Nuclear magnetic resonance analysis of female and male pre-hatching embryo metabolites at the embryo-maternal interface.

INTRODUCTION: Bovine female and male embryos differentially release metabolites with signalling effects to culture media. However, it is unknown if the embryo-maternal interface (EMI) metabolome is modified by embryonic sex. OBJECTIVE: To analyse using a combination of 1H NMR and a co-culture of endometrial cells the EMI. RESULTS: Twenty-six metabolites were identified and quantified in the EMI, nine metabolites reflected the sex of the embryo rather than their presence. CONCLUSIONS: 1H NMR is sensitive enough to perform quantitative analysis of sex-induced differences in the EMI. These results may help to understand the embryo-maternal dialogue on the basis of embryonic sex.

Short- and long-term outcomes of the absence of protein during bovine blastocyst formation in vitro.

In cattle, individual in vitro embryo culture after Day 6 benefits development, allowing non-invasive analysis of culture medium. However, undefined supplements in culture reduce analytical reliability. In this study we assayed the short- and long-term performance of embryos after bovine serum albumin removal over a 24-h period in individual culture. The absence of protein decreased embryo development and cell counts in the inner cell mass without affecting blastocyst sex ratio. However, the absence of protein produced embryos with an improved tendency to survive vitrification after 24h in culture (P=0.07). After transfer to recipients, birth rates of embryos that had been cultured with protein tended to decrease (P<0.06) mostly as a result of a higher number of miscarriages (P<0.013), reflecting lower viability. Birthweight, gestation length, height and thorax circumference did not differ between embryos cultured with or without protein. In fresh blastocysts cultured without protein, gene expression analysis showed higher abundance (P<0.05) of insulin-like growth factor 2 receptor (IGF2R; imprinting) and activating transcription factor 4 (ATF4) and DNA-damage-inducible transcript 3 (DDIT3; endoplasmic reticulum stress) transcripts, with DNA methyltransferase 3A (DNMT3A; imprinting) tending to increase (P=0.062). However, in hatched blastocysts that survived cryopreservation, glucose-6-phosphate dehydrogenase (G6PD) was overexpressed in embryos cultured without protein (P<0.01). The absence of protein results in fewer blastocysts but improved long-term viability after cryopreservation.

Metabolomic Profiling of Bos taurus Beef, Dairy, and Crossbred Cattle: A Between-Breeds Meta-Analysis.

Cattle breeds may differ substantially in their metabolism. However, the metabolomes of dairy and beef cattle are not well-known. Knowledge of breed-specific metabolic features is essential for biomarker identification and to adopt specific nutritional strategies. The muscle hypertrophy (mh), a beef cattle phenotype present in Asturiana de los Valles (AV) but absent in Asturiana de la Montaña (AM) and Holsteins, may underlie such differences. We compared the plasma metabolomes of Holstein, AV, AM, and crossbred cattle recipients selected for meta-analysis within an embryo transfer (ET) program. Blood samples were collected on day 0 (oestrus) and day 7 (prior to ET) (N = 234 samples × 2 days). Nuclear magnetic resonance quantified N = 36 metabolites in plasma, and more metabolic differences between breeds were found on day 0 (N = 19 regulated metabolites) than on day 7 (N = 5). AV and AM largely differed from Holstein cattle (N = 55 and 35 enriched metabolic pathways, respectively); however, AV and AM differed in N = 6 enriched pathways. Metabolic activity was higher in AV than in Holstein cattle, as explained in part by the mh phenotype. The metabolomic characterization of breeds facilitates biomarker research and helps to define the healthy ranges of metabolite concentrations.

Low serum concentration in bovine embryo culture enhances early blastocyst rates on Day-6 with quality traits in the expanded blastocyst stage similar to BSA-cultured embryos.

In bovine, single in vitro embryo culture in protein-free medium from Day-6 to Day-7 leads to expanded blastocyst (XB) with improved pregnancy and birth rates after cryopreservation. Under these conditions, early blastocysts (EB) progress to the XB stage at higher rates than morulae (M). However, embryo production with BSA in culture prior to Day-6 leads to low EB rates. We investigated whether a very low FCS concentration (0.1%) in culture from Day-1 to Day-6 would improve EB rates and, subsequently, increase XB rates on Day-7 after single culture in protein-free medium. The quality of embryos produced was evaluated in terms of survival to cryopreservation, apoptosis percentage, lipid accumulation and transfer to recipients. On Day-6, EB rates from embryos cultured with FCS were higher than with BSA (P=0.022). On Day-7, XB rates were higher in embryos from Day-6 EB than from Day-6M, both with and without FCS (P<0.005). After vitrification/warming of Day-7 XB, 100% embryos survived at 24h in all treatments, and total cell number and apoptosis percentage were not affected by the presence of FCS or embryonic stage on Day-6. Cryopreserved and fresh embryos produced with FCS until Day-6, and then deprived of protein and cultured individually, led to pregnancies after ET. In conclusion, minute FCS concentration improves EB rates on Day-6 leading, after one-day single culture without protein, to more XBs. The quality of XB produced with FCS compares well with XB produced with BSA in terms of apoptosis, lipid accumulation and pregnancy.

Non-invasive metabolomics for improved determination of embryonic sex markers in chemically defined culture medium.

Metabolic differences between early male and female embryos can be reflected in culture medium (CM). We used a single bovine embryo culture step (24h) supporting improved birth rates under chemically defined conditions (CDC) to investigate biomarker detection of embryonic sex in contrast to classical BSA-containing medium. In vitro matured slaughterhouse oocytes were fertilized in vitro with a single bull. Embryos were initially cultured in synthetic oviduct fluid with BSA. On day-6, morulae were cultured individually in droplets with (BSA) or without protein (CDC). On day-7, expanded blastocysts were sexed (amelogenin gene amplification) and CM was stored at -145°C until metabolomic analysis by UHPLC-TOF MS. N=10 embryos per group (i.e. male-protein; female-protein; male-non-protein; female-non-protein) were produced. Statistical analysis revealed N=6 metabolites with different concentrations in CM, N=5 in male embryos (methionine, tryptophan, N-stearoyl-valine, biotin and pipecolic acid), N=1 in female embryos (threonine) (P<0.05 in BSA; P<10-7 in CDC). Only the clear threshold between males and females in CDC allowed correct classification of 100% males and 91% females within 5 out of 6 biomarkers (one female outlier showing the male biomarker profile). The use of CDC represents a critical aspect in the efficient detection of embryonic sex biomarkers by metabolomics.