INTRODUCTION:Although the in vitro production of bovine embryos now exceeds in vivo production, the quality of in vitro-produced embryos remains inferior. Metabolomic analysis of the spent culture medium used for embryos is considered a relevant source of markers for subsequent developmental capacity, genetic status and sex. However, little is known about the sources of variability in this metabolome and their respective significance. OBJECTIVE:We compared bovine embryo spent culture media from in vivo developed (IVD) and in vitro produced (IVP) embryos, and analyzed how they varied with embryo stage, grade and sex. METHODS:Embryos were produced in vitro under two different conditions: SOF medium supplemented with 1% fetal calf serum or IVF Bioscience media, or developed in vivo. They were recovered at Day-6 and cultured individually for 26 hours. The culture medium was analyzed using 1H-NMR spectroscopy, and the embryos were staged, graded and sexed. RESULTS:In vitro production significantly reduced inter-embryo variability of the 24 metabolites measured. The mean variance of metabolite content was 5 to 10 times lower in IVP than in IVD embryos (depending on the IVP conditions). IVD embryo stages and grades at the start or end of the culture period contributed to 8% to 18% of variability in the media content, but only 1% of variability for IVP embryos. IVD embryo stage at the start of culture significantly impacted metabolite content. IVD embryo sex did not globally impact composition of the spent culture medium, but the kinetics of decrease of about half of the metabolites differed between male and female blastocysts. CONCLUSIONS:Standardization of the metabolic properties of IVP embryos invites new questions regarding the impact of embryo biotechnology. Differences in metabolism between male and female IVD embryos are highly transient during the morula-blastocyst stages.

We used metadata to explore the metabolic interplay between culture medium from in vitro-produced bovine embryos transferred fresh or frozen, recipient blood plasma, and calf fitness, alongside gene expression and methylation in calf lymphocytes. Principal component (PC) analysis (PCA) identified covariates that were depicted in Debiased Sparse Partial Correlation networks and analyzed as enriched pathways. Four PCs explained 13.77, 9.58, 7.73 and 5.84% variability. PC1 clustered only mother weight and two embryonic metabolites. PC2, PC3 and PC4 associated 10, 17, and 5 calf features with 10, 6, and 16 embryonic and 2, 20, and 5 recipient metabolites, respectively. Subsequently, gene methylation and expression, and calf fitness were analyzed by PCA. Three PCs covered 100% variability. PC1 associated acid-base balance, protein metabolism, Cl-, and Ca2+ with IGF2 and IL1R1 expression, and IL4 and IL12B methylation. PC2 linked H19 expression and methylation with growth and biochemical traits. PC3 clustered growth, hydration, and redox balance, with IGF2, IGF2R, IL1R1 and IL3 methylation, and H19, IGF2, IGF2R and IL12B expression. Gene methylation connected with embryo metabolites through networks via K+, Cl-, HCO3- and TCO2. Calf fitness parallels the early metabolic fingerprints of the embryo and recipient, allowing embryo transfer decision-making based on calf health.

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.

Today, dairy cattle farmers are seeking to optimize expenditure and productivity throughout the lives of their animals by focusing on efficiency at all levels. One strategy for bringing forward the start of a dairy cow’s profitability phase is to advance the onset of puberty and reduce the animal’s age at their first calving. Thus, one objective of this study was to feed two groups of Holstein dairy heifers the same diet but in different quantities, with the aim of generating a growth differential of at least 200 g/day between the two groups. Thirty-eight heifer calves were followed from birth through puberty using body morphometric measurements and quantitative data collected by automatic feeders, which enabled the monitoring of both feed intake and growth for individual heifers. Routine ultrasonography was used to examine changes in the muscle and adipose tissue compartments, and x-ray tomography was used to monitor bone mineralization and rumen development. At 12 weeks of age, heifers in the optimized feeding (OPT) group had greater cortical bone thickness in the tibia compared with the control (CON) group. At 18 weeks of age, OPT heifers also had more trabecular cancellous bone. In contrast, the ruminal volumes of the heifers in the CON group were greater than those of the OPT heifers at 12 weeks. The OPT heifers had greater indices of general, skeletal, and muscular development at 9 weeks, 6 months, and 12 months. Among the circulating plasma indicators measured in this study, non-esterified fatty acids were highest in the CON calves at 6 and 12 months of age and at the onset of puberty, whereas IGF1 was highest in the OPT calves at all ages. The age at puberty of the OPT calves was 8.4 ± 0.6 months compared with 11.2 ± 1.1 months for the CON calves. The OPT heifers had an average daily weight gain of 1018 g/day from birth to the onset of puberty, which enabled them to achieve increased body fattening and to reach puberty 3 months earlier compared with the CON heifers; the average daily weight gain of the CON heifers was 780 g/day over the same period. Taken together, this study defines new reference values for a multitude of morphometric indicators, thereby enabling precise monitoring of the growth of Holstein heifers from birth to puberty.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

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.

Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that improves fertility by increasing membrane fluidity. Moreover, embryos produced by donor females supplied with n-3 PUFA did not show any difference in terms of the lipid profile after 7 days of culture. The present study aimed to investigate the effects of DHA (20 and 100 μM) coupled with carnosine (5 mg/mL), an antioxidant, during oocyte maturation and embryo development on the developmental and cryosurvival rates and the number of pluripotent cells. Free fatty acid receptor-4 (FFAR4), which is able to bind DHA, was visualised by immunostaining. The addition of DHA in the in vitro development (IVD) medium decreased the percentage of pluripotent SOX2 positive cells compared with the control (8.4% vs. 10.9%) without affecting the number of cells (196.7 vs. 191.6 cells) or the developmental (20.9% vs. 23.9% blastocysts rate on D7) and cryosurvival rates (86.3% vs 86.2%). Such a decrease in pluripotent cells, relevant to the differentiation of the first lineage within the inner cell mass, represents an improvement in the embryo quality. On the contrary, embryos without any pluripotent SOX2-positive cells would not be able to achieve gestation. Future studies should follow up these results by carrying out embryo transfers to assess the beneficial effects of DHA supplementation.

Embryo lipid profile is affected by in vitro culture conditions that lead to an increase in lipids. Efforts have been made to optimize embryo lipid composition as it is associated with their quality. The objective of this study was to evaluate whether the diet supplementation of donor cows (n-3 or n-6 polyunsaturated fatty acids), or the slow freezing protocols (ethylene glycol sucrose vs. glycerol-trehalose), or the physiological stage of the donor (nulliparous heifers vs. primiparous lactating cows) may impact the bovine embryo lipid profile. Lipid extracts of 97 embryos were individually analyzed by liquid chromatography-high resolution mass spectrometry, highlighting 246 lipids, including 85% being overabundant in cow embryos compared to heifer embryos. Among 105 differential lipids, 72 were overabundant after ethylene glycol sucrose protocol, including a single glycerophosphate PA(32:1) representing 27.3% of the significantly modulated lipids, suggesting that it is degraded when glycerol-trehalose protocol is used. No lipids were different according to the n-3 or n-6 supplementation of the donor cows. In conclusion, the embryonic lipid profile was mainly affected by the physiological stage of the donors and the slow freezing protocols. The overabundance of lipids in lactating cow embryos and the resulting lower quality of these embryos are consistent with the lower pregnancy rate observed in cows compared to heifers. Unlike glycerol-trehalose protocol, ethylene glycol sucrose freezing allowed to preserve glycerophospholipids, potentially improving the slow freezing of in vitro-produced embryos. Further studies are required to modulate embryo quality and freezability by modulating the lipidome and by integrating all stages of embryonic production.

Reproduction, Fertility and Development is an international journal publishing original research , review and comment in the fields of reproduction and developmental biology in humans, domestic animals and wildlife

Currently, in vitro embryo production (IVP) is successfully commercially applied in cattle. However, the high sensitivity of embryos to cryopreservation in comparison to in vivo (IVD) embryos slows the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to in vitro culture conditions. The objective of this study was to evaluate the lipid composition of biopsied and sexed embryos, produced either in vivo or in vitro from the same Holstein heifers before and after a slow freezing protocol. Lipid extracts were analysed by liquid chromatography-high resolution mass spectrometry, which enabled the detection of 496 features. Our results highlighted a lipid enrichment of IVP embryos in triglycerides and oxidised glycerophospholipids and a reduced abundance in glycerophospholipids. The slow freezing process affected the lipid profiles of IVP and IVD embryos similarly. Lysophosphatidylcholine content was reduced when embryos were frozen/thawed. In conclusion, the embryonic lipid profile is impacted by IVP and slow freezing protocols but not by sex. Lysophosphatidylcholine seemed highly sensitive to cryopreservation and might contribute to explain the lower quality of frozen embryos. Further studies are required to improve embryo freezability by modulating the lipidome.

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.

Currently, invitro embryo production (IVP) is successfully applied commercially in cattle. However, the high sensitivity of embryos to cryopreservation compared with invivo-derived (IVD) embryos still impairs the dissemination of this biotechnology. Reduced cryotolerance is frequently associated with lipid accumulation in the cytoplasm mainly due to invitro culture conditions. The objective of this study was to evaluate the lipid content of fresh and frozen sexed bovine grade 1 IVP or IVD embryos. The same 8 Holstein heifers were used in a Latin square design for both IVP and IVD embryo production. Zygotes were cultured in synthetic oviductal fluid (SOF) supplemented with 1% oestrus cow serum. The same bull was used for IVP and IVD. All expanded Day 7 blastocysts (n=40 IVP and 40 IVD) were biopsied and sexed. Half of the embryos (n=20 in each group) was slow frozen (1.5M ethylene glycol, 0.1m sucrose) and thawed before lipid extraction. Remaining embryos underwent lipid extraction in the fresh state. Briefly, the liposoluble fraction of the embryos was extracted according to the Bligh and Dyer method using chloroform and methanol. Liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis was performed and operated in positive ionization mode. Lipids with variance intensities greater than 30% in quality control samples were removed as well as those identified as background noise. Partial least square discriminant analysis (PLS-DA) was used to show the relationship between variance in the data and difference among embryo origin (IVP vs. IVD), state before extraction (fresh vs. frozen), and sex of the embryos (male vs. female). The differentially lipid species groups were identified using Wilcoxon test, and considered significantly different when P<0.05. LC-HRMS analysis allowed us to identify 75 lipids. PLS-DA showed that embryo origin (IVP vs. IVD) and state before extraction (fresh vs. frozen) can be determined by LC-HRMS profiles by group in PLS-DA plot, despite slight overlaps. Sex of the embryos did not allow us to differentiate the lipid profile. However, 15 lipids varied significantly between male and female IVD, predominantly triglycerides (TG), whereas no lipid varied between the sexes in the IVP homologues. Moreover, 26 lipids varied significantly between IVP and IVD fresh embryos with enrichment of IVP embryos in TG, phosphatidyl choline, cholesteryl ester, and less diglyceride and lysophospholipid (LP) compared with IVD embryos. The comparison of the lipid profiles before and after freezing for IVP embryos showed that only 7 lipids varied significantly between fresh and frozen states with a decrease in LP for the frozen embryos. For the invivo counterparts, 13 lipids varied significantly, including the same LP as those identified for IVP embryos in the same way. Our results showed that the embryonic lipid profile is mainly affected by IVP and slow freezing protocols and, to a lesser extent, by sex. Further studies are needed to improve IVP protocols and optimize the cryotolerance of IVP embryos in cattle.

Developper des outils d’aide a la decision pour s’equiper en detecteurs automatises des chaleurs et tirer le meilleur parti des capteurs deja installes dans les elevages laitiers, voila les objets du projet MARIAGE, pilote par l'Institut de l'Elevage. Dans un contexte de developpement de l'elevage de precision, il a permis d'elaborer des outils techniques et economiques destines a aiguiller le choix des eleveurs pour equiper ou non leur troupeau en capteurs de detection des chaleurs. Par ailleurs, les donnees produites en continu par les capteurs HeatLive ® de Medria, installes dans plus de 1043 elevages, ont ete combinees aux donnees du Systeme National d’Information Genetique bovin. Le traitement de cette base de donnees a permis de caracteriser de nouveaux indicateurs d’expression des chaleurs (amplitude de la suractivite associee aux chaleurs, duree de la suractivite, aire sous la courbe de suractivite). Ces indicateurs sont influences par la race, la parite et le niveau de production des vaches. Il reste a verifier si ces caracteres d'expression des chaleurs pourrait donner lieu a selection genetique.

Blood biomarkers may help to predict pregnancy in recipients of in vitro produced (IVP) embryos. Using H-1 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.

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.