We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared (FTIR) metabolomics to predict pregnancy outcome. Individually cultured, in vitro-produced (IVP) blastocysts were transferred to recipients as fresh and vitrified-warmed. Spent CM and plasma samples were evaluated using FTIR. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the receiver operator characteristic curve (AUC). Within all IVP fresh embryos (birth rate = 52%), high AUC were obtained at birth, especially with expanded blastocysts (CM: 0.80 ± 0.053; plasma: 0.89 ± 0.034). The AUC of vitrified IVP embryos (birth rate = 31%) were 0.607 ± 0.038 (CM, expanded blastocysts) and 0.672 ± 0.023 (plasma, all stages). Recipient plasma generally predicted pregnancy outcome better than did embryo CM. Embryos and recipients with improved pregnancy viability were identified, which could increase the economic benefit to the breeding industry.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (France AUC = 0.751 ± 0.039, Spain AUC = 0.718 ± 0.024), while cumulative data decreased the AUC (AUC = 0.604  ±  0.029). Predictions for CM at birth were lower than Day 60 pregnancy. Predictions with plasma at birth improved cumulative over individual results (Day 0: France AUC = 0.690 ± 0.044; Spain AUC < 0.55; cumulative AUC = 0.747 ± 0.032). Plasma generally predicted pregnancy and birth better than CM. These first results show that FTIR metabolomics could allow the identification of embryos and recipients with improved pregnancy viability, which may contribute to increasing the efficiency of selection schemes based on ET.

No abstract

No abstract

High hydrostatic pressure (HHP) treatment of immature porcine oocytes improves embryo development rates and cell numbers (Pribenszky et al. 2008 Anim. Reprod. Sci. 106, 200–207). However, it is unknown if similar effects can be obtained with bovine oocytes and how HHP affects cryopreservation of the developed blastocysts. In this work, we analyzed the effect of an HHP treatment (Cryo-Innovation Ltd., Budapest, Hungary) on bovine cumulus–oocyte complex (COC) as determined by their developmental ability and embryo quality. Immature COC were submitted to a pressure treatment (200 bar, 1 h at 37°C; HHP group; n = 643) in HEPES-buffered TCM199. Simultaneously, a group of COC was held at 37°C for 1 h (T group; n = 304) in HEPES-buffered TCM199, while other COC were untreated (n = 1182). After in vitro maturation, COC were fertilized in vitro (IVF) and cultured in modified SOF + 6 g L–1 BSA (Holm et al. 1999 Theriogenology 52, 683–700), and embryo development was recorded (5 replicates). Day 7 and 8 excellent- and good-quality embryos were selected for vitrification (cryologic vitrification method; Trigal et al. 2012 Theriogenology 10.1016/j.theriogenology.2012.06.018). After warming, vitrified blastocysts were cultured in modified SOF + 6 g L–1 BSA + 10% FCS for 48 h (3 replicates). Those blastocysts hatching after warming (at 24 and 48 h) were fixed and stained for differential cell counts. Data were analyzed by ANOVA and REGWQ test and are presented as least squares means ± standard error. The HHP-treated oocytes showed increased development rates on Day 3 (Day 3 ≥5-cell embryos: 64.5 ± 2.9a, 53.4 ± 3.9b, 56.7 ± 2.2b for HHP, T, and untreated groups, respectively; a v. b: P < 0.05); however, D8 blastocyst rates were not affected by the pressure treatment (28.5 ± 1.6, 26.4 ± 2.2, and 27.8 ± 1.3 for HHP, T, and untreated groups, respectively). Treatment did not affect survival rates to vitrification (2-h re-expansion rates: 100 ± 6.7, 100 ± 6.7, and 95.4 ± 6.7; 48-h hatching rates: 58.1 ± 9.4, 71.2 ± 9.4, and 62.3 ± 9.4, for HHP, T, and untreated, respectively). Embryos that hatched after warming did not differ in inner cell mass and trophectoderm cell counts (inner cell mass: 15.0 ± 1.9, 12.7 ± 3.0, and 13.0 ± 2.0; trophectoderm: 133.6 ± 8.4, 137.3 ± 12.8, and 138.4 ± 8.6 for HHP, T, and untreated groups, respectively; P > 0.05). Complementary studies are needed to analyze the effects of a sublethal stress in bovine oocytes on the subsequent embryo production and quality. Species-specific mechanisms could underlie the differences in results obtained in bovine and porcine. RTA2011-00090 (FEDER-INIA). Muñoz, Trigal, and Correia are sponsored by RYC08-03454, Cajastur, and FPU2009-5265, respectively.

Multiple reaction monitoring (MRM) allows targeted quantitative proteomics with a wide dynamic range and limit of detection down to femtomoles. We used MRM to study uterine growth factors (GF) presumed to promote embryonic development. A validated experimental model was used to recover uterine fluid (UF) and analyse GF expression in the presence or absence of embryos. Briefly, Day-6 in vitro-produced embryos (n = 50) or vehicle (sham transfer) were transferred into the uteri of each oestrus-synchronized Holstein heifer (n = 14) during nonconsecutive cycles. Blood P4 concentrations were measured on Days 0 (oestrus), 6, and 8. On Day 8, UF was recovered from embryo and sham recipients. After retrieval, UF were centrifuged and supernatants stored at –145°C. Sham and embryo UF selected for MRM were from n = 10 animals (n = 20 samples). Uterine fluid, recovered after embryo transfer, contained on average n = 43.1 ± 5.2 total and n = 34.1 ± 3.7% viable embryos per recipient. For MRM, UF samples were concentrated, and protein was precipitated and resuspended in ammonium bicarbonate. Protein (20 μg) was reduced with DTT, trypsin-digested, and desalted. Proteotypic peptides for targeted GF were selected with MRM Pilot software (ABsciex, Farmingham, MA, USA), with 3 to 5 transitions programmed for each peptide. A control, unrelated synthetic peptide was spiked as an internal standard. The area of the larger transition for the control peptide was used to normalise the area values of each other peptide. The MRM experiments were performed on a 5500 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer (ABsciex) equipped with an Eksigent 1D+plus nanoLC chromatographic system. Data analysis was performed with Analyst 1.5.2 and MultiQuant 2.0.2 softwares (ABsciex). The area of most abundant transition for each analysed peptide was used for relative quantitation. Proteins studied were betacellulin, heparin-binding EGF-like growth factor, neuregulin, artemin, connective tissue growth factor, nerve growth factor, kit ligand, stanniocalcin-1 (STC1), early pregnancy factor (EPF), and hepatoma-derived growth factor (HDGF). Proteotypic peptides were identified in all samples for HDGF, kit ligand, STC1, and EPF (n = 1, n = 1, n = 1, and n = 3 peptides, respectively), which precluded the analysis of the remaining GF. No differences in relative abundance were detected between UF containing or not containing embryos for HDGF, kit ligand, STC1, and EPF (2.85 ± 0.6 v. 4.43 ± 0.6; 0.15 ± 0.02 v. 0.16 ± 0.02; 0.03 ± 0.00 v. 0.04 ± 0.00; and 1.20 ± 0.16 v. 1.09 ± 1.16, respectively). However, STC1 and Day 8 blood P4 were highly correlated (r = 0.71; P = 0.0004), suggesting P4 regulation of STC1. Multiple reaction monitoring-LC-MS/MS is a useful technique to identify some scarce GF in UF at different dynamic ranges. MICINN, project AGL2012-37772 and FEDER. E. C. was supported by MEC-FPU-AP2009-5265. The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception environment.

Tumor necrosis factor alpha (TNF), a pleiotropic cytokine that could be involved in early embryo-maternal interactions (Muñoz et al. 2012 J. Proteome Res. 11, 751–766), binds to receptors TNFR1 and TNFR2. The TNFR2 mediates apoptotic and survival processes (Fischer et al. 2011 Cell. Signal. 23, 161–170) and its expression is hormonally regulated (Okuda et al. 2010 Mol. Cell. Endocrinol. 330, 41–48). In this work we analyzed the expression of TNFR2 by Western blot (WB) and immunocytochemistry (ICQ) and its co-localization with TNF by ICQ in bovine embryos and endometrium. Heifers that were transferred with multiple in vitro produced (IVP) embryos (n = 3) or sham transferred (n = 3) on Day 5 to horn ipsilateral to the corpus luteum were slaughtered on Day 8. Embryos were flushed and endometrial samples were collected from caruncular and intercaruncular regions in the middle and cranial horn thirds. Endometrial samples and Day 8 IVP embryos were subjected to ICQ, and the immunostaining pattern of TNFR2 and TNF was examined by confocal microscopy. Endometrial samples were also subjected to WB. Expression of TNRF2 was quantified by densitometry (immunoblots) and blind assessment (immunostaining). Data were analyzed using the GLM procedure of SAS Version 9.2 (SAS Institute Inc., Cary, NC, USA) and REGWQ test for means. Trophectoderm (TF) cells from blastocysts and uterine epithelial and stromal cells showed TNFR2 expression. TNF and TNRF2 were predominantly co-localised in embryos and endometrial samples, although occasionally they were detected independently. The presence of embryos increased TNFR2 in the basal glandular epithelia (P ≤ 0.05). Moreover, TNFR2 was higher in the intercaruncular than in the caruncular luminal epithelium (P = 0.07). The presence of embryos did not affect TNFR2 expression between cranial and middle horn thirds. However, the TNFR2 low-molecular-weight isoform (Lmw) in the caruncles and in the middle third of the uterine horn tended to increase in the presence of embryos (P ≤ 0.1). Interestingly, TNFR2 Lmw was more abundant in the middle caruncular region than in other endometrial regions (P < 0.05). Our findings suggest that TNF can mediate embryo-maternal communication in the uterus, acting both in the embryonic and maternal sides. In addition, although implantation does not begin in ruminants until elongation is complete, early bovine embryos seem to show an ability to interact with caruncles. Project AGL2009-10059 (MICINN). M. Muñoz, A. Balseiro, B. Trigal, and E. Correia are sponsored by RYC08-03454, Contrato de Investigación para Doctores grant from INIA, Cajastur, and FPU (AP2009-5265), respectively.

Bilateral asymmetry in the cow affects ovarian function, uterine horn morphology, pregnancy, and embryonic sex. However, many aspects and molecular mechanisms of such laterality remain obscure. The objective of this work was identifying new traits of ovarian and uterine asymmetry, as based on oestrus and ovarian monitoring, P4 concentrations, early embryo development, flushing performance, and pregnancy outcomes after embryo transfer (ET). In addition, proteins identified in previous work by difference gel electrophoresis and mass spectrometry (DIGE-MS) in uterine fluid (UF) were reanalyzed in a horn-of-origin basis (n = 16 and n = 14 flushes from left and right horns, respectively; Muñoz et al. 2012 J. Proteome Res. 11, 751–766; Gómez et al. 2011 Reprod. Fert. Dev. 24, 152). Studies were performed in experimental herd and on field. Data were analyzed by Proc GLM of SAS/STAT (Version 9.2; SAS Inst. Inc., Cary, NC) and REGWQ test for means. In experimental herd, we analyzed ovarian and uterine asymmetry within animals (n = 25) monitored through different reproductive cycles (n = 109). Animals synchronised with progestagen + PGF2α were alternatively transferred with IVP embryos (n = 30–60) or vehicle (sham transfer) on Day 5 to ipsilateral horn. On Day 8, embryos and/or diluted UF were recovered by flushing with 30 or 45 mL PBS. Nonsignificant differences (P > 0.7) were obtained in ovulatory follicle diameters 48 h after PGF2α injection, onset oestrus time and recoverable total protein by flushing between animals ovulating in the left or in the right ovary. However, cows bearing the corpus luteum (CL) in the right ovary (i.e. right) had higher (26.3 ± 1.5) Day 8 P4 concentration than those showing a CL in the left ovary (i.e. left) (21.6 ± 1.8) (P = 0.03). Fluid recovery (%) was lower in the left (47.0 ± 6.3) than in the right (64.4 ± 5.0) horn when 30 mL were infused (P = 0.035); in contrast, 45 mL infused did not differ between horns (61.6 ± 4.1 v. 67.9 ± 4.1). Less total embryos were recovered from the left (14.6 ± 4.7) than the right (31.0 ± 3.7) horn (P < 0.02), although the relative proportions of viable embryos were conserved. Among 76 proteins analyzed, concentrations of VLCAD, KPYM, CFB, ALB, FGG, EZR, and ACTB were higher (P ≤ 0.05), while TWF1 and ENO1 were lower in the left horn. On field experiments (n = 184 ET in 286 synchronised animals from 39 farms; ≥3 ET per farm) confirmed on Day 7 the above differences in P4 (right: 8.3 ± 0.43 v. left: 6.1 ± 0.55; P = 0.0058). Pregnancy rates after ET did not differ between horns (51.0 ± 3.6, right v. 53.2 ± 4.7, left). However, P4 concentrations differ (P = 0.018) between pregnant and open animals in the left (15.9 ± 1.7 v. 8.3 ± 1.2) but not in the right horn (12.4 ± 1.3 v. 12.4 ± 1.2), respectively. Genital asymmetry in the cow has physical concerns (flushing and recoveries), while changes in P4 and/or proteins could operate to hold similar pregnancy rates between horns. Project AGL2009-10059 (MICINN). MM, BT and EC are sponsored by RYC08-03454, Cajastur and FPU2009-5265, respectively.