Tesi sul tema "Bovine embryonic"

Thesis (Ph. D.) -- University of Maryland, College Park, 2008.
Thesis research directed by: Dept. of Animal and Avian Sciences. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
A acetilação das histonas é um dos principais mecanismos de reprogramação epigenética do genoma dos gametas a fim de estabelecer um estado totipotente para o desenvolvimento normal. O objetivo deste trabalho foi avaliar o efeito da utilização da Tricostatina A (TSA) sobre os níveis de acetilação das histonas e o desenvolvimento embrionário, avaliando-se a contagem do número de células da massa celular interna (MCI), trofectoderma (TE), e células totais, e os níveis da reação de imunocitoquímica para H3K9ac. Para isso, três experimentos foram delineados. No primeiro experimento, verificou-se que o grupo tratado com 5nM de TSA por 144h durante o cultivo in vitro (CIV) aumentou (p<0,01) o nível de H3K9ac em relação ao grupo controle. A taxa de clivagem e o desenvolvimento embrionário foram avaliados em um segundo experimento, e o grupo tratado com 5nM de TSA não apresentou diferença na produção de embriões comparado ao grupo controle. No experimento 3, o tratamento com a TSA não apresentou efeitos significativos em relação ao número de células da MCI, porém houve redução (p<0,01) tanto do número de células da TE quanto de células totais comparado ao grupo controle. Portanto, conclui-se que o tratamento com TSA não altera o desenvolvimento embrionário pré-implantacional de embriões fêmeas de bovinos, mostrando efeito negativo sobre o desenvolvimento dos embriões tratados
Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genome to establish a totipotent state for normal development. The aim of this work was to evaluate the use of Trichostatin A (TSA) on the levels of histone acetylation and embryonic development, the assessment of counting the cell number of inner cell mass (ICM), trophectoderm (TE), and total cells, and the levels of H3K9ac for immunocytochemical reaction. Therefore, three experiments were designed. In the first experiment, it was verified that the group treated with TSA for 5nM for 144h during in vitro culture (IVC) increased (p <0.01) the level of H3K9ac in the control group. Cleavage rate and embryo development were evaluated in a second experiment, and the group treated with 5nM TSA showed no difference in embryo production compared to control. In Experiment 3, treatment with TSA had no significant effect on the cell number of ICM, but decreased (p <0.01) both the cell number of TE and total cells as compared to control. Therefore, it is concluded that treatment with TSA does not alter the preimplantation embryonic development female bovine embryos, showing a negative effect on the development of embryos treated

Resumo: A acetilação das histonas é um dos principais mecanismos de reprogramação epigenética do genoma dos gametas a fim de estabelecer um estado totipotente para o desenvolvimento normal. O objetivo deste trabalho foi avaliar o efeito da utilização da Tricostatina A (TSA) sobre os níveis de acetilação das histonas e o desenvolvimento embrionário, avaliando-se a contagem do número de células da massa celular interna (MCI), trofectoderma (TE), e células totais, e os níveis da reação de imunocitoquímica para H3K9ac. Para isso, três experimentos foram delineados. No primeiro experimento, verificou-se que o grupo tratado com 5nM de TSA por 144h durante o cultivo in vitro (CIV) aumentou (p<0,01) o nível de H3K9ac em relação ao grupo controle. A taxa de clivagem e o desenvolvimento embrionário foram avaliados em um segundo experimento, e o grupo tratado com 5nM de TSA não apresentou diferença na produção de embriões comparado ao grupo controle. No experimento 3, o tratamento com a TSA não apresentou efeitos significativos em relação ao número de células da MCI, porém houve redução (p<0,01) tanto do número de células da TE quanto de células totais comparado ao grupo controle. Portanto, conclui-se que o tratamento com TSA não altera o desenvolvimento embrionário pré-implantacional de embriões fêmeas de bovinos, mostrando efeito negativo sobre o desenvolvimento dos embriões tratados
Abstract: Histone acetylation is one of the major mechanisms of epigenetic reprogramming of gamete genome to establish a totipotent state for normal development. The aim of this work was to evaluate the use of Trichostatin A (TSA) on the levels of histone acetylation and embryonic development, the assessment of counting the cell number of inner cell mass (ICM), trophectoderm (TE), and total cells, and the levels of H3K9ac for immunocytochemical reaction. Therefore, three experiments were designed. In the first experiment, it was verified that the group treated with TSA for 5nM for 144h during in vitro culture (IVC) increased (p <0.01) the level of H3K9ac in the control group. Cleavage rate and embryo development were evaluated in a second experiment, and the group treated with 5nM TSA showed no difference in embryo production compared to control. In Experiment 3, treatment with TSA had no significant effect on the cell number of ICM, but decreased (p <0.01) both the cell number of TE and total cells as compared to control. Therefore, it is concluded that treatment with TSA does not alter the preimplantation embryonic development female bovine embryos, showing a negative effect on the development of embryos treated
Orientador: Jeffrey Frederico Lui
Coorientador: Joaquim Mansano Garcia
Banca: Gilson Hélio Toniollo
Banca: Sandro Henrique Antunes Ribeiro
Mestre

Tese de Doutoramento em Ciências Veterinárias. Especialidade de Clínica
The objectives of this thesis were to evaluate steroidogenic and prostanoid embryo-maternal interactions leading to embryonic development and survival in cattle, and to evaluate therapeutic strategies at embryo transfer (ET) designed to enhance embryo survival. In vitro experiments (three experimental chapters) - bovine early (Day 7) embryos i) had transcription of genes coding for enzymes progesterone (P4) and of prostaglandins (PGs) synthesis pathways (StAR, P450scc,3β-HSD, PTGS2, PGFS, PGES); ii) produced these mediators (P4, PGF2α, PGE2) into culture medium; iii) had a significant increase in transcription levels of the above genes (except StAR) associated to first embryonic cellular differentiation; iv) derived from pre-pubertal oocyte donors had transcription levels of the above genes similar to those of embryos derived from post-pubertal cyclic heifers (except for 3β-HSD, which tended to be higher in embryos from cyclic heifers). Additionally, v) in a developed luteal cells (LC) co-culture model, LC induced an embryotrophic effect, significantly increasing blastocyst yield and quality; however, this embryotrophic effect was not associated with an increase in embryonic gene transcription or production of P4, PGF2α, PGE2; vi) Embryos co-cultured with LC did not exert a luteotrophic effect upon the cells; and vii) oil overlaying of culture wells exerted an embryotrophic effect, but absorbed P4 from culture medium. In vivo experiments (two experimental chapters) - novel in-vivo models considering poor developmental competence embryos (demi-embryos) and either sub-normal fertility recipients (lactating high-yielding dairy cow) or high fertility recipients (virgin dairy heifers) were used to evaluate the effect of hCG and carprofen treatment at embryo transfer on embryo survival and on plasma P4 and PSPB concentrations of recipients. Conclusions were that: i) treatment with hCG induced formation of secondary CL, increased plasma P4 concentrations, survival rate of demi-embryos and pregnancy rate of recipients (only in cows). Embryos were rescued beyond maternal recognition of pregnancy (MPR), but later embryonic survival, growth until implantation and placental PSPB secretion until Day 63 of pregnancy (only tested in cows) were not affected; ii) embryonic survival following MRP is not under direct dependency of maternal P4 concentrations; iii) treatment with carprofen had no significant effect on plasma P4 concentrations and embryonic survival, but decreased the luteotrophic effect of hCG.
RESUMO - Interações embrio-maternas relevantes para o desenvolvimento e sobrevivência embrionários em bovinos – papel da progesterona e das prostaglandinas - Os objetivos desta tese foram: avaliar interações embrio-maternas esteroidogénicas e prostanoides no desenvolvimento e sobrevivência embrionárias; testar estratégias terapêuticas na transferência embrionária (TE) com vista ao aumento da sobrevivência embrionária. Experiências in vitro (três capítulos experimentais) – embriões bovinos (Dia 7): i) revelaram transcrição de genes codificantes das enzimas das vias sintéticas da progesterona (P4) e PGs (PTGS2, PGFS, PGES, StAR, P450scc,3β-HSD); ii) produziram estes mediadores (P4, PGF2α, PGE2) para o meio de cultura; iii) apresentaram um aumento significativo dos níveis de transcrição destes genes (à exceção da StAR) associado à primeira diferenciação celular embrionária; iv) derivados de dadoras de oócitos pré-púberes revelaram níveis de transcrição dos genes mencionados similares aos de embriões de dadoras cíclicas (à exceção dos níveis de transcrição para a 3β-HSD, tendencialmente mais elevados em embriões provenientes de fêmeas cíclicas). Adicionalmente, v) num modelo de co-cultura de células lúteas desenvolvido, estas exerceram um efeito embriotrófico, aumentando significativamente a taxa de desenvolvimento e qualidade embrionárias; porém, este efeito não foi associado a aumento na transcrição génica ou produção de P4, PGF2α, PGE2; vi) Embriões em co-cultura com células lúteas não exerceram um efeito luteotrófico nas células; e vii) o uso de óleo mineral na cobertura dos poços de cultura exerceu um efeito embriotrófico, mas absorveu P4 do meio. Experiências in vivo (dois capítulos experimentais) – novos modelos in vivo - embriões de baixa competência de desenvolvimento (hemi-embriões) e recetoras sub-férteis (vacas leiteiras de alta produção) ou com fertilidade alta (novilhas leiteiras virgens) - foram usados na avaliação do efeito na sobrevivência embrionária e nas concentrações plasmáticas de P4 e PSPB das recetoras, de tratamentos, na TE, com hCG ou carprofen. Concluiu-se que: i) o tratamento com hCG induziu a formação de CLs secundários, aumentou as concentrações plasmáticas de P4, a taxa de sobrevivência dos hemi-embriões e as taxas de gestação das recetoras (em vacas). Os embriões foram resgatados para além do reconhecimento materno da gestação (RMG), mas a sobrevivência embrionária posterior, o crescimento até à implantação e a secreção placentária de PSPB até ao Dia 63 de gestação (testados em vacas) não foram afetados; ii) a sobrevivência embrionária após o RMG não está diretamente dependente das concentrações de P4 maternas; iii) o tratamento com o carprofeno não afetou significativamente as concentrações de P4 ou a sobrevivência embrionária, mas diminuiu o efeito luteotrófico da hCG.

Fertilization and cleavage of bovine embryos depend not only on maternal involvement, but also on the paternal contributions that involve more than just providing the haploid male genome. Therefore, the overall objective of this project was to determine the impact of morphologically abnormal spermatozoa on fertilization, subsequent embryonic development, and embryo quality at the cellular level. Four experiments used morphologically abnormal semen samples collected and cryopreserved from four Holstein bulls before (Pre) and after a scrotal insulation (PI) period of 48 h. Zygotes were cultured for 8 d when a developmental score was assigned to each embryo; subpopulations were subjected to either the TUNEL or caspase assays to determine apoptosis. In the final experiment pronuclear decondensation for presumptive zygotes was evaluated by differential interference contrast microscopy at 3 h time intervals from 6 to 18 h post in vitro insemination (hpi). Morphological evaluation of semen samples revealed a decrease (P < 0.01) in the percentages of normal spermatozoa in the PI samples in comparison with the Pre samples for Bulls I and Bull III (74 to 22.3% and 67.7 to 0.5 %, respectively) and the scrotal insulation effects persisted from the time of cleavage through blastocyst formation for Bulls I and III and corresponded with a similar decrease in blastocyst development for PI samples in experiment 1 regardless of which semen separation method was used. Likewise, the overall pronuclear decondensation rate for the PI zygotes of Bull I and III showed no increase over time and remained predominantly at PN1 stage (1.5 ± 0.17; 1.8 ± 0.22, respectively). In contrast, the development for Bull II and Bull IV were unaffected. The embryo quality assessment revealed that the caspase intensity increased significantly for both Bull I (217 ± 147) and Bull III (229 ± 98) for the PI embryo groups compared to those of Bull II (98 ± 115) and Bull IV (90 ± 111). In conclusion, the tested separation methods used seemed inadequate in their ability to provide potentially competent sperm for IVF. The decrease in embryonic development appears to be multifaceted and related to the changes in head shape morphology and we suggest the failure in normal pronuclear formation is associated with an absence of normal decondensation of the penetrating spermatozoon. The inability to consistently measure apoptosis in early stage embryos complicates the assessment of differences in embryo quality. These observations support the hypothesis of uncompensable seminal traits in IVF with abnormal spermatozoa and provide compelling evidence that the effect of morphologically abnormal spermatozoa occurred prior to cleavage, thus is manifested during the early stages of fertilization.
Ph. D.

The cells from the inner cell mass (ICM) of an early embryo have the potential to differentiate into all the different cell types present in an adult organism. Cells from the ICM can be isolated and cultured in vitro, becoming embryonic stem cells (ESCs). ESCs have several properties that make them unique: they are unspecialized, can self-renew indefinitely in culture, and given the appropriate cues can differentiate into cells from all three germ layers (ecto-, meso-, and endoderm), including the germline, both in vivo and in vitro. Induced pluripotent stem cells (iPSCs) can be generated from adult, terminally differentiated somatic cells by transient exogenous expression of four transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) present normally in ESCs. It has been shown that iPSCs are equivalent to ESCs in terms of morphology, gene expression, epigenetic signatures, in vitro proliferation capacity, and in vitro and in vivo differentiation potential. However, unlike ESCs, iPSCs can be obtained from a specific individual without the need for embryos. This makes them a promising source of pluripotent cells for regenerative medicine, tissue engineering, drug discovery, and disease modelling; additionally, in livestock species such as the bovine, they also have applications in genetic selection, production of transgenic animals for agricultural and biomedical purposes, and species conservancy. Nevertheless, ESC and iPSC lines that meet all pluripotency criteria have, to date, only been successfully produced in mice, rats, humans, and non-human primates. In the first part of this dissertation, we attempted reprogramming of three types of bovine somatic cells: fetal fibroblasts (bFFs), adult fibroblasts (bAFs), and bone marrow-derived mesenchymal stem cells (bMSCs), using six different culture conditions adapted from recent work in mice and humans. Using basic mouse reprogramming conditions, we did not succeed in inducing formation of ESC-like colonies in bovine somatic cells. The combination of 2i/LIF plus ALK5 inhibitor II and ascorbic acid, induced formation of colony-like structures with flat morphology, that occasionally produced trophoblast-like structures. These trophoblast-like vesicles did not appear when an inhibitor of Rho-associated, coiled-coil containing protein kinase 1 (ROCK) was included in the medium. We screened for expression of exogenous OSKM vector with RT-PCR and found upregulation of OSKM vector 24h after Dox was added to the medium; however, expression was sharply decreased on day 2 after Dox induction, and was not detectable after day 3. In a separate experiment, we induced reprogramming of bFF and bAFs using medium supplemented with 50% of medium conditioned by co-culture with the bovine trophoblast CT1 line. These cells expressed both OCT4 and the OSKM vector 24h after Dox induction. However, similar to our previous observations, both markers decreased expression until no signal was detected after day 3. In summary, we were unable to produce fully reprogrammed bovine iPSCs using mouse and human protocols, and the exact cause of our lack of success is unclear. It is possible that a different method of transgene expression could play a role in reprogramming. However, these ideas would be driven by a rather empirical reasoning, extrapolating findings from other species, and not contributing in our understanding of the particular differences of pluripotecy in ungulates. Our inability to produce bovine iPSCs, combined with the only partial reprogramming observed by others, justifies the need for in depth study of bovine pluripotency mechanisms, before meaningful attempts to reprogram bovine somatic cells to plutipotency are made. Therefore, we focused on getting a better understanding of bovine nuclear reprogramming. This would allow us to rationally target the specific requirements of potential bovine pluripotent cells. Cell fusion is a process that involves fusion of the membrane of two or more cells to form a multinucleated cell. Fusion of a somatic cell to an ESC is known to induce expression of pluripotency markers in the somatic nucleus. In the second part of this dissertation, we hypothesized that fusion of bFFs to mouse ESCs (mESCs) would induce expression of pluripotency markers in the bFF nucleus. We first optimized a cell fusion protocol based on the use of polyethylene glycol (PEG), and obtained up to 11.02% of multinucleated cells in bFFs. Next, we established a method to specifically select for multinucleated cells originated from the fusion of mESCs with bFFs (heterokaryons), using indirect immunofluorescence. With this in place, flow cytometry was used to select 200 heterokaryons which were further analyzed using RNA-seq. We found changes in bovine gene expression patterns between bFFs and heterokaryons obtained 24h after fusion. Focusing on the bovine transcriptome, heterokaryons presented upregulation of early pluripotency markers OCT4 and KLF4, as well as hypoxia response genes, contrasted with downregulation of cell cycle inhibitors such as SST. The cytokine IL6, known to increase survival of early embryos in vitro, was upregulated in heterokaryons, although its role and mechanism of action is still unclear. This indicates that the heterokaryon cell fusion model recapitulates several of the events of early reprogramming, and can therefore be used for further study of pluripotency in the bovine. The cell fusion model presented here can be used as a tool to characterize early changes in bovine somatic nuclear reprogramming, and to study the effect of different reprogramming conditions on the bovine transcriptome.
Ph. D.

Embryonic stem cells are pluripotent cells isolated from morula stage embryos or the inner cell mass of blastocyst stage embryos. They are capable of differentiating into tissues of all three primary germ layers. In recent years pluripotent cell lines have been created from somatic cell types using various methods, the primary method being viral transduction of exogenous Oct4, Sox2, Klf4, and c-Myc or Oct4, Sox2, Nanog, and Lin28 transgene constructs. The resulting cell lines are termed induced pluripotency stem cells, and are similar to embryonic stem cells in many ways. However, these cell lines are not acceptable for clinical applications due to the use of both modified viral vectors and insertion of exogenous transgenes in their production. Recently the small molecule RepSox, a TGF-ß pathway inhibitor, was used to replace Sox2 during cellular reprogramming of murine embryonic fibroblasts. We evaluated the effects of RepSox on expression of pathways related to pluripotency in murine embryonic fibroblast, murine embryonic stem, and bovine embryonic fibroblast cells. Each cell type was treated with RepSox for 72 hours and subjected to standard qPCR for gene expression analysis. PCR arrays specific to stem cell pathways were used to initially evaluate the effects of RepSox on candidate genes. A subset of genes was then selected for further analysis based on these initial results. We report that RepSox inhibition of the TGF-ß pathway in murine embryonic fibroblasts results in significant upregulation of components of the Wnt, Notch, and Hedgehog signaling pathways, all of which have been linked to stem cell maintenance. In addition, we observed significant upregulation of genes associated with embryonic, mesenchymal, stem cell, and neural cell lineages, indicating that RepSox may be useful in direct reprogramming of murine cells to other somatic cell types. RepSox treatment of murine embryonic stem cells did not result in consistent upregulation of Wnt, Notch, or Hedgehog pathway components, but did result in upregulation of Sox2 and Klf4 expression. Lastly, RepSox treatment of bovine embryonic fibroblasts did not result in the same effects as seen in murine fibroblasts, indicating a need for further analysis to determine the effects of RepSox on bovine cells.

The aging phenomena is a process to which all organisms eventually succumb. The universality of this phenomena suggests that there may be one overwhelming factor involved. The exact biochemical basis of aging is still unclear. Free radicals such as the superoxide radical (O₂⁻) and the hydroxyl radical (OH⁻), formed in biological oxidation reactions may be responsible for cellular aging. Because of the high reactivity of the O₂⁻ and the OH⁻ they can produce extensive damage to lipids, proteins, and nucleic acids. In this study we have developed an in vitro quiescent model using density dependent bovine embryonic lung fibroblast (BELF). The effect of this process on the antioxidant defense enzymes such as, the superoxide dismutases, catalase, glutathione reductase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase, was studied. We have also extensively monitored the levels of free radical in the cell by both direct and indirect methods. The results indicate that no significant (p<0.05) changes in the activity of any of the major antioxidant enzymes in our quiescent model. Significant increases were observed in the intracellular levels of lipofuscin (age pigment) with time, but no changes in the generation of free radicals were observed using electron spin resonance spectrometry, cytochrome c reduction or spectrofluorometric techniques (caution should be placed in statistical interpretation of the data because of the small sample size in some experiments). The transcriptional and translational controls of the one of the major antioxidant defense enzymes (manganese superoxide dismutase) in bovine embryonic lung fibroblasts, human pulmonary artery endothelial cells (HPAE) and bovine PAE cell lines were also studied. Our preliminary data suggest that inhibitors of protein and RNA synthesis both cause a significant decrease in the induction of the manganese SOD in bovine pulmonary endothelial cells.
Master of Science

Lors de la fécondation, le sperme et l'ovule s'unissent pour former un zygote totipotent. Initialement, le zygote est transcriptionnellement inactif. Au cours des premiers clivages a lieu la mise en route du génome embryonnaire (EGA) et le développement passe alors sous le contrôle de l’information embryonnaire (au stade 8-16-cellules chez le bovin). Cette transition d’un contrôle maternel à un contrôle embryonnaire est appelée « maternal to embryonic transition (MET) ». De la même façon, lors du transfert nucléaire (clonage), un noyau de cellule somatique placé dans un ovocyte énucléé devient totipotent. Ce processus est appelé «reprogrammation nucléaire somatique?». En fait, la reprogrammation nucléaire lors du clonage est équivalente à la MET, toutefois, le clonage est très peu efficace. Les objectifs de cette étude chez les bovins sont a) d'explorer le processus de reprogrammation lors de la MET dans des embryons fécondés in vitro (FIV) et b) d’estimer l'efficacité de la reprogrammation génique après le transfert nucléaire lors du clonage. Nous émettons l'hypothèse que l'acquisition d'un profil d'expression génique correct pourrait être prédictif d’un potentiel de développement à terme de l'embryon, et pourrait être évalué dès juste après l'activation du génome embryonnaire (EGA) chez les bovins. Nous avons développé notre travail selon deux axes a) des analyses globales d'expression génique utilisant une puce dédiée à l’EGA et b) l’analyse du profil d'expression de gènes candidats par qRT-PCR dans les embryons fécondés et clonés. Dans un premier temps nous avons optimisé le protocole d'amplification d'ARNm pour l'analyse du transcriptome de matériels rares. Puis nous avons fait l'analyse du transcriptome avant et après EGA d’embryons issus d’ovocytes prélevés sur des vaches phénotypées comme « bonnes » ou « mauvaises » donneuses d’embryons. En outre, ces ovocytes ont été maturés soit in vivo soit in vitro. Nos analyses montrent que l'effet individuel est plus important que l'effet « bonne ou mauvaise donneuse » ou même que l’effet « conditions de maturation ». Nous avons ensuite analysé les expressions géniques de 5 types d'embryons clonés ayant différents potentiels de développement à terme en fonction de la lignée cellulaire utilisée comme source de cellules donneuses. Globalement, leur expression génique est proche de celle de morulae FIV, mais quelques gènes présentent une expression différente. Ces gènes varient avec la lignée de cellules donneuses et leur nombre n’est pas lié à l’aptitude au développement à terme. L’analyse d’un lien éventuel entre leur nature et cette aptitude devra être poursuivie. Dans un deuxième temps, nous avons analysé les profils d'expression spatio-temporelle des transcrits et des protéines des gènes de pluripotence (OCT4, SOX2 et NANOG) et les niveaux d'ARNm de certains de leurs cibles dans les ovocytes et les embryons précoces chez le bovin. Les profils d'expression de ces gènes ont aussi été analysés dans des embryons clonés présentant différents potentiels de développement à terme. Nos résultats montrent que (1) la triade de gènes de pluripotence n'est probablement pas impliquée dans l’EGA bovine. (2) les transcrits et protéines de SOX2 et de NANOG sont restreints au lignage pluripotent plus tôt que ceux de OCT4, (3) les embryons à faible taux de développement à terme ont un taux de transcription plus élevé, néanmoins, l’équilibre précaire entre les gènes de pluripotence est maintenue. Cet équilibre pourrait permettre un développement normal in vitro, mais le taux de transcription plus élevé pourrait avoir des conséquences délétères sur le développement ultérieur
In natural fertilization, sperm and ovum unite to form a totipotent zygote. Initially, the zygote is transcriptionally inactive and after few cleavages (8-16-cell stage in bovine) embryonic genome activation (EGA) takes place and embryo shifts from maternal to embryonic control, the process called maternal to embryonic transition (MET). Likewise, in nuclear transplantation (cloning) a somatic cell nucleus achieves totipotency when placed in an enucleated oocyte, the process called “nuclear reprogramming”. In fact, nuclear reprogramming in cloning experiments is equivalent to MET; however, this process is afflicted with low efficiency. The objectives of this study in bovine were a) to explore the process of MET reprogramming of in vitro fertilized (IVF) embryos and b) to estimate the efficiency of gene reprogramming after nuclear transfer in animal cloning. We hypothesized that the acquisition of a proper gene expression pattern could herald development potential of the embryos, which could be assessed as early as morula stage or after embryonic genome activation (EGA) in bovine. Here, we opted for a study plan consisting of two axes a) global gene expression analysis using an EGA-dedicated microarray and b) candidate gene expression profiling through qRT-PCR in the fertilized and cloned bovine embryos. Firstly, we optimized the protocol of mRNA amplification for transcriptome analysis which generates antisens-RNA (aRNA). Then we did transcriptomic analysis of the 4-cell and morulae derived from two genotypes having better and two genotypes having poorer in vitro embryonic development potentials. In addition, these oocytes were either matured in vivo or in vitro. We observed that the effect of individual genotype was more important than the effect of the phenotypic category (poorer or better) or conditions of oocyte maturation. Furthermore, we explored the expression patterns of 5 types of cloned embryos having different full term developmental potentials depending upon the donor cell line used. Their genes expression patterns closely resembled to the IVF morulae, except for few genes which present differences. These genes vary with the cell line used as somatic cell donor for SCNT and the number of these deregulated genes did not increase with the poorer developmental potential of the cloned embryos. The analysis of an eventual correlation between the potential for embryonic development to term and nature of the deregulated genes should be addressed. Secondly, we charted quantitative and/or qualitative spatio-temporal expression patterns of transcripts and proteins of pluripotency genes (OCT4, SOX2 and NANOG) and mRNA levels of some of their downstream targets in bovine oocytes and early embryos. Furthermore, to correlate expression patterns of these genes with term developmental potential, we used cloned embryos, instead of gene ablation, having similar in vitro but different full term development rates. We chose these genes to be analysed since pluripotency genes are implicated in mouse embryonic genome activation (EGA) and pluripotent lineage specification. Moreover, their expression levels have been correlated with embryonic term development. Our findings affirm: first, the core triad of pluripotency genes probably is not implicated in bovine EGA since their proteins were not detected during pre-EGA phase, despite the transcripts for OCT4 and SOX2 were present. Second, an earlier ICM specification of SOX2 and NANOG makes them better candidates of bovine pluripotent lineage specification than OCT4. Third, embryos with low term development potential have higher transcription rates; nevertheless, precarious balance between pluripotency genes is maintained. This balance presages normal in vitro development but, probably higher transcription rate disturbs it at later stage that abrogates term development

Até o momento a literatura não determinou qual o melhor estadio de maturação (imaturo ou maduro) para que o oocito mantenha sua competência para o desenvolvimento reprodutivo após a criopreservação. O objetivo deste estudo foi determinar em qual estadio meiótico (imaturo -VG (vesícula germinativa) ou maduro- MII (metáfase II)) o oócito é menos susceptível ao dano na criopreservação, utilizando modelo experimental bovino. Foram utilizados ovários de vacas abatidas em matadouro, após a aspiração dos folículos, os oócitos imaturos (VG) foram selecionados para a maturação in vitro e vitrificação, e foram divididos em três grupos: 1) oócitos maturados in vitro e não submetidos à vitrificação (CONTROLE); 2) oócitos vitrificados imaturos (VG), descongelados e submetidos à maturação in vitro (CRIO-MIV); 3) oócitos maturados in vitro (MII), vitrificados e descongelados (MIV-CRIO). Os oócitos foram avaliados quanto a: a)maturação nuclear pela técnica de orceína acética; b) integridade da zona pelúcida (ZP) através de microscopia de polarização; c) viabilidade oocitária pela técnica de DEAD-LIVE; d) desenvolvimento embrionário (taxa de clivagem, produção e eclosão de blastocistos) através da fertilização in vitro (FIV) e ativação partenogenética (AT). Não houve diferença na capacidade de maturação nuclear entre os oócitos frescos e descongelados no grupo CRIO-MIV (p=0,23). Em relação à zona pelúcida a totalidade dos oócitos (100%) nos três grupos apresentou leitura de zona pelúcida positiva, não havendo correlação com evolução embrionária posterior. Na análise de viabilidade celular pelo DEAD-LIVE verificou-se que houve redução da viabilidade do grupo MIV-CRIO (27%) quando comparado com controle (84%) (p<0,0001). Na análise do potencial de desenvolvimento embrionário o grupo controle apresentou melhores taxas de clivagem após FIV (80%) e AT (58%), do que os grupos CRIO-MIV (28%; p<0,0001; 28%; p=0,0002, respectivamente) e MIV-CRIO (26%; p<0,0001; 22%, p<0,0001,respectivamente). As taxas de formação de blastocisto e eclosão após FIV nos grupos CRIO-MIV, MIV-CRIO e após AT no grupo MIV-CRIO foram nulas. Houve a produção e eclosão de apenas um blastocisto no grupo CRIO-MIV após AT. No modelo experimental utilizado, o procedimento de vitrificação comprometeu parcialmente a viabilidade dos oócitos medida pela técnica de DEAD- LIVE e completamente o desenvolvimento embrionário subseqüente, independente do estadio de maturação meiótica (VG ou MII) durante a criopreservação. No entanto, oócitos vitrificados em estadio de VG e submetidos à MIV foram meioticamente competentes e progrediram até o estadio de MII, sugerindo que o dano não compromete a capacidade de maturação nuclear do oócito. Este estudo não conseguiu determinar qual o melhor estadio meiótico oocitário para criopreservação, já que os dois estadios meióticos (VG e MII) se mostraram igualmente prejudicados pela criopreservação em relação à capacidade reprodutiva.
Until the present literature has not achieved a consensus regarding the best maturation stage for oocyte to maintain their reproductive capacity after cryopreservation. The aim of this study was to determine, using an experimental bovine model, in which stage of development (VG stage, immature, or MII stage, post-maturation in vitro) the oocyte is less susceptible to damage during cryopreservation. Immature oocytes (VG) from the ovaries of slaughtered cows were selected for in vitro maturation or vitrification and divided into three groups. The first group (CONTROL) consisted of immature oocytes, matured in vitro without vitrification; the second group (CRYO-IVM) consisted of vitrified immature oocytes thawed and submitted to in vitro maturation; and the third group (IVM-CRYO) consisted of matured in vitro oocytes submitted to vitrification and thawing. The oocytes were evaluated for: nuclear maturation by acetic orcein staining; integrity of the zona pellucida using a polarized microscope; cell viability by the Dead-Live technique; and embryo development (cleavage, production and hatching rate) by in vitro fertilization and parthenogenetic activation. There was no difference in capacity of nuclear maturation between fresh and thawed oocytes (p=0.23). Regarding the zona pellucida (ZP), all oocytes (100%) of all three groups (control, CRYO-IVM and IVMCRYO) presented a positive ZP reading, with no correlation with later embryo evolution. DEAD-LIVE analysis of cell viability revealed reduction of viability in the IVM-CRYO group (27%) compared to control (84%) (p<0.0001) and to the CRYO-IVM group (56%) (p=0.017), with no difference between the last two groups (p=0.055). Analysis of the potential for embryo development by means of in vitro fertilization showed that the control group presented better cleavage and blastocyst formation rates than the CRYO-IVM (p<0.0001 and p<0.0001, respectively) and the IVM-CRYO (p<0.0001 and p=0.0004, respectively) groups. Analyzing the potential for embryo development the control group presented better cleavage by means of in vitro fertilization (80%) and parthenogenetic activation (58%) than the CRYOIVM (28%; p<0,0001; 28%; p=0,0002, respectively) and the IVM-CRYO groups (26%; p<0,0001; 22%, p<0,0001,respectively) Analysis of blastocyst formation rates and hatching after FIV and AT in CRYO-IVM and IVM-CRYO groups were null. Vitrification of bovine oocytes causes great impairment of their reproductive capacity regardless of the stage of maturation at the time of freezing. However the vitrified immature oocytes submitted to IVM maintained their capacity of nuclear maturation, as they achieved MII stage. This study was not able to determine which stage was better in reducing crio damage, as both stages (VG and MII) presented equally impaired by the process.

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Due to the importance of in vitro embryo production (IVEP) to accelerate genetic improvement is important the search for technological innovation that can enhance the in vitro embryo development. Mouse embryonic fibroblasts (MEFs) have been widely used as a feeder layer to support embryonic stem cells due to their release of growth factors. Mesenchymal stem cells (MSCs) from different sources were also found to release bioactive factors that can support cell growth. This study aims to investigate the effect of co-culture of MSC from rat bone marrow or MEF as a feeder source for in vitro production of bovine embryos. Cumulus oophorus complexes were matured into three groups: control (CTRL), co-culture with monolayer of mesenchymal cells of rats (MSC) or co-cultured with monolayer of embryonic fibroblasts of mice (MEF). Fertilization was performed in control condition for all groups, and in vitro fertilized embryos were cultured from fourth day on in CTRL, co-culture with MSC or co-cultured with MEF, so that the following groups were performed: (CTRL / CTRL) - maturation and embryo development in CTRL conditions; (CTRL / MSC) - maturation in CTRL and embryo development with MSC from the fourth day on after the beginning of the in vitro embryo culture; (CTRL / MEF) - maturation CTRL and embryo development with MEF from the fourth day on after the beginning of the in vitro embryo culture; (MSC / CTRL) - MSC during maturation and embryo development in CTRL; (MSC / MSC) - maturation and embryo development in MSC from the fourth day on after the beginning of the in vitro embryo culture; (MEF / CTRL) - maturation and embryo development in MEF and CTRL (MEF / MEF) - maturation and embryo development in MEF from the fourth day on after the beginning of embryo development in vitro. No significant difference was found among the oocytes matured in CTRL, MSC and MEF conditions for metaphase II and apoptosis rates and for cleavage rate in embryos at 4th day after the beginning of the in vitro culture. The number of cells in the inner cell mass, trophoblast cells, apoptotic cells and total cells were similar (P> 0.05) in the embryos from all experimental groups. The rates of blastocyst formation, expanded, hatched and the total of blastocysts did not differ among experimental groups (P> 0.05) at 7th day of embryo development. At eighth day of embryo culture we observed a difference (P <0.05) in hatched blastocyst rate which was higher in the CTRL /CTRL group when compared to MSC/MSC group, however, the proportion of blastocyst, expanded and total blastocysts was not different (P> 0.05). We conclude that there was no significant improvement in bovine embryo development using co-cultures of MSC from rats or MEF when compared to control culture system. However more studies investigating the use of stem cells from other sources or their conditioned medium are needed to better understand the effect of these cells on embryonic development.
Diante da importância da produção in vitro de embriões (PIVE) na expansão do melhoramento genético, é importante a busca de inovações tecnológicas que possam potencializar o desenvolvimento embrionário in vitro. Fibroblastos embrionários de camundongo (MEFs) têm sido amplamente utilizados como camada alimentadora para suportar as células-tronco embrionárias, devido à sua liberação de fatores de crescimento. As células-tronco mesenquimais (MSCs) identificadas a partir de diferentes fontes, também liberam fatores bioativos que possam suportar o crescimento celular. Este estudo tem como objetivo investigar o efeito da co-cultura de MSC de medula óssea de rato ou MEF como fonte alimentadora na produção in vitro de embriões bovinos. Complexo cumulus oophorus foram maturados em três grupos distintos: Controle (CTRL), co-cultura com monocamada de células mesenquimais de ratos (MSC) ou co-cultura com monocamada de fibroblastos embrionários de camundongos (MEF). A fecundação foi realizada em condição controle para todos os grupos e os embriões fecundados in vitro foram também cultivados a partir do quarto dia em CTRL, co-cultura com MSC ou co-cultura com MEF, formando os seguintes grupos experimentais: (CTRL/CTRL) – maturação e cultivo embrionário em condições CTRL; (CTRL/MSC) – maturação em CTRL e cultivo embrionário em MSC a partir do quarto dia após o início do cultivo embrionário in vitro; (CTRL/MEF) – maturação em CTRL e cultivo embrionário em MEF a partir do quarto dia após o início do cultivo embrionário in vitro; (MSC/CTRL) – maturação em MSC e cultivo embrionário em CTRL; (MSC/MSC) – maturação e cultivo embrionário em MSC a partir do quarto dia após o início do cultivo embrionário in vitro; (MEF/CTRL) – maturação em MEF e cultivo embrionário em CTRL e (MEF/MEF) – maturação e cultivo embrionário em MEF a partir do quarto dia após o início do cultivo embrionário in vitro. Nenhuma diferença significativa foi encontrada entre os oócitos dos grupos CTRL, MSC e MEF, na taxa de estruturas em metáfase II, apoptose e clivagem nos embriões de 4 dias após o início do cultivo in vitro. O número de células da massa celular interna, células do trofoblasto, células em apoptose e células totais foram iguais (P>0,05) entre os embriões dos diferentes grupos experimentais. As taxas de embriões em estágio de blastocisto, blastocisto expandido, blastocisto eclodido e blastocistos totais dos grupos experimentais não se diferiram (P>0,05) no sétimo dia de cultivo embrionário. No oitavo dia de cultivo embrionário houve diferença (P<0,05) da taxa de blastocisto eclodido, sendo maior no grupo CTRL/CTRL quando comparado ao grupo MSC/MSC; no entanto, a proporção de blastocisto, blastocisto expandido e blastocistos totais não foram diferentes (P>0,05) entre os grupos experimentais. Concluímos que não houve melhora significativa no desenvolvimento embrionário bovino utilizando co-culturas com MSC de ratos ou MEF de camundongos, quando comparado com sistema de cultura controle. Entretanto, mais estudos investigando o uso de células-tronco de outras fontes ou seu meio condicionado são necessários para se entender melhor o efeito destas células no desenvolvimento embrionário.

Les embryons bovins produits in vitro sont plus sensibles à la cryoconservation que ceux produits in vivo, en partie à cause de leur contenu lipidique, triglycérides et phospholipides. L’objectif de ce travail visait à comprendre les mécanismes moléculaires responsables de cette différence. Le profil transcriptomique de gènes impliqués dans le métabolisme lipidique a été établi. Le niveau d'expression génique de l’adipophiline obtenu indique qu’il peut être un marqueur spécifique de l'accumulation des triglycérides et de la cryorésistance des embryons. Ainsi, l’accumulation des triglycérides pourrait être liée à une absence de dégradation des lipides et non à une synthèse de novo uniquement. L’ajout d’acides gras polyinsaturés, C18:2, C18:3 ou DHA dans le milieu de développement, a régulé l'expression génique de SCD1 et de FADS2, deux enzymes qui désaturent les lipides, et ce, probablement via la régulation de SREBP1, ce qui pourrait être en lien direct avec les modifications de la balance acides gras saturés / insaturés et jouer sur la fluidité membranaire et la cryorésistance
In vitro produced embryos are more sensitive to cryopreservation than those in vivo derived, partly because of their fat content, triglycerides and phospholipids. The objective of this work was to understand the molecular mechanisms responsible for this difference. mRNA expression of genes involved in lipid metabolism has been established. Results of adipophilin mRNA level indicates that it maybe a specific marker for triglycerides accumulation and embryo cryorésistance. Thus, triglyceride accumulation could be related to a lack of lipids degradation rather than new lipids synthesis only. Polyunsaturated fatty acids supplementation, C18: 2 C18: 3 or DHA in culture media regulated mRNA expression of SCD1 and FADS2, two enzymes involved in lipids desaturation, probably through SREBP1 regulation, which could be directly linked to changes in the balance of saturated / unsaturated fatty acids and could contribute to change membrane fluidity and embryo cryoresistance

La production d'embryon in vitro (PIV) à partir d'ovocytes immatures implique trois étapes successives: la maturation (MIV), la fécondation (FIV) et le développement in vitro (DIV) et permet d'obtenir des embryons capables de donner une gestation après transfert. La maturation et, plus précisément, ses aspects cytoplasmiques dont le potentiel au développement après des ovocytes FIV et DIV reste son seul critère d'évaluation, représente une des étapes les plus limitantes de la PIV. L'effet de facteurs somatiques lors du DIV dans du milieu SOF (synthetic oviduct fluid) sur le taux et la qualité des blastocytes ont été testés. Pour la MIV, le milieu de base, permet seul un bon taux de développement, cependant, l'addition de sérum a provoqué le taux de développement. Récolté dans des follicules de taille et de qualité (degré d'atrésie) connues, le FF a provoqué le taux de développement (sauf le FF provenant de follicules en début d'atrésie). Des facteurs de croissance impliqués dans l'action du sérum et du FF ont été testés, sans succès pour les facteurs de la famille de l'insuline (insuline, insulin-like growth factor I et II) mais l'EGF (epidermal growth factor) a eu un effet positif. Des résultats récents montrent que l'origine peut déterminer la qualité ovocytaire (compétence in vitro). Un système pour la PIV à partir des ovocytes/embryons cultivés individuellement permet l'analyse de l'influence de caractéristiques ovocytaires mais pas à la qualité folliculaire. Le développement embryonnaire après PIV est influencé par la qualité de la maturation cytoplasmique des ovocytes qui elle-même dépend des conditions de MIV et de l'origine de l'ovocyte

A partir de 7000 ovaires récoltés aux abattoirs, au cours de 100 séances, 21042 ovocytes ont été récoltés produisant après fécondation 6422 blastocystes, afin de mettre au point un milieu de culture d’embryons entièrement synthétique. Le sérum de veau fœtal (SVF) et l’albumine sérique bovine (BSA) ont été remplacés par une association de facteurs de croissance (IGF-I, IGFII, bFGF, TGF-?1 et PDGF-BB) et cytokines (GMCSF et LIF) additionnée successivement de différentes molécules : ITS (insuline, transferrine, sélénium), PVP (polyvinylpyrrolidone), tween80, albumine recombinante et acide hyaluronique. Finalement l’association suivante donne les mêmes résultats que le milieu témoin par rapport au nombre de cellules du bouton embryonnaire et du trophoblaste et le taux de survie après décongélation, avec une amélioration du taux de blastocystes au 7ème et 8ème jour après fécondation ; FC et CYK : IGF-I, IGF-II, bFGF, GM-CSF, PDGF-BB, LIF, 50 ng/ml et TGF-?1, 100 ng/ml, additionné de 0. 15% d’albumine recombinante et 0. 5mg/ml d’acide hyaluronique. Les embryons obtenus ont permis d’obtenir 4 gestations sur 7 embryons transférés en frais et 1 gestation sur 3 après congélation.

Au cours des dernières années, la production laitière a été en croissance constante en raison de plusieurs facteurs tels que l’utilisation d’individus plus performants. L’augmentation du nombre d’enregistrements de vaches de la race Jersey confirme l’intérêt économique des producteurs pour cette race en raison de la production élevée de protéines et de gras du lait par rapport aux autres races. Cependant, les embryons de la race Jersey ont montré des difficultés dans le processus de cryopréservation lequel peut aider à une commercialisation massive de matériel génétique de cette race. Il a été observé que la race Jersey présente un faible taux de gestation lors du transfert des embryons après la cryopréservation en comparaison aux embryons de la race Holstein. Nous proposons que cette différence entre les deux races bovines soit due à des caractéristiques spécifiques de la race au niveau du phénotype et du génome. Dans un premier temps, les résultats de cette étude ont mis en évidence des différences au niveau du phénotype et du profil lipidique et génomique de l’embryon Jersey. Celui-ci est caractérisé par un contenu de lipides associé à une faible fonction mitochondriale laquelle déterminera le faible succès à la cryopréservation. Par la suite, nous avons évalué au niveau du phénotype et de la génomique, l’utilisation de la L-carnitine dans le milieu de culture in vitro afin de compenser cette caractéristique des embryons de la race Jersey. Cette étude sur la supplémentation de L-carnitine a permis d'identifier une faible réponse chez les embryons Jersey qui est expliqué par le faible effet de la L-carnitine sur l’activité mitochondriale. Afin de définir l’impact de la fonction mitochondriale sur la viabilité de l’embryon lors de nos études, nous avons mis au point une méthode pour compenser la dysfonction mitochondriale sur le développement embryonnaire chez le bovin. Enfin, l’application de la vitamine K2 dans le milieu de culture in vitro montre un impact positif sur la fonction mitochondriale qui a mené à des changements phénotypiques et génomiques chez les embryons bovins. En conclusion, ce projet a permis de caractériser et d’identifier la race comme un facteur qui limite la cryopréservation des embryons et peut influencer sur le métabolisme embryonnaire au niveau des mitochondries. La fonction mitochondriale est une caractéristique importante sur le développement embryonnaire bovin qui peut ouvrir sur des perspectives d’amélioration de la viabilité embryonnaire.
For the past decades, milk production has been increasing due to several factors such as the use of high genetic merit individuals. In this regard, Jersey cows have been of interest for the producers because of high protein and fat indexes in their milk compared to others breeds. However, there are some challenges associated with Jersey particularly poor results using embryo cryopreservation which could help to massively commercialize the genetic material of this breed. It was observed that the Jersey breed have low pregnancy rates following embryo transfer of cryopreserved Jersey embryos compared to the Holstein breed. Here, we hypothesised that those differences between these two breeds in embryo cryopreservation are due to specific phenotypic and genotypic characteristics at the embryo level. Initially, the results of this study showed differences on the phenotype, lipid profile and genomic differences of Jersey embryo characterized by the higher lipid droplets content associated with low mitochondrial function which will determine the low success with cryopreservation. Subsequently, we assessed the phenotype and genotype of embryos using L-carnitine supplementation in the in vitro embryo culture medium in order to compensate those characteristics in Jersey embryos. The results of this study revealed moderate beneficial effects of L-carnitine supplementation in Jersey embryos through low effect of L-carnitine on mitochondrial activity. To define the impact of mitochondrial function on the embryo viability during our study, we developed a method to compensate the mitochondrial dysfunction during early embryo development in bovine model. To do that, Vitamin K2 supplementation in the in vitro embryo culture medium was applied which showed a positive effect on the mitochondrial function leading to satisfactory phenotypic and genotypic changes in the embryos. In conclusion, this study resulted in identification and characterization of the cattle breeds effects as a critical factor on cryopreservation performance and embryonic metabolism of the mitochondria. Our results emphasized that mitochondrial function is an important feature of embryonic development in cattle, which can provide opportunities to improve embryonic viability.

L’oviducte joue un rôle central dans le transport et la préparation des gamètes, la fécondation et le développement précoce. Un dialogue embryo-maternel s’établit pour assurer le succès du développement de l’embryon et de son transport vers le site d’implantation. L’objectif principal de ce travail était de confirmer l’existence d’un dialogue moléculaire et fonctionnel précoce grâce à la coculture d’embryons bovins produits in vitro sur des cellules épithéliales tubaires bovines (BOEC). Nous avons montré que les BOEC ont un effet important sur le développement embryonnaire précoce, particulièrement pendant les 4 premiers jours. Cet effet ce traduit par un clivage accéléré, une modulation des gènes exprimés après l’activation du génome embryonnaire, un taux plus élevé de développement au stade de blastocyste et une adaptation de l’expression génique de ces blastocystes. En retour, les embryons induisent dans les BOEC, des changements d’expression de facteurs impliqués dans la réponse à l’interféron. Une spécificité régionale des profils d’expression a également été observée dans l’oviducte
The oviduct plays a pivotal role in gametes transport and final capacitation, as well as in fertilization and early embryo development. An embryo-maternal communication takes place to ensure the successful early embryo development and transport towards its implantation site. The principal aim of this research was to confirm the existence of such early embryo-maternal molecular and functional dialogue using bovine oviduct epithelial cells (BOEC) as coculture to support the development of in vitro produced bovine embryos. We showed that BOEC had an important effect on early embryo development, especially during the first 4 days. This effect translates into accelerated cleavage kinetics, modulation of gene expression after embryonic genome activation, increased rate of embryo development to the blastocyst stage and improved gene expression profile. Moreover the embryos are triggering a BOEC response by upregulating genes related to interferon signaling. A regional specificity of gene expression profile in the oviduct has also been detected

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The objective of this study was to evaluate the addition of antioxidants in different cryopreservation diluents on bovine semen quality after thawing and the in vitro production of embryos. First, the effects of adding different concentrations of glutathione (1.5 and 2.5 mM) and melatonin (0.5 and 1.0 mM) in egg yolk semen cryopreservation diluents (Bovimix®) and soy lecithin (Andromed®) were evaluated. The results showed that the addition of the antioxidant glutathione did not improve the maintenance of sperm characteristics of bovine semen. Melatonin had a negative effect on the spermatic parameters evaluated. The diluent Andromed® obtained better parameters of spermatic kinetics when compared with the diluent Bovimix®. However, in relation to the membrane integrity analyzes of the Bovimix® diluent showed better performance in the maintenance of these parameters. The effect of the cryopreservation diluents and the addition of glutathione in the vitro production of bovine embryos (PIVE) was then evaluated. The rate of cleavage, blastocyst rate and hatching were evaluated eight and nine days after IVF, as well as the correlation between sperm characteristics and PIVE. The diluent Andromed® provided a better cleavage rate, with no effect of the addition of glutathione in it. However, the addition of 2.5 mM glutathione in the Bovimix® diluent improved the cleavage rate. There was a significant correlation of high and low magnitude between some sperm characteristics with the rate of cleavage. It was concluded that glutathione did not improve sperm viability. Melatonin worsened the maintenance of sperm characteristics. Andromed® diluent was more efficient in the in vitro production of bovine embryos with no glutathione effect observed in this diluent. The addition of 2.5 mM glutathione in the egg yolk Bovimix® diluent gave a higher cleavage rate. In vitro evaluation of semen quality is not the best method to predict the fertilizer potential of the same in PIVE, because only significant low to moderate magnitude correlations were observed between some parameters of semen quality and fertilization rate.
Objetivou-se avaliar a adição de antioxidantes em diferentes diluentes de criopreservação sobre a qualidade do sêmen bovino pós-descongelação e a produção in vitro de embriões. Primeiramente, avaliou-se os efeitos da adição de diferentes concentrações de glutationa (1,5 e 2,5 mM) e melatonina (0,5 e 1,0 mM) em diluentes de criopreservação de sêmen à base de gema de ovo (Bovimix®) e lecitina de soja (Andromed®). Os resultados demonstraram que a adição do antioxidante glutationa não melhorou a manutenção das características espermáticas do sêmen bovino. A melatonina reduziu a qualidade das características espermáticas avaliadas. O diluente Andromed® obteve melhores variáveis de cinética espermática quando comparado com o diluente Bovimix®. No entanto, com relação às análises de integridade de membrana o diluente Bovimix® demonstrou melhor desempenho na manutenção dessas variáveis. Posteriormente, avaliou-se o efeito dos diluentes de criopreservação e a adição de glutationa sobre a produção in vitro de embriões bovinos (PIVE). Avaliou-se a taxa de clivagem, taxa de blastocisto e eclosão oito e nove dias após a fertilização (FIV), assim como a correlação entre as características espermáticas e as taxas de desenvolvimento embrionário. O diluente Andromed® proporcionou melhor taxa de clivagem, sem a necessidade da adição de glutationa no mesmo. Entretanto, a adição de 2,5 mM de glutationa no diluente Bovimix® melhorou a taxa de clivagem. Houve correlação significativa de alta e baixa magnitude entre algumas características espermáticas com a taxa de clivagem. Conclui-se que a glutationa não melhorou a viabilidade espermática. A melatonina reduziu a manutenção das características espermáticas. O diluente Andromed® foi mais eficiente na produção in vitro de embriões bovinos não sendo observado efeito da glutationa neste diluente. A adição de glutationa na concentração de 2,5 mM no diluente Bovimix®, à base de gema de ovo, proporcionou maior taxa de clivagem. A avaliação in vitro da qualidade do sêmen não é o melhor método para predizer o potencial fertilizante do mesmo na PIVE, pois só foram observadas correlações significativas de baixa a moderada magnitude entre algumas características de qualidade do sêmen e a taxa de fertilização.

Na inativação do cromossomo X (ICX) um dos dois cromossomos X presentes nas fêmeas de mamíferos placentários é silenciado transcricionalmente. Esse é um mecanismo de compensação de dose que assegura que a quantidade dos produtos gênicos oriundos do cromossomo X esteja em equilíbrio entre machos e fêmeas. A ICX pode ocorrer de modo aleatório, onde cada célula escolhe ao acaso qual será o cromossomo X inativado: cromossomo X paterno ou cromossomo X materno; ou de forma \"imprintada\" (termo adaptado do inglês imprinted), ou seja, dependente da origem parental do cromossomo X. Enquanto nas fêmeas marsupiais a inativação ocorre de forma \"imprintada\", sendo o X paterno inativado em todos os tecidos, nos mamíferos eutérios a ICX nos tecidos somáticos ocorre de modo aleatório. Porém alguns eutérios mantiveram o mecanismo \"imprintado\" de ICX exclusivamente nos tecidos extraembrionários, como ratos e camundongos. Em humanos, o estado controverso da ICX em tecidos extraembrionários foi reavaliado por nosso grupo utilizando uma análise mais ampla e identificou-se um padrão aleatório (Moreira de Mello et al., 2010), demonstrando a importância de se realizar uma análise global para se determinar o perfil de atividade do cromossomo X. Em bovinos o padrão de ICX em placenta não está claro. Ele foi verificado analisando-se a expressão de um único gene, e os autores concluíram que o padrão era \"imprintado\" (Xue et al., 2002). Porém a análise de um único gene pode não representar o estado epigenético de um cromossomo inteiro. Assim o padrão de ICX em tecidos extraembrionários bovinos se mostra uma questão importantíssima para ser esclarecida. No presente trabalho o cromossomo X bovino foi analisado em busca de SNPs (polimorfismos de base única) localizados em regiões codificadoras em genes expressos no tecido extraembrionário, permitindo assim através da análise da expressão alelo-específica determinar o padrão de expressão do cromossomo X. Os resultados apresentados neste trabalho mostram um padrão de expressão bialélica, indicando que em populações diferentes de células, diferentes cromossomos X estavam ativos. Portanto a ICX em tecidos extraembrionários bovinos ocorre de modo aleatório, padrão semelhantes àquele encontrado em humanos, e diferente daquele encontrado em ratos e camundongos. Este trabalho mostra a importância de uma análise global da expressão gênica no cromossomo X, permitindo assim traçar um perfil de atividade mais próximo possível da realidade.
In X chromosome inactivation (XCI), one of the two X chromosomes present in female mammals is transcriptionally silenced, resulting in a dosage compensation mechanism. The XCI can occur randomly, so that each cell chooses randomly which one will be the inactivated X chromosome: paternal (pX) or maternal (mX); or dependent on parental origin of X chromosome, ie, imprinted. While in female marsupials the inactivation occurs in an imprinted fashion, with the Xp inactivated in all tissues, both somatic and extra-embryonic, in the mammalian eutherians XCI in the somatic tissues occurs randomly. However some eutherians still retain the imprinted XCI mechanism exclusively in extra-embryonic tissues, such as rats and mice. In humans, the controversy of the XCI in placenta was re-evaluated by our group. Using a broader analysis, a random pattern was identified, in contrast to the previously published works. It demonstrated the importance of conducting a comprehensive analysis to determine the profile of X chromosome (Moreira de Mello et al., 2010). In cattle the pattern of XCI in bovine placenta is unclear. It was verified by analyzing the expression of a single gene, and the authors concluded that the pattern was imprinted (Xue et al., 2002). Because the analysis of a single gene may not represent the epigenetic state of an entire chromosome, the pattern of XCI in cattle extra-embryonic tissues is an important issue to be clarified. In the present study the cattle X chromosome was analyzed searching for SNPs (single nucleotide polymorphisms) located in coding regions of genes expressed in extra-embryonic tissue. So that, by analyzing the allele-specific expression it is possible to determine the X chromosome expression patter. The preset results show a bi-allelic expression pattern. This indicates that in different cells populations, different X chromosomes are active. Thus, the XCI in extra-embryonic tissues of bovines occurs randomly, similar to the human pattern but different to that verified in rats and mice. This work shows the importance of a global analysis of the gene expression in X chromosome, through which it can trace the closest activity profile as possible to reality.

La purification des facteurs de croissance du fibroblaste (fgf) acide et basique chez le poulet au cours du developpement embryonnaire a permis de confirmer la grande conservation des fgf au cours de l'evolution. Le photorecepteur est la cellule qui contient la plus grande activite mitogenique specifique en fgf

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Células tronco embrionárias (CTE) são caracterizadas pelas capacidades de auto-renovação e diferenciação. No entanto, os mecanismos moleculares que regulam estes dois processos são mal compreendidos. CTE de camundongos foram originalmente isoladas a partir da massa celular interna (MCI) de blastocistos produzidos in vitro e in vivo e mantidas em cultura sem perda da pluripotência, originando tecidos das três camadas germinativas. Há profundo interesse em conhecer os processos envolvidos na proliferação e diferenciação das células embrionárias contribuindo, no futuro, para engenharia de tecidos e clonagem terapêutica. Objetivos: Comparar o potencial gerador de CTE de embriões bovinos produzidos in vitro em meio contendo alta e baixa concentração de soro, assim como avaliar a manutenção da pluripotência das células em cultivo através da ação de dois fatores, a LIF e o bFGF, utilizados isoladamente ou em conjunto, no cultivo in vitro de CTEbov. Foram utilizados blastocistos bovinos com 9 dias de desenvolvimento in vitro para remoção da massa celular interna (MCI) e posterior cultivo das mesmas em placas tratadas com monocamada de fibroblastos bloqueados. As colônias celulares semelhantes a células-tronco foram analisadas através da identificação da expressão in sito dos fatores de indiferenciação Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1- 60, TRA-1-81. Os blastocistos bovinos com 9 dias de idade também foram submetidos à marcação imunocitoquímica. Adicionalmente foi avaliado o potencial de diferenciação in vitro das CTEbov para linhagens celulares de origem endodermal, mesodermal e ectodermal. Em média, 525 embriões de cada um dos dois grupos (2,5% e 10% de soro fetal bovino, respectivamente) foram selecionados para o isolamento da MCI. Foram utilizados 300 blastocistos iniciais...
Embryonic stem cells (ESC) are characterized by their capacity for selfrenewal and differentiation. However, the molecular mechanisms which regulate these two processes are poorly understood. ESC of mice were originally isolated from the inner cell mass (ICM) of blastocysts in vitro and in vivo and maintained in culture without loss of pluripotency, yielding three germ layers of tissue. There is keen interest in learning about the processes involved in proliferation and differentiation of embryonic cells contribute in the future, tissue engineering and therapeutic cloning. To compare the potential generator ESC of bovine embryos produced in vitro in medium containing high and low concentrations of serum, as well as evaluating the maintenance of pluripotency of the cells in culture through the action of two factors, the LIF and bFGF, used singly or together, in vitro cultivation of ESCbov. We used bovine blastocysts to nine days of in vitro development to remove the inner cell mass (ICM) and further cultivation of the same plates treated with fibroblast monolayer blocked. The cell colonies similar to stem cells were analyzed by in situ identification of the expression of differentiation factors Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 . The bovine blastocysts with 9 days of age were also subjected to immunocytochemical labeling. Additionally it was evaluated the potential of differentiation in vitro of cell lines to ESCbov endodermal origin, mesodermal and ectodermal. The average 525 embryos from each of the two groups (2.5% and 10% fetal bovine serum, respectively) were selected for isolation of the ICM. Early blastocysts were used 300, 160 expanded blastocysts and 45 hatched blastocysts per group. However, only expanded blastocysts adhered to the monolayer of fibroblasts and developed into colonies similar to... (Complete abstract click electronic access below)

Orientador: Fernanda da Cruz Landim
Banca: Claudia Barbosa Fernandes
Banca: Flávia Karina Dlella
Resumo: Células tronco embrionárias (CTE) são caracterizadas pelas capacidades de auto-renovação e diferenciação. No entanto, os mecanismos moleculares que regulam estes dois processos são mal compreendidos. CTE de camundongos foram originalmente isoladas a partir da massa celular interna (MCI) de blastocistos produzidos in vitro e in vivo e mantidas em cultura sem perda da pluripotência, originando tecidos das três camadas germinativas. Há profundo interesse em conhecer os processos envolvidos na proliferação e diferenciação das células embrionárias contribuindo, no futuro, para engenharia de tecidos e clonagem terapêutica. Objetivos: Comparar o potencial gerador de CTE de embriões bovinos produzidos in vitro em meio contendo alta e baixa concentração de soro, assim como avaliar a manutenção da pluripotência das células em cultivo através da ação de dois fatores, a LIF e o bFGF, utilizados isoladamente ou em conjunto, no cultivo in vitro de CTEbov. Foram utilizados blastocistos bovinos com 9 dias de desenvolvimento in vitro para remoção da massa celular interna (MCI) e posterior cultivo das mesmas em placas tratadas com monocamada de fibroblastos bloqueados. As colônias celulares semelhantes a células-tronco foram analisadas através da identificação da expressão in sito dos fatores de indiferenciação Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1- 60, TRA-1-81. Os blastocistos bovinos com 9 dias de idade também foram submetidos à marcação imunocitoquímica. Adicionalmente foi avaliado o potencial de diferenciação in vitro das CTEbov para linhagens celulares de origem endodermal, mesodermal e ectodermal. Em média, 525 embriões de cada um dos dois grupos (2,5% e 10% de soro fetal bovino, respectivamente) foram selecionados para o isolamento da MCI. Foram utilizados 300 blastocistos iniciais... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Embryonic stem cells (ESC) are characterized by their capacity for selfrenewal and differentiation. However, the molecular mechanisms which regulate these two processes are poorly understood. ESC of mice were originally isolated from the inner cell mass (ICM) of blastocysts in vitro and in vivo and maintained in culture without loss of pluripotency, yielding three germ layers of tissue. There is keen interest in learning about the processes involved in proliferation and differentiation of embryonic cells contribute in the future, tissue engineering and therapeutic cloning. To compare the potential generator ESC of bovine embryos produced in vitro in medium containing high and low concentrations of serum, as well as evaluating the maintenance of pluripotency of the cells in culture through the action of two factors, the LIF and bFGF, used singly or together, in vitro cultivation of ESCbov. We used bovine blastocysts to nine days of in vitro development to remove the inner cell mass (ICM) and further cultivation of the same plates treated with fibroblast monolayer blocked. The cell colonies similar to stem cells were analyzed by in situ identification of the expression of differentiation factors Oct-4, Nanog, SSEA-1, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 . The bovine blastocysts with 9 days of age were also subjected to immunocytochemical labeling. Additionally it was evaluated the potential of differentiation in vitro of cell lines to ESCbov endodermal origin, mesodermal and ectodermal. The average 525 embryos from each of the two groups (2.5% and 10% fetal bovine serum, respectively) were selected for isolation of the ICM. Early blastocysts were used 300, 160 expanded blastocysts and 45 hatched blastocysts per group. However, only expanded blastocysts adhered to the monolayer of fibroblasts and developed into colonies similar to... (Complete abstract click electronic access below)
Mestre

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cows treated with recombinant bovine somatotropin (bST) have higher conception rate. It was found that maternal and embryonic components are positively affected by the design favoring bST. However, few investigations have been done on beef cows. It was hypothesized that beef cows treated with bST, the day timed AI (TAI) or seven days after TAI, have higher conception rates and the morphology of the endometrial glands and the proportion of steroidogenic luteal cells are altered by such treatment. The present study aimed to evaluate the effect of bST at a dose of 250 or 500mg, the day of TAI, the conception rate at 30 days of gestation (Experiment 1); verify the effects of application of 500mg of bST, seven days after TAI, the conception rate at 30 days of gestation (Experiment 2) and investigate the effects of bST in the proportion of steroidogenic luteal cells and morphometry of the endometrial glands (Experiment 3). In Experiment 1, Nelore cows (n=587) received an intravaginal device containing progesterone (1g; DIB®) associated with an intramuscular (IM) injection of estradiol benzoate (2mg; Gonadiol®). The devices were removed after eight days, when cows were treated with D-cloprostenol (0.530 mg; Ciosin®) and equine chorionic gonadotropin (300UI; Novormon®) and after 24 hours received estradiol benzoate (1 mg; Gonadiol®), via IM. After 30 hours of the last injection (D0) females were inseminated and received 5ml of saline (Control Group; n=194) or somatotropin (Boostin®) at a dose of 250mg (Group bST 250; n=197) or 500mg (Group bST 500; n=196), subcutaneously (SC). Pregnancy diagnosis was performed 30 days after TAI by ultrasonography. In Experiment 2, Nelore cows (n=243) received the Experimento1 similar protocol and were inseminated on D0. In D7 received 5ml of saline (Control Group; n=124) or somatotropin (Boostin®) at a dose of 500mg (Group bST 500; n =119) subcutaneously...(Complete abastract click eletrononic acess below)

Orientador:Flávia Lombardi Costa
Co-orientador:Cláudia Maria Bertan Membrive
Banca:Caliê Castilho
Banca:Ricardo da Fonseca
Resumo:Fêmeas bovinas leiteiras tratadas com somatotropina recombinante bovina (bST) apresentam maior taxa de concepção. Verificou-se que componentes maternais e embrionários são positivamente afetados pela bST favorecendo a concepção. Entretanto, poucas investigações foram realizadas em fêmeas de corte. Hipotetizou-se que vacas de corte tratadas com bST, no dia da inseminação artificial em tempo fixo (IATF) ou sete dias após a IATF, apresentam maiores taxas de concepção e que a morfometria das glândulas endometriais e a proporção de células esteroidogênicas luteais são alteradas por tal tratamento. No presente estudo objetivou-se avaliar o efeito da aplicação de bST, na dose de 250 ou 500mg, no dia da IATF, na taxa de concepção aos 30 dias de gestação (Experimento 1); verificar os efeitos da aplicação de 500mg de bST, sete dias após a IATF, na taxa de concepção aos 30 dias de gestação (Experimento 2) e averiguar os efeitos da bST na proporção de células esteroidogênicas luteais e na morfometria das glândulas endometriais (Experimento 3). No Experimento 1, vacas Nelore (n=587) receberam um dispositivo intravaginal contendo progesterona (1g; DIB®) associado a uma injeção intramuscular (IM) de benzoato de estradiol (2mg; Gonadiol®). Os dispositivos foram removidos oito dias após, quando as vacas foram tratadas com D-cloprostenol (0,530μg; Ciosin®) e gonadotrofina coriônica equina (300UI; Novormon®) e 24 horas após receberam benzoato de estradiol (1mg; Gonadiol®), via IM. Após 30 horas da última injeção (D0) as fêmeas foram submetidas à IATF e receberam 5ml de solução salina (Grupo Controle; n= 194) ou bST (Boostin) na dose de 250mg (Grupo bST 250; n=197) ou 500mg (Grupo bST 500; n=196), via subcutânea (SC). O diagnóstico de gestação foi realizado 30 dias após a IATF por ultrassonografia. No Experimento 2, vacas Nelore (n=243) receberam o... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract:cows treated with recombinant bovine somatotropin (bST) have higher conception rate. It was found that maternal and embryonic components are positively affected by the design favoring bST. However, few investigations have been done on beef cows. It was hypothesized that beef cows treated with bST, the day timed AI (TAI) or seven days after TAI, have higher conception rates and the morphology of the endometrial glands and the proportion of steroidogenic luteal cells are altered by such treatment. The present study aimed to evaluate the effect of bST at a dose of 250 or 500mg, the day of TAI, the conception rate at 30 days of gestation (Experiment 1); verify the effects of application of 500mg of bST, seven days after TAI, the conception rate at 30 days of gestation (Experiment 2) and investigate the effects of bST in the proportion of steroidogenic luteal cells and morphometry of the endometrial glands (Experiment 3). In Experiment 1, Nelore cows (n=587) received an intravaginal device containing progesterone (1g; DIB®) associated with an intramuscular (IM) injection of estradiol benzoate (2mg; Gonadiol®). The devices were removed after eight days, when cows were treated with D-cloprostenol (0.530 mg; Ciosin®) and equine chorionic gonadotropin (300UI; Novormon®) and after 24 hours received estradiol benzoate (1 mg; Gonadiol®), via IM. After 30 hours of the last injection (D0) females were inseminated and received 5ml of saline (Control Group; n=194) or somatotropin (Boostin®) at a dose of 250mg (Group bST 250; n=197) or 500mg (Group bST 500; n=196), subcutaneously (SC). Pregnancy diagnosis was performed 30 days after TAI by ultrasonography. In Experiment 2, Nelore cows (n=243) received the Experimento1 similar protocol and were inseminated on D0. In D7 received 5ml of saline (Control Group; n=124) or somatotropin (Boostin®) at a dose of 500mg (Group bST 500; n =119) subcutaneously...(Complete abastract click eletrononic acess below)
Mestre

Ce travail porte sur l’identification, la fonction et la régulation des molécules maternelles d’ARNm qui dirigent la compétence développementale juste après la fécondation chez les bovins. Tout d’abord, en utilisant le modèle du temps écoulé jusqu’au premier clivage zygotique et à travers l’évaluation du transcriptome des embryons à 2-cellules, il fut possible de déterminer la signature moléculaire des niveaux extrêmes de compétence au développement et sélectionner des molécules candidates pour des études postérieures. Les résultats ont montré que les embryons de capacité développementale variable diffèrent dans certaines fonctions comme la réparation de l’ADN, le traitement de l’ARN, la synthèse de protéines et l'expression génique définies par des ARNm synthétisés par l’ovocyte. Pour obtenir une confirmation fonctionnelle, une paire de transcrits maternels (l’un détecté dans notre sondage précédent et l’autre étant une molécule reliée) ont été inhibés par « knock-down » dans des ovocytes. Les effets du knock-down de ces facteurs de transcription sont apparus avant la formation des blastocystes dû à une diminution de la capacité au clivage et celle à progresser après le stage de 8-cellules. L’analyse moléculaire des embryons knock-down survivants suggère qu’un de ces facteurs de transcription est un contrôleur crucial de l’activation du génome embryonnaire, qui représente une fenêtre développementale dans l’embryogenèse précoce. Dans la dernièr étude, nous avons testé si les facteurs de transcription d'intérêt sont modulés au niveau traductionnel. Des ARNm rapporteurs couplés à la GFP (Protéine fluorescente) contenant soit la version courte ou la version longue de la séquence 3’-UTR des deux molécules furent injectées dans des zygotes pour évaluer leur dynamique traductionnelle. Les résultats ont montré que les éléments cis-régulateurs localisés dans les 3’-UTRs contrôlent leur synchronisation traductionnelle et suggèrent une association entre la compétence développementale et la capacité de synthèse de ces protéines. Ceci conduit à l’idée que ces facteurs de transcription cruciaux sont aussi contrôlés au niveau traductionnel chez les embryons précoces. Les connaissances acquises ont joué un rôle essentiel pour définir le contrôle potentiel des molécules maternelles sur les embryons au début de leur développement. Cette étude nous montre aussi une utilisation potentielle de cette information ainsi que les nouveaux défis présents dans le secteur des technologies reproductives.
This work explores the identity, the function, and the regulation of maternal mRNA molecules that drive developmental competence shortly after fertilization in cattle. First of all, by using the model of the time of first zygotic cleavage and assessing the transcriptome of 2-cell embryos, it was possible to determine the molecular fingerprint of extreme levels of developmental competence and select candidate molecules for further monitoring. Data implied that early embryos of variable developmental capacity differ in functions including DNA repair, RNA processing, protein synthesis, and gene expression that are dictated by oocyte-synthesized mRNA. To obtain a functional confirmation, a pair of maternal transcripts (one detected in our previous survey and other related molecule) were knocked-down in oocytes that were further cultured. The effects of ablating these transcription factors were evident before blastocyst formation due to a decrease in cleavage capacity, as well as progression past the 8-cell stage. The molecular analysis of surviving knocked-down embryos suggested that one of these transcription factors is a pivotal orchestrator of the activation of the embryonic genome, a critical developmental window in early embryogenesis. In the last survey, we asked whether the transcription factors of interest are modulated at the translational level. Reporter mRNAs containing either short or long versions of the 3’-UTR sequences of both molecules were injected in zygotes to look at their translational dynamics. Results showed that cis-acting elements located in the 3’-UTRs govern their timely translation and suggested an association between developmental competence and protein synthesis capacity. This led to the notion that these crucial transcription factors are also controlled at the translational level in early embryos. The acquired knowledge was instrumental to define the possible control operated by maternal molecules on embryos at the onset of their development, as well as some of the challenges and potential use of this information in the field of reproductive technologies.

L'évaluation de la capacité de développement des spermatozoïdes fécondants devrait permettre de choisir les semences pouvant potentiellement augmenter le nombre de blastocystes produits in vitro. Nous avons d'abord défini des conditions standards de production in vitro chez le cerf sika du Japon (cervus nippon nippon) et le cerf élaphe (cervus elaphus) à partir de protocoles utilisés chez les ruminants domestiques. Le rendement de blastocystes peut varier en fonction de la semence utilisée alors que les pourcentages d'ovocytes fécondés sont identiques. Des travaux réalisés sur des ovocytes bovins fecondés in vitro par des semences de taureaux produisant des pourcentages différents de blastocystes (bonne ou mauvaise fertilité in vitro) ont permis de lier la capacité de développement à des événements suivant la fécondation. Le début de la réplication de l'ADN (phase s) dans les deux pronoyaux puis le premier clivage sont plus précoces quand les ovocytes sont fécondés par une semence permettant un pourcentage élèvé de blastocystes. L'effet paternel observé détermine l'entrée en réplication des deux pronoyaux pendant la phase G1 qui précède l'entrée en phase S. Apres fécondation in vitro d'ovocytes bovins depellucidés avec différents lots de semences de cervidés, nous avons observé que le spermatozoïde de cerf dirige l'entrée en première phase S des deux pronoyaux et qu'il existe également un rapport entre le moment d'entrée en première phase S et le rendement de blastocystes obtenus. Chez les bovins, la caractérisation de cet effet paternel montre que la formation du pronoyau mâle s'accompagne d'une forte augmentation de la voie des pentoses phosphates après fécondation avec des taureaux de bonne fertilité in vitro. L'effet paternel observé déterminerait donc le moment de l'entrée en première phase S sur les deux pronoyaux et ainsi le rendement de blastocystes par le biais de facteurs issus du métabolisme glucidique. En stimulant artificiellement le métabolisme glucidique après fécondation avec des mâles de mauvaises fertilité, l’amélioration du taux de blastocystes serait dons envisageable.

La reprise de méiose et le début du développement embryonnaire nécessitent un contrôle très précis de la stabilité et de la traduction des ARN accumulés dans le cytoplasme de l’ovocyte, y compris des transcrits ovocyte-spécifiques. Les travaux menés dans le cadre de cette thèse visaient à améliorer la connaissance des aspects moléculaires de la qualité de l’ovocyte chez le bovin. D’une part, nous avons caractérisé de nouveaux gènes exprimés préférentiellement dans l’ovocyte, en particulier le gène Stem loop binding protein 2, mis en évidence pour la première fois chez les mammifères, qui pourrait réguler la synthèse des histones comme chez le xénope. D’autre part, en combinant des approches globale et ciblée, nous avons montré que les transcrits maternels subissent une dégradation modérée au cours de la maturation mais présentent des profils de déadénylation contrastés. La plupart des gènes ovocyte-spécifiques ne sont pas activés dans l’embryon. Ces travaux ouvrent la voie à la caractérisation d’un profil d’expression associé à la compétence de l’ovocyte à soutenir le développement de l’embryon
Meiotic resumption and early embryo development rely on the precise control of the stability and translation of transcripts accumulated in the oocyte cytoplasm, including oocyte-specific transcripts. This work was to improve the understanding of molecular aspects of oocyte quality in bovine. In one hand, we have characterized novel genes expressed preferentially in the oocyte. In particular, we report expression of Stem loop binding protein 2 for the first time in mammals; it could be involved in controlling histone synthesis as in Xenopus. In the other hand, using both global and targeted approaches, maternal transcripts were shown to undergo moderate degradation during maturation, but they displayed contrasted deadenylation profiles. Most oocyte-specific genes were not reactivated in the embryo. Overall, this work paves the way for the characterization of an expression profile associated with the oocyte competence to support the embryo development

Chez les mammifères, l’oviducte est essentiel à la fonction de reproduction. Les interactions cellulaires et moléculaires entre le jeune embryon et l’oviducte permettent d’ajuster le microenvironnement tubaire à l’évolution de ses besoins spécifiques. L’objectif de cette thèse est de caractériser ce dialogue embryo-maternel chez les ruminants en condition in vitro. L’étude des modes d’action des Boec sur le développement embryonnaire précoce bovin par l’étude de leur transcriptome a montré que celles-ci avaient un impact positif sur les paramètres quantitatifs et qualitatifs du développement embryonnaire bovin. De plus, le système de co-culture mis au point a indiqué que la présence des Boec augmentait aussi les paramètres quantitatifs de survie embryonnaire caprins. Enfin, la recherche d’autres gènes candidats potentiellement impliqués dans le développement embryonnaire préimplantatoire par hybridation de microarrays a permis de mettre en évidence de nouveaux gènes impliqués dans les 3 grands modes d’action des Boec
In mammals, oviducte play an essential role in the reproductive process. Cellular and molecular interactions between the zygoteand and the oviducte lead to adjust the tubal microenvironment to the evolution of the embryo needing. The main objective of this thesis work was to characterize the embryo-maternal dialogue actors in ruminants in in vitro conditions. First, characterization of the way of share of Bovine oviduct epithelial cells (Boec) on bovine early embryo development by a trsncriptomic approach revealed that these cells had a positive impact on quantitative and qualitative parameters of bovine embryonic development. Moreover, this coculture system indicated that Boec also enhanced quantitave parameters of goat embryonic survival. Finally, the search for other genes potentially implicated in early embryonic development by microarrays hybridization described a new set of genes implicated in the 3 main ways of action of Boec, thus contributing to the validation of our work hypothesis

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
Na produÃÃo in vitro (PIV) de embriÃes bovinos vÃrios fatores contribuem para as variÃveis na produÃÃo e qualidade dos oÃcitos e embriÃes. Avaliou-se o uso parenteral de vitamina A (VA) e vitamina E (VE) na produÃÃo de oÃcitos colhidos por aspiraÃÃo folicular (OPU) e embriÃes por produÃÃo in vitro (PIV) em de vacas (n=22), sendo: Simental (S) (n=2); Nelore (N) (n=4); Brahma (B) (n=5) e Gir (G) (n=11). Todos os animais foram alocados na fase prÃ-tratamento (F1) (n=22) (nÃo receberam vitaminas) e os mesmos animais utilizados para a fase pÃs-tratamento (F2) (receberam 1.000.000 UI de vitamina A e 1g de vitamina E). A primeira OPU foi na F1, logo em seguida foi aplicado 1.000.000 UI de VA e 1g de VE, e apÃs 12 dias realizou-se nova OPU para fazer a F2. Os oÃcitos (CCO) foram maturados, fecundados e cultivados in vitro. As 44 OPU produziram 520 oÃcitos, 217 (F1) e 303 (F2), havendo efeito significativo, com acrÃscimo de 86 oÃcitos, obtendo mÃdia e desvio padrÃo 9,86Â5,53 F1 e 13,77Â2,0 F2, (*p<0,0219). Quando separada as raÃas NBG (Nelore, Brahma e Gir) (n=20) houve acrÃscimo de 95 oÃcitos, obtendo mÃdia e desvio padrÃo 9,90Â5,81 F1-NBG e 14,65Â9,44 F2-NBG, (*p<0,0085). As 44 PIV produziram 224 embriÃes, sendo 93 F1 e 131 F2, obtendo mÃdia e desvio padrÃo 4,23Â3,09 F1 e 5,95Â4,05 F2, (*p<0,0228). Quando separada as NBG a produÃÃo foi de 214 embriÃes, havendo acrÃscimo de 38 embriÃes, obtendo valores de 4,45Â3,15 F1 e 6,25Â4,09 F2, (*p<0,0285). Houve um efeito significativo na quantidade produzida de oÃcitos (n=22) e oÃcitos NBG (n=20). Houve efeito na produÃÃo de embriÃes de todas as raÃas (n=22) e embriÃes NBG (n=20). A suplementaÃÃo com VA e VE aumentou o nÃmero de oÃcitos totais (1,7Â0,7); oÃcitos NBG (1,8Â0,8); embriÃes totais (3,9Â1,6) e embriÃes NBG (4,7Â1,6). A resposta da F2 comparado com a F1 na produÃÃo de oÃcitos e embriÃes foi significativa quando todas as raÃas estavam agrupadas e tambÃm quando foi agrupado apenas as Bos taurus indicus (NBG). O uso das vitaminas A e E pode ser usada para maior recuperaÃÃo oÃcitÃria e embrionÃria em raÃas ZebuÃnas.
The in vitro (IVP) bovine embryos production has several factors that contribute to the variables in the production and quality of oocytes and embryos. We evaluated the parenteral use of vitamin A (VA) and vitamin E (VE) in the production of oocytes collected by follicular aspiration (OPU) and embryos by in vitro production (IVP) in cows (n = 22), where: Simmental (S) (n = 2), Nelore (N) (n = 4), Brahma (B) (n = 5) and Gir (G) (n = 11). All animals were allocated in the pre-treatment (F1) (n = 22) (not receiving vitamins) and the same animals used for post-treatment (F2) (received 1,000,000 IU of vitamin A and vitamin 1g E). The first OPU was in F1, soon after 1,000,000 IU was administered 1 g of VE and VA, and after 12 days was held to make the new OPU F2. Oocytes (COC) were matured, fertilized and cultured in vitro. The OPU 44 yielded 520 oocytes, 217 (F1) and 303 (F2), with significant effect, an increase of 86 oocytes, obtaining mean and standard deviation 9.86 Â 5.53 13.77 Â 2.0 F1 and F2, (*p <0.0219). When separate races NBG (Nelore, Brahman and Gir) (n = 20) there was an increase of 95 oocytes, obtaining mean and standard deviation 9.90 Â 5.81 and 14.65 NBG F1-F2-NBG Â 9.44, (*p <0.0085). The 44 IVP embryos produced 224, 93 F1 and 131 F2, getting mean and standard deviation 4.23 Â 3.09 5.95 Â 4.05 F1 and F2, (*p <0.0228). When separated from the NBG production was 214 embryos, with an increase of 38 embryos, obtaining values of 4.45 Â 3.15 6.25 Â 4.09 F1 and F2, (*p <0.0285). There was a significant effect on the quantity produced of oocytes (n = 22) and NBG oocytes (n = 20). It was an increased in all breeds embryos production (n = 22) and NBG embryos (n = 20). Supplementation with VE and VA increased the total number of oocytes (1.7 Â 0.7); NBG oocytes (1.8 Â 0.8); total embryos (3.9 Â 1.6) and embryos NBG (4 7 Â 1.6). The response of the F2 compared to F1 in the production of oocytes and embryos was significant when all races were grouped together and also when it was grouped only Bos taurus indicus (NBG). The use of vitamins A and E can be used to greater oocyte recovery and embryo in Zebu breeds.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Recentemente, tem-se dado atenção crescente aos efeitos deletérios dos radicais livres sobre os oócitos e embriões cultivados in vitro em mamíferos. No entanto, poucos estudos foram conduzidos sobre a ação desses agentes sobre a interação entre o espermatozóide-oócito durante a fertilização in vitro (FIV). Com o intuito de melhorar os resultados da produção in vitro de embriões bovinos, este trabalho teve por objetivo avaliar os efeitos da suplementação do meio de fecundação in vitro com antioxidantes cisteamina (Cist) e 2-Mercaptoetanol (2-ME) sobre a capacitação espermática, fecundação, qualidade dos zigotos e competência no desenvolvimento embrionário. Os espermatozóides apresentaram o DNA íntegro quase na totalidade entre os diferentes tratamentos e durante o tempo no processo da FIV.
Recently, deleterious effects of free radicals over oocytes and embryos cultivated in vitro in mammals have received increasing attention. Nonetheless, few studies were conducted about the action of those agents over the interaction between the sperm-oocyte during in vitro fertilization (IVF). Aimed at improving results of in vitro production of bovine embryos, this work had as objective to evaluate the effects of supplementation of the in vitro fertilization environment with antioxidants cisteamine (Cist) and 2-Mercaptoethanol (2-ME) over the spermatic capacitation, fertilization, quality of zygotes and competency in the embryonic development. Spermatozoas exhibited an integral DNA almost in the totality of the different treatments and along the time in the IVF process.

Orientador: Gisele Zoccal Mingoti
Banca: Joaquim Mansano Garcia
Banca: Rubens Paes de Arruda
Resumo: Recentemente, tem-se dado atenção crescente aos efeitos deletérios dos radicais livres sobre os oócitos e embriões cultivados in vitro em mamíferos. No entanto, poucos estudos foram conduzidos sobre a ação desses agentes sobre a interação entre o espermatozóide-oócito durante a fertilização in vitro (FIV). Com o intuito de melhorar os resultados da produção in vitro de embriões bovinos, este trabalho teve por objetivo avaliar os efeitos da suplementação do meio de fecundação in vitro com antioxidantes cisteamina (Cist) e 2-Mercaptoetanol (2-ME) sobre a capacitação espermática, fecundação, qualidade dos zigotos e competência no desenvolvimento embrionário. Os espermatozóides apresentaram o DNA íntegro quase na totalidade entre os diferentes tratamentos e durante o tempo no processo da FIV.
Abstract: Recently, deleterious effects of free radicals over oocytes and embryos cultivated in vitro in mammals have received increasing attention. Nonetheless, few studies were conducted about the action of those agents over the interaction between the sperm-oocyte during in vitro fertilization (IVF). Aimed at improving results of in vitro production of bovine embryos, this work had as objective to evaluate the effects of supplementation of the in vitro fertilization environment with antioxidants cisteamine (Cist) and 2-Mercaptoethanol (2-ME) over the spermatic capacitation, fertilization, quality of zygotes and competency in the embryonic development. Spermatozoas exhibited an integral DNA almost in the totality of the different treatments and along the time in the IVF process.
Mestre

L'objectif de cette thèse, a été tout d'abord, d'essayer de mettre en évidence l'influence de la femelle donneuse sur la qualité de l'ovocyte chez la vache. Afin d'isoler le rôle de l'ovocyte dans le taux de développement embryonnaire, l'influence du tractus génital femelle a été supprimée par collecte des ovocytes dans les follicules (par Ovum Pick-Up ; OPU) puis par maturation, fécondation et culture in vitro. Ce protocole expérimental a été appliqué à deux situations physiologiques permettant d'explorer l'influence maternelle sous plusieurs approches. Tout d'abord, nous avons utilisé des génisses issues de clonage embryonnaire. Dans un second temps, ce protocole expérimental a été appliqué à 10 vaches d'origines familiales différentes (non apparentées). Ce sperme ne pouvait donc pas être considéré comme le facteur limitant du taux de développement. Nous avons ainsi démontré scientifiquement l'existence d'un effet maternel, c'est-à-dire d'une influence des facteurs maternels ovocytaires, sur le développement embryonnaire (in vitro). Ce travail a permis d'isoler des vaches donneuses d'ovocytes de " bonne " et " mauvaise " qualité. Nos recherches ont ensuite porté sur le(s) mécanisme(s) pouvant être responsable(s) de ces différences d'aptitude au développement in vitro entre femelles. Cette recherche a été plus particulièrement axée sur le métabolisme énergétique : réserves d'ATP, quantité de mitochondries (d'ADN mitochondrial) et séquence de l'ADN mitochondrial. Seules les mutations de l'ADN mitochondrial ont réellement permis de différencier les deux phénotypes de qualité ovocytaire. Ces travaux de thèse ont donné lieu à la rédaction de 3 articles (2 publiés, 1 soumis)
The main objective of this thesis was to show the influence that the oocyte donor cow has over the oocyte quality measured by the embryo production in vitro. In order to isolate the effect of the oocyte on embryo production, we used the techniques of ovum pick-up (OPU), in vitro fertilisation (IVF) and culture (IVC) of embryos. These procedures were used to test the influence of oocytes in two distinct situations. Initially, the purpose was to test the in vitro embryo production in cloned cows (embryonic cloning). A second purpose was performed using ten cows with diverse genetic origins. In order to avoid the sperm induced variation on embryo production, only semen with good IVF rates from the same bull and from the same ejaculate was used. The results of this second study demonstrated scientifically the existence of a maternal effect over in vitro embryo production. This work allowed the identification of cows that produce oocytes of "good" and "bad" quality. After showing the existence of a maternal effect over in vitro embryo production, the research focused on the exploration of mechanisms that could be involved in such differences. Thus, the third experiment centered on the energetic metabolism of the oocyte, studying the oocytes' ATP reserves and the quantity of mitochondrial DNA and polymorphisms in the control region of the mitochondrial DNA. Only the study of mutations of the mitochondrial DNA control region was successful in differentiating the phenotypic variation in oocyte quality. This work resulted in the publication of two articles and a third one that was submitted for publication in international pier-reviewed journals

Avec l'arrivée des techniques de reproduction assistée et de la génomique, le progrès génétique chez les bovins laitiers est plus rapide que jamais, favorisant maintenant l'utilisation d'animaux de plus en plus jeunes pour la reproduction. Cette situation aurait possiblement un impact sur la qualité des embryons, affectant potentiellement la génisse de la génération suivante. Ce projet vise à documenter l'effet de l'âge sur la qualité de l'embryon et, en l'occurence, à identifier des pistes pour corriger la situation. Dix jeunes femelles Holstein ont subi trois cycles de stimulation ovarienne (8, 11, 14 mois). Les ovules ont ensuite été fécondés in vitro (semence d'un même taureau adulte), générant trois lots d’embryons par animal. Grâce à la plateforme EmbryoGENE, il fut possible de mesurer l'expression génique ainsi que l'état de méthylation de l'ADN au stade blastocyste. En premier lieu, l'analyse transcriptomique des contrastes selon l'âge (8 vs 14 mois et 11 vs 14 mois) a permis de dénombrer 242 gènes différentiellement exprimés pour le premier contraste et 296 pour le deuxième. Parmi les voies géniques affectées par l'âge, on retrouve notamment les voies mTOR et PPAR, ainsi que la voie de réponse au stress oxydatif médiée par NRF2. Pour sa part, l'analyse épigénétique a permis d'identifier 5787 régions différentiellement méthylées pour le premier contraste et 3658 pour le deuxième. Il est possible d'observer une tendance à l'hyperméthylation chez les embryons obtenus à partir de donneuses de 8 mois, alors qu'une hypométhylation du génome plus marquée est notée chez les embryons provenant des donneuses de 11 mois. Le premier constat est que les embryons sont marginalement affectés par l'âge de la donneuse et que la qualité s’avère très bonne dès 8 mois. Les résultats suggèrent une causemétabolique pour expliquer les différences observées, trahissant un impact plus grand des conditions in vitrosur les embryons produits par les plus jeunes donneuses.

Les objectifs de cette étude étaient d’évaluer le risque de transmission de Coxiella burnetii, agent d’une zoonose la fièvre Q, (i) selon une voie vertical, in utero à l’embryon ou au fœtus et (ii) selon une voie horizontal, lors des opérations de transferts d’embryons produits in vitro et in vivo, afin de définir les possibilités de production de matériels génétiques indemnes. Les résultats obtenus montrent, pour la première fois, la présence de charges importantes de Coxiella burnetti dans l’appareil génital, dans les liquides de lavage des oviductes et des cornes utérines et dans les tissus de l’appareil génital de chèvres, non gravides. De même, suite à l’infection expérimentale (infection in vitro) ces travaux démontrent clairement que Coxiella burnetii possède une forte tendance à adhérer sur les embryons protégés par leur zone pellucide qu’ils soient caprins produits in vitro et in vivo ou bovins produits in vitro, et que la procédure de lavage recommandée par l’International Embryo Transfer Society (IETS) pour l’embryon bovin est inefficace pour l’éliminer. La visualisation de Coxiella burnetii par immunohistochimie chez l’embryon entouré de sa zone pellucide confirme sa position extra embryonnaire. Par ailleurs, les traitements enzymatiques utilisés pour décontaminer les embryons caprins produits in vitro et in vivo et infectés in vitro, sont inefficaces. En absence d’étude complémentaire, notamment selon un modèle d’infection naturelle de Coxiella burnetii lors du transfert embryonnaire semble réel.

O estradiol secretado pelo folículo dominante (DOM) desempenha importante papel na luteólise da vaca. Em adição, o reconhecimento materno da prenhez (MRP) requer um ambiente uterino otimizado, o qual por sua vez depende da função luteínica e de adequadas concentrações de progesterona circulante. Os objetivos do presente estudo foi testar diferentes estratégias para otimizar a função luteínica e prevenir a influência de um DOM durante o período crítico (CP) para o MRP (de D13 a D20 após o estro o estro). Diferentes abordagens foram testadas. No exp.1, 23 vacas Nelore foram tratadas com o protocolo ovsynch para induzir um cio sincronizado (D0). As vacas receberam: Gc (n=7) - nada mais; ThCG (n=5) - 3000 IU de hCG no D5; TE2 (n=6) - 5mg de 17b-estradiol (E2) no D12; ThCG/E2 (n=5) - hCG/D5 + E2/D12. Ultra-sonografias e dosagens de progesterona plasmática durante o ciclo estral permitiram determinar que o E2 reprogramou o ciclo ovariano ao prevenir a presença (P⁢0,05) de um DOM durante quase todo CP (0,6±0,7 dias entre D15 e D20), porém o E2 induziu a luteólise. As vacas que receberam a hCG desenvolveram corpo lúteo acessório e tiveram [P4] mais alta até o D13 (P⁢0,05). Portanto, a fase luteínica não foi prolongada No exp.2, os mesmos tratamentos foram impostos a 220 vacas Nelore (55 por grupo) após uma inseminação artificial em tempo fixo (TAI). As taxas de prenhez (PR) à TAI ou às inseminais de repasse durante uma estação com 64 dias de duração foram diminuídas (P⁢0,05) quando o E2 foi usado e a hCG não foi capaz de aumentar aquelas PR. No exp.3, vacas Red Angus no pós-parto tiveram seu estro sincronizado (D0) e receberam: nada mais (GCT, n=5) ou 200mg de gonadorrelina no D5 mais 3000 IU hCG no D13 (GRF, n=5); ou ablação de todos folículos ³7mm através de aspiração folicular em D14, D17 e D20 (GRM, n=5). GRF teve luteólise retardada (18,2±1,0b dias, 23,6±1,0a dias e 18,7±1,2b dias para GCT, GRF e GRM, respectivamente) e maior [P4] que os outros grupos. Folículos maiores que 7mm foram observados quando das aspirações. Foi possível concluir que: a) E2 permitiu consistentemente reprogramar o desenvolvimento folicular, porém causou luteólise e o seu uso trouxe efeitos negativos sobre as PR; b) a hCG melhorou a função luteínica mas não aumentou as PR; c) a ablação dos folículos ³7mm não preveniu a presença do DOM no CP para o MRP e d) a associação GnRH/hCG otimizou a função luteínica, retardou a luteólise e prolongou a fase luteínica de modo que todo o CP esteve sob influência da progesterona
Estradiol secreted from the dominant follicle (DOM) plays a key role in triggering luteolysis in the cow. In addition, maternal recognition of pregnancy (MRP) requires an optimum uterine environment, which directly depends on luteal function and adequate levels of circulating progesterone. The aim of this study was to test different strategies to optimize luteal function and prevent the influence of a DOM throughout the critical period (CP) for MRP (from D13 to D20 after estrus). Different approaches were tested. In exp.1, 23 Nelore cows were treated with the ovsync protocol to induce a synchronized estrus (D0). Cows received: Gc (n=7) - nothing else; ThCG (n=5) - 3000 IU of hCG five days (D5) after estrus; TE2 (n=6) - 5mg of 17b-estradiol (E2) on D12; ThCG/E2 (n=5) - hCG/D5 + E2/D12. Ultrasound evaluation and plasmatic progesterone concentration ([P4]) throughout estrous cycle allowed to conclude: E2 reprogrammed ovarian cycle by preventing the presence (P⁢.05) of a DOM during almost all CP (0.6±.7 days within the D15 to D20 interval) but induced luteolysis; cows receiving hCG developed accessory corpus luteum and had higher [P4] up to D13 (P⁢.05). Therefore, luteolysis was not delayed and luteal phase was not prolonged. In exp.2, same treatments were imposed to 220 Nelore cows (55 per group) after a timed artificial insemination (TAI). Pregnancy rates (PR) at TAI or at AIs thereafter over a 64-day period were reduced (P⁢0.05) by using E2 and hCG was not capable to improve those PR. In exp.3, postpartum Red Angus cows were estrus synchronized (D0) and received: nothing else (GCT, n=5) or 200mg of gonadorrelin on D5 plus 3000 IU hCG on D13 (GRF, n=5); or ablation of all follicles ³7mm through follicular aspiration on D14, D17 and D20 (GRM, n=5). GRF had delayed luteolysis (18.2±1.0b days, 23.6±1.0a days, 18.7±1.2b days for GCT, GRF, GRM, respectively) and higher [P4] than other groups. Follicles larger than 7mm were observed in all occasions of aspiration. In could be concluded that: a) E2 allowed to consistently reschedule follicular development but caused luteolysis and its use was detrimental to PR; b) hCG improved luteal function but did not increase PR; c) ablation of 7mm follicles did not prevent a DOM throughout CP for MRP and d) GnRH/hCG association optimized luteal function, delayed luteolysis and prolonged luteal phase in such a way that all CP was under progesterone influence

En début de lactation, la vache subit un stress important occasionné par l’impossibilité de combler l’ensemble de ses besoins énergétiques par sa consommation exogène. Cette période spécifique se caractérise par une balance énergétique négative, entraîne une utilisation excessive des réserves corporelles de l’animal et représente un défi métabolique important. Ironiquement, depuis maintenant plus de 40 ans, le système incite les producteurs laitiers à effectuer l’insémination au jour 60 post-partum, c’est-à-dire au moment où la vache rencontre un déficit métabolique. Ce déficit au moment de la conception aurait un impact chez la progéniture, notamment au niveau épigénétique. Ce projet consiste à documenter l’effet de la balance énergétique négative sur la qualité de l’embryon et, en l’occurrence, à proposer des pistes afin d’améliorer la fertilité des bovins laitiers. La mesure du bêta-hydroxybutyrate (BHB) a été effectuée à partir d’échantillons sanguins entre 45 et 60 jours post-partum sur dix-huit vaches de race Holstein. Selon la mesure obtenue, la vache fut classée comme étant faible ou élevée en BHB, afin d’avoir au moins six vaches par groupe. Après un processus de stimulation ovarien, chaque vache fut inséminée et les embryons, récoltés. Pour chaque vache, deux embryons ont été transférés dans deux primipares, afin de déterminer subséquemment la persistance des marqueurs dans le matériel biologique. Grâce à la plate-forme EmbryoGENE, il fut possible de déterminer l’expression génique ainsi que l’état de méthylation de l’ADN des embryons récoltés. Les résultats obtenus soutiennent l’existence d’une altération du métabolisme énergétique au niveau embryonnaire, notamment par la modification de la voie de signalisation de mTOR ainsi que celle des sirtuines. Cette altération semble se traduire par une dysfonction mitochondriale et une inhibition de la transcription, entraînant un freinage au niveau cellulaire, probablement dû à la programmation de l’embryon à utiliser ses réserves lipidiques lors de conditions importantes de stress.
In early lactation, the cow undergoes an important stress generated by the impossibility of filling its entire energetic needs by exogenous consumption. This is characterized by a negative energy balance, excessive use of animal body reserves and represents an important metabolic challenge. Ironically, for more than 40 years now, the system has been encouraging dairy farmers to inseminate on day 60 postpartum, when the cow has a metabolic deficit. This deficit at the time of conception could impact the offspring, especially at the epigenetic level. This project is meant to document the effect of the negative energy balance on the quality of the embryo and to identify ways to improve the fertility of dairy cows. The beta hydroxybutyrate (BHB) measure was done from blood samples between day 45 and 60 postpartum on eighteen Holstein cows. According to the measure obtained, each cow was classified as low or high in BHB, so as to have at least six cows per group. After an ovarian stimulation process, each cow was inseminated and the embryos were harvested. For each cow, two embryos were transferred in two primiparous cows in order to subsequently determine the persistence of the markers in the biological material. With the EmbryoGENE platform, it was possible to determine the gene expression as well as the methylation status of DNA embryos. The results obtained support the existence of an alteration of the energetic metabolism at the embryonic level, especially by the modification of the signaling pathway of mTOR as well as those of the sirtuins. This alteration appears to result in mitochondrial dysfunction and inhibition of transcription, leading to a reduced activity at a cellular level, probably due to programming of the embryo to use its lipid reserves during severe stress conditions.

Pour ce projet, nous avons développé une plateforme pour l’analyse pangénomique de la méthylation de l’ADN chez le bovin qui est compatible avec des échantillons de petites tailles. Cet outil est utilisé pour étudier les caractéristiques génétiques et épigénétiques (méthylation de l'ADN) des gamètes soumis aux procédures de procréation médicalement assisitée et des embryons précoces. Dans un premier temps, une plateforme d’analyse de biopuces spécifiques pour l’étude de la méthylation de l’ADN chez l’espèce bovine a été développée. Cette plateforme a ensuite été optimisée pour produire des analyses pangénomiques de méthylation de l'ADN fiables et reproductibles à partir d’échantillons de très petites tailles telle que les embryons précoces (≥ 10 ng d'ADN a été utilisé, ce qui correspond à 10 blastocystes en expansion). En outre, cet outil a permis d’évaluer de façon simultanée la méthylation de l’ADN et le transcriptome dans le même échantillon, fournissant ainsi une image complète des profils génétiques et épigénétiques (méthylation de l’ADN). Comme preuve de concept, les profils comparatifs de méthylation de l'ADN spermatique et de blastocystes bovins ont été analysés au niveau de l'ensemble du génome. Dans un deuxième temps, grâce à cette plateforme, les profils globaux de méthylation de l'ADN de taureaux jumeaux monozygotes (MZ) ont été analysés. Malgré qu’ils sont génétiquement identiques, les taureaux jumeaux MZ ont des descendants avec des performances différentes. Par conséquent, l'hypothèse que le profil de méthylation de l'ADN spermatique de taureaux jumeaux MZ est différent a été émise. Dans notre étude, des différences significatives entre les jumeaux MZ au niveau des caractéristiques de la semence ainsi que de la méthylation de l’ADN ont été trouvées, chacune pouvant contribuer à l’obtention de performances divergentes incongrues des filles engendrées par ces jumeaux MZ. Dans la troisième partie de ce projet, la même plateforme a été utilisée pour découvrir les impacts d’une supplémentation à forte concentration en donneur de méthyle universel sur les embryons précoces bovins. La supplémentation avec de grandes quantités d'acide folique (AF) a été largement utilisée et recommandée chez les femmes enceintes pour sa capacité bien établie à prévenir les malformations du tube neural chez les enfants. Cependant, plus récemment, plusieurs études ont rapporté des effets indésirables de l’AF utilisé à des concentrations élevées, non seulement sur le développement de l'embryon, mais aussi chez les adultes. Au niveau cellulaire, l’AF entre dans le métabolisme monocarboné, la seule voie de production de S-adénosyl méthionine (SAM), un donneur universel de groupements méthyles pour une grande variété de biomolécules, y compris l’ADN. Par conséquent, pour résoudre cette controverse, une forte dose de SAM a été utilisée pour traiter des embryons produits in vitro chez le bovin. Ceci a non seulement permis d’influencer le phénotype des embryons précoces, mais aussi d’avoir un impact sur le transcriptome et le méthylome de l’ADN. En somme, le projet en cours a permis le développement d'une plateforme d'analyse de la méthylation de l'ADN à l’échelle du génome entier chez le bovin à coût raisonnable et facile à utiliser qui est compatible avec les embryons précoces. De plus, puisque c’est l'une des premières études de ce genre en biologie de la reproduction bovine, ce projet avait trois objectifs qui a donné plusieurs nouveaux résultats, incluant les profils comparatifs de méthylation de l'ADN au niveau : i) blastocystes versus spermatozoïdes ; ii) semence de taureaux jumeaux MZ et iii) embryons précoces traités à de fortes doses de SAM versus des embryons précoces non traités.
Pour ce projet, nous avons développé une plateforme pour l’analyse pangénomique de la méthylation de l’ADN chez le bovin qui est compatible avec des échantillons de petites tailles. Cet outil est utilisé pour étudier les caractéristiques génétiques et épigénétiques (méthylation de l'ADN) des gamètes soumis aux procédures de procréation médicalement assisitée et des embryons précoces. Dans un premier temps, une plateforme d’analyse de biopuces spécifiques pour l’étude de la méthylation de l’ADN chez l’espèce bovine a été développée. Cette plateforme a ensuite été optimisée pour produire des analyses pangénomiques de méthylation de l'ADN fiables et reproductibles à partir d’échantillons de très petites tailles telle que les embryons précoces (≥ 10 ng d'ADN a été utilisé, ce qui correspond à 10 blastocystes en expansion). En outre, cet outil a permis d’évaluer de façon simultanée la méthylation de l’ADN et le transcriptome dans le même échantillon, fournissant ainsi une image complète des profils génétiques et épigénétiques (méthylation de l’ADN). Comme preuve de concept, les profils comparatifs de méthylation de l'ADN spermatique et de blastocystes bovins ont été analysés au niveau de l'ensemble du génome. Dans un deuxième temps, grâce à cette plateforme, les profils globaux de méthylation de l'ADN de taureaux jumeaux monozygotes (MZ) ont été analysés. Malgré qu’ils sont génétiquement identiques, les taureaux jumeaux MZ ont des descendants avec des performances différentes. Par conséquent, l'hypothèse que le profil de méthylation de l'ADN spermatique de taureaux jumeaux MZ est différent a été émise. Dans notre étude, des différences significatives entre les jumeaux MZ au niveau des caractéristiques de la semence ainsi que de la méthylation de l’ADN ont été trouvées, chacune pouvant contribuer à l’obtention de performances divergentes incongrues des filles engendrées par ces jumeaux MZ. Dans la troisième partie de ce projet, la même plateforme a été utilisée pour découvrir les impacts d’une supplémentation à forte concentration en donneur de méthyle universel sur les embryons précoces bovins. La supplémentation avec de grandes quantités d'acide folique (AF) a été largement utilisée et recommandée chez les femmes enceintes pour sa capacité bien établie à prévenir les malformations du tube neural chez les enfants. Cependant, plus récemment, plusieurs études ont rapporté des effets indésirables de l’AF utilisé à des concentrations élevées, non seulement sur le développement de l'embryon, mais aussi chez les adultes. Au niveau cellulaire, l’AF entre dans le métabolisme monocarboné, la seule voie de production de S-adénosyl méthionine (SAM), un donneur universel de groupements méthyles pour une grande variété de biomolécules, y compris l’ADN. Par conséquent, pour résoudre cette controverse, une forte dose de SAM a été utilisée pour traiter des embryons produits in vitro chez le bovin. Ceci a non seulement permis d’influencer le phénotype des embryons précoces, mais aussi d’avoir un impact sur le transcriptome et le méthylome de l’ADN. En somme, le projet en cours a permis le développement d'une plateforme d'analyse de la méthylation de l'ADN à l’échelle du génome entier chez le bovin à coût raisonnable et facile à utiliser qui est compatible avec les embryons précoces. De plus, puisque c’est l'une des premières études de ce genre en biologie de la reproduction bovine, ce projet avait trois objectifs qui a donné plusieurs nouveaux résultats, incluant les profils comparatifs de méthylation de l'ADN au niveau : i) blastocystes versus spermatozoïdes ; ii) semence de taureaux jumeaux MZ et iii) embryons précoces traités à de fortes doses de SAM versus des embryons précoces non traités.
In this project, we developed a bovine genome-wide DNA methylation platform compatible with small sample size to study genetic and epigenetic (DNA methylation) makeup of ART-treated bovine gametes and early embryos. Initially, a bovine-specific array-based DNA methylation analysis platform was developed. This platform was subsequently optimized to produce reliable and reproducible genome-wide DNA methylation analysis from very small sample sizes, e.g. bovine early embryos (≥ 10 ng gDNA input, corresponding to 10 expanded blastocysts). In addition, this platform permitted concurrent assessment of both DNA methylation and transcription in the same sample, thereby providing a very complete picture of genetic and epigenetic (DNA methylation) profiles. As proof of concept, for the first time, comparative DNA methylation profiles of bovine sperm and blastocysts were analysed at a genome-wide level. Using this platform, global DNA methylation profiles of monozygotic (MZ) twin bulls were analysed. Despite being geneticially identical, MZ twin bulls consistently have different progeny performance. Therefore, it was hypothesised that the DNA methylation profile of sperm from MZ twin bulls is different. In our study, there were significant differences between MZ twin for semen end points, as well as for the sperm epigenome (DNA methylation), all of which would be expected to contribute to incongruous divergent performances of daughters sired by MZ twins.In the next part of this project, using the developed platform, impacts of supplementation of a high-concentration global methyl donor on bovine early embryos was investigated. Supplementation with large amounts of folic acid (FA) has been extensively used and recommended in pregnant women for its well-established ability to prevent neural tube defects in children. However, more recently, several studies reported adverse effects of high FA concentrations, not only on embryo development, but also in adults. At the cellular level, FA enters one-carbon metabolism, the only pathway to produce S-adenosyl methionine (SAM) as the global methyl donor for a wide variety of biomolecules, including DNA. Therefore, to address this controversy, a high dose of SAM was used to treat in vitro -produced bovine embryos. This not only affected early embryo phenotypes, but also the transcritome and genome-wide DNA methylome. Overall, the current project resulted in development of a user-friendly and cost-effective bovine genome-wide DNA methylation analysis platform, which is compatible with small cell number such as early embryos. In addition, as one of the first studies of its kind in bovine reproductive biology, this project had three objectives which yielded several novel results, including comparative genome-wide DNA methylation profiles of: i) bovine blastocysts versus sperm; ii) sperm from monozygotic twin bulls and iii) high dose SAM-treated versus non-treated bovine early embryos.
In this project, we developed a bovine genome-wide DNA methylation platform compatible with small sample size to study genetic and epigenetic (DNA methylation) makeup of ART-treated bovine gametes and early embryos. Initially, a bovine-specific array-based DNA methylation analysis platform was developed. This platform was subsequently optimized to produce reliable and reproducible genome-wide DNA methylation analysis from very small sample sizes, e.g. bovine early embryos (≥ 10 ng gDNA input, corresponding to 10 expanded blastocysts). In addition, this platform permitted concurrent assessment of both DNA methylation and transcription in the same sample, thereby providing a very complete picture of genetic and epigenetic (DNA methylation) profiles. As proof of concept, for the first time, comparative DNA methylation profiles of bovine sperm and blastocysts were analysed at a genome-wide level. Using this platform, global DNA methylation profiles of monozygotic (MZ) twin bulls were analysed. Despite being geneticially identical, MZ twin bulls consistently have different progeny performance. Therefore, it was hypothesised that the DNA methylation profile of sperm from MZ twin bulls is different. In our study, there were significant differences between MZ twin for semen end points, as well as for the sperm epigenome (DNA methylation), all of which would be expected to contribute to incongruous divergent performances of daughters sired by MZ twins.In the next part of this project, using the developed platform, impacts of supplementation of a high-concentration global methyl donor on bovine early embryos was investigated. Supplementation with large amounts of folic acid (FA) has been extensively used and recommended in pregnant women for its well-established ability to prevent neural tube defects in children. However, more recently, several studies reported adverse effects of high FA concentrations, not only on embryo development, but also in adults. At the cellular level, FA enters one-carbon metabolism, the only pathway to produce S-adenosyl methionine (SAM) as the global methyl donor for a wide variety of biomolecules, including DNA. Therefore, to address this controversy, a high dose of SAM was used to treat in vitro -produced bovine embryos. This not only affected early embryo phenotypes, but also the transcritome and genome-wide DNA methylome. Overall, the current project resulted in development of a user-friendly and cost-effective bovine genome-wide DNA methylation analysis platform, which is compatible with small cell number such as early embryos. In addition, as one of the first studies of its kind in bovine reproductive biology, this project had three objectives which yielded several novel results, including comparative genome-wide DNA methylation profiles of: i) bovine blastocysts versus sperm; ii) sperm from monozygotic twin bulls and iii) high dose SAM-treated versus non-treated bovine early embryos.

Depuis plus de 40 ans, il est connu que les fœtus de mammifères sont sensibles aux conditions métaboliques de la mère durant la gestation. On commence à comprendre aujourd’hui les principes moléculaires de ces observations épidémiologiques. L’épigénétique définit cette nouvelle réalité et semble être la voie par laquelle l’environnement influence l’expression des gènes à plus ou moins long terme. Cette réalité est aussi présente en production laitière où les vaches en plus de montrer une production de lait accrue doivent soutenir le développement d’un fœtus. La forte mobilisation des réserves graisseuses associées à cette condition peut venir modifier la composition du milieu ovarien ainsi qu’utérin, et par le fait même, créer une dysfonction du métabolisme mitochondriale qui provoquera une augmentation du stress oxydatif. Cette modification peut pousser l’ovule ou l’embryon à modifier considérablement sa programmation épigénétique dans le but de s’adapter à ce signe de déficit métabolique. Dans cette étude, nous avons fait subir un stress métabolique à des embryons bovins in vitro afin de valider l’impact d’une telle perturbation sur l’épigénome embryonnaire. Les résultats obtenus ont permis de mettre en évidence une tendance à l’hypométhylation dans les régions télomériques de la majorité des chromosomes ainsi que des modifications sur des gènes reliés au métabolisme énergétique. Il devient donc important d’étudier ces modifications sur le développement et les performances futures de l’embryon et ce afin de mieux comprendre les impacts que certains types de rations ou habitudes de régie peuvent avoir sur le potentiel productif et reproductif des animaux de relève. Ces connaissances nous permettront d’adapter notre régie afin de maximiser le potentiel productif des animaux de l’industrie laitière québécoise et de conserver notre place parmi les leaders mondiaux de ce secteur de production.

Chez les mammifères, le passage d’une génération à une autre nécessite la reprogrammation du génome. Cette reprogrammation nécessite la déméthylation de l’ADN parternel et maternel ainsi que celle des lysines des histones suite à la fécondation. Des familles d’enzymes ont récemment été associées à ces processus : les déaminases, notamment Aicda (activation-induced cytosine deaminase), et les Tet (Ten-eleven translocation) désoxygénase, Tet1, Tet2 et Tet3. Plusieurs déméthylases des lysines des histones (KDM) ont été identifiées au cours des dernières années mais très peu d’informations sont disponibles à leur sujet, particulièrement en ce qui a trait à l’embryon bovin. Notre étude s’est attardée à dresser un profil d’expression spatiotemporel de ces familles d’enzymes lors des différents stades embryonnaires précoces chez la vache. Nous suggérons que Tet3 participe activement à la déméthylation de l’ADN, possiblement assisté par Tet2, mais sans Tet1 ni Aicda. Nous montrons également que KDM3A, KDM4A, KDM4C et KDM5B sont présents à des stades et à des endroits précis de l’embryon suggérant ainsi un rôle dans certains processus clés du développement embryonnaire. Ces informations ouvrent la voie à de nouvelles recherches afin de comprendre les modifications de l’épigénome et de réduire les anomalies épigénétiques rencontrées chez les animaux issus de certains protocoles de reproduction assistée.
In mammals, the transition from one generation to the next requires genomic reprogramming. Such epigenetic change is mediated by paternal and maternal DNA demethylation as well as histone lysines demethylation after fertilization, which is a poorly understood process. Some family of enzymes have recently been associated to those process: the deaminases, like Aicda (activation-induced cytosine deaminase), and Tet (Ten-eleven translocation) dioxygenases, Tet1, Tet2 and Tet3. Many lysine specific histone demethylases (KDM) have been identified in the past few years but little is known about their roles in mammalian embryo. The objective of this study was to develop of a spatiotemporal expression profile of those proteins at different preimplantation stage of bovine embryo. We suggest an active participation of Tet3 in DNA methylation, possibly supported by Tet2 but without Tet1 or Aicda. We also demonstrate the presence and specific localization of KDM3A, KDM4A, KDM4C and KDM5B which may suggest a role during the different embryonic stages. This information opens up the possibilities for further research in order to reduce epigenetic abnormalities associated to assisted reproduction technologies.

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O crescente avanço da produção in vitro de embriões bovinos intensificou a utilização de outras biotecnologias da reprodução tais como a micro-manipulação embrionária e o diagnóstico genético pré-implantacional, sendo a identificação do sexo embrionário utilizada na rotina comercial de laboratórios de produção in vitro. O objetivo deste trabalho foi avaliar as interações de diferentes fatores sobre a taxa de mortalidade embrionária e a proporção do sexo de embriões bovinos submetidos ao processo de sexagem. Foi realizado levantamento no banco de dados da Transfix – Transplante de Embriões Ltda, Patrocínio Paulista / Brasil, referente a 4.650 embriões produzidos in vitro e sexados entre 2005 e 2007. Os embriões foram submetidos à micro-manipulação pela técnica de micro-aspiração, e as biópsias à reação em cadeia pela polimerase (PCR). Somente as fêmeas foram transferidas para receptoras previamente sincronizadas. O diagnóstico de gestação e a determinação do sexo fetal foram realizados por ultra-sonografia. As variáveis foram classificadas de acordo com o sexo dos embriões (macho, fêmea e indeterminado), cinco laboratórios (A, B, C, D e E), seis raças bovinas (Nelore, Brahman, Girolando, Simental, Holandês e Jersey), estágio embrionário (MO, BI, BL, BX e BE), qualidade embrionária (1, 2 e 3) e qualidade da biópsia (“dentro do padrão” e “fora do padrão”). As análises estatísticas foram realizadas pelos testes 2 de associação, 2 de aderência para proporção 1:1 e pela análise de regressão logística com o método de Hosmer-Lameshow utilizando o procedimento logístico (PROC LOGISTIC) programa computacional SAS. A PCR apresentou eficiência de 93,3%, acurácia de 93,2% e taxa de machos e fêmeas de 52,9% e 47,1%, respectivamente. A taxa de mortalidade dos embriões...
Crescent progress of in vitro bovine embryo production has improved the use of other reproductive biotechnologies, as embryo micromanipulation and preimplantation genetic diagnosis, being embryo sexing used in commercial routine of in vitro embryo production laboratories. The present study aimed to evaluate the interactions among different factors on the mortality rate and sex ratio of in vitro produced bovine embryos. A survey was performed in the Transfix – Transplante de Embriões Ltda, Patrocínio Paulista / Brazil data base, referring to 4.650 in vitro produced bovine embryos sexed during years 2005/2007. Embryos were submited to the biopsy by the microaspiration technique, and biopsies to the polymerase chain reaction (PCR). Only female embryos were transferred to synchronized recipients. Pregnancy diagnosis and fetal sex determination were carried out by ultrasound. The variables were classified according embryo sex (male, female and indeterminate), five laboratories (A, B, C, D and E), six bovine breeds (Nellore, Brahman, Girolando, Simmental, Holstein and Jersey), embryo stage (MO, EB, BL, XB and HB), embryo quality (1, 2, and 3) and biopsy quality (“standard” and “non standard”). The statistical analysis was carried out by association 2 test, 2 for 1:1 ratio and logistic regression analysis with Hosmer- Lameshow method using logistic procedure (PROC LOGISTIC) of SAS package. The PCR showed 93.3% efficiency, 93.2% accuracy and male and female ratio of 52.9% and 47.1%, respectively. Mortality rate of biopsied embryos was 10.3% and pregnancy rate was 31.7%. Although no significant differences were observed between male and female ratio, indeterminate embryos possess greater possibility to die after micromanipulation. For quality 2 and 3 embryo mortality rate after biopsy was 3.19 and 11.37 fold higher, respectively, than for quality 1 embryo. For those whose biopsy... (Complete abstract click electronic access below)