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

In cattle, vitrified/warmed (V/W) and frozen/thawed (F/T), in vitro-produced (IVP) embryos, differ in their physiology and survival from fresh embryos. In this study, we analyzed the effects of embryo cryopreservation techniques on the offspring. IVP embryos cultured with albumin and with or without 0.1% serum until Day 6, and thereafter in single culture without protein, were transferred to recipients on Day 7 as F/T, V/W, or fresh, resulting in N = 24, 14, and 13 calves, respectively. Calves were clinically examined at birth, and blood was analyzed before and after colostrum intake (Day 0), and subsequently on Day 15 and Day 30. On Day 0, calves from V/W and F/T embryos showed increased creatinine and capillary refill time (CRT) and reduced heartbeats. Calves from F/T embryos showed lower PCO2, hemoglobin, and packed cell volume than calves from V/W embryos while V/W embryos led to calves with increased Na+ levels. Colostrum effects did not differ between calves from fresh and cryopreserved embryos, indicating similar adaptive ability among calves. However, PCO2 did not decrease in calves from V/W embryos after colostrum intake. Serum in culture led to calves with affected (P < 0.05) temperature, CRT, HCO 3 - , base excess (BE), TCO2, creatinine, urea, and anion gap. On Day 15, the effects of embryo cryopreservation disappeared among calves. In contrast, Day 30 values were influenced by diarrhea appearance, mainly in calves from V/W embryos (i.e., lower values of TCO2, HCO 3 - , and BE; and increased glucose, anion gap, and lactate), although with no more clinical compromise than calves from fresh and F/T embryos. Diarrhea affected PCO2 and Na+ in all groups. Embryo cryopreservation, and/or culture, yield metabolically different calves, including effects on protein and acid-base metabolism.

Non-Invasive Identification of Sex in Cultured Bovine Embryos by UHPLC-MS/MS Metabolomics.

INTRODUCTION: Different gene expression between male and female bovine embryos leads to metabolic differences. OBJECTIVE: We used UHPLC-MS/MS to identify sex metabolite biomarkers in embryo culture medium (CM). METHODS: Embryos were produced in vitro under highly variable conditions, i.e., fertilized with 7 bulls, two breeds, and cultured with BSA or BSA + serum until Day-6. On Day-6, embryos were cultured individually for 24 h. CM of Day-7 embryos (86 female and 81 male) was collected, and Day-6 and Day-7 embryonic stages recorded. RESULTS: A study by sample subsets with fixed factors (culture, bull breed, and Day-6 and Day-7 stages) tentatively identified 31 differentially accumulated metabolites through 182 subsets. Day-6 and Day-7 stage together affected 13 and 11 metabolites respectively, while 19 metabolites were affected by one or another stage and/or day. Culture supplements and individual bull changed 19 and 15 metabolites, respectively. Single bull exerted the highest influence (20 metabolites with the significantly highest p values). Lipid (93 subsets; 11 metabolites) and amino acid (55 subsets; 13 metabolites) were the most relevant classes for sex identification. CONCLUSIONS: Single biomarker led to inefficient sex diagnosis, while metabolite combinations accurately identified sex. Our study is a first in non-invasive sex identification in cattle by overcoming factors that induce metabolic variation.

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

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.

Metabolites Secreted by Bovine Embryos In Vitro Predict Pregnancies That the Recipient Plasma Metabolome Cannot, and Vice Versa.

This work describes the use of mass spectrometry-based metabolomics as a non-invasive approach to accurately predict birth prior to embryo transfer (ET) starting from embryo culture media and plasma recipient. Metabolomics was used here as a predictive platform. Day-6 in vitro produced embryos developed singly in modified synthetic oviduct fluid culture medium (CM) drops for 24 h were vitrified as Day-7 blastocysts and transferred to recipients. Day-0 and Day-7 recipient plasma (N = 36 × 2) and CM (N = 36) were analyzed by gas chromatography coupled to the quadrupole time of flight mass spectrometry (GC-qTOF). Metabolites quantified in CM and plasma were analyzed as a function to predict pregnancy at Day-40, Day-62, and birth (univariate and multivariate statistics). Subsequently, a Boolean matrix (F1 score) was constructed with metabolite pairs (one from the embryo, and one from the recipient) to combine the predictive power of embryos and recipients. Validation was performed in independent cohorts of ETs analyzed. Embryos that did not reach birth released more stearic acid, capric acid, palmitic acid, and glyceryl monostearate in CM (i.e., (p < 0.05, FDR < 0.05, Receiver Operator Characteristic-area under curve (ROC-AUC) > 0.669)). Within Holstein recipients, hydrocinnamic acid, alanine, and lysine predicted birth (ROC-AUC > 0.778). Asturiana de los Valles recipients that reached birth showed lower concentrations of 6-methyl-5-hepten-2-one, stearic acid, palmitic acid, and hippuric acid (ROC-AUC > 0.832). Embryonal capric acid and glyceryl-monostearate formed F1 scores generally >0.900, with metabolites found both to differ (e.g., hippuric acid, hydrocinnamic acid) or not (e.g., heptadecanoic acid, citric acid) with pregnancy in plasmas, as hypothesized. Efficient lipid metabolism in the embryo and the recipient can allow pregnancy to proceed. Changes in phenolics from plasma suggest that microbiota and liver metabolism influence the pregnancy establishment in cattle.

Metabolomic identification of pregnancy-specific biomarkers in blood plasma of BOS TAURUS beef cattle after transfer of in vitro produced embryos.

Blood biomarkers may help to predict pregnancy in recipients of in vitro produced (IVP) embryos. Using 1H nuclear magnetic resonance, we quantified 36 metabolites in the blood plasma of recipients (90% heifers, healthy, 1.95 years on average at the time of 1st embryo transfer -ET-) collected at Day-0 (estrus) and Day-7 (before ET time). First, IVP embryos were transferred to Asturiana de los Valles recipients as fresh (F) (N = 26) and vitrified/warmed (V/W) (N = 48) (discovery groups). Only at estrus, we discovered 4, 11, and 5 (F-ET), and 2, 2, and 4 (V/W-ET) metabolites that predicted pregnancy on Day-40, Day-62 and calving time, respectively (ROC-AUC > 0.700; P < .05). Thereafter, validation was performed in independent samples (N = 67 F and N = 63 V/W) of three cattle breeds by an index of overall classification accuracy (OCA>0.650, P < .05). The numbers of candidate biomarkers validated were 2, 9 and 1 (F-ET) and 2, 2, and 3 (V/W-ET) on Day 40, Day-62 and calving time. Relevant metabolites were validated at the three (2-Oxoglutaric acid (F-ET), and 2-Hydroxybutyric acid and Dimethylamine (V/W-ET)) and two pregnancy endpoints (Ketoleucine (F-ET); Day-40 and Day-62) analysed. Fatty acid degradation and oxidative metabolism were enriched in pregnant recipients. The candidate biomarkers identified can improve embryo-recipient selection. SIGNIFICANCE: We identified, for the first time, reliable pregnancy and birth candidate metabolite biomarkers for fresh and vitrified IVP embryos in blood of beef cattle recipients. Our findings can help to improve embryo-recipient selection, which is usually carried out in a way that females that will not become pregnant are not well differentiated.

Blood Plasma Metabolomics Predicts Pregnancy in Holstein Cattle Transferred with Fresh and Vitrified/Warmed Embryos Produced in Vitro.

Metabolomics may identify biomarkers in blood that differentiate pregnant from open embryo recipients. Fresh and vitrified/warmed, in vitro-produced embryos were transferred to Holstein recipients (discovery group). Recipient blood plasma collected on Day-0 (estrus) and Day-7 (before embryo transfer) were analyzed by nuclear magnetic resonance (N = 36 metabolites quantified). Metabolites whose concentrations differed between open and pregnant recipients were analyzed [(P < 0.05); false discovery rate (FDR) (P < 0.05)]. Biomarkers were identified in Day-7 plasma (receiver operator characteristic-area under curve (ROC-AUC) > 0.650; t-test P < 0.05; random forests, mean decrease accuracy) and cross-validated in independent Holstein, beef, and crossbred recipients (overall classification accuracy -OCA-; P < 0.05). Recipients with fresh embryos showed N = 6 biomarkers consistently on Day-40, Day-62, and at birth. Recipients with vitrified embryos showed N = 5 biomarkers on Day-40 and Day-62 but only one biomarker at birth. The most predictive biomarkers identified at birth within fresh embryos were oxoglutaric acid (ROC-AUC = 0.709; OCA = 0.812) and ornithine (ROC-AUC = 0.731; OCA = 0.727), while l-glycine was identified in vitrified embryos (ROC-AUC = 0.796; OCA = 0.667) together with other predictive biomarkers not identified at birth (Day-62: l-glutamine ROC-AUC = 0.757; OCA = 0.767) and l-lysine (Day-62: ROC-AUC = 0.680; OCA = 0.767). Pathway enrichment analysis distinguished between pregnant recipients for fresh (enriched energy oxidative metabolism from fat) and vitrified (lower lipid metabolism) embryos. Metabolomics can select individuals that will become pregnant in a defined cycle.