Effect of vitrification using the Cryotop method on the gene expression profile of in vitro-produced bovine embryos
ABSTRACT: Changes in gene expression induced by the Cryotop vitrification procedure in bovine blastocysts using Agilent EmbryoGENE microarray slides. Bovine in vitro-produced embryos at the blastocyst stage (144 to 156 hours post insemination) were vitrified using the Cryotop system and compared with non-vitrified (control) embryos. After vitrification, the embryos were warmed and cultured for an additional 4 hours. Embryos that re-expanded or developed to the expanded blastocyst stage were used for microarray analysis. Four pools of vitrified embryos were hybridized against four pools of control embryos in a dye-swap design.
Project description:Transcriptome analysis of in vitro-produced bovine 2-cell embryos according to their time to the first cleavage. Three time points were established in order to collect all the 2-cell embryos present at each moment to contrast only the two exteme embryo subpopulations: fast against slow. 4 biological replicates of fast (29.5 hpf) vs. slow (46 hpf) 2-cell embryos were contrasted. Each replicate was subjected to a dye-swap procedure.
Project description:The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared to in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker coloured cytoplasm which could be a consequence of impaired mitochondrial function. In this study, L-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breed collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also accounted for general appearance, lipid composition, mitochondrial activity and gene expression. Adding L-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. L-carnitine vs controls, in 4 replicates for each breed, with dye-swaps.
Project description:Study the effect of in vitro maturation system on the transcriptomics of bovine cumulus cells during incubation of first 6 hrs. 3 biological replicates of cumulus cells derived from cumulus oocyte complexes (COCs) after 6 hrs of in vitro matutaion(Ct-6H) vs. cumulus cells before maturation (Ct-0H, REFERENCE) were contrasted. Each replicate was subjected to a dye-swap procedure.
Project description:Study the effect of recombinant human FSH supplementation in the in vitro maturation medium for an incubation of 6 hrs on the transcriptomics of bovine cumulus cells. 3 biological replicates of cumulus cells derived from cumulus oocyte complexes (COCs) supplemented with FSH (CC-FSH) vs. cumulus cells without FSH (Ct-6H, REFERENCE) were contrasted. Each replicate was subjected to a dye-swap procedure.
Project description:Some embryos display better survival potential to cryopreservation than others. The cause of such phenotype is still unclear and might be due to cell damage during cryopreservation, resulting from over-accumulation and composition of lipids. In cattle embryos, in vitro culture conditions have been shown to impact the number of lipid droplets within blastomeres. So far, the impact of breed on embryonic lipid content has not yet been studied. In this study were compared the colour, lipid droplet abundance, lipid composition, mitochondrial activity, and gene expression of in vivo collected Jersey breed embryos which are known to display poor performance post-freezing and in vivo Holstein embryos which have good cryotolerance. Holstein in vivo day 6 embryos vs Jersey in vivo day 6 embryos: 4 replicates of each breed, with dye-swap.
Project description:Study the effect of forskolin, dipyridamol and IBMX (here called FID) supplementation in the in vitro maturation medium for an incubation of 6 hrs on the transcriptomics of bovine cumulus cells. 3 biological replicates of cumulus cells derived from cumulus oocyte complexes (COCs) supplemented with FID (CC-FID) vs. cumulus cells without FID (Ct-6H, REFERENCE) were contrasted. Each replicate was subjected to a dye-swap procedure.
Project description:Background The domestic pig is an important livestock species for meat production worldwide and is becoming an established biomedical research model. As a result, there is a strong interest in the factors that affect the efficient production of viable embryos and offspring in this species using either in vivo or in vitro production methods. A limited understanding of the molecular mechanisms involved in this critical physiological process has inhibited our ability to fully elucidate these factors. The use of next generation deep sequencing and microarray technology are powerful tools for delineation of molecular pathways during early embryonic development of mammals. Here, we report on the assessment of a porcine-embryo-specific microarray platform created from a large expressed sequence tag (EST) analysis generated by Roche/454 next-generation sequencing of cDNAs constructed from critical stages of in vivo or in vitro porcine preimplantation embryos. Results Two cDNA libraries constructed from in vitro and in vivo produced preimplantation porcine embryos were normalized and sequenced using the 454 Titanium pyrosequencing technology. Treatment of cDNA libraries with BAL 31 nuclease digestion resulted in a 2 fold improvement of sequencing quality compared with untreated libraries. Over one million high quality EST sequences were obtained from this process and used to create an augmented porcine genome catalogue. Using the resulting dataset the EMbryogene Porcine Version 1 (EMPV1) microarray was developed and is composed of 43,795 probes printed onto a 4 × 44 K Agilent array. Based on the initial probe sequences annotation, the EMPV1 featured 17,409 protein-coding, 473 pseudogenes, 46 retrotransposed, 2,359 non-coding RNA (snRNA, snoRNA, etc.), 4,121 splice variants in 2,862 genes and a total of 12,324 Novel Transcript Regions (NTR). After re-annotation, the total unique genes increased from 11,961 to 16,281 and 1.9% of them belonged to a large olfactory receptor (OR) gene family. Quality control of EMPV1 was performed using porcine cumulus–oocyte complexes (COC) as well as early developmental stages of embryos. This revealed an even distribution of ten clusters of spike-in control spots and array to array (dye-swap) correction was 0.97. Further bioinformatics analysis revealed that our microarray probes hybridized with more developmental related transcripts from embryonic labelled targets when compared to COC. Conclusions Using next-generation deep sequencing we have produced a large EST dataset to provide the selection of probe sequences for the development of the EMPV1 microarray platform. The quality of this embryo- specific array was confirmed with the high level of reproducibility using current Agilent microarray technology. Despite the current limitations for full NTR annotation, due to the incomplete porcine genome sequencing project, a significant number of NTR were annotated using Version 10 of porcine genome and human RefSeq RNA database to enrich the orthologous genes with unique gene symbol (GS) for Gene Ontology (GO) search. GO terms confirmed that many are related relevant developmental processes. With more than an estimated 20 thousands unique genes represented on the EMPV1, this platform will provide the foundation for future research into the in vivo and in vitro factors that affect the viability of the porcine embryos, as well as the effects of these factors on the live offspring that result from these embryos. Two biological samples.
Project description:Early bovine cleavage (day 0 to 3) was treated with high glucose (5mM) or control glucose (0.2mM) then total day 3 embryos were cultured in control condition until day 5 when gene expression was assessed in developping morula by microarray 4 replicates of 10 morulae/rep was produced for high glucose and control treatment. According to a dye swap design, 4 arrays were used, one array for each comparison between HG and control, i.e replicate 1 from HG were compared to replicate 1 from CTL, rep 2 from HG were compared to rep 2 from CTL, rep 3 HG vs rep 3 CTL, rep 4 HG vs rep 4 CTL.
Project description:The main objective of superovulatory treatment is to achieve multiple ovulations and produce multiple embryos. The technique is intended to rescue a cohort of follicles within a follicular wave that would otherwise regress. Superstimulory treatments change the intra-follicular and systemic hormonal milieu, but it is not clear how and to what extent treatment alters gene expression of follicular cells. In this study, a genome-wide bovine oligo-microarray was used to compare the gene expression of granulosa cells from post-LH preovulatory dominant follicle with those from follicles of the same status after superstimulatory treatment. Cows were allocated randomly to two groups (superstimulation group and control group, n=6 cows per group). A new follicular wave was induced by transvaginal ultrasound-guided ablation of follicles ≥5 mm in diameter, and a progesterone-releasing device (CIDR) was placed in vagina. The superstimulation group was given eight doses of 25 mg FSH im at 12-h intervals starting from the day of wave emergence (Day 0), whereas the control group was not given any FSH treatment. Both groups were given prostaglandin F2α im twice, 12 h apart, on Day 3 and the CIDR was removed at the second injection; 25 mg pLH was given im 24 h after CIDR removal, and cows were ovariectomized 24 h later. Granulosa cells were collected for RNA extraction, amplification and microarray hybridization. To translate microarray results to a physiological context, a list of differentially expressed transcripts were biologically annotated. A total of 190 genes were down-regulated and 280 genes were up-regulated in superstimulated group when compared with the reference (non-superstimulated control) group. straight comparison of superstimulation group (the treatment; group 1) versus non-superstimulated ( the reference; group 4) using 3 different aninals (biological replicates) in each group and performed dye swap. For example on array 1: group1 cow 1 versus group 4 cow 1 (cy3 vs cy5) and on array 4 is the dey swap group 4 cow 1 versus group 1 cow 1 (cy3 vs cy5).
Project description:In order to understand how in vitro culture affects embryonic quality, we analyzed survival and global gene expression in bovine blastocysts after exposure to increased oxidative stress conditions of IVC. A pro-oxidant agents that act extra-cellularly by promoting ROS production (0.01 mM 2,2'-azobis (2-amidinopropane) dihydrochloride [AAPH]) was added from days 3 to 7, and transcriptomic analysis was then performed in resulting blastocysts. Precisely, after in vitro maturation and fertilization, bovine zygotes were culture in vitro in normal condition, then at day 3, embryos were allocated into culture in control or supplemented with AAPH (0.01 mM) until day 7. At this time, blastocysts were harvested and analyzed. Our hypothesis was that AAPH treatment will affect blastocyst survival and gene expression associated with low embryo quality 4 replicates of 10 blast/rep was produced for AAPH and control treatment. According to a dye swap design with 2 colors, 4 arrays were used to compare AAPH (green) against CTL (red) and 4 arrays were used to compared AAPH (red) againt CTL (green).