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.

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.

Effect of DHA on the quality of In vitro produced bovine embryos.

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.

Effects of the donor factors and freezing protocols on the bovine embryonic lipid profile†.

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.

Lipid profile of bovine grade-1 blastocysts produced either in vivo or in vitro before and after slow freezing process.

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.

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.

Oviduct Fluid Extracellular Vesicles Change the Phospholipid Composition of Bovine Embryos Developed In Vitro.

Oviduct fluid extracellular vesicles (oEVs) have been proposed as bringing key molecules to the early developing embryo. In order to evaluate the changes induced by oEVs on embryo phospholipids, fresh bovine blastocysts developed in vitro in the presence or absence of oEVs were analyzed by intact cell MALDI-TOF (Matrix assisted laser desorption ionization-Time of flight) mass spectrometry (ICM-MS). The development rates, cryotolerance, and total cell number of blastocysts were also evaluated. The exposure to oEVs did not affect blastocyst yield or cryotolerance but modified the phospholipid content of blastocysts with specific changes before and after blastocoel expansion. The annotation of differential peaks due to oEV exposure evidenced a shift of embryo phospholipids toward more abundant phosphatidylcholines (PC), phosphatidylethanolamines (PE), and sphingomyelins (SM) with long-chain fatty acids. The lipidomic profiling of oEVs showed that 100% and 33% of the overabundant masses in blastocysts and expanded blastocysts, respectively, were also present in oEVs. In conclusion, this study provides the first analysis of the embryo lipidome regulated by oEVs. Exposure to oEVs induced significant changes in the phospholipid composition of resulting embryos, probably mediated by the incorporation of oEV-phospholipids into embryo membranes and by the modulation of the embryonic lipid metabolism by oEV molecular cargos.

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.

Genetic merit for fertility alters the bovine uterine luminal fluid proteome†.

Over the last decades, fertility of dairy cows has declined due to selection strategies focusing on milk yield. To study the effect of genetic merit for fertility on the proteome of the bovine uterine luminal fluid, Holstein heifers with low- and two groups of heifers with high-fertility index (high-fertility Holstein and Montbéliarde) were investigated. To focus on the maternal effect, heifers from all groups were synchronized and received on Day 7 high-quality embryos. Uterine luminal fluid from Day 19 pregnant heifers was analyzed in a holistic proteomic approach using nano-LC-MS/MS analysis combined with a label-free quantification approach. In total, 1737 proteins were identified, of which 597 differed significantly in abundance between the three groups. The vast majority of proteome differences was found comparing both high-fertility groups to the low-fertility Holstein group, showing that the genetic predisposition for fertility is prevalent regarding the uterine luminal fluid proteome. Evaluation of this dataset using bioinformatic tools revealed an assignment of higher abundant proteins in low-fertility Holstein to several metabolic processes, such as vitamin metabolic process, which comprises folate receptor alpha (FOLR1) and retinol-binding protein, indicating an involvement of disturbed metabolic processes in decreased fertility. Moreover, immune system-related proteins - lactotransferrin and chromogranin A - were enriched in low-fertility cows together with interferon tau 3 h and interferon tau-2. Our results indicate that the genetic merit for fertility leads to substantial quantitative differences at the level of proteins in uterine fluid of pregnant animals, thus altering the microenvironment for the early conceptus.

Intraoviductal concentrations of steroid hormones during in vitro culture changed phospholipid profiles and cryotolerance of bovine embryos.

The objective of this study was to evaluate the effect of progesterone (P4), estradiol (E2), and cortisol (CO) at intraoviductal concentrations on bovine embryo development and quality in vitro. After fertilization of in vitro matured oocytes, zygotes were cultured for 8 days in synthetic oviductal fluid, supplemented with 55 ng/ml P4, 120 pg/ml E2, 40 ng/ml CO, or their combination (ALL). Control embryos were cultured with vehicle (0.1% ethanol). Exposure to steroids did not affect the embryo developmental rate nor the mean number of cells per blastocyst. However, at 24 hr after vitrification-warming, exposure to P4 improved the proportion of embryos that re-expanded and were viable while exposure to CO decreased the proportion of viable embryos. By intact cell MALDI-TOF mass spectrometry, a total of 242 phospholipid masses of 400-1000 m/z were detected from individual fresh blastocysts. Exposure to ALL induced the highest and most specific changes in embryo phospholipids, followed by P4, E2, and CO. In particular, the m/z 546.3 and 546.4 attributed to lysophosphatidylcholines were found less abundant after exposure to P4. In conclusion, exposure of bovine embryos to intraoviductal concentrations of steroid hormones did not affect in vitro development but changed blastocyst quality in terms of cryotolerance and phospholipid profiles.

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.

A missense mutation in PFAS (phosphoribosylformylglycinamidine synthase) is likely causal for embryonic lethality associated with the MH1 haplotype in Montbéliarde dairy cattle.

A candidate mutation in the sex hormone binding globulin gene was proposed in 2013 to be responsible for the MH1 recessive embryonic lethal locus segregating in the Montbéliarde breed. In this follow-up study, we excluded this candidate variant because healthy homozygous carriers were observed in large-scale genotyping data generated in the framework of the genomic selection program. We fine mapped the MH1 locus in a 702-kb interval and analyzed genome sequence data from the 1,000 bull genomes project and 54 Montbéliarde bulls (including 14 carriers and 40 noncarriers). We report the identification of a strong candidate mutation in the gene encoding phosphoribosylformylglycinamidine synthase (PFAS), a protein involved in de novo purine synthesis. This mutation, located in a class I glutamine amidotransferase-like domain, results in the substitution of an arginine residue that is entirely conserved among eukaryotes by a cysteine (p.R1205C). No homozygote for the cysteine-encoding allele was observed in a large population of more than 25,000 individuals despite a 6.7% allelic frequency and 122 expected homozygotes under neutrality assumption. Genotyping of 18 embryos collected from heterozygous parents as well as analysis on nonreturn rates suggested that most homozygous carriers died between 7 and 35 d postinsemination. The identification of this strong candidate mutation will enable the accurate testing of the reproducers and the efficient selection against this lethal recessive embryonic defect in the Montbéliarde breed.

Effect of different superovulation stimulation protocols on adenosine triphosphate concentration in rabbit oocytes.

Ovarian stimulation protocols are used usually to increase the number of oocytes collected. The determination of how oocyte quality may be affected by these superovulation procedures, therefore, would be very useful. There is a high correlation between oocyte ATP concentration and developmental competence of the resulting embryo. The aim of this study was to evaluate the effect of follicle stimulating hormone (FSH) origin and administration protocols on oocyte ATP content. Rabbit does were distributed randomly into four groups: (i) a control group; (ii) the rhFSH3 group: females were injected, every 24 h over 3 days, with 0.6 µl of rhFSH diluted in polyvinylpyrrolidone (PVP); (iii) the pFSH3 group: females were injected every 24 h over 3 days with 11.4 µg of pFSH diluted in PVP; and (iv) the pFSH5 group: females were injected twice a day for 5 days with 11.4 µg of pFSH diluted in saline serum. Secondly, the effect of pFSH5 protocol on developmental potential was evaluated. Developmental competence of oocytes from the control and pFSH5 groups was examined. Differences in superovulation treatments were found for ATP levels. In the pFSH5 group, the ATP level was significantly lower than that of the other groups (5.63 ± 0.14 for pFSH group versus 6.42 ± 0.13 and 6.19 ± 0.15 for rhFSH3 and pFSH3, respectively; P < 0.05). In a second phase, only 24.28% of pFSH5 ova developed into hatched blastocysts compared with 80.39% for the control group. A negative effect on oocyte quality was observed in the pFSH5 group in ATP production, it is possible that, after this superovulation treatment, oocyte metabolism would be affected.

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.

Determination of anti-Müllerian hormone concentrations in blood as a tool to select Holstein donor cows for embryo production: from the laboratory to the farm.

High between-animal variability in the number of embryos produced by multiple ovulation and embryo transfer (MOET) and ovum pick-up and in vitro production (OPU-IVP) methods remains a major limit to the development of embryo biotechnologies in cattle. The measurement of anti-Müllerian hormone (AMH) endocrine concentrations in cows can help to predict their follicular and ovulatory responses to gonadotrophin treatment. The present study aimed to provide practical information for a simple prognostic method based on AMH measurement in Holstein cows. Accurate AMH concentrations could be measured with ELISA in blood or plasma. In cows undergoing repeated OPU protocols over 1 year, the AMH concentrations measured in plasma samples collected before each gonadotrophin treatment were found to be highly repeatable and were tightly correlated with follicular responses. From data obtained at both an experimental station and farm settings, it was possible to propose AMH cut-off values to identify low-responding cows. Gonadotrophin-stimulated cows producing fewer than 15 large follicles at oestrus and fewer than 10 embryos in MOET protocols could be discarded efficiently with plasma AMH concentrations below 87 and 74 pg mL(-1), respectively. In conclusion, we propose a prognostic method based on a single AMH measurement to improve the results of embryo biotechnologies.

Structural, metabolic and developmental evaluation of ovulated rabbit oocytes before and after cryopreservation by vitrification and slow freezing.

The cryopreservation of oocytes is valuable for the preservation of women's fertility and might also be an interesting tool to preserve animal genetic biodiversity but it is not often used because of the very poor fertility recovered after thawing, especially in rabbit species. The objective of our study was to evaluate the effect of slow-freezing and vitrification on the structural integrity of ovulated rabbit oocytes, their ATP contents, and their developmental competence. Results show that, whatever the method is used, cryopreservation has a dramatic effect on the metabolic integrity, the structural integrity, and the developmental ability of the oocytes. Vitrification and slow freezing both impair the rabbit oocytes viability after thawing but the processes act differently. Further studies are needed to improve the cryopreservation techniques in rabbit species. Moreover, we underlined that morphology and maintenance of the structural integrity of the oocytes are not suitable enough to assess the potential for further development of cryopreserved M(II) oocytes. The assessment of ATP metabolism allows efficient evaluation of the viability of the frozen or vitrified oocytes. It should be used in addition to parthenogenesis to better assess the potential for further development.

Effect of the luteinizing hormone on embryo production in superovulated rabbit does.

For most domestic animals, the responses to superovulation treatments are not controlled as a consequence of the lack of knowledge on exogenous gonadotrophins effects on the ovarian function. The role of luteinizing hormone (LH) on the number and quality of embryos produced was evaluated on rabbit does superovulated with porcine FSH (pFSH). Parameters of embryos recovery, in vitro and in vivo embryo development rates after freezing/thawing were compared. We used three experimental groups: (1) control group without superovulation treatment, (2) "pFSH+pLH" and (3) "pFSH" groups where females were treated with pFSH, respectively, with (20%) or without (0%) porcine LH supplementation. The number of corpora lutea and the number of embryos produced were significantly higher (p<0.001) in superovulated does than in control group (27.1, 26.7 versus 11.9 corpora lutea and 20.3, 21.2 versus 9.6 embryos produced for pFSH+pLH, pFSH and control group, respectively). However, both gonadotrophins administrations (groups 2 and 3) led to defaults of ovulation when compared with untreated does. No significant difference was observed between the number and quality of the embryos produced by does treated with pFSH+pLH or with pFSH alone. Moreover, we observed no significant difference between results of in vivo and in vitro viability assays after thawing. We concluded that pFSH alone seems to be sufficient to stimulate the follicles growth and that exogenous pLH administrated has no effect on the quantity and quality of embryos. Further studies are needed to evaluate the hormonal patterns before and after the gonadotrophins injections in the rabbit species.

Effect of antibiotics on thermodynamic properties of freezing media in rabbit species: A first calorimetric approach.

Semen freezing is an effective and safe solution for the cryopreservation of animal genetic resources and for the diffusion of the genetic progress. Actually, these techniques are not yet under control for the rabbit species partly because methods are not clearly defined. Thus, the aim of this work is to study the effect of antibiotics (Penicillin G, Streptomycin) routinely used in freezing semen on the thermodynamic properties of freezing media mainly used in rabbit species. Measurements realized by differential scanning calorimetry show that these antibiotics may change the temperature of crystallization and the quantity of ice formed in the freezing media considered. Our calorimetric approach underlined that the composition and the properties of the cryoprotective solutions should be studied more precisely.