 0.9 FABP3 $$-$$2.84 ± 0.27 $$-$$2.55 ± 0.31 1.2 0.1 STAR $$-$$7.95 ± 0.19 $$-$$8.11 ± 0.21 0.9 0.3 PLIN2 $$-$$3.67 ± 0.17 $$-$$3.97 ± 0.14 0.8 0.04 Immunoregulation CPN1 $$-$$5.55 ± 0.23 $$-$$5.90 ± 0.18 0.8 0.9 TCF7 $$-$$5.44 ± 0.11 $$-$$5.47 ± 0.07 1.0 0.4 Wnt signaling SFRP2 $$-$$4.57 ± 0.35 $$-$$4.54 ± 0.26 1.0 0.9 WNT11 $$-$$6.00 ± 0.16 $$-$$5.97 ± 0.12 1.0 0.5 Signal transduction YWHAG $$-$$1.44 ± 0.07 $$-$$1.50 ± 0.06 1.0 0.04 SPAG9 $$-$$2.61 ± 0.06 $$-$$2.72 ± 0.05 0.9 0.03 View Large Table 4. Luteal relative gene expression (mean ± SEM) and mean fold-changes of pregnant heifers as measured via qPCR. ΔCt ω6 FA diet (n = 6) ΔCt ω3 FA diet (n = 12) Mean fold- change (ω3 vs ω6) P-value HSD3B1 2.80 ± 0.16 2.73 ± 0.13 0.95 0.8 STAR 2.43 ± 0.28 2.58 ± 0.14 1.11 0.6 PTGFR 0.57 ± 0.11 0.73 ± 0.13 1.12 0.4 AKR1C3 $$-$$4.13 ± 0.08 $$-$$4.07 ± 0.05 1.04 0.5 CASP8 $$-$$5.45 ± 0.10 $$-$$5.39 ± 0.08 1.04 0.7 CASP3 $$-$$11.88 ± 0.24 $$-$$11.97 ± 0.06 0.94 0.6 PPARG $$-$$8.22 ± 0.28 $$-$$8.50 ± 0.11 0.82 0.3 ΔCt ω6 FA diet (n = 6) ΔCt ω3 FA diet (n = 12) Mean fold- change (ω3 vs ω6) P-value HSD3B1 2.80 ± 0.16 2.73 ± 0.13 0.95 0.8 STAR 2.43 ± 0.28 2.58 ± 0.14 1.11 0.6 PTGFR 0.57 ± 0.11 0.73 ± 0.13 1.12 0.4 AKR1C3 $$-$$4.13 ± 0.08 $$-$$4.07 ± 0.05 1.04 0.5 CASP8 $$-$$5.45 ± 0.10 $$-$$5.39 ± 0.08 1.04 0.7 CASP3 $$-$$11.88 ± 0.24 $$-$$11.97 ± 0.06 0.94 0.6 PPARG $$-$$8.22 ± 0.28 $$-$$8.50 ± 0.11 0.82 0.3 View Large Table 4. Luteal relative gene expression (mean ± SEM) and mean fold-changes of pregnant heifers as measured via qPCR. ΔCt ω6 FA diet (n = 6) ΔCt ω3 FA diet (n = 12) Mean fold- change (ω3 vs ω6) P-value HSD3B1 2.80 ± 0.16 2.73 ± 0.13 0.95 0.8 STAR 2.43 ± 0.28 2.58 ± 0.14 1.11 0.6 PTGFR 0.57 ± 0.11 0.73 ± 0.13 1.12 0.4 AKR1C3 $$-$$4.13 ± 0.08 $$-$$4.07 ± 0.05 1.04 0.5 CASP8 $$-$$5.45 ± 0.10 $$-$$5.39 ± 0.08 1.04 0.7 CASP3 $$-$$11.88 ± 0.24 $$-$$11.97 ± 0.06 0.94 0.6 PPARG $$-$$8.22 ± 0.28 $$-$$8.50 ± 0.11 0.82 0.3 ΔCt ω6 FA diet (n = 6) ΔCt ω3 FA diet (n = 12) Mean fold- change (ω3 vs ω6) P-value HSD3B1 2.80 ± 0.16 2.73 ± 0.13 0.95 0.8 STAR 2.43 ± 0.28 2.58 ± 0.14 1.11 0.6 PTGFR 0.57 ± 0.11 0.73 ± 0.13 1.12 0.4 AKR1C3 $$-$$4.13 ± 0.08 $$-$$4.07 ± 0.05 1.04 0.5 CASP8 $$-$$5.45 ± 0.10 $$-$$5.39 ± 0.08 1.04 0.7 CASP3 $$-$$11.88 ± 0.24 $$-$$11.97 ± 0.06 0.94 0.6 PPARG $$-$$8.22 ± 0.28 $$-$$8.50 ± 0.11 0.82 0.3 View Large Plasma progesterone was affected by dietary FA In both groups, the concentration of plasma P4 increased from day 3 to day 15 (Figure 2). Animals from the ω3 group had higher P4 concentrations compared to animals from the ω6 group. The average higher concentration of P4 in the ω3 group was due to the animals from which embryos >8 cm in length were recovered. Figure 2. View largeDownload slide Plasma progesterone concentrations were higher in ω3 FA supplemented heifers than in ω6 FA supplemented heifers. Values are given as means ± SEM. Figure 2. View largeDownload slide Plasma progesterone concentrations were higher in ω3 FA supplemented heifers than in ω6 FA supplemented heifers. Values are given as means ± SEM. Luteal gene expression of enzymes involved in P4 synthesis did not differ on day 15 between diet groups The luteal gene expression of neither hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), steroidogenic acute regulatory protein (STAR), prostaglandin F receptor (PTGFR), aldo-keto reductase family 1 member C3 (AKR1C3; previously known as hydroxysteroid (17-beta) dehydrogenase 5 (HSD17B5)), caspase 3 (CASP3), caspase 8 (CASP8) or PPARG differed between diet groups (Table 4). Dietary FA neither impacted on plasma LDL nor on plasma nor endometrial α- and γ-tocopherol concentrations The LDL cholesterol concentration in plasma did not differ between the two diet groups (ω6 group: 0.24 ± 0.03 mmol/l; ω3 group: 0.23 ± 0.03 mmol/l) on day 15 of pregnancy. Despite liver concentrations of α- and γ-tocopherol being significantly lower in the ω3 group compared to the ω6 group, neither plasma nor endometrial concentrations of α- and γ-tocopherol differed between groups, respectively (Supplementary Table S4). Discussion The present study is the first to report differences in embryo elongation in heifers supplemented with ω3 FA-rich fish oil and ω6 FA-rich sunflower oil, respectively. The main findings were that (1) day 15 embryos were significantly longer in ω3 compared to ω6 fed animals; (2) the concentration of AA was significantly lower, while the concentration of EPA was significantly higher and DHA was unaffected in the endometrium of ω3 compared to ω6 fed animals; (3) the concentrations of uterine prostaglandins did not differ between diet groups despite significantly different endometrial amounts of their precursor AA; and (4) increased plasma P4 concentration in ω3 compared to ω6 fed animals during luteal growth was associated with filamentous embryos present on day 15 of pregnancy. There is no general agreement on how to categorize embryos according to their length, specifically at the beginning of elongation. The classification of embryos differs between published studies [63–67]. Bovine embryos are suggested to have an ovoid (day 12), a tubular (day 14), or a filamentous (day 16–18) shape, and the term “elongation” is generally applied to embryos with an ovoid to slightly tubular shape [63, 68]. The day 15 embryos of the present study showed a great variance in length from the ovoid to the filamentous forms. The embryos recovered from ω3 FA supplemented animals were not only significantly longer than those recovered from ω6 FA supplemented animals, but also the variance in embryo length was greater in the ω3 group than in the ω6 group. Staples et al. [69] reported bovine embryo lengths on day 15 of pregnancy ranging from 0.6 to 17 cm, whereas Betteridge et al. [70] found day 15 embryo lengths ranging between 0.3 and 4 cm. Following superovulation, an enormous range of variation in embryo length (e.g. 0.4–4 cm on day 14) was observed even within a single donor [70]. Therefore, it remains difficult to state with certainty whether ω3 FA promoted or ω6 FA delayed embryo elongation in comparison to the respective other supplementation, or both. Dietary PUFA normally undergo partial biohydrogenation by ruminal microorganisms. Our FA supplements were coated with hydrogenated rapeseed oil for rumen protection. We chose heifers during their postpubertal growing phase to maximize the incorporation of dietary PUFA into the body lipid pools. The plasma and the endometrial FA profiles of our heifers confirm the successful rumen protection of ω3 FA. The higher EPA and DHA concentration in plasma of ω3 compared to ω6 supplemented animals is in accordance with the literature, where a stronger increase in EPA compared to DHA concentration as well as an increase in total ω3 PUFA lacking a difference in total ω6 PUFA has been shown [71, 72]. Increased plasma levels of supplemented ω3 FA led to an increased endometrial EPA concentration compared to the supplementation with ω6 FA. We observed an incorporation of dietary ω3 FA into endometrial tissue. This is in line with previously published studies [55, 71, 72], leading to a decreased ω6/ω3 ratio [55, 72, 73]. The latter has also been observed for luteal tissue [72]. During early embryonic development, PUFA play an important role by affecting membrane fluidity and permeability. They also serve as essential precursors for signaling molecules such as PG. Childs et al. [55] did not find any difference in plasma AA concentrations after 50 days of ω3 FA supplementation compared to an unsupplemented control group. This is in accordance with our findings, though we compared to a supplementation with ω6 FA. The supplementation with ω3 FA increased EPA almost threefold and decreased AA concentration in the UF of beef heifers compared to an ω6 supplementation, leading to significantly different ω6/ω3 ratios in the UF at day 7 of pregnancy [54]. Even though we did not analyze the FA in the UF, we have observed similar changes in EPA and AA in the endometrium. Consequently, we assume that our rumen-protected ω3 and ω6 FA supplementation has most likely resulted in a similar FA profile in the UF. Interestingly, Childs et al. [54] did not detect differences in DHA concentrations in the UF when comparing ω3 and ω6 supplemented animals. Accordingly, despite significant differences in DHA concentrations in plasma, we neither observed differing DHA concentrations in the endometrium of the two diet groups. Furthermore, the concentration of EPA was lower in endometrium compared to plasma, although endometrial AA and DHA concentrations were higher than those in plasma. Thus, we assume that AA and DHA have preferentially been incorporated into the endometrium. These results point toward a selective uptake of specific FA into the endometrium and the UF, respectively. We hypothesize that this regulatory mechanism might be due to specific needs of the developing embryo. It has been hypothesized that ω3 FA may act synergistically with IFNT to inhibit endometrial production of PGF2α and thus luteolysis [74]. In our study, we could not observe any diet-induced differences in endometrial gene expression of enzymes involved in PG synthesis. Moreover, with regard to the significantly decreased endometrial availability of AA in ω3 compared to ω6 FA supplemented animals, we hypothesized to find decreased series-2 PG in the UF. Surprisingly, this was not case. In several animal cell culture systems including bovine endometrial cells, incubation with ω3 FA decreased PGF2α production and secretion [74–77]. In addition, the PG-reducing effect of ω3 FA in the bovine reproductive tract has repeatedly been reported, especially in cyclic animals [78]. However, this was assessed by determining circulating PGFM. Our data show that irrespective of decreased AA, the local intrauterine series-2 PG concentrations during preimplantation development remained unaffected. We doubt whether the AA shift induced by a maternal diet can be pronounced enough at all to impact on the intrauterine hormonal signaling of series-2 PG. Most studies that report an effect of PUFA supplementation on PG concentrations were performed with cyclic animals. Therefore, we cannot rule out an effect of dietary ω3 and ω6 PUFA on PG concentrations in nonpregnant animals. Increasing plasma P4 concentrations might present a mechanism by which ω3 FA increase pregnancy rates in cows [79]. Intervention studies showed that higher P4 concentrations during the early stage of pregnancy were associated with a greater embryo elongation [18, 20–22, 80–82]. The importance of the P4 regulatory effect has been demonstrated in synchronous versus asynchronous embryo transfer [83, 84]. However, dietary ω3 and ω6 FA effects on plasma P4 concentrations led to contradictory results. In lactating Holstein cows, the supplementation of ω3 FA slightly increased mean P4 concentrations in some studies [85, 86], but lacked to show an effect in others [85, 87, 88]. Robinson et al. [89] observed lower plasma P4 concentrations when dairy cows were supplemented with FA. However, the effect was irrespective of a ω3 or ω6 FA supplementation. The incubation of bovine luteal cells with ω3 FA decreased P4 synthesis [90], whereas ω6 FA increased P4 in bovine CL cells [91]. Dietary ω3 FA are suggested to prevent the regression of the CL by decreased endometrial PGF2α released into the circulation (indicated by reduced PGFM), which may result in a prolonged P4 secretion [13, 92]. Indeed, a fish meal supplementation to lactating animals resulted in P4 concentrations >1 ng/ml 2 days after PGF2α injection. This led to the assumption of an inhibitory effect of ω3 FA on PGF2α-induced CL regression [13]. Dietary supplementation with flaxseed containing ALA led to higher P4 values on the day of AI in lactating Holstein cows compared to sunflower seed, also suggesting a delay in luteolysis [87]. In our experiment, we did not determine the timing of luteolysis and ovulation. If ω3 FA supplementation had caused a delay in luteolysis in our animals as described in the literature [13, 87], a delayed ovulation would be expected to result in less elongated embryos because of a relative younger embryonic age. However, we observed the opposite, thereby rather strengthening the hypothesis of promoted embryo elongation via ω3 FA supplementation. In our data, the heterogeneity in P4 was higher in the ω3 than in the ω6 group. Those heifers carrying an elongated embryo on day 15 had higher P4 from day 6 onwards. The supplementation with dietary ω3 FA has been associated with reduced plasma cholesterol compared to dietary ω6 FA [89]. While our data indicate the lack of change in plasma cholesterol, we found higher plasma P4 concentrations in the ω3 FA supplemented animals compared to the ω6 FA supplemented animals during luteal growth. However, this appeared due to those animals in the ω3 group where a filamentous embryo was present. Clearly, the rise in P4 of these latter ones could have caused the enhanced embryo elongation. However, it is unclear why only some animals responded to the ω3 treatment with an increase in P4 while others of the same treatment group did not. A direct effect of the dietary ω3 FA on P4 synthesis and luteal function will thus need further substantiation. We further analyzed whether the supplementation with ω3 compared to ω6 FA altered the response of the endometrium to P4. Specifically, gene expression changes via FA-sensitive transcription factors were of interest. In general, ω3 FA seem to be more effective in modifying gene expression than ω6 FA or SFA and increased EPA plasma levels are associated with increased PPAR gene expression [93, 94]. Interestingly, the endometrial gene expression of PPARs did not differ in the present study. Waters et al. [49] demonstrated that feeding ω3 FA led to differential gene expression of a variety of genes in the endometrium. We could confirm only some of these reported gene expression changes in our study. However, the control group in Waters et al. [49] received palmitic acid and not ω6 FA as reference. With the exception of alanine, neither the concentration of amino acids in the UF, the expression of amino acid transporters, the presence of endometrial α- and gamma-tocopherol, or uterine cortisol was affected by the diet. Surprisingly, even the gene expression of ISGs did not significantly differ between animals with embryos of different lengths. Because day 7 embryos already induce endometrial ISG15 ubiquitin-like modifier (ISG15; previously known as interferon, alpha-inducible protein (clone IFI-15K) (G1P2)), 2΄-5΄-oligoadenylate synthetase 1 (OAS1), and MX dynamin like GTPase 1 (MX1) gene expression [95], we assume that embryos may have induced the respective gene expression to a maximum already at an earlier stage of elongation. This would explain the lack of an observable difference. Taken together, endometrial gene expression differences in the present study in general were very scarce, even though the endometrial concentration of ω3 and ω6 FA was significantly altered. It remains to be analyzed to which extent gene expression and PG concentration differed before day 15 of pregnancy and may hereby had affected embryo elongation. Despite a significant difference in embryo length between the ω3 and ω6 FA groups, we cannot identify with certainty a mechanism by which ω3 and ω6 FA differentially affected embryo elongation. Our study rules out (1) a direct effect of ω3 FA altering endometrial embryotropic series-2 PG as well as (2) a direct or indirect effect of ω3 FA altering uterine amino acids, α- and γ-tocopherol, or cortisol furthering embryo elongation. In the present study, we neither investigated possible effects of dietary FA on uterine series-3 PG nor FA effects on the ovaries and thus follicles and oocytes. The latter remains a specifically interesting target because dietary ω3 and ω6 FA have been shown to affect follicular development and oocyte competency [96–98]. Therefore, further research is needed to understand the functional link between dietary ω3 FA and reproductive outcome. Supplementary data Supplementary data are available at BIOLRE online. Supplementary Table S1 Proximate (mean of pooled diet samples) and fatty acid composition of diets (n = 3) adapted from [57]. Values are given as means ± standard deviation. Supplementary Table S2 Sequences of primers. Supplementary Table S3 Amino acid concentrations in the uterine fluid. Values are given as means ± SEM. Supplementary Table S4 Plasma, liver and endometrial concentrations of alpha- and gamma-tocopherol. Values are given as means ± SEM. Acknowledgments We are thankful for the kind support of Marcus Wysshaar (ErboSpraytech), Michael Kreuzer for expert consulting, as well as the staff of the former ETH research station Chamau. The authors are active members of the European Union COST action CA16119 CELLFIT. References 1. Diskin MG , Murphy JJ , Sreenan JM . Embryo survival in dairy cows managed under pastoral conditions . Anim Reprod Sci 2006 ; 96 ( 3-4 ): 297 – 311 . Google Scholar CrossRef Search ADS PubMed 2. Moore K , Thatcher WW . Major advances associated with reproduction in dairy cattle . J Dairy Sci 2006 ; 89 : 1254 – 1266 . Google Scholar CrossRef Search ADS PubMed 3. Dobson H , Smith R , Royal M , Knight C , Sheldon I . The high-producing dairy cow and its reproductive performance . Reprod Domest Anim 2007 ; 42 ( s2 ): 17 – 23 . Google Scholar CrossRef Search ADS PubMed 4. Diskin MG , Parr MH , Morris DG . Embryo death in cattle: an update . Reprod Fertil Dev 2012 ; 24 : 244 – 251 . Google Scholar CrossRef Search ADS 5. Filant J , Spencer TE . Uterine glands: biological roles in conceptus implantation, uterine receptivity and decidualization . Int J Dev Biol 2014 ; 58 : 107 – 116 . Google Scholar CrossRef Search ADS PubMed 6. Ashworth CJ , Toma LM , Hunter MG . Nutritional effects on oocyte and embryo development in mammals: implications for reproductive efficiency and environmental sustainability . Philos T Roy Soc B Biol Sci 2009 ; 364 : 3351 – 3361 . Google Scholar CrossRef Search ADS 7. Mattos R , Staples CR , Thatcher WW . Effects of dietary fatty acids on reproduction in ruminants . Rev Reprod 2000 ; 5 : 38 – 45 . Google Scholar CrossRef Search ADS PubMed 8. Lopes CN , Scarpa AB , Cappellozza BI , Cooke RF , Vasconcelos JL . Effects of rumen-protected polyunsaturated fatty acid supplementation on reproductive performance of Bos indicus beef cows . J Anim Sci 2009 ; 87 : 3935 – 3943 . Google Scholar CrossRef Search ADS PubMed 9. Juchem SO , Cerri RLA , Villasenor M , Galvao KN , Bruno RGS , Rutigliano HM , DePeters EJ , Silvestre FT , Thatcher WW , Santos JEP . Supplementation with calcium salts of linoleic and trans -octadecenoic acids improves fertility of lactating dairy cows . Reprod Dom Anim 2010 ; 45 : 55 – 62 . Google Scholar CrossRef Search ADS 10. Colazo MG , Kastelic JP , Martinez MF , Whittaker PR , Wilde R , Ambrose JD , Corbett R , Mapletoft RJ . Fertility following fixed-time AI in CIDR-treated beef heifers given GnRH or estradiol cypionate and fed diets supplemented with flax seed or sunflower seed . Theriogenology 2004 ; 61 : 1115 – 1124 . Google Scholar CrossRef Search ADS PubMed 11. Bork NR , Schroeder JW , Lardy GP , Vonnahme KA , Bauer ML , Buchanan DS , Shaver RD , Fricke PM . Effect of feeding rolled flaxseed on milk fatty acid profiles and reproductive performance of dairy cows . J Anim Sci 2010 ; 88 : 3739 – 3748 . Google Scholar CrossRef Search ADS PubMed 12. Richardson GF , McNiven MA , Petit HV , Duynisveld JL . The effects of dietary omega fatty acids on pregnancy rate, plasma prostaglandin metabolite levels, serum progesterone levels, and milk fatty-acid profile in beef cows . Can J Vet Res 2013 ; 77 : 314 – 318 . Google Scholar PubMed 13. Burke JM , Staples CR , Risco CA , de la Sota RL , Thatcher WW . Effect of ruminant grade Menhaden fish meal on reproductive and productive performance of lactating dairy cows . J Dairy Sci 1997 ; 80 : 3386 – 3398 . Google Scholar CrossRef Search ADS PubMed 14. Gulliver CE , Friend MA , King BJ , Clayton EH . The role of omega-3 polyunsaturated fatty acids in reproduction of sheep and cattle . Anim Reprod Sci 2012 ; 131 : 9 – 22 . Google Scholar CrossRef Search ADS PubMed 15. Spencer TE , Ott TL , Bazer FW . tau-Interferon: pregnancy recognition signal in ruminants . Exp Biol Med 1996 ; 213 : 215 – 229 . Google Scholar CrossRef Search ADS 16. Roberts RM , Ealy AD , Alexenko AP , Han CS , Ezashi T . Trophoblast interferons . Placenta 1999 ; 20 : 259 – 264 . Google Scholar CrossRef Search ADS PubMed 17. Hansen TR , Austin KJ , Perry DJ , Pru JK , Teixeira MG , Johnson GA . Mechanism of action of interferon-tau in the uterus during early pregnancy . J Reprod Fertil Suppl 1999 ; 54 : 329 – 339 . Google Scholar PubMed 18. Garrett JE , Geisert RD , Zavy MT , Morgan GL . Evidence for maternal regulation of early conceptus growth and development in beef cattle . Reproduction 1988 ; 84 : 437 – 446 . Google Scholar CrossRef Search ADS 19. Mann GE , Lamming GE . Relationship between maternal endocrine environment, early embryo development and inhibition of the luteolytic mechanism in cows . Reproduction 2001 ; 121 : 175 – 180 . Google Scholar CrossRef Search ADS PubMed 20. Satterfield MC , Bazer FW , Spencer TE . Progesterone regulation of preimplantation conceptus growth and galectin 15 (LGALS15) in the ovine uterus . Biol Reprod 2006 ; 75 : 289 – 296 . Google Scholar CrossRef Search ADS PubMed 21. Carter F , Forde N , Duffy P , Wade M , Fair T , Crowe MA , Evans AC , Kenny DA , Roche JF , Lonergan P . Effect of increasing progesterone concentration from Day 3 of pregnancy on subsequent embryo survival and development in beef heifers . Reprod Fertil Dev 2008 ; 20 : 368 – 375 . Google Scholar CrossRef Search ADS PubMed 22. Forde N , Carter F , Fair T , Crowe MA , Evans AC , Spencer TE , Bazer FW , McBride R , Boland MP , O’Gaora P , Lonergan P , Roche JF . Progesterone-regulated changes in endometrial gene expression contribute to advanced conceptus development in cattle . Biol Reprod 2009 ; 81 : 784 – 794 . Google Scholar CrossRef Search ADS PubMed 23. Mesquita FS , Pugliesi G , Scolari SC , Franca MR , Ramos RS , Oliveira M , Papa PC , Bressan FF , Meirelles FV , Silva LA , Nogueira GP , Membrive CM et al. Manipulation of the periovulatory sex steroidal milieu affects endometrial but not luteal gene expression in early diestrus Nelore cows . Theriogenology 2014 ; 81 : 861 – 869 . Google Scholar CrossRef Search ADS PubMed 24. Dorniak P , Welsh TH Jr , Bazer FW , Spencer TE . Cortisol and interferon tau regulation of endometrial function and conceptus development in female sheep . Endocrinology 2013 ; 154 : 931 – 941 . Google Scholar CrossRef Search ADS PubMed 25. Brooks K , Burns G , Spencer TE . Conceptus elongation in ruminants: roles of progesterone, prostaglandin, interferon tau and cortisol . J Anim Sci Biotechnol 2014 ; 5 : 53 . Google Scholar CrossRef Search ADS PubMed 26. Bazer FW , Wu G , Spencer TE , Johnson GA , Burghardt RC , Bayless K . Novel pathways for implantation and establishment and maintenance of pregnancy in mammals . Mol Hum Reprod 2010 ; 16 : 135 – 152 . Google Scholar CrossRef Search ADS PubMed 27. Forde N , Lonergan P . Transcriptomic Analysis of the bovine endometrium: what is required to establish uterine receptivity to implantation in cattle? J Reprod Dev 2012 ; 58 : 189 – 195 . Google Scholar CrossRef Search ADS PubMed 28. Charpigny G , Reinaud P , Tamby JP , Creminon C , Martal J , Maclouf J , Guillomot M . Expression of cyclooxygenase-1 and -2 in ovine endometrium during the estrous cycle and early pregnancy . Endocrinology 1997 ; 138 : 2163 – 2171 . Google Scholar CrossRef Search ADS PubMed 29. Simmons RM , Satterfield MC , Welsh TH Jr , Bazer FW , Spencer TE . HSD11B1, HSD11B2, PTGS2, and NR3C1 expression in the peri-implantation ovine uterus: effects of pregnancy, progesterone, and interferon tau . Biol Reprod 2010 ; 82 : 35 – 43 . Google Scholar CrossRef Search ADS PubMed 30. Wathes DC , Lamming GE . The oxytocin receptor, luteolysis and the maintenance of pregnancy . J Reprod Fertil Suppl 1995 ; 49 : 53 – 67 . Google Scholar PubMed 31. Ulbrich SE , Schulke K , Groebner AE , Reichenbach HD , Angioni C , Geisslinger G , Meyer HH . Quantitative characterization of prostaglandins in the uterus of early pregnant cattle . Reproduction 2009 ; 138 : 371 – 382 . Google Scholar CrossRef Search ADS PubMed 32. Dorniak P , Bazer FW , Spencer TE . Prostaglandins regulate conceptus elongation and mediate effects of interferon tau on the ovine uterine endometrium . Biol Reprod 2011 ; 84 : 1119 – 1127 . Google Scholar CrossRef Search ADS PubMed 33. Charpigny G , Reinaud P , Tamby JP , Creminon C , Guillomot M . Cyclooxygenase-2 unlike cyclooxygenase-1 is highly expressed in ovine embryos during the implantation period . Biol Reprod 1997 ; 57 : 1032 – 1040 . Google Scholar CrossRef Search ADS PubMed 34. Lewis GS , Waterman RA . Effects of endometrium on metabolismof arachidonic acid by bovine blastocysts . Prostaglandins 1983 ; 25 : 881 – 889 . Google Scholar CrossRef Search ADS PubMed 35. Lewis GS , Waterman RA . Metabolism of arachidonic acid in vitro by ovine conceptuses recovered during early pregnancy . Prostaglandins 1985 ; 30 : 263 – 283 . Google Scholar CrossRef Search ADS PubMed 36. Bartol FF , Thatcher WW , Bazer FW , Kimball FA , Chenault JR , Wilcox CJ , Roberts RM . Effects of the estrous cycle and early pregnancy on bovine uterine, luteal, and follicular responses . Biol Reprod 1981 ; 25 : 759 – 776 . Google Scholar CrossRef Search ADS PubMed 37. Schallenberger E , Schams D , Meyer HH . Sequences of pituitary, ovarian and uterine hormone secretion during the first 5 weeks of pregnancy in dairy cattle . J Reprod Fertil Suppl 1989 ; 37 : 277 – 286 . Google Scholar PubMed 38. Arosh JA , Banu SK , Kimmins S , Chapdelaine P , Maclaren LA , Fortier MA . Effect of interferon-tau on prostaglandin biosynthesis, transport, and signaling at the time of maternal recognition of pregnancy in cattle: evidence of polycrine actions of prostaglandin E2 . Endocrinology 2004 ; 145 : 5280 – 5293 . Google Scholar CrossRef Search ADS PubMed 39. Emond V , MacLaren LA , Kimmins S , Arosh JA , Fortier MA , Lambert RD . Expression of cyclooxygenase-2 and granulocyte-macrophage colony-stimulating factor in the endometrial epithelium of the cow is up-regulated during early pregnancy and in response to intrauterine infusions of interferon-tau . Biol Reprod 2004 ; 70 : 54 – 64 . Google Scholar CrossRef Search ADS PubMed 40. Krishnaswamy N , Chapdelaine P , Tremblay JP , Fortier MA . Development and characterization of a simian virus 40 immortalized bovine endometrial stromal cell line . Endocrinology 2009 ; 150 : 485 – 491 . Google Scholar CrossRef Search ADS PubMed 41. Mattos R , Staples CR , Williams J , Amorocho A , McGuire MA , Thatcher WW . Uterine, ovarian, and production responses of lactating dairy cows to increasing dietary concentrations of menhaden fish meal . J Dairy Sci 2002 ; 85 : 755 – 764 . Google Scholar CrossRef Search ADS PubMed 42. Needleman P , Raz A , Minkes MS , Ferrendelli JA , Sprecher H . Triene prostaglandins: Prostacyclin and thromboxane biosynthesis and unique biological properties . Proc Natl Acad Sci USA 1979 ; 76 : 944 – 948 . Google Scholar CrossRef Search ADS PubMed 43. Ringbom T , Huss U , Stenholm A , Flock S , Skattebol L , Perera P , Bohlin L . COX-2 inhibitory effects of naturally occurring and modified fatty acids . J Nat Prod 2001 ; 64 : 745 – 749 . Google Scholar CrossRef Search ADS PubMed 44. Desvergne B , Wahli W . Peroxisome proliferator-activated receptors: nuclear control of metabolism . Endocr Rev 1999 ; 20 : 649 – 688 . Google Scholar PubMed 45. Calder PC , Yaqoob P . Understanding omega-3 polyunsaturated fatty acids . Postgrad Med 2009 ; 121 : 148 – 157 . Google Scholar CrossRef Search ADS PubMed 46. Lim H , Gupta RA , Ma WG , Paria BC , Moller DE , Morrow JD , DuBois RN , Trzaskos JM , Dey SK . Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta . Genes Dev 1999 ; 13 : 1561 – 1574 . Google Scholar CrossRef Search ADS PubMed 47. Waters SM , Kelly JP , O’Boyle P , Moloney AP , Kenny DA . Effect of level and duration of dietary n-3 polyunsaturated fatty acid supplementation on the transcriptional regulation of Delta9-desaturase in muscle of beef cattle . J Anim Sci 2009 ; 87 : 244 – 252 . Google Scholar CrossRef Search ADS PubMed 48. Berger J , Moller DE . The mechanisms of action of PPARs . Annu Rev Med 2002 ; 53 : 409 – 435 . Google Scholar CrossRef Search ADS PubMed 49. Waters SM , Coyne GS , Kenny DA , MacHugh DE , Morris DG . Dietary n-3 polyunsaturated fatty acid supplementation alters the expression of genes involved in the control of fertility in the bovine uterine endometrium . Physiol Genomics 2012 ; 44 : 878 – 888 . Google Scholar CrossRef Search ADS PubMed 50. Brooks K , Spencer TE . Biological roles of interferon tau (IFNT) and type I IFN receptors in elongation of the ovine conceptus . Biol Reprod 2015 ; 92 : 47 . Google Scholar PubMed 51. Mattos R , Staples CR , Arteche A , Wiltbank MC , Diaz FJ , Jenkins TC , Thatcher WW . The effects of feeding fish oil on uterine secretion of PGF2alpha, milk composition, and metabolic status of periparturient holstein cows . J Dairy Sci 2004 ; 87 : 921 – 932 . Google Scholar CrossRef Search ADS PubMed 52. Filley SJ , Turner HA , Stormshak F . Plasma fatty acids, prostaglandin F-2alpha metabolite, and reproductive response in postpartum heifers fed rumen bypass fat . J Anim Sci 2000 ; 78 : 139 – 144 . Google Scholar CrossRef Search ADS PubMed 53. Petit HV , Germiquet C , Lebel D . Effect of feeding whole, unprocessed sunflower seeds and flaxseed on milk production, milk composition, and prostaglandin secretion in dairy cows . J Dairy Sci 2004 ; 87 : 3889 – 3898 . Google Scholar CrossRef Search ADS PubMed 54. Childs S , Carter F , Lynch CO , Sreenan JM , Lonergan P , Hennessy AA , Kenny DA . Embryo yield and quality following dietary supplementation of beef heifers with n-3 polyunsaturated fatty acids (PUFA) . Theriogenology 2008 ; 70 : 992 – 1003 . Google Scholar CrossRef Search ADS PubMed 55. Childs S , Hennessy AA , Sreenan JM , Wathes DC , Cheng Z , Stanton C , Diskin MG , Kenny DA . Effect of level of dietary n-3 polyunsaturated fatty acid supplementation on systemic and tissue fatty acid concentrations and on selected reproductive variables in cattle . Theriogenology 2008 ; 70 : 595 – 611 . Google Scholar CrossRef Search ADS PubMed 56. Fuentes MC , Calsamiglia S , Sanchez C , Gonzalez A , Newbold JR , Santos JEP , Rodriguez-Alcala LM , Fontecha J . Effect of extruded linseed on productive and reproductive performance of lactating dairy cows . Livestock Sci 2008 ; 113 : 144 – 154 . Google Scholar CrossRef Search ADS 57. Wolf C , Ulbrich SE , Kreuzer M , Berard J , Giller K . Differential partitioning of rumen-protected n –3 and n –6 fatty acids into muscles with different metabolism . Meat Sci 2018 ; 137 : 106 – 113 . Google Scholar CrossRef Search ADS PubMed 58. Firl N , Kienberger H , Hauser T , Rychlik M . Determination of the fatty acid profile of neutral lipids, free fatty acids and phospholipids in human plasma . Clin Chem Lab Med 2013 ; 51 : 799 – 810 . Google Scholar CrossRef Search ADS PubMed 59. Giesbertz P , Padberg I , Rein D , Ecker J , Hofle A , Spanier B , Daniel H . Metabolite profiling in plasma and tissues of ob/ob and db/db mice identifies novel markers of obesity and type 2 diabetes . Diabetologia 2015 ; 58 : 2133 – 2143 . Google Scholar CrossRef Search ADS PubMed 60. Pfaffl MW , Tichopad A , Prgomet C , Neuvians TP . Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper – Excel-based tool using pair-wise correlations . Biotechnol Lett 2004 ; 26 : 509 – 515 . Google Scholar CrossRef Search ADS PubMed 61. Ruhmann B , Giller K , Hankele A , Ulbrich SE , Schmicke M . Interferon-tau induced gene expression in hepatocytes during early pregnancy in Angus heifers . Reprod Domest Anim 2017 ; 52 : 45 – 45 . Google Scholar CrossRef Search ADS PubMed 62. Grebenstein N , Frank J . Rapid baseline-separation of all eight tocopherols and tocotrienols by reversed-phase liquid-chromatography with a solid-core pentafluorophenyl column and their sensitive quantification in plasma and liver . J Chromatogr A 2012 ; 1243 : 39 – 46 . Google Scholar CrossRef Search ADS PubMed 63. Hue I , Degrelle SA , Campion E , Renard J-P . Gene expression in elongating and gastrulating embryos from ruminants . Soc Reprod Fertil Suppl 2007 ; 64 : 13 . Google Scholar PubMed 64. Degrelle SA , Jaffrezic F , Campion E , Le Cao KA , Le Bourhis D , Richard C , Rodde N , Fleurot R , Everts RE , Lecardonnel J , Heyman Y , Vignon X et al. Uncoupled embryonic and extra-embryonic tissues compromise blastocyst development after somatic cell nuclear transfer . PLoS One 2012 ; 7 : e38309 . Google Scholar CrossRef Search ADS PubMed 65. Degrelle SA , Campion E , Cabau C , Piumi F , Reinaud P , Richard C , Renard JP , Hue I . Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts . Dev Biol 2005 ; 288 : 448 – 460 . Google Scholar CrossRef Search ADS PubMed 66. Barnwell CV , Farin PW , Ashwell CM , Farmer WT , Galphin SP , Farin CE . Differences in mRNA populations of short and long bovine conceptuses on Day 15 of gestation . Mol Reprod Dev 2016 ; 83 : 424 – 441 . Google Scholar CrossRef Search ADS PubMed 67. Bazer FW , Song GH , Thatcher WW . Roles of conceptus secretory proteins in establishment and maintenance of pregnancy in ruminants . Asian Australas J Anim Sci 2012 ; 25 : 1 – 16 . Google Scholar CrossRef Search ADS PubMed 68. Hue I . Determinant molecular markers for peri-gastrulating bovine embryo development . Reprod Fertil Dev 2016 ; 28 : 51 – 65 . Google Scholar CrossRef Search ADS PubMed 69. Staples RE , Hansel W , Mcentee K . Luteal function as related to pituitary and ovarian cytology and embryo development in the bovine . J Dairy Sci 1961 ; 44 : 2049 – 2057 . Google Scholar CrossRef Search ADS 70. Betteridge KJ , Eaglesome MD , Randall GC , Mitchell D . Collection, description and transfer of embryos from cattle 10-16 days after oestrus . Reproduction 1980 ; 59 : 205 – 216 . Google Scholar CrossRef Search ADS 71. Burns PD , Engle TE , Harris MA , Enns RM , Whittier JC . Effect of fish meal supplementation on plasma and endometrial fatty acid composition in nonlactating beef cows . J Anim Sci 2003 ; 81 : 2840 – 2846 . Google Scholar CrossRef Search ADS PubMed 72. White NR , Burns PD , Cheatham RD , Romero RM , Nozykowski JP , Bruemmer JE , Engle TE . Fish meal supplementation increases bovine plasma and luteal tissue omega-3 fatty acid composition . J Anim Sci 2012 ; 90 : 771 – 778 . Google Scholar CrossRef Search ADS PubMed 73. Moussavi ARH , Gilbert RO , Overton TR , Bauman DE , Butler WR . Effects of feeding fish meal and n-3 fatty acids on ovarian and uterine responses in early lactating dairy cows . J Dairy Sci 2007 ; 90 : 145 – 154 . Google Scholar CrossRef Search ADS PubMed 74. Mattos R , Guzeloglu A , Badinga L , Staples CR , Thatcher WW . Polyunsaturated fatty acids and bovine interferon-tau modify phorbol ester-induced secretion of prostaglandin f2 alpha and expression of prostaglandin endoperoxide synthase-2 and phospholipase-a2 in bovine endometrial cells . Biol Reprod 2003 ; 69 : 780 – 787 . Google Scholar CrossRef Search ADS PubMed 75. Levine L , Worth N . Eicosapentaenoic acid: Its effects on arachidonic acid metabolism by cells in culture . J Allergy Clin Immunol 1984 ; 74 : 430 – 436 . Google Scholar CrossRef Search ADS PubMed 76. Achard F , Gilbert M , Benistant C , Ben Slama S , DeWitt DL , Smith WL , Lagarde M . Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1 expression in bovine aortic endothelial cells . Biochem Biophys Res Commun 1997 ; 241 : 513 – 518 . Google Scholar CrossRef Search ADS PubMed 77. Caldari-Torres C , Rodriguez-Sallaberry C , Greene ES , Badinga L . Differential effects of n-3 and n-6 fatty acids on prostaglandin F2 alpha production by bovine endometrial cells . J Dairy Sci 2006 ; 89 : 971 – 977 . Google Scholar CrossRef Search ADS PubMed 78. Wathes DC , Abayasekara DR , Aitken RJ . Polyunsaturated fatty acids in male and female reproduction . Biol Reprod 2007 ; 77 : 190 – 201 . Google Scholar CrossRef Search ADS PubMed 79. Mann GE , Lamming GE . The influence of progesterone during early pregnancy in cattle . Reprod Domest Anim 1999 ; 34 : 269 – 274 . Google Scholar CrossRef Search ADS 80. Rizos D , Carter F , Besenfelder U , Havlicek V , Lonergan P . Contribution of the female reproductive tract to low fertility in postpartum lactating dairy cows . J Dairy Sci 2010 ; 93 : 1022 – 1029 . Google Scholar CrossRef Search ADS PubMed 81. O’Hara L , Forde N , Kelly AK , Lonergan P . Effect of bovine blastocyst size at embryo transfer on day 7 on conceptus length on day 14: Can supplementary progesterone rescue small embryos? Theriogenology 2014 ; 81 : 1123 – 1128 . Google Scholar CrossRef Search ADS PubMed 82. Silva JCE , da Costa LL , Silva JR . Embryo yield and plasma progesterone profiles in superovulated dairy cows and heifers . Anim Reprod Sci 2002 ; 69 : 1 – 8 . Google Scholar CrossRef Search ADS PubMed 83. Ledgard AM , Berg MC , McMillan WH , Smolenski G , Peterson AJ . Effect of asynchronous transfer on bovine embryonic development and relationship with early cycle uterine proteome profiles . Reprod Fertil Dev 2012 ; 24 : 962 – 972 . Google Scholar CrossRef Search ADS PubMed 84. Randi F , Kenny DA , Lonergan P . The use of fixed time artificial insemination in beef suckler herds . Cattle Practice 2015 ; 23 : 235 – 236 . 85. Petit HV , Twagiramungu H . Conception rate and reproductive function of dairy cows fed different fat sources . Theriogenology 2006 ; 66 : 1316 – 1324 . Google Scholar CrossRef Search ADS PubMed 86. Thangavelu G , Colazo MG , Ambrose DJ , Oba M , Okine EK , Dyck MK . Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows . Theriogenology 2007 ; 68 : 949 – 957 . Google Scholar CrossRef Search ADS PubMed 87. Ambrose DJ , Kastelic JP , Corbett R , Pitney PA , Petit HV , Small JA , Zalkovic P . Lower pregnancy losses in lactating dairy cows fed a diet enriched in alpha-linolenic acid . J Dairy Sci 2006 ; 89 : 3066 – 3074 . Google Scholar CrossRef Search ADS PubMed 88. Bilby TR , Sozzi A , Lopez MM , Silvestre FT , Ealy AD , Staples CR , Thatcher WW . Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows: i. ovarian, conceptus, and growth hormone–insulin-like growth factor system responses . J Dairy Sci 2006 ; 89 : 3360 – 3374 . Google Scholar CrossRef Search ADS PubMed 89. Robinson RS , Pushpakumara PGA , Cheng Z , Peters AR , Abayasekara DRE , Wathes DC . Effects of dietary polyunsaturated fatty acids on ovarian and uterine function in lactating dairy cows . Reproduction 2002 ; 124 : 119 – 131 . Google Scholar CrossRef Search ADS PubMed 90. Hinckley T , Clark RM , Bushmich SL , Milvae RA . Long chain polyunsaturated fatty acids and bovine luteal cell function . Biol Reprod 1996 ; 55 : 445 – 449 . Google Scholar CrossRef Search ADS PubMed 91. Weems YS , Kim L , Humphreys V , Tsuda V , Blankfein R , Wong A , Weems CW . Effect of luteinizing hormone (LH), pregnancy-specific protein B (PSPB), or arachidonic acid (AA) on secretion of progesterone and prostaglandins (PG) E (PGE; PGE1 and PGE2) and F2 alpha (PGF2 alpha) by ovine corpora lutea of the estrous cycle or pregnancy in vitro . Prostaglandins Other Lipid Mediat 2007 ; 84 : 163 – 173 . Google Scholar CrossRef Search ADS PubMed 92. McCracken JA , Samuelsson B , Goding JR , Glew ME , Green K , Carlson JC , Baird DT . Prostaglandin F2-Alpha identified as a Luteolytic Hormone in Sheep . Nat New Biol 1972 ; 238 : 129 – 134 . Google Scholar CrossRef Search ADS PubMed 93. MacLaren LA , Guzeloglu A , Michel F , Thatcher WW . Peroxisome proliferator-activated receptor (PPAR) expression in cultured bovine endometrial cells and response to omega-3 fatty acid, growth hormone and agonist stimulation in relation to series 2 prostaglandin production . Domest Anim Endocrinol 2006 ; 30 : 155 – 169 . Google Scholar CrossRef Search ADS PubMed 94. Clarke SD , Jump DB . Dietary polyunsaturated fatty acid regulation of gene transcription . Annu Rev Nutr 1994 ; 14 : 83 – 98 . Google Scholar CrossRef Search ADS PubMed 95. Sponchiado M , Gomes NS , Fontes PK , Martins T , del Collado M , Pastore AD , Pugliesi G , Nogueira MFG , Binelli M . Pre-hatching embryo-dependent and -independent programming of endometrial function in cattle . PLoS One 2017 ; 12 : e0175954 . Google Scholar CrossRef Search ADS PubMed 96. Marei WFA , De Bie J , Mohey-Elsaeed O , Wydooghe E , Bols PEJ , Leroy JLMR . Alpha-linolenic acid protects the developmental capacity of bovine cumulus–oocyte complexes matured under lipotoxic conditions in vitro . Biol Reprod 2017 ; 96 : 1181 – 1196 . Google Scholar CrossRef Search ADS PubMed 97. Oseikria M , Elis S , Maillard V , Corbin E , Uzbekova S . N-3 polyunsaturated fatty acid DHA during IVM affected oocyte developmental competence in cattle . Theriogenology 2016 ; 85 : 1625 – 1634.e2 . Google Scholar CrossRef Search ADS PubMed 98. Hughes J , Kwong WY , Li DF , Salter AM , Lea RG , Sinclair KD . Effects of omega-3 and -6 polyunsaturated fatty acids on ovine follicular cell steroidogenesis, embryo development and molecular markers of fatty acid metabolism . Reproduction 2011 ; 141 : 105 – 118 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of Society for the Study of Reproduction. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Biology of Reproduction – Oxford University Press

Published: Apr 12, 2018

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Bovine embryo elongation is altered due to maternal fatty acid supplementation

Bovine embryo elongation is altered due to maternal fatty acid supplementation

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Bovine embryo elongation is altered due to maternal fatty acid supplementation

Bovine embryo elongation is altered due to maternal fatty acid supplementation

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