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

In cattle, lateral asymmetry affects ovarian function and embryonic sex, but the underlying molecular mechanisms remain unknown. The plasma metabolome of recipients serves to predict pregnancy after embryo transfer (ET). Thus, the aim of this study was to investigate whether the plasma metabolome exhibits distinct lateral patterns according to the sex of the fetus carried by the recipient and the active ovary side (AOS), i.e., the right ovary (RO) or the left ovary (LO). We analyzed the plasma of synchronized recipients by 1H+NMR on day 0 (estrus, n = 366) and day 7 (hours prior to ET; n = 367). Thereafter, a subset of samples from recipients that calved female (n = 50) or male (n = 69) was used to test the effects of embryonic sex and laterality on pregnancy establishment. Within the RO, the sex ratio of pregnancies carried was biased toward males. Significant differences (P < 0.05) in metabolite levels were evaluated based on the day of blood sample collection (days 0, 7 and day 7/day 0 ratio) using mixed generalized models for metabolite concentration. The most striking differences in metabolite concentrations were associated with the RO, both obtained by multivariate (OPLS-DA) and univariate (mixed generalized) analyses, mainly with metabolites measured on day 0. The metabolites consistently identified through the OPLS-DA with a higher variable importance in projection score, which allowed for discrimination between male fetus- and female fetus-carrying recipients, were hippuric acid, l-phenylalanine, and propionic acid. The concentrations of hydroxyisobutyric acid, propionic acid, l-lysine, methylhistidine, and hippuric acid were lowest when male fetuses were carried, in particular when the RO acted as AOS. No pathways were significantly regulated according to the AOS. In contrast, six pathways were found enriched for calf sex in the day 0 dataset, three for day 7, and nine for day 7/day 0 ratio. However, when the AOS was the right, 20 pathways were regulated on day 0, 8 on day 7, and 13 within the day 7/day 0 ratio, most of which were related to amino acid metabolism, with phenylalanine, tyrosine, and tryptophan biosynthesis and phenylalanine metabolism pathways being identified throughout. Our study shows that certain metabolites in the recipient plasma are influenced by the AOS and can predict the likelihood of carrying male or female embryos to term, suggesting that maternal metabolism prior to or at the time of ET could favor the implantation and/or development of either male or female embryos.

This study explored how the active ovary side (AOS, i.e., left or right) and the sex of the calf carried by the recipient relate to the plasma metabolome in blood. For this purpose, we analyzed blood samples from heifers at two specific times: the day of the estrus and the day of the embryo transfer. We found significant differences in the sex ratio of pregnancies carried in the right ovary, and in the levels of certain metabolites depending on whether the active ovary was on the right or left and whether the calf was male or female. As examples, the concentrations of hydroxyisobutyric acid, propionic acid, l-lysine, methylhistidine, and hippuric acid were lowest when male calves were carried, in particular when the right ovary was active. Interestingly, the calf sex also influenced certain metabolic pathways, especially in the right AOS, several of them related to amino acid metabolism. However, no significant metabolic pathway changes were observed based solely on which ovary was active. Overall, the study suggests that the metabolism of the recipient, influenced by the AOS, might play a role in the successful implantation and development of embryos of a certain sex. This insight could potentially help to predict and improve pregnancy outcomes in cattle through embryo transfer techniques.

Alteration of the embryonic microenvironment and sex-specific responses of the preimplantation embryo related to a maternal high-fat diet in the rabbit model.

The maternal metabolic environment can be detrimental to the health of the offspring. In a previous work, we showed that maternal high-fat (HH) feeding in rabbit induced sex-dependent metabolic adaptation in the fetus and led to metabolic syndrome in adult offspring. As early development representing a critical window of susceptibility, in the present work we aimed to explore the effects of the HH diet on the oocyte, preimplantation embryo and its microenvironment. In oocytes from females on HH diet, transcriptomic analysis revealed a weak modification in the content of transcripts mainly involved in meiosis and translational control. The effect of maternal HH diet on the embryonic microenvironment was investigated by identifying the metabolite composition of uterine and embryonic fluids collected in vivo by biomicroscopy. Metabolomic analysis revealed differences in the HH uterine fluid surrounding the embryo, with increased pyruvate concentration. Within the blastocoelic fluid, metabolomic profiles showed decreased glucose and alanine concentrations. In addition, the blastocyst transcriptome showed under-expression of genes and pathways involved in lipid, glucose and amino acid transport and metabolism, most pronounced in female embryos. This work demonstrates that the maternal HH diet disrupts the in vivo composition of the embryonic microenvironment, where the presence of nutrients is increased. In contrast to this nutrient-rich environment, the embryo presents a decrease in nutrient sensing and metabolism suggesting a potential protective process. In addition, this work identifies a very early sex-specific response to the maternal HH diet, from the blastocyst stage.

Biomarker metabolite mating of viable frozen-thawed in vitro-produced bovine embryos with pregnancy-competent recipients leads to improved birth rates.

Selection of competent recipients before embryo transfer (ET) is indispensable for improving pregnancy and birth rates in cattle. However, pregnancy prediction can fail when the competence of the embryo is ignored. We hypothesized that the pregnancy potential of biomarkers could improve with information on embryonic competence. In vitro-produced embryos cultured singly for 24 h (from d 6 to 7) were transferred to d 7 synchronized recipients as fresh or after freezing and thawing. Recipient blood was collected on d 0 (estrus; n = 108) and d 7 (4-6 h before ET; n = 107) and plasma was analyzed by nuclear magnetic resonance (1H+NMR). Spent embryo culture medium (CM) was collected and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry in a subset of n = 70 samples. Concentrations of metabolites quantified in plasma (n = 35) were statistically analyzed as a function of pregnancy diagnosed on d 40, d 62 and birth. Univariate analysis with plasma metabolites consisted of a block study with controllable fixed factors (i.e., embryo cryopreservation, recipient breed, and day of blood collection; Wilcoxon test and t-test). Metabolite concentrations in recipients and embryos were independently analyzed by iterations that reclassified embryos or recipients using the support vector machine. Iterations identified some competent embryos, but mostly competent recipients that had a pregnancy incompetent partner embryo. Misclassified recipients that could be classified as competent were reanalyzed in a new iteration to improve the predictive model. After subsequent iterations, the predictive potential of recipient biomarkers was recalculated. On d 0, creatine, acetone and l-phenylalanine were the most relevant biomarkers at d 40, d 62, and birth, and on d 7, l-glutamine, l-lysine, and ornithine. Creatine was the most representative biomarker within blocks (n = 20), with a uniform distribution over pregnancy endpoints and type of embryos. Biomarkers showed higher abundance on d 7 than d 0, were more predictive for d 40 and d 62 than at birth, and the pregnancy predictive ability was lower with frozen-thawed (F-T) embryos. Six metabolic pathways differed between d 40 pregnant recipients for fresh and F-T embryos. Within F-T embryos, more recipients were misclassified, probably due to pregnancy losses, but were accurately identified when combined with embryonic metabolite signals. After recalculation, 12 biomarkers increased receiver operator characteristic-area under the curve (>0.65) at birth, highlighting creatine (receiver operator characteristic-area under the curve = 0.851), and 5 new biomarkers were identified. Combining metabolic information of recipient and embryos improves the confidence and accuracy of single biomarkers.

Analytical Methodology for a Metabolome Atlas of Goat's Plasma, Milk and Feces Using 1H-NMR and UHPLC-HRMS.

Metabolomics has been increasingly used in animal and food sciences. Animal health is one of the most important factor that can also alter animal integrity and welfare. Some studies have already investigated the link between health and metabolic profile of dairy animals. These studies in metabolomics often consider a single type of sample using a single analytical platform (nuclear magnetic resonance or mass spectrometry). Only few studies with multi-platform approaches are also used with a single or a multi type of sample, but they mainly consider dairy cows' metabolome although dairy goats present similar diseases, that it could be interesting to detect early to preserve animal health and milk production. This study aims to create a metabolic atlas of goat plasma, milk and feces, based on healthy animals. Our study describes a standard operating procedure for three goat matrices: blood plasma, milk, and feces using multiple platforms (NMR (1H), UHPLC (RP)-MS and UHPLC (HILIC)-MS) that follows a unique sample preparation procedure for each sample type to be analyzed on multi-platforms basis. Our method was evaluated for its robustness and allowed a better characterization of goat metabolic profile in healthy conditions.

Optimization of Sample Preparation for Metabolomics Exploration of Urine, Feces, Blood and Saliva in Humans Using Combined NMR and UHPLC-HRMS Platforms.

Currently, most clinical studies in metabolomics only consider a single type of sample such as urine, plasma, or feces and use a single analytical platform, either NMR or MS. Although some studies have already investigated metabolomics data from multiple fluids, the information is limited to a unique analytical platform. On the other hand, clinical studies investigating the human metabolome that combine multi-analytical platforms have focused on a single biofluid. Combining data from multiple sample types for one patient using a multimodal analytical approach (NMR and MS) should extend the metabolome coverage. Pre-analytical and analytical phases are time consuming. These steps need to be improved in order to move into clinical studies that deal with a large number of patient samples. Our study describes a standard operating procedure for biological specimens (urine, blood, saliva, and feces) using multiple platforms (1H-NMR, RP-UHPLC-MS, and HILIC-UHPLC-MS). Each sample type follows a unique sample preparation procedure for analysis on a multi-platform basis. Our method was evaluated for its robustness and was able to generate a representative metabolic map.

Metabolomic analysis of oviduct fluid on day 3 post-estrus in Holstein heifers.

The metabolites in the oviduct fluid (OF) of both oviducts were analyzed by proton nuclear magnetic resonance (1H-NMR) in Holstein heifers on day 3 after synchronized estrus. Twenty-six metabolites were quantified, among which lactate, glycine and myoinositol were the most abundant. Six metabolites including glycine and myoinositol varied in amount according to the proximity to the corpus luteum. Glucose and histidine were among the most variable metabolites among heifers while threonine and lactate were among the most stable ones, suggesting different mechanisms of homeostasis in the OF.

Metabolomic differences in blastocoel and uterine fluids collected in vivo by ultrasound biomicroscopy on rabbit embryos†.

The success of embryo development and implantation depends in part on the environment in which the embryo evolves. However, the composition of the uterine fluid surrounding the embryo in the peri-implantation period remains poorly studied. In this work, we aimed to develop a new strategy to visualize, collect, and analyze both blastocoelic liquid and juxta-embryonic uterine fluid from in vivo peri-implantation rabbit embryos. Using high-resolution ultrasound biomicroscopy, embryos were observed as fluid-filled anechoic vesicles, some of which were surrounded by a thin layer of uterine fluid. Ultrasound-guided puncture and aspiration of both the blastocoelic fluid contained in the embryo and the uterine fluid in the vicinity of the embryo were performed. Using nuclear magnetic resonance spectroscopy, altogether 24 metabolites were identified and quantified, of which 21 were detected in both fluids with a higher concentration in the uterus compared to the blastocoel. In contrast, pyruvate was detected at a higher concentration in blastocoelic compared to uterine fluid. Two acidic amino acids, glutamate and aspartate, were not detected in uterine fluid in contrast to blastocoelic fluid, suggesting a local regulation of uterine fluid composition. To our knowledge, this is the first report of simultaneous analysis of blastocoelic and uterine fluids collected in vivo at the time of implantation in mammals, shedding new insight for understanding the relationship between the embryo and its local environment at this critical period of development.

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.

Metabolomic Profile of Oviductal Extracellular Vesicles across the Estrous Cycle in Cattle.

Oviductal extracellular vesicles (oEVs) have been proposed as key modulators of gamete/embryo maternal interactions. The aim of this study was to examine the metabolite content of oEVs and its regulation across the estrous cycle in cattle. Oviductal EVs were isolated from bovine oviducts ipsilateral and contralateral to ovulation at four stages of the estrous cycle (post-ovulatory stage, early and late luteal phases, and pre-ovulatory stage). The metabolomic profiling of EVs was performed by proton nuclear magnetic resonance spectroscopy (NMR). NMR identified 22 metabolites in oEVs, among which 15 were quantified. Lactate, myoinositol, and glycine were the most abundant metabolites throughout the estrous cycle. The side relative to ovulation had no effect on the oEVs' metabolite concentrations. However, levels of glucose-1-phosphate and maltose were greatly affected by the cycle stage, showing up to 100-fold higher levels at the luteal phase than at the peri-ovulatory phases. In contrast, levels of methionine were significantly higher at peri-ovulatory phases than at the late-luteal phase. Quantitative enrichment analyses of oEV-metabolites across the cycle evidenced several significantly regulated metabolic pathways related to sucrose, glucose, and lactose metabolism. This study provides the first metabolomic characterization of oEVs, increasing our understanding of the potential role of oEVs in promoting fertilization and early embryo development.

Metabolomic profiling of bovine oviductal fluid across the oestrous cycle using proton nuclear magnetic resonance spectroscopy.

In the present study we tested whether regulation of the metabolome in bovine oviductal fluid depended on the stage of the oestrous cycle, the side relative to ovulation and local concentrations of steroid hormones. Luminal fluid samples from both oviducts were collected in the preovulatory, postovulatory, mid- and late luteal phases, from cyclic cows at a local abattoir (18-27 cows per stage and side). The metabolomes were assessed by proton nuclear magnetic resonance spectroscopy (H-NMR). In all, 39 metabolites were identified, among which the amino acid glycine and the energy substrates lactate and myoinositol were the most abundant at all stages. The concentrations of 14 metabolites varied according to the stage of the oestrous cycle in at least one side relative to ovulation, of which four (choline, glucose-1-phosphate, glycine and pyruvate) were correlated with intraoviductal progesterone or oestradiol concentrations. Glucose-1-phosphate was most affected by the stage of the cycle, with four- to sixfold higher levels in luteal than periovulatory stages. These results provide new knowledge on the regulation of secretory activity in the oviduct and may help optimise culture media for gamete maturation, IVF and embryo production.

Comparison of three devices for the automated detection of estrus in dairy cows.

Considerable technological advances have been made in the automated detection of estrus in dairy cattle, but few studies have evaluated their relative performance on the same animals or assessed cow-related factors that affect their performance. Our objective was to assess the performance and reliability of three devices commercially available in France for cow estrus detection. The devices were a pedometer (PM; Afitag) and two activity meters (AM1; Heatime-RuminAct, and AM2; HeatPhone). Two algorithms were tested for AM2. We fitted 63 lactating Holstein cows with the three detectors from calving to 90 days after calving. The onset and pattern of cyclicity were monitored from 7 to 90 days postpartum measuring progesterone concentration in milk twice weekly. A total of 211 ovulations were identified. Cyclicity was classified as normal in 60% of cows (38/63). Calculated over the operating period of all the devices (179 periods of estrus), the sensitivities and positive predictive values were, respectively, 71% and 71% for PM, 62% and 84% for AM1, 61% and 67% for the first algorithm of AM2, and 62% and 87% for the second algorithm of AM2. Both activity meters had a lower sensitivity but a higher positive predictive value than the PM (P < 0.05). For all devices, the performance in estrus detection was much poorer at the first postpartum ovulation than at subsequent ovulations (P < 0.05). Lactation rank and milk production affected some devices (P < 0.05). These devices could be used to reinforce visual observations, especially after 50 days postpartum, the minimum recommended delay to insemination. However, their full benefit remains to be verified in different farming systems and taking into account the specific objectives of the dairy farmer.