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, 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.

Recently, it has been postulated that oviductal extracellular vesicles (oEV) might act as natural nanoshuttles bringing key components (small noncoding RNAs and proteins) of the oviduct into gametes and embryos. Furthermore, co-incubation of frozen-thawed oEV with invitro-produced bovine embryos was reported to increase blastocyst rate and quality (Almiñana et al. 2017 Reproduction 154, 153-168). The objective of this study was to determine the dose-dependent effect of oEV supplementation of embryo culture medium on the invitro development and cryotolerance of embryos. Briefly, oEV were isolated by ultracentrifugation from a pool of oviductal fluids (8 cows/sample) collected at the slaughterhouse at the post-ovulatory stage and ipsilateral to ovulation and stored at −80°C until used. Slaughterhouse-derived bovine oocytes were invitro matured and fertilised with frozen-thawed semen from one bull (4 replicates; 194 presumptive zygotes per group), according to our standard procedures. After IVF, groups of presumptive zygotes (n=20/drop) were cultured under humidified air with 5% CO2, 5% O2 at 38.8°C for 7 days in 30µL of synthetic oviductal fluid-bovine serum albumin supplemented with oEV at different protein concentrations: 0.5, 0.05, or 0.005mgmL−1 and without (control). Cleavage rates were evaluated on Day 2 and blastocyst rates were assessed on Days 6 and 7 (IVF as Day 0). At Day 7, expanded grade 1 blastocysts were evaluated (International Embryo Technology Society classification) and embryos at the expanded grade 1 blastocyst stage were slow frozen in 1.5M ethylene glycol + 0.1M sucrose and stored in liquid nitrogen. For cryotolerance evaluation, embryos were thawed and cultured for 48h in synthetic oviductal fluid-bovine serum albumin + 1% estrous cow serum. Hatching rates were assessed at 48h post-thawing. Data were analysed by a logistic regression mixed model (SAS, SAS Institute Inc.; Glimmix procedure) followed by post-hoc Tukey for multiple comparisons. Differences were considered significant at P<0.05. No differences were observed among the different oEV concentrations tested for cleavage and Day 6 blastocysts. A tendency (P=0.0535) was observed for Day 7 blastocyst rates (19.1±2.8, 29.4±3.3, 16.0±2.6, and 20.6±2.9 for 0.5, 0.05, 0.005mgmL−1, and control, respectively) in favour of the 0.05mgmL−1 group. However, a significant difference (P<0.0288) for Day 7 grade 1 expanded blastocyst rates in favour of the 0.05mgmL−1 group was observed (5.2±1.6, 12.9±2.4, 3.1±1.2, and 9.8±2.2 for 0.5, 0.05, 0.005mgmL−1, and control, respectively). For cryopreserved embryos, hatching rates of frozen-thawed embryos were not significant among experimental groups (81.6±10.2 (n=19), 89.6±6.6 (n=27), 77.2±12.2 (n=10), and 60.2±13.6 (n=23) for 0.5, 0.05, 0.005mgmL−1, and control, respectively). In conclusion, under our experimental conditions, the supplementation of the embryo culture medium with frozen-thawed post-ovulatory oEV at the protein concentration of 0.05mgmL−1 increased the Day 7 grade 1 expanded blastocyst rate. Moreover, we showed a tendency to improve Day 7 blastocyst rates but with no apparent effects on the cryotolerance of embryos. This work was supported by APIS GENE.

Beneficial effects of n-3 polyunsaturated fatty acid (PUFA) supplementation on dairy cow reproduction have been previously reported. The objectives of the present study were to assess whether n-3 PUFA supplementation would affect in vitro embryo production (IVP) after ovarian stimulation. Holstein cows received a diet with 1% dry matter supplementation of either n-3 PUFA (n = 18, microencapsulated fish oil) or a control, n-6 PUFA (n = 19, microencapsulated soy oil). Both plasma and follicular fluid FA composition showed integration of total PUFA through the diet. All cows underwent an IVP protocol consisting of ovarian stimulation, ultrasound-guided transvaginal oocyte retrieval (ovum pick-up, OPU, five per cow) followed by in vitro maturation, fertilisation and 7 days of embryo development. A tendency toward an increase in the blastocyst rate (diet effect, P = 0.0865) was observed in n-3 cows, with 49.6 ± 5.5% vs 42.3 ± 5.5% in control n-6 cows. A significant increase (diet effect, P = 0.0217) in the good-quality blastocyst rate (freezable blastocysts) was reported in n-3 cows (42.2 ± 7.7%) compared to control n-6 cows (32.7 ± 7.7%). A significant difference in lipid composition was shown in the oocytes recovered by OPU from n-3 and n-6 treated cows, by intact single-oocyte MALDI-TOF mass spectrometry. The 42 differentially abundant identified lipids were mainly involved in cell membrane structure. In conclusion, n-3 PUFA supplementation enhanced oocyte quality and modified their lipid composition. Further studies are necessary to investigate the potential link of these lipid modifications with enhanced oocyte quality.

The objective of the present study was to determine the effect of supplementation of culture medium with carnosine (β-alanyl-l-histidine; Sigma, St-Quentin Fallavier, France), a reactive oxygen species scavenger, on in vitro bovine embryo development and survival following cryopreservation. Abattoir-derived bovine oocytes (4 replicates) were in vitro matured and fertilized with frozen-thawed semen of one bull, according to our standard procedures. In Experiment 1, 20 h after IVF, groups of presumptive zygotes were cultured in 30 μL of SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 205) or 5 μg mL−1 of carnosine (n = 209) under humidified air with 5% CO2, 5% O2, and 88% N2. Cleavage rates were determined on Day 2, and the blastocyst rates and grade were assessed on Day 7 according to IETS classification. Day 7 grade 1 expanded blastocysts (n = 25 control and n = 27 carnosine) were frozen in 1.5 M ethylene glycol + 0.1 M sucrose. Embryos were thawed and then cultured for 72 h in SOF-BSAaa + 1% oestrus cow serum for re-expansion and hatching rate assessments at +24 h, +48 h, and +72 h post-thawing. In Experiment 2, presumed zygotes were cultured in SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 48) or 5 μg mL−1 of carnosine (n = 48) in a WOW dish and observed with Time Laps Cinematography (Primo Vision®, VitroLife, Göteborg, Sweden). Images were recorded every 15 min for up to 168 h post-insemination. For embryos that reached the blastocyst stage, mean timing of the first cleavage (C1; 2-cell stage), second cleavage (C2; 4-cell stage), second cleavage to compaction (C3), and blastocoel cavity appearance (B4) were recorded. Chi-square test for Experiment 1 and Student’s t-test for Experiment 2 were used, and differences were considered significant at P < 0.05. In Experiment 1, no differences were observed in cleavage rate, blastocyst rate on Day 7, and grade 1 blastocyst rate between both control and carnosine groups (84.0 ± 4.2 v.85.2 ± 3.8, P = 0.7; 46.9 ± 7.1 v. 45.0 ± 7.5, P = 0.7; 24.1 ± 2.0 v. 24.0 ± 6.5, P = 0.6; respectively). After thawing, the re-expansion at +24 h was not different between groups (74.1 v. 48.0% for carnosine and control groups, respectively; P = 0.06). However, at +48 h and +72 h, the survival rate of carnosine treated blastocysts was significantly higher than that of blastocysts in the control group: 70.4 ± 4.5% v. 40.0 ± 3.8% and 59.3 ± 3.8% v. 24.0 ± 3.6%, respectively. Results from Experiment 2 indicated no difference between control and carnosine groups for C1 (32.1 ± 3.9 v. 33.8 ± 6.1; P = 0.3), C2 (8.2 ± 8.9 v. 8.9 ± 0.9; P = 0.07), and B4 (147.0 ± 9.5 v. 145.4 ± 11.6; P = 0.6), whereas C3 was significantly different within groups: 59.9 ± 9.6 v. 51.8 ± 6.7 (P = 0.008). In conclusion, bovine blastocysts derived from zygotes cultured in the presence of 5 μg mL−1 carnosine possess a significantly faster kinetic from 4-cell stage to compaction and show a higher post-thawing viability. However, further analyses are still needed to clarify the relationship between the reactive oxygen species intracellular levels after carnosine treatment and in vitro bovine embryo quality. This work was supported by FECUND European project (grant agreement number 312097).

This review aims at giving an overview on the physiological events leading to puberty onset in mammals and more specifically in cattle. Puberty is an important developmental milestone in mammals involving numerous changes in various physiological regulations and behaviors. It is a physiological unique event integrating several important central regulations at the crossroad of adaptation to environment: reproductive axis, feeding behavior and nutritional controls, growth, seasonal rhythm and stress. Puberty onset is also an important economic parameter in replacement heifer program and in genomic selection (genomic bulls). The quest for advanced puberty onset should be carefully balanced by its impact on physiological parameters of the animal and its offspring. Thus one has to carefully consider each step leading to puberty onset and set up a strategy that will lead to early puberty without being detrimental in the long term. In this review, major contributions in the understanding of puberty process obtained in rodents, primates and farm animals such as sheep and cattle are discussed. In the first part we will detail the endocrine events leading to puberty onset with a special focus on the regulation of GnRH secretion. In the second part we will describe the neural mechanisms involved in silencing and reactivating the GnRH neuronal network. These central mechanisms are at the crossroad of the integration of environmental factors such as the nutritional status, the stress and the photoperiod that will be discussed in the third part. In the fourth part, we will discuss the genetic determinants of puberty onset and more particularly in humans, where several pathologies are associated with puberty delay or advance and in cattle where several groups have now identified genomic regions or gene networks associated with puberty traits. Last but not least, in the last part we will focus on the embryologist point of view, how to get good oocytes for in vitro fertilization and embryo development from younger animals.

SensiTemp, a new in vitro maturation (IMV) bull straw concept, presents the advantage of colour changing while the straw is thawed. The colour of frozen straws is blue and straws start to become white when the temperature reaches 33°C, with a complete change of colour at 37°C. The objective of this study is to assess sperm quality after thawing of semen frozen in SensiTemp from 2 bulls, by analysing, in experiment 1, sperm motility and membrane integrity using computer-assisted semen analysis (CASA) and flow cytometry (FC), and, in experiment 2, the in vitro embryo production (IVP) using IVP technologies [IVM, IVF, and in vitro culture (IVC)]. The ejaculates of 2 bulls, selected during preliminary experiments on high in vitro fertility, were harvested at CIA L’Aigle, France, and split ejaculates were frozen in experimental (SensiTemp) and conventional (control) straws. In experiment 1 after thawing semen from the 2 types of straws (5 pooled straws each; 2 replicates), motility was assessed using the IVOS CASA system (Hamilton Thorne Inc., Beverly, MA, USA) and membrane integrity was evaluated through FC with Cytosoft software (Millipore-Guava Technologies Inc., Hayward, CA, USA). In experiment 2, IVF was used to evaluate the non-toxicity of SensiTemp and control straws. Cumulus-oocyte complexes (COC; n = 1178; 4 replicates) collected from slaughterhouse ovaries were matured in IVM medium (TCM-199 with bicarbonate, Sigma-Aldrich, Saint Quentin Fallavier, France; 10 µg mL–1 FSH-LH, Reprobiol, Liège, Belgium; and 10% FCS, Thermo Fisher, Illkirch, France) for 22 h. After fertilization, presumptive zygotes of each group (SensiTemp and control for each bull) were cultured in synthetic oviduct fluid medium (SOF, Minitube, Tiefenbach, Germany) with 1% estrous cow serum (ECS) and 0.6% BSA (Sigma-Aldrich, France) up to 8 days. All cultures were conducted at 38.5C in 5% CO2, and 5% O2. The cleavage and blastocysts rates were evaluated on Days 3 and 7, respectively, for each group. Embryo quality was recorded on Day 7 according to the IETS evaluation. Data from each bull were analysed separately using the chi-squared test (P < 0.05). In experiment 1, neither sperm motility from bull 1 (61.2 and 60.5%) and bull 2 (66.2 and 66.5%) nor membrane integrity from bull 1 (58.6 and 52.2%) and bull 2 (61.0 and 61.9%) were different between SensiTemp and control, respectively. Results from experiment 2 showed no difference (P > 0.05) in cleavage rate between SensiTemp and control for the 2 bulls: 92.1 and 91.7% for bull 1 and 94.2 and 94.6% for bull 2 respectively. The blastocysts rate on Day 7 did not differ (P > 0.05) among groups (47.5, 47.1 and 51.3, 50.4% for SensiTemp and control bull 1 and bull 2, respectively) nor the quality of embryos retrieved in the different groups: 25.4, 23.3, and 30.8, 29.6% in grade 1 embryo for SensiTemp and control bull 1 and bull 2, respectively. Those results demonstrate, in vitro, that the new SensiTemp straws were non-toxic and did not affect the semen quality after thawing nor did the SensiTemp straws affect the ability of sperm cells to fertilize oocytes and produce 8-day-old embryos.

Genomic tools are now available for most livestock species and are used routinely for genomic selection (GS) in cattle. One of the most important developments resulting from the introduction of genomic testing for dairy cattle is the application of reasonably priced low-density single nucleotide polymorphism technology in the selection of females. In this context, combining genome testing and reproductive biotechnologies in young heifers enables new strategies to generate replacement and elite females in a given period of time. Moreover, multiple markers have been detected in biopsies of preimplantation stage embryos, thus paving the way to develop new strategies based on preimplantation diagnosis and the genetic screening of embryos. Based on recent advances in GS, the present review focuses on new possibilities inherent in reproductive technologies used for commercial purposes and in genetic schemes, possible side effects and beneficial impacts on reproductive efficiency. A particular focus is on the different steps allowing embryo genotyping, including embryo micromanipulation, DNA production and quality assessment.

INTRODUCTION:Alteration of expression of various genes including extracellular matrix components, have been suggested to play major role in the placental pathologies after somatic cloning in mammals. The objectives of the present study were to analyze pattern of expression (mRNA and protein) of the small leucine-rich proteoglycan, Decorin in association with Type I Collagen and Fibronectin in bovine placental tissues from normal and clone pregnancies.METHODS:Genotyping and allelic expression of Decorin were determined by Sanger sequencing. The expression patterns of Decorin, Type I collagen and Fibronectin 1 were analyzed by quantitative RT-qPCR and combined in situ hybydization (ISH) and immunohistochemistry (IHC) in endometrial and placental tissues from D18 to term from artificially inseminated and somatic cloning pregnancies.RESULTS:The expression levels of DCN increased in the AI endometrial stroma and chorionic mesenchyme during implantation and declined during placentome growth until term. Combined ISH and IHC revealed an unexpected discrepancy mRNA and protein tissue distribution. Moreover, Decorin was maintained in the placentome tissues from SCNT pregnancies while both mRNA and protein were absent in AI derived placenta.DISCUSSION:In bovine, the pattern of expression of Decorin exhibits significant changes during placental formation. Downregulation of Decorin is associated with proliferation, remodeling and vascularization of placental tissues. These observations reinforces the putative role of Decorin in these processes.CONCLUSIONS:These observations suggest that Decorin is involved in placental growth and that dysregulation of its expression is associated with placental abnormalities in SCNT derived pregnancy.

Reprogramming the differentiated cell to totipotency can be achieved following the introduction of its nucleus into an enucleated oocyte, a procedure known as cloning. We used cattle clones as a pertinent model to assess the inter-individual epigenetic variability and its consequences on phenotypes, including agronomically relevant traits and developmental pathologies. Indeed, the developmental defects frequently associated with cloning could be related to the insufficient extent of reprogramming, leading to perturbations of the nuclear microenvironment of the early embryo, with long-term consequences on the phenotype. Immunoprecipitation of methylated DNA following hybridization on a new bovine-specific tiling array (MeDIP-chip) was used to describe the epigenomic patterns affected by incomplete reprogramming. We first focused on the liver, because overgrowth of this organ is, to a certain extent, bound to global foetal overgrowth, which is often observed in clones. The microarray represents the 21 416 bovine genes currently annotated (UMD3.1 genome assembly). Each gene was tiled by 34 probes, on average, spanning upstream regions from –2000 to +1360 bp relative to the transcription start site. This microarray was hybridized with MeDIP samples from livers of normal Holstein animals obtained by AI (4 perinatal controls and 8 adults: 2 males and 10 females) and livers of female Holstein clones (7 perinatal clones from 2 different genotypes, either stillborn or suffering from severe pathologies, and 7 adult clones from 3 different genotypes, with normal to pathological phenotypes). After normalization of the data, enriched probes were identified using the ChIPmix method (Martin-Magniette et al. 2008 Bioinformatics) and located on the Ensembl Genome Browser. Results of exploratory analysis, including correlation clustering, principal components analysis (PCA), and independent components analysis (ICA), will be presented. A statistical test based on differences in the spatial distribution of the enriched probes along promoters was applied to the data, to associate epigenetic signatures to specific parameters (cloning, phenotype, stage, and genotype). Most promoters with more than 5 enriched probes across individuals showed a clustered distribution of the enriched probes. This local enrichment was highly conserved among individuals for 96% of the promoters, suggesting that most of the methylated regions were common to all animals. More interestingly, the distributions of the enriched probes showed inter-individual variability for 4% of the promoters in all samples. The identification and validation of these promoters is currently in progress. Funding was provided by ANR-09-GENM-012-01 and ACI-PHASE-INRA-2010.

Genomic tools are now available for most livestock species and are used routinely for genomic selection in cattle. Recently, biopsies of pre-implantation-stage embryos were genotyped for multiple markers. This strategy provides the opportunity to estimate breeding values for traits of particular interest and the presence of genetic abnormalities, thus allowing selection of embryos before transfer (Le Bourhis et al. 2011 Reprod. Fertil. Dev. 23, 197 abst). The present work aimed to compare the genotype and the breeding values from biopsied bovine embryos with the corresponding calves. Bovine embryos were obtained after superovulation (8 cows) and flushing at Day 6 or 7. A total of 11 embryos (1 or 2 Grade 1 embryos per flush per cow) were washed and biopsied using a microblade. Biopsies of 5 to 10 cells were transferred individually as dry samples in tubes and frozen before whole genome amplification (WGA). The genomic DNA of each biopsy was amplified using a WGA kit according to the manufacturer's instructions (WGA; QIAGEN REPLI-g® Mini Kit, Qiagen, Valencia, CA, USA). Biopsied embryos were transferred either frozen or fresh to synchronized recipients. At birth a blood sample was taken from the calf for subsequent genotyping. Genotyping was done using the Illumina BovineSNP50TM beadchip. Only embryos with call rate (CR) higher than 80% were selected for breeding values comparisons. Because of allele dropout, heterozygous markers are turned into homozygous makers artificially. Only markers that were still heterozygous in genotypes of an embryo were selected and error rates were calculated with the same markers from the corresponding calf. Imputation was done using Beagle and taking into account the 50 k genotyping results of the parents. Breeding values (milk production and morphological traits) were calculated and compared with those of the 7 corresponding calves. From 11 embryos analysed, only 1 (9%) gave a CR lower than 80% and a higher percentage error with calf genotype (Table 1). These results indicate that, using embryonic DNA after WGA, genotyping errors between embryo and calf are low and correlated with the CR of embryos. For embryos with CR higher than 84, the concordance of genomic values between embryo and calf is very high. More embryo–calf pairs are needed to assess the reliability of this method and to validate the breeding value at the embryo stage. This new technique offers new possibilities in managing breeding schemes by selecting the embryos before transfer. Table 1.Embryo call rate (CR) and genotyping and breeding value concordances between embryos and calves

Since the first success in sheep, the production of viable cloned offspring by somatic cell nuclear transfer (SCNT) is still doomed by a high incidence of pregnancy failure. In cattle, most gestation losses initially take place during the peri-implantation period. Then, abnormal placentation associated with fetal overgrowth (abnormal offspring syndrome) occurs in late pregnancy. As a consequence, IETS recommendations include regular ultrasound evaluation of SCNT pregnancies, with qualitative ultrasound evaluation of the placentomes, fetal movements and fluids being proposed. The objective of this study was to evaluate the use of new, quantitative parameters for the assessment of feto-placental development in AI and SCNT bovine pregnancies. Twenty-two heifers of 4 different breeds were used as SCNT recipients and 11 Holstein heifers were used as AI controls (C). All SCNT fetuses were produced as previously published in the laboratory, using the same fibroblast donor. Animals were scanned every 2 weeks from Day 150 using a Voluson-i (GE Medical Systems) with a transabdominal multifrequency probe (2.2 to 6.5 MHz), allowing automatic 3D volume and Doppler acquisition. Each time, 7 placentomes were scored from 0 to 3 according to echogenicity, general appearance and degree of oedema and their 2D surface was measured. Fetal intercostal width (ICW) was measured in a coronal view just behind the heart. Doppler velocimetry indices, pulsatility index (PI) and resistance index (RI) of one of the two fetal umbilical arteries were obtained and the diameters of the umbilical vessels were measured (UD). The 3D power Doppler was performed to obtain vascular index (VI), flow index (FI) and vascular flow index (VFI). Data were analysed by ANOVA with GraphPad Prism®. So far, 27 heifers have either delivered (n = 10 SCNT and 7 C) or pregnancies have been terminated because of abnormal offspring syndrome (n = 10 SCNT). One thousand five hundred and 197 placentomes have been analysed for 2D and 3D analyses, respectively. The mean placentome score is significantly higher (less normal) in SCNT compared with C (P < 0.0001) but does not vary according to gestational age. The placentome surface is also significantly larger in SCNT vs C (P < 0.0001) but also in SCNT that did not go to term vs those that were alive at birth (P < 0.002). Fetal ICW and UD are consistently larger in SCNT vs C (P < 0.0001) at all stages of pregnancy and there is a significant correlation (r2 = 0.81) between ICW at 15 days before term and birth weight. The PI and RI were not different between SCNT and C. Intra-operator reproducibility of 3D analyses was very high (intra-class correlation coefficient: 80 to 95% for a 95% confidence interval). There was no significant difference for VI, FI, or VFI between SCNT and C. Ongoing work taking into account pregnancy outcomes indicates that placentome scores are useful indicators of pregnancy outcome in SCNT pregnancies. In contrast, the abnormal vascularization observed by histology in SCNT placentomes does not appear to be accompanied by abnormal placental blood flow when analysed using quantitative 3D Doppler. This project received financial support from ANR (ref. PCS-09-GENM-022).

Genomic tools are now available for most livestock species and are used routinely for marker-assisted selection (MAS) and genomic selection (GS) in cattle. Recently, multiple-marker detection has been achieved from biopsies of preimplantation stage embryos, thus allowing embryos to be selected before transfer (Le Bourhis et al. 2009 Reprod. Fertil. Dev. 21, 192 abst). This strategy provides the opportunity to estimate some traits of particular interest, the presence of genetic abnormalities, or both. The present work aimed to assess the efficiency of MAS/GS evaluation from biopsied bovine embryos by using the bovine 50K single nucleotide polymorphism (SNP) Illumina chip. A biopsy of 5 to 10 cells was obtained under laboratory conditions, using a microblade under a stereomicroscope, from 29 in vitro-cultured morulae and blastocysts. Biopsies were transferred individually as dry samples in tubes and sent frozen (n = 13) or at room temperature (n = 16) to the genotyping laboratory. The genomic DNA of each biopsy was amplified using a whole-genome amplification (WGA) kit according to the manufacturer’s instructions (Qiagen REPLI-g® Mini Kit, Qiagen, Valencia, CA). Following WGA, DNA concentration was determined by using PicoGreen. For subsequent genotyping, a custom CRV 50K Illumina chip was used. Call rates were calculated from 50 905 SNP. Percentage of allele drop-out (%ADO), which was estimated from the number of heterozygous markers [%ADO = (calculated hetero – observed hetero)/calculated hetero]. Parentage error was estimated from 12 embryos by using the genotypes of the parents of the embryos. Both groups of transport conditions were compared using Student’s t-test. Results are presented as mean ± SEM. A greater quantity of DNA was obtained after amplification of biopsies that were sent frozen to the laboratory when compared with those at room temperature (P < 0.05). However, the SNP call rate, %ADO, and parentage error did not differ between groups. These results indicate that genotyping from embryo biopsies following WGA can be achieved with good efficiency when using high-density marker chips. To validate the use of MAS/GS from early embryos in breeding schemes, a larger number of in vivo embryos are currently genotyped under field conditions. This will allow the reliability of this method to be assessed and the correlation between embryo and calf genetic evaluation to be quantified with the current WGA efficiency. Table 1.Amount of DNA after WGA and genotyping results

The polled and multisystemic syndrome (PMS) is a genetic abnormality observed in the progeny of a unique bull affected by a large chromosomal deletion and cellular mosaicism. Hemizygous females, representing 15% of total progeny, are hornless and show organ malformations, including of the ovaries. Hemizygous males are assumed to die in early fetal development. This study was initiated to produce sufficiently affected fetuses using the semen of the sire as a unique genetic resource. Oocytes from slaughterhouse ovaries were in vitro matured, fertilized with the same bull and cultured in SOF medium using standard in vitro procedures currently performed in the laboratory. On Day 7, grade 1 to 3 embryos were biopsied (5 to 10 cells) and frozen using a conventional glycerol procedure. Whole-biopsy amplification of genomic DNA was performed using a Qiagen Repli-g® Mini Kit (Qiagen, Valencia, CA, USA). Sex determination was done by PCR (UNCEIA Sexing Kit, UNCEIA, Paris, France). The PMS status was indirectly determined using both tetra-primer amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism procedures to genotype a single SNP located within the deleted region. Because the sire was both homo- and hemizygous for this SNP, 3 categories of progeny were defined according to their genotypes: unaffected (heterozygous), potentially affected (homo- or hemizygous for the same allele as their sire) and affected (hemizygous for the alternate allele). Thirteen affected and 21 potentially affected female embryos were thawed and transferred (1 ≤ n ≤ 4) into 17 Day 7 recipients. Pregnant females were slaughtered on Day 9 and fetuses were recovered; sex and PMS status were then verified. From 2133 inseminated oocytes (7 replicates), 64% cleaved and 10% (n = 216) developed to the blastocyst stage on Day 7. This was significantly lower than the 87% cleavage and 25% blastocyst development rates observed with a control bull used to inseminate 368 oocytes from the same batches. Finally, 174 embryos were biopsied and 169 were frozen. Fifty percent were sexed as female (n = 87). Among them, 15% (n = 13) were affected and 29% (n = 25) were potentially affected. The 24-h survival rate averaged 67% from 54 thawed, unaffected embryos, whereas the hatching rate at 72 h was 59%. Seven female fetuses were recovered from 6 recipient cows. Clinical examination revealed an absence of horn buds for 3 of them. This status was subsequently confirmed by a non-Mendelian inheritance study based on Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA, USA) genotyping data. This combination of in vitro procedures allowed us to increase the number of affected fetuses from 15 to 43% (3/7) in the mosaic bull progeny. In addition, it enabled us to produce valuable material from a very limited resource to perform clinical and functional studies. Finally, it demonstrates the feasibility of a pre-implantation genetic diagnosis combined with freezing and transfer of IVP embryos.

Genomic tools have now become available for most livestock species and are being used routinely for marker-assisted selection in cattle. One major challenge in bovine selection is the possibility to detect multiple markers from biopsies of pre-implantation stage embryos which allows to transfer only selected embryos following genotyping. Preliminary studies have shown that 2 ng of DNA collected from 200 embryonic cells (hatched blastocyst) may be sufficient for genotyping based on few markers (<100). However, the present genotyping techniques are much more demanding in terms of DNA. The aim of this work was to test different in vitro culture conditions of biopsied cells issued from bovine blastocysts to produce a large number of cells for genotyping. Bovine embryos were produced in vitro according to a standard protocol (Menck M et al. 1997 Reprod. Nutr. Dev. 37, 141-150). Only grade 1 embryos were biopsied using a microblade under a stereomicroscope. Biopsies had from 5 to 10 cells. Biopsied embryos were in vitro cultured in B2 + 2.5% FCS seeded with VERO cells for 48 h to assess the survival rate. Individual biopsies were cultured in vitro in 4-well culture dishes (Nunc) coated with collagen type 1 at 39°C in a humidified air atmosphere and 5% CO2 under 3 medium conditions. Intact hatched Days 8 to 10 blastocysts were cultured under the same conditions as controls. In condition 1, 43 biopsies and 35 control blastocysts were cultured in DMEM/F12 + 10% FCS and 0.25% ITS (insulin, rransferrin, selenium). In condition 2, 30 biopsies and 35 control blastocysts were cultured in DMEM/F12 + 20% FCS supplemented with 1 mM sodium pyruvate, 1 μg mL-1 of heparin, and 1 μg mL-1 of FGF4. In condition 3, 30 biopsies and 43 control blastocysts were cultured in a complex medium composed of 30% of [DMEM/F12 + 20% FCS] and 70% [DMEM/F12 + 20% FCS conditioned medium using mitomycined VERO cells] supplemented with 1 mM sodium pyruvate, 1.5 μg mL-1 of heparin, and 1.5 μg mL-1 of FGF4 (adapted from Oda et al. 2006 Methods Enzymol. 419, 387-400). Medium was replaced every 3 days. Outgrowths were physically detached and isolated cells were cultured using condition 3. For further passages, monolayers were trypsinized (0.025%) and cells were analyzed by immunofluorescence using anti-cytokeratin 1-8 antibodies. After biopsy and 48 h of in vitro culture, 97.1% (100/103) of embryos survived. For all culture conditions, none of the biopsied cells attached to the coated dishes and no colony were observed after culture. Control intact blastocysts adhered and formed significantly lower rate of outgrowths for condition 1 v. 2 and 3: 77.1% v. 85.7% and 93%, respectively (P < 0.05). After several passages, 3 cell lines were produced and we observed a network of cytokeratin filaments by immunofluorescence suggesting an epithelial cell type for this network. These results show that production of a large number of cells from biopsies was not efficient enough for genotyping. However, the 3 tested culture conditions are favorable for the production and multiplication of cells from intact bovine blastocysts and condition 3 seems to be a suitable medium condition for embryonic cell culture.

Genome reprogramming is the ability of a nucleus to modify its epigenetic characteristics and gene expression pattern when placed in a new environment. Low efficiency of mammalian cloning is attributed to the incomplete and aberrant nature of genome reprogramming after somatic cell nuclear transfer (SCNT) in oocytes. To date, the aspects of genome reprogramming critical for full-term development after SCNT remain poorly understood. To identify the key elements of this process, changes in gene expression during maternal-to-embryonic transition in normal bovine embryos and changes in gene expression between donor cells and SCNT embryos were compared using a new cDNA array dedicated to embryonic genome transcriptional activation in the bovine. Three groups of transcripts were mostly affected during somatic reprogramming: endogenous terminal repeat (LTR) retrotransposons and mitochondrial transcripts were up-regulated, while genes encoding ribosomal proteins were downregulated. These unexpected data demonstrate specific categories of transcripts most sensitive to somatic reprogramming and likely affecting viability of SCNT embryos. Importantly, massive transcriptional activation of LTR retrotransposons resulted in similar levels of their transcripts in SCNT and fertilized embryos. Taken together, these results open a new avenue in the quest to understand nuclear reprogramming driven by oocyte cytoplasm.

In cattle, reliable methods for whole genome amplification (WGA) have been implemented for DNA pre-amplification and subsequent genotyping from embryo biopsy containing 5 cells or more. In France, these methods are now tested under field conditions. Several studies report pregnancy rates after direct transfer of biopsied frozen–thawed grade 1 embryos similar to that intact frozen ones. Even so, grade 2 and 3 embryos represent 25.5% of the transferable embryos (AETE data, 2007) and may limit the use of the above mentioned techniques if results are not satisfactory. The objective of this study was to investigate the impact of the embryo quality on the pregnancy rates after single direct transfer of biopsied frozen–thawed embryos. Embryos were collected on 12 donor cows after 15 sessions of superovulation treatment using FSH injected twice daily in decreasing doses over 4 days. Cows were inseminated at 12 and 24 hours after onset of estrus. Embryos were recovered 7 days post-insemination and evaluated according to IETS standards. Biopsies were performed on stage 4 to 7 grade 1 to 3 embryos using a microblade. Embryonic cells from the biopsy were dry deposited in microtubes and frozen before WGA (QIAGEN REPLI-g® Mini kit, Valencia, CA, USA) and multi-genotyping (GeneMapper software® – Applied Biosystems Europe). Each biopsied embryo was equilibrated for 10 min in 1.5 m Ethylene Glycol and then loaded into straw containing two columns of F1 medium separated by a central column of 1.5 m EG with the embryo. The freezing sequence was: –7°C directly; seeding; held for 10 min; 0.5°C min–1 until –35°C before plunging into liquid nitrogen. Embryos were thawed (straws 10 s in air and 20 s in water at 20°C) and directly transferred into synchronized recipient heifers. Pregnancy was diagnosed by ultrasonography at 35 and 90 days. Effect of embryonic stage and quality on pregnancy rates were analysed by log linear models (Proc CATMOD, SAS Institute Inc). A total of 58 embryos were micromanipulated. All grade 1 and 2 embryos were successfully biopsied and frozen and, 0.8% of grade 3 (2/25) were discarded due to their low quality after biopsy. No significant effect of embryo stage and quality on pregnancy rates was found after direct transfer (Table 1). These preliminary results suggest that high pregnancy rates can be achieved after direct transfer of biopsied frozen–thawed embryos of Grade 1, 2 and 3 allowing most of the embryos to be involved in the genotyping process. Table 1.Pregnancy rates following single direct transfer of biopsied frozen–thawed G1 to 3 bovine embryos This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) supported by FRT/ANR and Apis-Genes.

Genomic tools are now available for most livestock species and used routinely for marker-assisted selection (MAS) in cattle. The detection of a large number of markers that are widespread over the genome is generally limited by the amount of genomic DNA available in an embryo biopsy of a small size not to be detrimental to embryonic survival. Amplification of DNA from such a biopsy is then necessary. In this study, the efficiency of embryo genotyping for 45 microsatellites (MS) following whole-genome amplification (WGA) was evaluated from samples of a variable number of cells isolated from cattle embryos. In a second part, this work aims to test the reliability of the MAS method for 45 MS and 13 single nucleotide polymorphisms (SNP) from bovine embryo biopsies under field conditions. In experiment 1, in vitro bovine morulae (n = 10) were produced, and 1, 5, and 10 embryonic cells were removed from each morula. Cells were dry frozen in tubes before further processing. Whole-genome amplification was performed using the commercial Qiagen REPLI-g® Mini Kit according to the manufacturer instructions (Qiagen, Valencia, CA, USA). WGA solution was then diluted, processed by PCR with 45 markers, and the resulting data were genotyped with GeneMapper software® (Applied Biosystems Europe). Accuracy and reliability of genotyping were assessed using different samples of cells from the same embryo. In experiment 2, after superovulation (10 cows), bovine embryos were in vivo-produced and collected at day 6 or day 7 of pregnancy. Only grade 1 embryos were washed and biopsied using a microblade. Biopsied embryos were either frozen or transferred back to synchronized recipients. Individual biopsies were transferred as dry samples to the laboratory. Genomic DNA was amplified using WGA, and embryos were genotyped. The results of experiment 1 clearly indicate that a conventional biopsy of 5 to 10 cells was sufficient for multi-markers detection after whole-genome amplification as 98% of the 45 markers were detected compared to 45% of marker detection using 1 cell (P < 0.01). In experiment 2, from 123 collected embryos, 79 were classified as grade I or II transferable embryos (64.2%) and 57 were biopsied (34 were classified as stage 4–5 and 23 as stage 5–6, according to the IETS criteria). Using the stereomicroscopic analysis, 44 biopsies had a number of cells ranging from 4 to 7 (5.6 ± 1.4) and 13 biopsies from 8 to 10 (8.4 ± 1.6). Overall, at least 95% of markers (MS + SNP) were detected in 49.1% of biopsies (28/57). The total detection rate for SNP was significantly higher than for MS; 70.2% (40/57) v. 31.6% (18/57), respectively, (chi-square, P < 0.01). The detection rate of the markers was not significantly affected by the embryo stage or the biopsy size. Our results confirm that genotyping a large number of markers from biopsy samples after whole-genome amplification is possible under field conditions. A larger number of biopsies is required to assess the reliability of this method that may allow the development of MAS from early embryo. This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) with the financial support of FRT/ANR and Apis-Genes.