The Metabolic Signature of In Vitro Produced Bovine Embryos Helps Predict Pregnancy and Birth after Embryo Transfer

Abstract

In vitro produced (IVP) embryos show large metabolic variability induced by breed, culture conditions, embryonic stage and sex and gamete donors. We hypothesized that the birth potential could be accurately predicted by UHPLC-MS/MS in culture medium (CM) with the discrimination of factors inducing metabolic variation. Day-6 embryos were developed in single CM (modified synthetic oviduct fluid) for 24 h and transferred to recipients as fresh (28 ETs) or frozen/thawed (58 ETs) Day-7 blastocysts. Variability was induced with seven bulls, slaughterhouse oocyte donors, culture conditions (serum + Bovine Serum Albumin [BSA] or BSA alone) prior to single culture embryonic stage records (Day-6: morula, early blastocyst, blastocyst; Day-7: expanding blastocyst; fully expanded blastocysts) and cryopreservation. Retained metabolite signals (6111) were analyzed as a function of pregnancy at Day-40, Day-62 and birth in a combinatorial block study with all fixed factors. We identified 34 accumulated metabolites through 511 blocks, 198 for birth, 166 for Day-62 and 147 for Day-40. The relative abundance of metabolites was higher within blocks from non-pregnant (460) than from pregnant (51) embryos. Taxonomy classified lipids (12 fatty acids and derivatives; 224 blocks), amino acids (12) and derivatives (3) (186 blocks), benzenoids (4; 58 blocks), tri-carboxylic acids (2; 41 blocks) and 5-Hydroxy-l-tryptophan (2 blocks). Some metabolites were effective as single biomarkers in 95 blocks (Receiver Operating Characteristic – Area Under the Curve [ROC-AUC]: 0.700–1.000). In contrast, more accurate predictions within the largest data sets were obtained with combinations of 2, 3 and 4 single metabolites in 206 blocks (ROC-AUC = 0.800–1.000). Pregnancy-prone embryos consumed more amino acids and citric acid, and depleted less lipids and cis-aconitic acid. Big metabolic differences between embryos support efficient pregnancy and birth prediction when analyzed in discriminant conditions.

Metabolic signature of spent culture media shows lipid metabolism as a determinant of pregnancy outcomes

“…It is important to note that, as pointed by our results and others (Gimeno et al, 2021; Gomez et al, 2021), lipid metabolism is an unequivocal marker of embryo's ability to establish and maintain pregnancy, and this occurs beyond the metabolization of fatty acids per se. For instance, (Gimeno et al, 2021) described that embryos with lower ability to establish pregnancy presented a more active lipid metabolism together with a higher incorporation of amino acids from culture media, such as glutamine, suggesting the use of other classes of biomolecules to maintain the energetic homeostasis during the early development.…”

“…It is important to note that, as pointed by our results and others (Gimeno et al, 2021; Gomez et al, 2021), lipid metabolism is an unequivocal marker of embryo's ability to establish and maintain pregnancy, and this occurs beyond the metabolization of fatty acids per se. For instance, (Gimeno et al, 2021) described that embryos with lower ability to establish pregnancy presented a more active lipid metabolism together with a higher incorporation of amino acids from culture media, such as glutamine, suggesting the use of other classes of biomolecules to maintain the energetic homeostasis during the early development. Indeed, the glutamine supplementation during embryo culture enhances development of IVP embryos in several species as porcine, murine and human by increasing the mitochondrial activity and diminishing the rate of apoptotic cells in the blastocysts (Chen et al, 2018; Devreker et al, 1998; Rezk et al, 2004).…”

The recipient metabolome explains the asymmetric ovarian impact on fetal sex development after embryo transfer in cattle

“…General procedures for in vitro embryo production in Spain have been described in previous studies ( Gómez et al, 2020c ; Gimeno et al, 2021 ). The following are descriptions in brief.…”

Fitness of calves born from in vitro-produced fresh and cryopreserved embryos

“…For these experimental purposes, the calves available for analysis at Day 0 were N = 13 (fresh), N = 14 (V/W), and N = 24 (F/T) (refer to Table 1 , for details). The readers are referred to our articles ( 4 , 24 , 47 ) for a complete description of oocyte collection from slaughterhouse ovaries, in vitro maturation (IVM) and in vitro fertilization (IVF), which was performed with frozen/thawed semen from single seven individual bulls [three Holstein and four Asturiana de los Valles (AV)]. Pregnancy rates from each procedure were also described ( 4 , 24 ).…”

“…Extended in vitro culture (IVC) procedures were described ( 47 ). Briefly, after IVF, presumed zygotes were loaded into the embryo culture medium (CM) which consisted of modified synthetic oviduct fluid (mSOF) with MEM non-essential amino acids (M7145), BME amino acids (B6766), citrate (0.1 μg/ml), myo-inositol (0.5 μg/ml), and Bovine serum albumin (BSA) (A3311) (6 mg/ml) with or without 0.1% (v/v) FCS (SIGMA F4135), under mineral oil at 38.7°C, 5% CO 2 , 5% O 2 , 90% N 2 , and saturated humidity.…”