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Wooldridge, Lydia Katherine. "Supplementing Bovine Embryo Culture Media to Improve the Production and Quality of In Vitro Produced Bovine Embryos." Diss., Virginia Tech, 2020. http://hdl.handle.net/10919/105143.
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Initial studies in this work explored the role of interleukin-6 (IL6) and leukemia inhibitory factor (LIF) in preimplantation bovine embryos. Neither cytokine affected the total percentage of embryos which developed to the blastocyst stage in vitro. However, supplementation of IL6 increased blastocyst inner cell mass (ICM) cell number without affecting trophectoderm (TE) cell number. Additionally, we found that IL6 activated signal transducer and activator of transcription 3 (STAT3) specifically within ICM cells. LIF, however, did not affect ICM cell number or activate STAT3 in ICM cells, and was not pursued further. This increase in ICM cell number by IL6 was largely comprised of hypoblast (GATA6+:NANOG-) cells, and most IL6-responsive cells in day 9 blastocysts were hypoblast cells (as measured by STAT3 activation). However, some epiblast (NANOG+) cells were also IL6-responsive, and IL6 appeared to initially slow epiblast differentiation. Finally, IL6-treated blastocysts also had increased transcripts of hypoblast/primitive endoderm (PE) markers. These results indicate that IL6 may improve pregnancy retention of IVP embryos by improving yolk sac development, but further work is needed to confirm this theory.
Activation of STAT3 by IL6 could be blocked with a chemical Janus kinase 2 (JAK2) inhibitor (AZD1480). JAK2 inhibition from day 5 to 8 resulted in blastocyst ICMs with fewer than 10% the normal cell number, regardless of IL6 supplementation. This indicates that STAT3 is critical for bovine ICM development. Further analysis revealed that inhibition of JAK2/STAT did not prevent ICM formation but disrupted its maintenance.
Additionally, we assessed the suitability of zinc sulfate and a bovine embryonic stem cell culture media (TeSR) for improving bovine embryo development in vitro. Zinc sulfate increased day 8 blastocyst total and ICM cell number. Therefore, zinc sulfate appears to improve blastocyst quality. The TeSR medium improved embryo development beyond day 8. In normal synthetic oviduct fluid, blastocysts degenerated after day 8, while blastocysts moved to TeSR had greatly increased cell numbers, and even exhibited PE migration out from the ICM, a phenomenon that has not been reported in vitro. This indicates that extended blastocyst culture is possible with TeSR media.Doctor of Philosophy
Bovine embryos have been produced in vitro for the purpose of being transferred to recipient cattle to produce a calf since the 1980s. This practice allows cattle breeders to increase the number of offspring from their best females each year, and also allows for more rapid progress in generational genetic improvement. However, only approximately 10% of bovine oocytes survive and produce a calf. This poor efficiency of bovine in vitro embryo production negatively impacts the procedure's widespread use. A significant portion of these embryo losses are likely a result of inadequate in vitro culture conditions, particularly of the embryo culture media, the fluid in which embryos are grown. This media is often called "synthetic oviduct fluid," or SOF, because it is designed to mimic the fluid present in the cow's oviduct, where the embryo would normally reside. However, SOF is much simpler in nature than actual cow oviduct fluid, and this leads to reduced embryonic survival of in vitro produced embryos. Unfortunately, we know very little of what molecules control and promote bovine embryo development. Therefore, one major goal of bovine embryo research is to identify these factors and add them to SOF. The goal of this work was to examine the ability of three molecules, interleukin-6 (IL6), leukemia inhibitory factor (LIF), and zinc sulfate, to increase the number and quality of blastocysts produced through in vitro culture techniques. Additionally, I tested the replacement of SOF with a complex cell culture media, known as TeSR. This medium is more complex than SOF, and therefore should better promote embryo development. This work revealed that IL6, but not LIF, improves in vitro produced (IVP) bovine blastocyst quality. Unfortunately, neither IL6 nor LIF affected the percentage of embryos which survived to the blastocyst stage. However, IL6, but not LIF, increased the number of cells in the inner cell mass (ICM) of the blastocysts. ICM cells are the portion of the embryo which will produce the future calf. IVP bovine embryos are known to have fewer cells than normal, in vivo derived, blastocysts, and this issue is believed to cause some embryonic death after embryo transfer. Therefore, treatment with IL6 may increase the percentage of embryos which will survive after transfer and produce a calf. We also found the addition of zinc sulfate to SOF to benefit embryo quality. None of the concentrations of zinc significantly improved the percentage of embryos which survived to the blastocyst stage, but 2 µM zinc did increase ICM cell number. Like IL6, this may improve embryo survival after transfer. The use of the TeSR media as a replacement for SOF had some benefits. Unfortunately, this media is unusable for producing embryos for transfer to recipients, as we discovered early embryos could not survive in the media. However, blastocyst-stage embryos thrived in it, and could be cultured in vitro for a longer period of time as a result. Therefore, this media will be a useful tool for studying bovine embryo development in vitro, however it is unlikely to benefit calf production. In summary, this work provides evidence that zinc sulfate and IL6 are beneficial additions to SOF. However, future work is needed to determine if embryos produced with these factors are more able to produce a calf. Additionally, we discovered that TeSR is a superior extended blastocyst culture medium.
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Simmet, Kilian Manuel Verfasser], and Eckhard [Akademischer Betreuer] [Wolf. "Chimeric bovine embryo multiplication with OCT4 knockout host embryos / Kilian Manuel Simmet ; Betreuer: Eckhard Wolf." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1155407822/34.
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Jooste, Frans. "The association between foot-and-mouth disease virus and bovine oocytes and embryos during in vitro embryo production." Diss., University of Pretoria, 2005. http://upetd.up.ac.za/thesis/available/etd-03022006-120630/.
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Jousan, Frank Dean. "Effects of Differences in Dietary Protein and Varying the Interval from Collection of Bovine Embryos to Freezing on Embryo Quality and Viability." Thesis, Virginia Tech, 2002. http://hdl.handle.net/10919/33788.
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High levels of dietary protein may be detrimental to reproductive performance in cattle. The objective of Exp. 1 was to determine the effects of differences in dietary protein on the production and quality of bovine embryos collected from superovulated donors. Angus cows were randomly assigned to receive one of three experimental diets: a daily ration of 5.7 kg poultry litter,
2.0 kg hay, 3.1 kg corn, and 0.5 kg peanut hulls (LITTER; n = 15); a daily ration of 6.2 kg peanut hulls, 2.2 kg soybean meal, 2.0 kg hay, 0.5 kg corn, and 0.4 kg dicalcium phosphate (SBM; n = 15); or a daily ration of 6.2 kg peanut hulls, 2.0 kg hay, and 3.1 kg corn (CON; n = 19). Diets differed in the amount of total, soluble and degradable protein, but were comparable in energy. After 30 d on the diets, all cows were treated to induce superovulation (28.8 mg FSH/cow, Folltropin) and synchronize estrus. After the detection of estrus each cow was inseminated with semen from one of four Holstein bulls. Embryos were collected 7 d after estrus and evaluated for quality (according to the International Embryo Transfer Society (IETS) standards) and stage of development. Prior to treatment to induce superovulation, blood samples were collected 6 h after feeding. Samples were analyzed to assess dietary effects on plasma urea nitrogen (PUN). Mean levels of PUN were higher
(P < 0.01) in cows fed the LITTER or SBM diet
(16.3 mg/dL, LITTER; 21.8 mg/dL, SBM; 9.7 mg/dL, CON) than in cows fed the CON diet. Additionally, concentration of PUN was higher in cows fed SBM than in those fed LITTER (P < 0.01). An average of 9.2 transferable embryos (Grade 1, 2 and 3) was collected from each cow and there were no significant differences in the number of transferable embryos collected among groups
(9.2, LITTER; 9.3, SBM; 9.1, CON). The number of degenerate embryos or unfertilized ova did not differ among dietary groups. High-protein diets elevated PUN, but did not affect the number or quality of embryos collected from superovulated donors.
Cryopreservation of bovine embryos is an important aspect of a successful embryo transfer program. The objective of Exp. 2 was to evaluate the post-thaw viability of bovine embryos collected in Exp. 1 in an in vitro culture system after the embryos had been held at room temperature or refrigerated for 2 to 12 h prior to freezing. Upon embryo recovery, each embryo was randomly assigned to be placed in holding media for 2, 6 or 12 h prior to freezing. During this interval, one-half of the embryos were maintained in a refrigerated environment (5 °C), while the remaining half of the embryos were held at room temperature (20.5 to
22 °C) until freezing. Immediately prior to freezing, embryos were removed from the holding media, transferred to a well containing ethylene glycol (10%) in ovum culture media and loaded individually into a 0.25-mL plastic straw. Straws were then placed in a freezer unit (-6 °C) and seeded to induce ice crystal formation through all columns of the straw. The temperature of the freezer was then decreased 0.6 °C/min to -32 °C, and straws were loaded into canes and plunged into a liquid nitrogen tank (-196 °C). After storage, each straw was exposed to a 5-s air thaw and placed in a water bath at 35 °C for 20 s. Each embryo was then washed to remove excess ethylene glycol prior to in vitro culture. Embryos were individually cultured in Ham's F-10 media supplemented with
4% fetal bovine serum for 72 h. Embryos were evaluated at 24 h intervals throughout the culture period and assigned a stage of development and quality grade score (according to IETS standards). The percentage of embryos that developed to the expanded blastocyst stage and hatched from the zona pellucida was greater for embryos held 2 or 6 h prior to freezing (P < 0.05) than for embryos held for 12 h after collection before being frozen (62.9, 52.0 and 31.1%, respectively). The percentage of embryos that degenerated during in vitro culture was lower for embryos held 2 or 6 h prior to freezing (20.4 and 26.6%; P < 0.05) than for embryos held for 12 h before freezing (50.8%). Furthermore, embryo quality grade was more desirable for embryos held for 2 or 6 h (1.5 and 1.7; P < 0.05) than for those held for 12 h before freezing (2.1). The semen used to inseminate donors and the diet fed to donors for 4 wk prior to embryo collection did not influence the proportion of embryos that hatched or degenerated during the 72 h of in vitro culture. Additionally, holding embryos in a refrigerated environment from the time of collection until freezing did not enhance embryonic development during post-thaw culture. Thus, embryonic viability may be impaired when embryos are held longer than 6 h following embryo recovery before being frozen; however, the storage temperature during the interval from collection to freezing does not influence embryonic development post-thaw.Master of Science
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Ben, Amor Hanene. "Chromosome abnormalities in preimplantation bovine embryos." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111790.
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Note:Studies suggest that chromosomal abnormalities notably mosaicism consisting of normal and abnormal cells is a common feature observed in mammalian preimplantation embryos. The data on chromosome abnormalities in bovine embryos however, are limited. The principal aim of this study was to investigate chromosome abnormalities and their effect on the development of bovine embryos produced in vitro. 193 embryos were evaluated for chromosomal abnormalities, using dual fluorescent in situ hybridization (FISH) with developed DNA probes for X and Y chromosomes. Our results demonstrate that uniformly abnormal embryos were found mostly at the early cleavage stages, and embryos with extensive chromosome abnormalities were usually arrested by the morula stage. Chromosomal mosaicism was observed at the 2- cell stage and increased steadily with subsequent stages of development. By the blastocyst stage, chromosomal mosaicism was the main abnormality observed and affected 95% of the blastocysts. Most of the mosaic blastocysts comprised of diploid and tetraploid cells. In the second part, a detailed analysis of 121 day 7 and days 9-10 blastocysts, demonstrated that the proportion of polyploid cells in most of the morphologically good quality embryos was less than 15%, which was significantly lower than in poor quality embryos. [...]
II a ete suggere que des anomalies chromosomiques particulierement le mosaicism sont frequemment rencontres chez les embryons des bovins produit in vitro, cependant les donnees disponibles sont tres limitees. Le but principal de cette etude est d'evaluer les anomalies chromosomiques particulierement le mosaicism au different stades de developpement embryonnaire par FISH en utilisant des probes 'ADN pour les chromosomes X et Y. Nos resultats demontrent que des embryons uniformement anormales ont ete surtout trouves aux premiers stades de cleavage, temoignant que les embryons avec une vaste anomalie affectant la totalite des embryons sont souvent arretes au stade du morula. Le mosaicism chromosomique a ete rencontre dans tous les stades de developpement et il a augmente emarquablement pendant le developpement embryonnaire. Ainsi, au stade du blastocyst, le mosaicism chromosomique etait l'anomalie principale observee avec 95 % de blastocysts analyses devenant mosaiques. [...]
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Annes, Kelly. "Caracterização do metabolismo de lipídeos no desenvolvimento inicial de embriões bovinos produzidos in vitro com diferentes cinéticas de desenvolvimento." reponame:Repositório Institucional da UFABC, 2015.
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Orientadora: Prof. Dra. Marcella Pecora Milazzotto.Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2015.
A morfologia e as taxas de clivagem e de blastocistos têm sido critérios utilizados para avaliação da competência embrionária. Entretanto, com o advento de novas biotecnologias tem-se tornado claro que a competência embrionária pode ser severamente comprometida sem alterações morfológicas perceptíveis. Estudos em embriões humanos propuseram avaliações morfológicas adicionais relacionadas ao momento das primeiras divisões celulares embrionárias (rápida e lenta) que parecem estar relacionadas com a viabilidade do embrião. No entanto, ainda não existem muitos dados dessa análise morfocinética em bovinos. A viabilidade embrionária também pode ser severamente comprometida pelo acúmulo de lipídeos nos embriões PIV, podendo inclusive prejudicar aplicações comerciais como a criopreservação. Com isso, o objetivo desse estudo foi caracterizar em embriões bovinos de cinéticas diferentes de desenvolvimento (rápido e lento) o padrão de metabolismo lipídico. Para tal, embriões produzidos in vitro foram analisados quanto a quantidade de lipídeos totais e caracterização de lipídeos de membrana nos estádios iniciais de clivagem (22hpi e 96hpi) e blastocisto. Para o estádio de blastocisto também foi incluído um grupo de embriões in vivo. Foi possível evidenciar menor quantidade de lipídeos totais pela coloração SUDAN BLACK B nos grupos lentos. As análises de MALDI-MS evidenciaram lipídeos de membrana com padrões distintos nos grupos rápidos e lentos nos estádios de clivagem e mórula. Já nos estádios de blastocistos os dados nos permitem inferir que o grupo de blastocisto lento parece estar mais próximo do grupo in vivo, pela semelhança na abundância/intensidade relativa no maior número de íons revelados pelas análises multivariadas. No entanto, o grupo lento ainda mostra alguma semelhança com o grupo rápido devido a exposição ao mesmo ambiente in vitro.
Embryo viability and competence have been evaluated by criteria such as morphology and cleavage and blastocyst rates. However, the advent and application of new biotechnologies have demonstrated that embryonic competence can be severely compromised without noticeable morphological changes. Human embryo studies proposed the use of additional morphological evaluations related to the moment of the first embryonic cell divisions and its kinetic (fast and slow), which appear to be relevant to the embryo viability. Nevertheless, there are still not enough data available related to the morphokinetic analysis of embryos in bovine cattle. Embryo viability can also be severely compromised by lipid accumulation in IVP (in vitro produced) embryos and can even harm commercial applications such as cryopreservation. Therefore, the aim of this study was to evaluate and characterize the pattern of lipid metabolism on bovine embryos with different developmental kinetics (fast and slow). For this goal, IVP embryos were analyzed considering the lipids total amount and membrane lipids characterization during the cleavage early stages (22hpi and 96hpi) and blastocyst stage. The study also included a group of in vivo embryos at the blastocyst stage. The results, using SUDAN BLACK B staining technique, showed a smaller amount of total lipids in the slow groups. The MALDI-MS analysis results showed different patterns of membrane lipids in the fast and slow groups in the cleavage and morulae stages. The data obtained at the blastocyst stage allow us to infer that the slow group is more similar to the in vivo group, since the results showed similarity in relative quantity/intensity in a greater number of ions revealed by multivariate analysis. However, the slow group still shows some similarity with the fast group due to the exposure to the same in vitro environment.
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Bilodeau-Goeseels, Sylvie. "Changes in RNA abundance in early bovine embryos." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq20726.pdf.
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McDougall, Kathryn Elizabeth. "Alkaline phosphatase isozyme expression in preattachment bovine embryos." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/NQ35804.pdf.
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Yang, Ming Yuan. "Studies on apoptosis in bovine follicles and embryos." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0019/NQ56648.pdf.
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Cebrián, Serrano Alberto. "Factors affecting the in vitro embryo production in cattle associated to ovum pick up sistem." Doctoral thesis, Universitat Politècnica de València, 2013. http://hdl.handle.net/10251/27646.
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La producción de embriones mediante la recuperación de ovocitos inmaduros por ovum pick up (OPU), y su posterior maduración, fecundación y cultivo en el laboratorio in vitro, presenta numerosos beneficios para optimizar el potencial reproductivo, tanto de hembras como de machos. Además, frente a la superovulación convencional mediante tratamiento hormonal y la recogida de embriones in vivo, la producción in vitro de embriones (PIVE) con ovocitos de OPU ofrece considerables ventajas. Sin embargo, actualmente la PIVE continua siendo ineficiente e incapaz de producir embriones de calidad similar a los in vivo, lo cual ha limitado una aplicación más amplia de esta tecnología. Así pues, el objetivo de esta tesis fue la optimización de la PIVE en ganado vacuno, condicionado por las peculiaridades y deficiencias de la PIVE cuando los ovocitos son recuperados por la técnica de OPU. Con este fin, cinco experimento se llevaron a cabo en esta tesis. En el primero de ellos se estudió el efecto del fluido oviductal bovino (FOb) sobre el desarrollo y la calidad embrionaria (Experimento 1). Las fases del proceso de PIVE en las cuales el cultivo de ovocitos/embriones, bien individualmente o bien en número reducido, pudiera perjudicar el posterior desarrollo hasta el estadio de blastocisto y/o a su calidad, se estudiaron en el Experimento 2. En el Experimento 3 se testó si el desarrollo y la calidad de embriones cultivados in vitro en número reducido podría ser mejorada con la adición conjunta de factor de crecimiento epidérmico, insulina, transferrina y selenio (FCE-ITS) o por el sistema de cultivo de embriones llamado well of well (WOW). Las propiedades protectoras de la melatonina frente a los daños causados por el estrés oxidativo, subsecuentes de las condiciones de PIVE o de un estrés térmico durante la maduración ovocitaria, fueron evaluadas en el Experimento 4. Por último, en el Experimento 5 usamos ovocitos recolectados por OPU para evaluar el efecto del semen sexado sobreCebrián Serrano, A. (2013). Factors affecting the in vitro embryo production in cattle associated to ovum pick up sistem [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/27646
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Ménétrey, Frédéric. "Multiple preimplantation genetic diagnosis and analysis of bovine embryos /." Zürich : ETH, 2007. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=16581.
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Choi, Inchul. "Effects of oocyte on epigenetic reprogramming of bovine SCNT embryos." Thesis, University of Nottingham, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479364.
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Matwee, Christie Nicole. "Apoptosis in the pre-attachment bovine embryo." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0021/MQ55691.pdf.
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Partridge, Robert James. "The biochemistry of the bovine preimplantation embryo." Thesis, University of York, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245866.
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Gibson, Bethany Gale. "Improving the development of bovine in vitro produced embryos cultured individually." Thesis, Virginia Tech, 2014. http://hdl.handle.net/10919/49694.
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Previous research in bovine embryology has found that embryos cultured individually have limited ability to develop compared to their counterparts cultured in a group of other embryos. This investigation aimed to find if any of three different interventions over two experiments would increase development of individually cultured embryos to that of group cultured embryos. In the first experiment both the addition of serum/serum replacer and a co-culture with bovine granulosa cells were applied to individually cultured embryos in a 3x2 design. None of the interventions was found to be significantly different from the others, and all resulted in significantly lower development than embryos cultured as a group (avg. 4.7 +/- 1.93% individual vs. 21.7 +/- 3.76% group). However, a significant difference was found in the hatching rate between blastocysts cultured in media including cells (71.4 +/- 17.07%) and those cultured without cells (18.1 +/- 11.63%). In the second experiment, embryos were either cultured in standard droplets or microwells made at the bottom of culture droplets either in groups or individually for a 2x2 design. This experiment experienced poor development in all treatments including the group control, and none of the treatments were found to be significantly different from each other. However, the hatching rate of blastocysts cultured in multiple microwells was significantly higher than those cultured individually in droplets. To summarize, none of the treatments increased the development rate, but embryos cultured with granulosa cell co-cultures and in group microwells showed improvements in hatching rates.Master of Science
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Marley, Mylissa Shonda Divina Givens Maurice Daniel. "Assessment of methods to minimize transmission of bovine herpesvirus associated with embryos." Auburn, Ala., 2007. http://hdl.handle.net/10415/1332.
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Roberts, Melissa Ann. "Metabolic Regulation and Cryotolerance of In Vitro-Produced Holstein Embryos." DigitalCommons@CalPoly, 2016. https://digitalcommons.calpoly.edu/theses/1693.
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In vitro production and transfer of embryos has become a common practice within the dairy industry to efficiently breed superior animals and meet the consumption demand of the growing population. Cyropreservation is necessary for the application of commercialized embryo transfer, however, in vitro-produced embryos show morphological and physiological defects which negatively impact their ability to withstand cryopreservation in comparison to their in vivo counterparts. These artifacts result from culture conditions that cause stress to the embryo during development, leading to an accumulation of intracellular lipids, mitochondrial dysfunction, and ultimately poor ability to withstand freezing and thawing. The objective of these studies was to examine the effects of various metabolic regulators on the viability and cryotolerance of in vitro-produced embryos. Pilot studies revealed that evaluating early (stage 6) versus late (stage 7) blastocysts did not affect the trend seen in results, nor did culturing embryos in continuous versus sequential media. From the main experiment performed, it was concluded that a combination of metabolic regulators decreased lipid content, improved cryopreservation survival, and lowered the percentage of apoptotic cells present after thawing. Conditioned media increased the blastocyst percentage, but did not produce superior quality embryos as measured by cryotolerance. Research concerning the metabolic needs of the preimplantation embryo must continue to determine more relevant markers of embryo quality in vitro.
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Rocha, Nathália Alves de Souza [UNESP]. "Efeitos de antioxidantes e da atmosfera gasosa em diferentes etapas da produção in vitro sobre o desenvolvimento e criotolerância de embriões bovinos." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/98170.
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O estudo foi conduzido com o objetivo de avaliar os efeitos da suplementação com antioxidantes intracelulares e extracelulares em diferentes etapas da PIV (MIV e/ou CIV), e da tensão de oxigênio durante o CIV sobre o desenvolvimento e criotolerância de embriões bovinos. No Exp.1 foi realizada a suplementação com antioxidantes {0,6 mM cisteína (CIST), 0,6 mM cisteína associado á 100 μM de cisteamina (C+C) e 100 UI catalase (CAT)} durante todo o período de CIV, em diferentes atmosferas gasosas {5% CO2 em ar (20% O2) ou atmosfera controlada (7% O2, 5% CO2 e 88% N2)}. Já, no Exp. 2 foi realizada a suplementação com antioxidantes {0,6 mM cisteína (CIST), 100 UI catalase (CAT) e 100 μM de β-mercaptoetanol (β-ME)} durante 72 horas de CIV, nas diferentes atmosferas gasosas. Posteriormente, após definir a tensão de oxigênio, bem como, o período de suplementação adequado para o CIV, foi realizada a adição de antioxidantes durante a maturação in vitro (MIV) e/ou 72 horas de CIV (Exp.3). No Exp.1, a taxa de desenvolvimento embrionário foi adversamente afetada (P<0,05) pelos tratamentos CIST (11,2%) e C+C (1,4%), em relação ao Controle (26,6%), e pela tensão de oxigênio (17,2% e 11,1%; 20 e 7% O2, respectivamente). Em relação à taxa de re-expansão, após reaquecimento e cultivo in vitro por 24 horas, não houve diferença significativa (P>0,05) entre os tratamentos avaliados (66,7% a 100%). No Exp.2, as taxas de blastocistos não foram afetadas (P>0,05) pelos tratamentos CIST, β-ME e CAT (43,7% a 48,5%), porém a baixa tensão de oxigênio afetou adversamente (P<0,05) o desenvolvimento embrionário (52,1% e 38,4%; 20 e 7% O2 respectivamente). A mensuração dos níveis intracelulares de ROS não foi afetada (P>0,05) pelas variáveis tratamentos (0,95 a 0,78) e tensão de oxigênio...
This study was conducted to evaluate the effects of intracellular and extracellular antioxidants supplementation, in different stages of IVP (IVM and/or IVC), and oxygen tension during IVC on development, quality and cryotolerance of bovine embryos. Exp.1 was performed with the supplementation with antioxidants {0.6 mM cysteine (CIST); 0.6 mM cysteine associated to 100 μM cysteamine (C+C); 100 UI catalase (CAT)} during entire period of IVC in different gaseous atmospheres {5% CO2 in air (20% O2) or controlled atmosphere (7% O2, 5% CO2 and 88% N2)}. Already, in Exp.2 was performed the antioxidant supplementation {0.6 mM cysteine (CIST); 100 μM β-mercaptoethanol (β-ME); 100 UI catalase (CAT)} for 72 hours of IVC in different gaseous atmospheres. Later, after setting the oxygen tension as well as the supplementation period suitable for IVC, was carried out the addition of antioxidants during in vitro maturation (IVM) and/or 72 hours of IVC (Exp.3). In Exp.1, the rate of embryo development was adversely affected (P<0.05) by the treatments CIST (11.2%) and C+C (1.4%), compared to Control (26.6%), and oxygen tension (17.2% and 11.1%, 20 and 7%O2, respectively). Regarding the re-expansion rate after warming and in vitro culture for 24 hours, no difference (P>0.05) between the treatments were found (66.7% to 100%). In Exp.2, blastocysts rates were not affected (P>0.05) by treatments CIST, β-ME and CAT (43.7% to 48.5%), but the low oxygen tension adversely affected (P<0.05) embryo development (52.1% to 38.4%, 20 and 7%O2, respectively). The quantification of intracellular levels of ROS was not affected (P>0.05) by the variables treatments (0.95 to 0.78) and oxygen tension... (Complete abstract click electronic access below)
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Rocha, Nathália Alves de Souza. "Efeitos de antioxidantes e da atmosfera gasosa em diferentes etapas da produção in vitro sobre o desenvolvimento e criotolerância de embriões bovinos /." Jaboticabal : [s.n.], 2012. http://hdl.handle.net/11449/98170.
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Orientador: Gisele Zoccal MingotiBanca: Joaquim Mansano Garcia
Banca: Juliana Corrêa Borges Silva
Resumo: O estudo foi conduzido com o objetivo de avaliar os efeitos da suplementação com antioxidantes intracelulares e extracelulares em diferentes etapas da PIV (MIV e/ou CIV), e da tensão de oxigênio durante o CIV sobre o desenvolvimento e criotolerância de embriões bovinos. No Exp.1 foi realizada a suplementação com antioxidantes {0,6 mM cisteína (CIST), 0,6 mM cisteína associado á 100 μM de cisteamina (C+C) e 100 UI catalase (CAT)} durante todo o período de CIV, em diferentes atmosferas gasosas {5% CO2 em ar (20% O2) ou atmosfera controlada (7% O2, 5% CO2 e 88% N2)}. Já, no Exp. 2 foi realizada a suplementação com antioxidantes {0,6 mM cisteína (CIST), 100 UI catalase (CAT) e 100 μM de β-mercaptoetanol (β-ME)} durante 72 horas de CIV, nas diferentes atmosferas gasosas. Posteriormente, após definir a tensão de oxigênio, bem como, o período de suplementação adequado para o CIV, foi realizada a adição de antioxidantes durante a maturação in vitro (MIV) e/ou 72 horas de CIV (Exp.3). No Exp.1, a taxa de desenvolvimento embrionário foi adversamente afetada (P<0,05) pelos tratamentos CIST (11,2%) e C+C (1,4%), em relação ao Controle (26,6%), e pela tensão de oxigênio (17,2% e 11,1%; 20 e 7% O2, respectivamente). Em relação à taxa de re-expansão, após reaquecimento e cultivo in vitro por 24 horas, não houve diferença significativa (P>0,05) entre os tratamentos avaliados (66,7% a 100%). No Exp.2, as taxas de blastocistos não foram afetadas (P>0,05) pelos tratamentos CIST, β-ME e CAT (43,7% a 48,5%), porém a baixa tensão de oxigênio afetou adversamente (P<0,05) o desenvolvimento embrionário (52,1% e 38,4%; 20 e 7% O2 respectivamente). A mensuração dos níveis intracelulares de ROS não foi afetada (P>0,05) pelas variáveis tratamentos (0,95 a 0,78) e tensão de oxigênio... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This study was conducted to evaluate the effects of intracellular and extracellular antioxidants supplementation, in different stages of IVP (IVM and/or IVC), and oxygen tension during IVC on development, quality and cryotolerance of bovine embryos. Exp.1 was performed with the supplementation with antioxidants {0.6 mM cysteine (CIST); 0.6 mM cysteine associated to 100 μM cysteamine (C+C); 100 UI catalase (CAT)} during entire period of IVC in different gaseous atmospheres {5% CO2 in air (20% O2) or controlled atmosphere (7% O2, 5% CO2 and 88% N2)}. Already, in Exp.2 was performed the antioxidant supplementation {0.6 mM cysteine (CIST); 100 μM β-mercaptoethanol (β-ME); 100 UI catalase (CAT)} for 72 hours of IVC in different gaseous atmospheres. Later, after setting the oxygen tension as well as the supplementation period suitable for IVC, was carried out the addition of antioxidants during in vitro maturation (IVM) and/or 72 hours of IVC (Exp.3). In Exp.1, the rate of embryo development was adversely affected (P<0.05) by the treatments CIST (11.2%) and C+C (1.4%), compared to Control (26.6%), and oxygen tension (17.2% and 11.1%, 20 and 7%O2, respectively). Regarding the re-expansion rate after warming and in vitro culture for 24 hours, no difference (P>0.05) between the treatments were found (66.7% to 100%). In Exp.2, blastocysts rates were not affected (P>0.05) by treatments CIST, β-ME and CAT (43.7% to 48.5%), but the low oxygen tension adversely affected (P<0.05) embryo development (52.1% to 38.4%, 20 and 7%O2, respectively). The quantification of intracellular levels of ROS was not affected (P>0.05) by the variables treatments (0.95 to 0.78) and oxygen tension... (Complete abstract click electronic access below)
Mestre
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Santos, Érika Cristina dos. "Metabolômica para avaliação não invasiva de embriões bovinos produzidos in vitro." reponame:Repositório Institucional da UFABC, 2015.
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Orientadora: Prof. Dra. Marcella Pecora Milazzotto.Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2015.
A bioenergia destaca-se como um substituto relevante para os combustíveis fósseis e os A capacidade de selecionar o embrião com maior viabilidade para transferência às receptoras é um componente crucial para o sucesso das técnicas de reprodução assistida. Atualmente, avaliações morfológicas são utilizadas para selecionar os embriões com maior potencial de implantação, e apesar de se tratar de um método não invasivo e relativamente bem sucedido, ainda apresenta uma série de limitações. Nos últimos anos, o desenvolvimento de novas tecnologias tem possibilitado análises quantitativas e qualitativas para determinação da viabilidade dos embriões para melhoria da seleção embrionária. Em especial as tecnologias espectroscópicas e espectrométricas permitiram o desenvolvimento de novos métodos para a caracterização embrionária e predição do potencial de estabelecimento da prenhez. Assim, o objetivo deste trabalho foi a caracterização não invasiva de embriões bovinos pela análise dos perfis espectroscópicos e espectrométricos dos meios de cultivo de embriões com diferentes cinéticas de desenvolvimento visando a obtenção de padrões específicos baseados no fenótipo embrionário. Para isso, embriões bovinos foram produzidos in vitro por protocolos convencionais. Os zigotos foram transferidos para meios de cultivo individualmente e classificados em dois grupos: rápido (4 células-40hpi) e lento (2 ou 3 células-40hpi). Os meios de cultura foram coletados às 40, 96 e 168 horas pós-inseminação (hpi), tranferidos para criotubos e congelados a -80ºC até o momento das análises. A análise do metaboloma embrionário foi realizada por espectroscopia Raman. Para tal, gotas de meios de cultura foram cobertas com óleo mineral, sendo escaneadas por um sistema Raman triplo. Os dados foram normalizados e analisados por Análises de Componentes Principais (PCA), de Agrupamentos (Clusters) e por Loading plot. A análise do secretoma embrionário foi feita por MALDI-MS-TOF, através da extração dos metabólitos, sendo os espectros adquiridos pelo espectrômetro em modo de ionização positiva. Foram feitas analises estatísticas univariadas e multivariadas pelo software online Metaboanalyst. Os resultados obtidos pela espectroscopia Raman e espectrometria de massas demonstram que embriões com diferentes cinéticas de desenvolvimento possuem diferentes perfis espectroscópicos e espectrométricos ao longo do desenvolvimento embrionário, indicando que estes embriões consomem e/ou produzem diferencialmente metabólitos nos meios de cultura. Com base em nossos resultados, propomos com este estudo que a análise dos meios de cultura por espectroscopia Raman pode ser utilizada para fins de diagnóstico embrionário, pois permite uma caracterização rápida e global dos perfis embrionários relacionados a cinética, enquanto a espectrometria de massas pode ser utilizada para caracterização qualitativa dos embriões, permitindo a identificação do secretoma de embriões durante o cultivo in vitro.
The ability to select embryo with greater viability to transfer to the receptors is a crucial component to the success of assisted reproduction techniques. Currently, morphological assessments are used to select embryos with the highest implantation potential, although it¿ s a noninvasive and relatively successful, has still a number of limitations. In recent years, the development of new technologies has enabled quantitative and qualitative analyzes for the determination of viability of embryos to improve embryo selection. In particular spectroscopic and spectrometric technologies have permitted the development of new methods for embryo characterization and prediction of potential establishment of pregnancy. The objective of this study was non-invasive characterization of bovine embryos by analysis of spectroscopic and spectrometric profiling of embryo culture media with different kinetics of development in order to obtain specific patterns based on embryonic phenotype. For this, bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast (4 cells-40hpi) and slow (2 or 3 cells-40hpi). The culture media were collected at 40, 96 and 168 hours post-insemination (hpi) tranfered to cryotubes and frozen at -80 until the time of analysis. The analysis of the metabolome embryo was made by Raman spectroscopy. For this, droplets of culture media were overlaid with mineral oil being scanned by a triple Raman system. The data were normalized and analyzed by Principal Component Analysis (PCA), clusters and Loading plot. Analysis of embryonic secretome was made by MALDI-TOF-MS, through the extraction of the metabolites, and the spectra acquired by the spectrometer in positive ionization mode. Analyzes were performed univariate and multivariate statistics by the online software Metaboanalyst. The results obtained by Raman spectroscopy and mass spectrometry demonstrated that the development of embryos with different kinetics have different spectroscopic and spectrometric profiles during embryonic development, indicating that these embryos consume and /or produce differentially metabolites in the culture media. Based on our results, we propose that the analysis of Raman spectroscopy culture media may be used for diagnostic purposes embryo, since it allows a fast and comprehensive characterization of embryonic profiles related to kinetics, while mass spectrometry can be used for qualitative characterization of the embryo, allowing the identification of secretome embryo during in vitro culture.
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Demant, Myriam. "Qualitative and quantitative proteome analyses of bovine oocytes and early embryos." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-142446.
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Rodriguez-Osorio, Nelida. "MOLECULAR REPROGRAMMING IN BOVINE EMBRYOS AFTER SERIAL SOMATIC CELL CHROMATIN TRANSFER." MSSTATE, 2008. http://sun.library.msstate.edu/ETD-db/theses/available/etd-04022008-095409/.
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Somatic Cell Nuclear Transfer (SCNT), commonly known as cloning, is the transfer of a somatic nucleus into an enucleated oocyte to produce a clone. The chromatin structure of somatic cells permits the expression of certain genes, while silencing the rest of the genome. The cytoplasm of oocytes can reprogram a somatic nucleus by reactivating the genes necessary for embryonic development and silencing the somatic genes. However, the low efficiency of SCNT indicates that successful nuclear reprogramming is a rare event. The objectives of this study were determine the extent of transcriptional reprogramming in bovine blastocysts produced by serial rounds of chromatin transfer (from first and fourth generations), using blastocysts produced by in vitro fertilization (IVF) as controls, to identify cumulative errors in the transcriptome profile. Differentially expressed genes were studied further to determine their function in embryonic development. We identified a set of transcripts consistently misregulated in cloned blastocyst, some of which had a more marked misregulation in the embryos produced by 4 successive rounds of cloning. Among the genes significantly upregulated in both CT groups compared to IVF blastocysts were both de novo DNA methylation enzymes DNMT3A and DNMT3B. Expression patterns, structural and functional analyses were performed for DNA methyltransferases. A high structural and functional conservation was observed for DNA methyltransferases among human, mouse, and bovine species. A set of genes that participate in early embryonic development, chromatin remodeling and DNA methylation were differentially regulated in cloned embryos and had not been fully annotated at the time of the analysis. We annotated those genes and submitted them to the Bovine Genome Sequencing Consortium database. These results have important implications for the selection of models for the study of DNA methylation during early development. The present study provides a valuable data set for identifying possible cumulative errors in somatic cell chromatin transfer that could hinder nuclear reprogramming shedding light on the epigenetic role in reprogramming and cell plasticity.
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McHughes, Courtney Elizabeth Prather Randall S. "Identification and quantification of differentally represented transcripts in preimplantation bovine embryos." Diss., Columbia, Mo. : University of Missouri--Columbia, 2007. http://hdl.handle.net/10355/5020.
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The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on September 15, 2009). Thesis advisor: Dr. Randall Prather Includes bibliographical references.
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Anderson, Bret L. "Effects of Antioxidants on Development of In Vitro Fertilized Bovine Embryos." DigitalCommons@USU, 1995. https://digitalcommons.usu.edu/etd/3920.
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Free radicals are short-lived molecules that can cause decreased embryonic development in vitro. Antioxidants are molecules that block free radical formation or guard against their harmful effects. Many studies have linked exposure of media to light and culturing of embryos in high (20%) oxygen concentrations to free radical production. Some of the antioxidants used in culture media are superoxide dismutase (SOD), catalase, zinc (II), ethylenedinitrilo tetraacetic acid (EDTA), mannitol, vitamin E, dimethyl sulfide, and taurine. Most research involving antioxidants and embryonic development has been conducted on non-farm animals, particularly mouse and rabbit. Studies have shown that antioxidants in vitro culture improved embryo development to the blastocyst stage.
In this study, we evaluated the effects of SOD and catalase on bovine embryo development. Four concentrations of SOD (0, 1500, 3000, 6000 IU/ml) and catalase (0, 75, 100, 125 μg/ml) and combinations of the two antioxidants were evaluated through maturation, fertilization, and culture. SOD and catalase were first reconstituted in water and then diluted to their final concentrations. Oocytes were matured in M-199 plus 0.5 μg/ml LH, 5 μg/ml FSH, and 10% FBS at 39°C in 5% CO2 for 24 hours. They were then placed in fertilization-TALP with heparin and 1 x 106/ml sperm. Embryos were cultured in CR2 medium supplemented with alanine, glycine, and 3 mg/ml of fatty-acid free bovine serum albumin in modular incubators with 5% CO2, 5% O2, and 90% N2. Embryo development was evaluated on day 8. Three replicates with approximately 50 embryos per treatment were used to evaluate the effects of SOD and catalase. The control had better embryo development than all treatments. The treatment that was most similar to the control was treatment 2, which consisted of no SOD and 75 μg/ml catalase.
Based on these observations, levels of both SOD and catalase were lowered to 0, 100, 250, and 500 IU/ml and 0, 10, 25, and 50 μg/ml, respectively. Although these levels appeared to improve embryo development, there were no statistical differences. Based on the culture system and media currently used along with the precautions against light and oxygen concentration, we did not find any beneficial effects of supplementing medium with SOD or catalase.
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Byrne, Annette Therese. "Analysis of apoptosis in the preimplantation mammalian embryo." Thesis, University of York, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310908.
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Arnold, Daniel Robert. "Regulation of trophoblast development in the bovine embryo." Thèse, [Montréal] : Université de Montréal, 2005. http://proquest.umi.com/pqdweb?index=0&did=1221731981&SrchMode=1&sid=1&Fmt=6&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1191513626&clientId=48948.
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Thèse (Ph. D.)--Université de Montréal, 2006.Titre de l'écran-titre (visionné le 4 oct. 2007). "Thèse présentée à la Faculté des études supérieures en vue de l'obtention du grade de Philosophiae Doctor (Ph.D.) en sciences vétérinaires option reproduction" Paraît aussi en version papier et en version microforme.
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Lott, Whitney Meghan. "Influence of Growth Factors on Bovine Embryo Development." Thesis, Virginia Tech, 2008. http://hdl.handle.net/10919/34481.
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Many attempts have been made to improve the in vitro production of cattle embryos by refining in vitro maturation (IVM) and culture systems. Cysteine supplementation to IVM media of bovine oocytes increases cellular glutathione production, which reduces reactive oxygen species (ROS). Similarly, beneficial effects of growth factors for improving the rate of blastocyst development have been reported, but combined effects are unknown. This study was conducted to determine the additive effect of the antioxidant cysteine with epidermal growth factor (EGF) and/or insulin-like growth factor-I (IGF-I) on subsequent embryo development. Bovine oocytes from slaughterhouse ovaries were matured in TCM-199 (control), with or without the addition of 0.6 mM cysteine (C) at 0 or 12 h of maturation. After in vitro fertilization, embryos were allocated to culture treatments containing synthetic oviductal fluid medium. Culture treatments included fetal calf serum (FCS, 4%) alone; IGF-I (100 ng/mL); EGF (10 ng/mL); and IGF-I+EGF (100 ng/mL+10 ng/mL) for all IVM treatments. Although rates for blastocysts development were not different among treatments, an increased proportion of embryos attaining morula formation was achieved when cysteine was added to the IVM media (12 h C IGF-I+EGF, 41.4%; 0 h C EGF, 40.0%) as compared to control (FCS: 34.6%). When cysteine treatments were combined, percent cleavage was greater for IGF-I+EGF (70.8%) compared to FCS (61.2%). The abundance of mRNA from the apoptotic genes, Bax and Bcl-2, and the oxidative stress genes, copper (Cu)-zinc (Zn) superoxide dismutase (SOD; SOD1) and manganese (Mn) SOD (SOD2) in embryos was assessed. No significant treatment effect was observed on the expression of apoptotic and oxidative stress genes. Bax was expressed strongly (4-fold) in morulae with the addition of IGF-I, but was less prevalent in all other morula and blastocyst groups relative to FCS. There was slightly less expression of both SOD1 and SOD2 with treatments compared to FCS in morulae and blastocysts, indicative of low mitochondrial activity and/or a low level of oxidative stress in treatments. There was no significant treatment effect on total cell number, apoptotic nuclei, or apoptotic index. In conclusion, supplementation of cysteine during IVM of oocytes, in conjunction with growth factors could effectively be used as a replacement for FCS.Master of Science
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Jimenez, Escobar Claudia. "Association of bovine viral diarrhea virus with day-7 bovine embryos produced under different culture conditions." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2002. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ66111.pdf.
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Al, Darwich Abdulrahman. "Métabolisme lipidique et cryorésistance des embryons dans l’espèce bovine." Thesis, Tours, 2009. http://www.theses.fr/2009TOUR4031/document.
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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 cryorésistanceIn 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
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Kawarsky, Sheldon Jerald. "Effects of elevated temperature on bovine oocytes and embryos cultured in vitro." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0001/NQ40376.pdf.
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Pichugin, Andrey. "Nuclear architecture in normal and cloned bovine early embryos : dynamics of heterochromatin." Versailles-St Quentin en Yvelines, 2008. http://www.theses.fr/2008VERS0005.
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La reprogrammation au cours de la fécondation et du développement pré-implantatoire de l’embryon fécondé in vitro (IVF) chez les mammifères est un processus complexe qui permet le retour à un état indifférencié des cellules hautement spécialisées que sont les gamètes lors de la syngamie, puis leur différenciation progressive en différent lignages cellulaires dans l’embryon. Ces processus comprennent des changements dans la structure de la chromatine, dans les modifications épigénétiques et dans l’architecture nucléaire des noyaux embryonnaires. L’objectif de cette étude était de mettre en place des approches pour la compréhension de ces processus dans les embryons précoces. Nous avons tiré parti de la comparaison entre embryons normaux (IVF) et issus de transfert de noyau (NT) dans l’espèce bovine où le développement pré-implantatoire s’étant sur une période suffisamment longue pour caractériser la dynamique des événements de reprogrammation nucléaire avant la mise en route de l’activation majeure du génome embryonnaire. Dans une première approche, nous avons étudié la mise en place de l’hétérochromatine spécifique à l’embryon au cours de la période de reprogrammation dans les embryons bovins issus de fécondation et de NT. Nous avons utilisé deux marqueurs de l’hétérochromatine péricentrique : la protéine hétérochromatine 1 (HP1-beta) et l’histone H3 tri-méthylée (H3K9me3) ; et un marqueur des centromères (CENP A/B), de façon à caractériser les paramètres structuraux de l’hétérochromatine constitutive dans les premiers stades de développement embryonnaire. En utilisant des techniques d’immunofluorescence, nous avons pu observer les changements dynamiques dans l’organisation de l’hétrochromatine en relation avec l’origine des embryons. Dans les embryons IVF et une partie des embryons NT, l’hétérochromatine apparaît dans un état dispersé, et ceci jusqu’au stade 8-cellule ; puis sous une forme condensée qui correspond à l’association bien caractéristique de blocs d’hétérochromatine et d’H3K9me3 avec les centromères, connues sous le nom de chromocentres. Cependant, une partie significative des embryons NT montrent une altération de la dynamique d’hétérochromatinisation révélée par une condensation précoce, dès le stade 2-cellule, de HP1 et H3K9-me3. Dans une seconde approche, nous avons utilisé des cellules somatiques au stade de la sénescence comm cellules donneuses de noyau de façon à tester l’influence de l’organisation structurale du noyau donneur sur l’organisation structurale des noyaux dans les embryons clonés. Les arrangements tridimensionnels à large échelle de l’hétérochromatine dans les noyaux sénescents NT récapitulent la dynamique observée dans les embryons IVF ou NT issus de noyau de cellules non sénescentes. Notamment, les pourcentages d’embryons avec une hétérochromatine dispersée ou condensée prématurément sont comparables avec les études précédentes. Ces résultats suggèrent l’existence d’un processus de reprogrammation épigénétique robuste piloté par l’ovocyte dans les embryons précoces de bovins. Les cinétiques de restructuration du génome altérées dans une partie des embryons NT pourraient être liées à l’activation précoce de la transcription et l’augmentation du taux de méthylation qui ont été rapportées dans d’autres études. Le présent travail nous a permis de mettre en évidence une distinction entre des embryons NT avec une reprogrammation normale et des embryons NT avec une anomalie, au moins temporelle, de la reprogrammtion épigénétique. Egalement, nous avons démontré que l’impact de l’organisation nucléaire de la cellule donneuse au niveau de l’hétérochromatine sur l’organisation structurale du noyau dans l’embryon NT est relativement modéréDevelopmental reprogramming during mammalian fertilization and pre-implantation development in in vitro fertilized (IVF) embryos is a complex process that allows the highly differentiated gametes to revert to undifferentiated cell types following syngamy and then gradually differentiate into individual cell lineages. These processes involve changes in chromatin structure, in global epigenetic modifications and in nuclear architecture of embryonic nuclei. The objective of the present study was to develop approaches for understanding these series of phenomena in early embryos. We used the possibility to compare normal (IVF) and nuclear tranfer (NT) embryos in bovine species where the pre-implantation development is long enough to assess the nuclear dynamics of reprogramming events before major embryonic genome activation. In a first approach, we studied the establishment of heterochromatin pattern specific for embryo during the reprogramming period in IVF and NT bovine embryos. We applied two markers of pericentric heterochromatin: heterochromatin protein 1 (HP1beta) and tri methylated histone H3 (H3K9me3); and a marker of centromeres (CENPA/B), in order to characterize structural parameters of constitutive heterochromatin in early stages of development. Using immunofluorescence technique, we were able to observe dynamic changes in heterochromatin organization in connection with embryos origin. In IVF and some NT embryos, heterochromatin was observed in dispersed state up to the 8-cell stage, and then in a condensed pattern corresponding to the well characterized chromocenters constituted by blocks of HP1beta and H3K9me3 associated with centromeres. However, a significant part of NT embryos underwent an altered dynamics of heterochromatinization characterized by a precocious heterochromatin condensation as soon as the 2-cell stage. In a second approach, we used senescent somatic cells as donors for nuclear transfer experiments in order to assess the influence of structural organization of donor cell nucleus on structural organization of nuclei in cloned embryos. Surprisingly, the large-scale three-dimensional arrangement of heterochromatin within the senescent-NT nucleus recapitulated the dynamics observed in IVF or somatic non-senescent NT embryos, with comparable percentages of embryos with dispersed and precociously condensed heterochromatin similar to those observed in the previous investigation. These results suggest that a robust process of epigenetic reprogramming is piloted by bovine oocyte in early embryos. The altered kinetics in genome restructuring in some NT embryos might be linked to the precocious transcriptional activation and precocious increase of DNA methylation level reported in other studies. The present work allowed us to point out the distinction between NT embryos with correct developmental reprogramming and abnormal, at least temporally, epigenetic reprogramming, as well as to demonstrate that the impact of nuclear organization of heterochromatin in the donor cell on structural organization of nucleus in NT embryos is relatively low. This may account for the relatively high development efficiency in bovine cloning as compared to other species
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Nociti, Ricardo Perecin. "Transcrição em embriões bovinos produzidos in vitro /." Jaboticabal, 2018. http://hdl.handle.net/11449/157385.
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Orientador: Vera Fernanda Martins Hossepian de LimaResumo: O processo transcricional em embriões extremamente complexo, nosso trabalho estimou o impacto de perturbações nos processos de transcricionais, durante as fases de ativação do genoma embrionário sobre o desenvolvimento embrionário in vitro de embriões; analisamos dados de sequenciamento de rna (RNA-seq) depositados nos bancos públicos (GEO) desde o estágio de oóocito até o dia 19 do desenvolvimento embrionário; Isolamos e caracterizamos a massa celular interna (ICM) e a trofectoderma (TE) do sexo masculino e feminino, oriundos de um mesmo blastocisto produzido in vitro com espermatozoides sexados (X e Y) e com sêmen convencional e caracterizamos e exploramos o transcriptoma desses isolados celulares. Concluímos então que a EGA menor é essencial para o desenvolvimento embrionário bovino, blastocistos possuem a maior atividade transcricional de um total de 6457 genes diferentemente expressos entre os contrastes avaliado encontramos; 2065 genes diferencialmente expressos entre a ICM e a TE, enquanto a ICM está voltada para a manutenção da pluripotência, a TE está voltada ao metabolismo energético. Os nossos dados sugerem que os embriões fêmeas são mais sensíveis ao cultivo in vitro.
Abstract: Transcription process in embryos is a complex process, our work estimated the impact of perturbations in the transcriptional processes during genome activation of vitro produced bovine embryos on their development; we analyzed public data (GEO) from rna sequencing data (RNA-seq) of oocyte up to the 19th day of embryonic development; We’d performed isolation and characterization of male and female inner cell mass (ICM) and trofectoderma (TE) from the same blastocyst produced in vitro with sorted semen (X and Y) and with conventional semen. We did the characterization and exploratory analysis of the transcriptome of these cells. We conclude that minor EGA is essential for bovine embryonic development. Blastocysts possess the highest transcriptional activity of 6457 differentially expressed genes among analyzed contrasts. We found 2065 genes differentially expressed between ICM and TE, while ICM is maintaining pluripotency, TE is focused on energy metabolism. Our data suggest that female embryos are more sensitive to in vitro culture.
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Menges, Suzanne Lynn. "The use of laser-assisted hatching in bovine in vitro produced embryos to improve pregnancy rate." Thesis, [College Station, Tex. : Texas A&M University, 2008. http://hdl.handle.net/1969.1/ETD-TAMU-3049.
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Nganvongpanit, Korakot. "Functional analysis of genes during bovine preimplantation embryo development." [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=980875153.
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Gopichandran, Nadia. "The development and metabolism of the bovine preimplantation embryo." Thesis, University of York, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.428455.
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Ferreira, Marcos Brandão Dias [UNESP]. "Obtenção de oócitos e produção in vitro de embriões em doadoras lactantes da raça Gir (Bos taurus indicus)." Universidade Estadual Paulista (UNESP), 2011. http://hdl.handle.net/11449/105910.
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ferreira_mbd_dr_jabo.pdf: 786611 bytes, checksum: 3c4c4423ce0ad7536fd2c581e6e11536 (MD5)Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)
Raças zebuínas (Bos taurus indicus) e seus cruzamentos têm papel fundamental na pecuária brasileira, e a raça Gir, em especial, acrescenta rusticidade e produtividade nas suas descendentes leiteiras. A produção in vitro de embriões bovinos é uma biotécnica de alto valor econômico, que, aliada à utilização de sêmen sexado para cromossoma X, possibilita a multiplicação com fêmeas de valor genético superior. Foram realizados dois experimentos com o objetivo de avaliar a produção in vitro (PIV) de embriões de doadoras da raça Gir na Fazenda Experimental da EPAMIG, em Uberaba, MG. O experimento 1 (EXP 1) visou verificar o efeito da ablação do folículo dominante sobre os resultados da PIV. No experimento 2 (EXP 2) os efeitos da estação do ano, idade da doadora, DNA mitocondrial materno e efeito do touro sobre a PIV foram estudados. No EXP 1, 42 multíparas e primíparas foram submetidas à aspiração de oócitos (OPU) a partir dos 15 dias pós-parto e a intervalos de 21 dias, sendo efetuada (65 sessões de OPU) ou não (115 sessões de OPU) a ablação do folículo dominante 3 dias antes da aspiração. Das 198 aspirações realizadas foram coletados 3.884 oócitos viáveis, que resultaram em 1.114 blastocistos. O número médio de oócitos viáveis aspirados e de blastocistos por sessão foi de 20,0 ± 10,6 e de 5,70 ± 4,9, respectivamente. Não foram identificados efeito da ordem de parto e aspiração, do dia após o parto para início da coleta e da condição corporal da doadora nos resultados da PIV. No entanto, a produção de blastocistos por sessão foi superior (5,65±1,02 vs. 3,78±0,97) nas vacas que sofreram ablação do folículo dominante (p<0,05). No EXP 2 foram avaliadas 363 aspirações de 85 doadoras fertilizadas com 23 touros diferentes que geraram 6.084 oócitos viáveis, 2.537 embriões, 1.105 gestações...
Zebu breeds (Bos taurus indicus) and its crosses have an essential role on the Brazilian cattle industry, and the Gyr breed, especially, incorporates hardiness and productivity onto its dairy descendants. The in vitro production of bovine embryos is a biotechnique of high economic value, which, combined to the use of sex-sorted semen bearing X chromosomes, allows for the multiplication of superior genetic value dams. Two experiments were conducted to evaluate the in vitro production (IVP) of embryos from Gyr donor cows at the EPAMIG Research Farm in Uberaba, MG, Brazil. Experiment 1 (EXP 1) aimed to verify the effect of ablation of the dominant follicle on IVP results. In experiment 2 (EXP 2), the effects of season, donor age, mitochondrial DNA and sire on IVP were studied. In EXP 1, 42 multiparous and primiparous cows underwent ovum pick up (OPU) after removal (65 OPU sessions- DFR) or non removal (115 OPU sessions- Control) of the dominant follicle 3 days before aspiration, starting from 15 days postpartum at approximately 21 day-intervals. Of the 180 OPU sessions performed, 3,884 viable oocytes were collected, which resulted in 1,114 blastocysts. The overall average numbers of oocytes aspirated and viable blastocysts per session were 20.0 ± 10.6 ± 4.9 and 5.70, respectively. OPU session, parity, calving to OPU interval and donor body condition did not influence IVP results. However, the production of blastocysts per session was higher (5.65 ± 1.02 vs. 3.78 ± 0.97) in DFR-cows (P <0.05). In EXP 2, a total of 363 OPU sessions from 85 donors was studied. Semen from 23 different bulls was used for in vitro fertilization, yielding 6,084 viable oocytes, 2,537 embryos and 1,105 pregnancies overall. Thirty and sixty-day day pregnancy rates after embryo transfer were 41.7% and 39.5%, respectively... (Complete abstract click electronic access below)
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Bruyère, Pierre. "Evaluation thermodynamique et biologique d’un substituant synthétique aux produits d’origine animale dans les solutions de cryoconservation pour embryons de mammifères." Thesis, Lyon 1, 2012. http://www.theses.fr/2012LYO10145/document.
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Plusieurs composés présents dans les solutions de cryoconservation pour embryons sontsource de préoccupations : soit du point de vue sanitaire à cause de leur origine animale, soitdu fait de leur rôle potentiellement mutagène, notamment pour certains cryoprotecteurspénétrants. Leur retrait ou leur substitution par des composés chimiquement définis pourraitdonc constituer une amélioration sensible des techniques de cryoconservation des embryons.C’est dans ce cadre de réflexion que s’inscrit notre travail.Souvent, la conception et la mise en oeuvre des protocoles de cryoconservation sontempiriques. Il en résulte de nombreuses variations entre les études qui rendent lacomparaison des résultats obtenus d’autant plus difficile. Dans notre démarche, deuxapproches complémentaires ont été associées :•La première, physique, s’appuie sur l’utilisation de la calorimétrie différentielle à balayage(DSC) pour standardiser la comparaison des différentes solutions de congélation lente.Ainsi, les propriétés thermodynamiques de solutions contenant un substituant défini ontété caractérisées et comparées à celles de solutions contenant des produits de référence(sérum de veau foetal ou albumine bovine sérique) ;• La seconde, biologique, a consisté à congeler des embryons de lapins produits in vivo etdes embryons bovins produits in vitro, puis à analyser les taux de survie après culture invitro et/ou transferts. Cette approche a permis d’objectiver les propriétés biologiques dessolutions ainsi définies.Nos résultats confirment qu’il est judicieux d’utiliser une approche thermodynamiquepour sélectionner des molécules chimiquement différentes des composés habituellementutilisésSeveral compounds in embryo cryopreservation solutions are a source of concern:products of animal origin because of the sanitary risks, and the permeating cryoprotectantsbecause of their potential mutagenic effect. Removing or substituting these compounds withchemically defined products might improve embryo cryopreservation technics.Conception and use of cryopreservation protocols are often empirical. This empiricismleads to many variations between the studies which make a comparison between results allthe more difficult. In our study, two complementary approaches were associated:• The first approach (physical) consisted of using the differential scanning calorimetry tostandardize the comparison between different slow-freezing solutions. So, thethermodynamic properties of solutions containing a potential substitute were characterizedand compared to those obtained with solutions containing reference products (fetal calfserum and bovine serum albumin) ;• The second approach (biological) consisted of using freezing of in vivo-produced rabbitembryos or freezing of in vitro-produced bovine embryos in order to evaluate survival
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Kramer, Joseph Michael. "Gene expression in bovine in vitro produced embryos, nuclear transfer-derived embryos, and donor cells using real-time reverse transcription polymerase chain reaction." [Gainesville, Fla.] : University of Florida, 2005. http://purl.fcla.edu/fcla/etd/UFE0010362.
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Bastos, Michele Ricieri [UNESP]. "Influência da ingestão de matéria seca e da condição corporal na produção in vitro de embriões bovinos." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/98215.
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bastos_mr_me_botfmvz.pdf: 790909 bytes, checksum: 06b5f7cf1b70d4316f61ff63d1907d62 (MD5)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Objetivou-se avaliar o efeito da condição corporal e/ou da alta ingestão de matéria seca (AIMS) na produção in vivo de embriões de fêmeas bovinas superovuladas. Em um primeiro experimento, 14 vacas Simental x Nelore não-lactantes com elevado escore de condição corporal (ECC) foram divididas em grupos de Manutenção=M ou alta ingestão de matéria seca=AIMS. As vacas do grupo AIMS receberam dieta com 180% da manutenção, entre 7 dias antes do início da superovulação (SOV) e o final das aplicações de FSH. O grupo M recebeu dieta de manutenção. O número de folículos recrutados e ovulados não diferiu entre os grupos (P>0,10). Entretanto, os números de estruturas totais e embriões viáveis colhidos foram maiores no grupo M (P<0,05). Em um segundo experimento avaliou-se a influência do ECC associado ou não da AIMS na produção embrionária em 36 novilhas Nelore. AIMS ocorreu por 14 dias antes do início da SOV. Após colheita, os embriões viáveis foram congelados para posterior cultivo até eclosão. Não houve diferença entre os grupos na população folicular ao início da SOV, na resposta superestimulatória ou superovulatória, nem no número ou qualidade dos embriões colhidos. As novilhas com
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Flood, Mark Randall. "Effect of Various Growth-Promoting Factors on Preimplantation Bovine Embryo Development in Vitro." DigitalCommons@USU, 1992. https://digitalcommons.usu.edu/etd/4044.
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The purpose of this research was to define the effects of various growth-promoting factors on in vitro embryonic development of in vitro matured and in vitro fertilized bovine embryos. The control medium was a chemically defined medium which improves the possibility of closely determining the in vivo conditions the embryo is actually exposed to. The growth-promoting factors tested in this experiment included transferrin, IGF-I (insulin-like growth factor-one), IGF-II (insulin-like growth factor-two), TGF-a (transforming growth factor-alpha) , TGF-B1 (transforming growth factor-beta1) , PDGF (platelet derived growth factor), EGF (epidermal growth factor), NGF (nerve growth factor), and bFGF (basic fibroblast growth factor). Transferrin was included at 10 micrograms/milliliter , while all other factors were utilized at 10 nanograms/milliliter in the control medium.
Bovine cumulus-oocytes were retrieved from slaughterhouse ovaries and were matured i n Medium-199 containing 10% feta l bovine serum for 24 hours at 39°C in a 5% C02 atmosphere. Frozen-thawed bull spe r m were s wim-up separated and capacitated in medium containing heparin for 3 hours prior to insemination. Gametes were co- incubated fo r 18 hours and then cumulus cells were stripped from the ova. Ova which did not cleave were removed from culture 36 hours after insemi nati on and were stained for evidence of fertilization. Embryos were cultured in one of the 10 conditions (including control) described above. A total of 150 total oocy.t.es were cultured per treatment for a tota l of 10 days. EGF improved embryo development, while TGF-Bl and TGF-a only slightly improved embryo development compared to the control. All other factors tested did not have a beneficial effect on embryo development in this culture medium.
In summary, EGF improved in vitro development of bovine embryos obtained from in vitro maturated and in vitro fertilized bovine oocytes. Other factors which were t est ed did not significantly improve in vitro bovine embryo development. Further experiments are necessary fo r determining the requirements of bovine embryos in vitro.
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Moreira, Vanessa. "Improving biosecurity of bovine in vitro embryo production and cryopreservation." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=25708.
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Harvey, Alexandra Juanita. "Expression of hypoxia-inducible factors during bovine preimplantation embryo development /." Title page, abstract and table of contents only, 2003. http://web4.library.adelaide.edu.au/theses/09PH/09phh3410.pdf.
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Ferguson, Elizabeth Mary. "Endogenous energy stores in the bovine oocyte and early embryo." Thesis, University of York, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.423763.
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Sedano, Rodolfo Canseco. "Effect of immunoglobulins on early bovine embryo development in vitro." Thesis, Virginia Tech, 1985. http://hdl.handle.net/10919/41575.
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Bovine morulae (day 6: n=257) were obtained to evaluate
the [effect of immunoglobulins (Ig) on early bovine embryo
development in vitro. Fifty-four cows superovulations were
conducted in 36 cows with follicle stimulating hormone.
Embryos were collected by non-surgical procedures and
morphologically evaluated and randomly assigned to culture.
Embryos were cultured in Ham's F-10 containing 10%
(6.4 mg/ml) steer serum (SS), 1% (.64 mg/ml) bovine gamma .
globulins (GG), 1% (.64 mg/ml) bovine IgG, 1% (.64 mg/ml)
bovine 1gM, 10% SS plus 1% GG, 10% SS plus 1% 1gG, or 10% SS
plus 1% 1gM. Embryos were cultured to the hatched blastocyst stage or degeneration and evaluated at 12 h intervals.Master of Science
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Cordova, Amanda. "Co-culture d'embryons bovins et de cellules épithéliales d'oviducte : un modèle in vitro pour la compréhension du dialogue embryo-maternel précoce." Thesis, Tours, 2013. http://www.theses.fr/2013TOUR4030.
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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’oviducteThe 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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ANJOS, Rafael Soares dos. "Produção in vitro de embriões e expressão gênica de IGF-I e IGF-II em embriões de fêmeas bovinas da raça nelore submetidas a administrações de somatotropina recombinante bovina." Universidade Federal Rural de Pernambuco, 2014. http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/5064.
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In vitro production of bovine embryos (IVP) is a tool that serves to increase reproductive potential of females considered exceptional, especially, as an instrument to accelerate the progress of animal selection programs. In order to improve results, exogenous synthetic hormones are being used routinely in the animal reproduction industry. Currently there is a research using recombinant bovine somatotropin (rbST), as a substance that causes the increase in receptors for insulin-like growth factor (IGF). Both IGFs type I and type II and its receptors (IGF-IR and IGF-IIR) are expressed in in vitro produced bovine and ovine oocytes and embryos from which one can prove the close relation of IGF system with the IVP. Researches have proven a trend in oocyte quality improvement in animals treated with rbST, as well as increased fertilization rate and embryo quality improvement. This experiment aimed to study the effect of rbST on quantity and quality of in vivo aspirated oocytes, embryo production and gene expression of IGF-I and IGF-II in vitro produced embryos of bovine females of Nelore breed. Five cows were treated with two administrations of 2mL of saline solution as a placebo, with an interval of 14 days between them, being the first administration performed 19 and the second 5 days before follicular aspirations; 30 days after these aspirations, in crossover system, same protocol was used in these five animals being, in this case, the saline solution replaced by 500mg of rbST in order to compare treatments. It was held to IVP of oocytes recovered from this research, being all the steps that involve this process evaluated with subsequent achievement of polymerase chain reactions in real time (qPCRs) for gene expression of IGF-I and IGF-II in the blastocysts produced in the experiment. No difference was observed on quantity and quality of aspirated oocytes in vivo, embryo production and gene expression of IGF-I and IGF-II in vitro produced embryos of bovine females of Nelore breed under the influence of rbST.
A produção in vitro de embriões (PIVE) bovinos constitui-se em uma ferramenta que serve para aumentar o potencial reprodutivo de fêmeas consideradas excepcionais, principalmente, como instrumento para acelerar o progresso dos programas de seleção animal. Com o intuito de melhorar taxas e índices, sintéticos exógenos vêm sendo utilizados corriqueiramente na área da reprodução animal. Pesquisa-se atualmente a utilização da somatotropina recombinante bovina (rbST), tida como substância que acarreta o aumento de receptores para o fator de crescimento semelhante à insulina (IGF). Tanto os IGFs tipos I e II como seus receptores (IGF-IR e IGF-IIR) são expressos em oócitos e embriões bovinos e ovinos produzidos in vitro - o que pode comprovar a relação do sistema IGF com a PIVE. Estudos tem comprovado tendência em melhora da qualidade oocitária nos animais tratados com rbST, bem como aumento da taxa de fecundação e melhoria na qualidade de embriões. Este experimento objetivou estudar a influência da rbST sobre a quantidade e qualidade de oócitos aspirados in vivo, produção embrionária e expressão gênica de IGF-I e IGF-II em embriões produzidos in vitro de fêmeas bovinas da raça Nelore. Cinco vacas foram tratadas com duas administrações de 2mL de solução salina como placebo, com intervalo de catorze dias entre elas, sendo a primeira administração realizada dezenove e a segunda cinco dias antes das aspirações foliculares; decorridos trintas dias após estas aspirações, em sistema crossover, o mesmo protocolo foi utilizado nesses cinco animais sendo, neste caso, a solução salina substituída por 500mg de rbST, com o intuito de efetuar-se comparações entre os tratamentos. Realizou-se a PIVE a partir dos oócitos recuperados nesta pesquisa, sendo todas as etapas que envolvem este processo analisadas e com posterior realização de reações em cadeia da polimerase em tempo real (qPCRs) para expressões gênicas de IGF-I e IGF-II nos blastocistos produzidos. Observou-se que a rbST não exerceu influência significativa sobre a quantidade e qualidade de oócitos aspirados in vivo, produção embrionária e expressão gênica de IGF-I e IGF-II em embriões produzidos in vitro de fêmeas bovinas da raça Nelore.
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Pavani, Krishna Chaitanya. "Optimization of a specific messenger RNA extraction protocol for fresh and vitrified bovine oocytes to gene expression studies : Specific mRNA extraction protocol for bovine oocytes." Thesis, University of the Azores, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-6900.
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To understand bovine oocytes meiotic maturation, developmental potential, gene expression is required. The gene expression studies in the preimplantation bovine oocytes has been difficult, because the procedures that are being employed for extracting total RNA are not specific for bovine oocytes and so far is not providing the required amount for further procedures. Quantification of genes generally requires large amounts of total RNA in order to overcome the problem of low amount of mRNA present, so a standardized specific protocol is recommended. These days most of the researchers are using commercial Kit protocols without knowing the significance of chemicals and how they are acting on cells. In present project a standardized protocol (modified trizol) was designed for bovine oocytes, which was specific and less expensive. The efficiency of this protocol compared with Pure Link (Kit Protocol), GNTC (Guanidinium thiocyanate) for extraction of total RNA from fresh oocytes, vitrified oocytes with PROH (1,2 propanediol) and DMSO (dimethylsulfoxide) cryoprotectans was much better. The RNA (absorbance 260/280) purity levels of the standardized protocol was ranging (1.50-2.10), whereas for GNTC protocol (1.05-1.36), Pure Link (kit protocol) (2.05-2.7). Amplification of housekeeping genes (SDHA and GAPDH gene) showed the specificity and efficiency of the standardized protocol over other protocols.
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Tejomurtula, Jyothsna. "Identification of a novel importin [alpha] predominantly expressed in bovine oocytes and early embryos." Morgantown, W. Va. : [West Virginia University Libraries], 2007. https://eidr.wvu.edu/etd/documentdata.eTD?documentid=5488.
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Thesis (M.S.)--West Virginia University, 2007.Title from document title page. Document formatted into pages; contains vi, 45 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 40-45).
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Reis, Alexandra. "Fatty acid and antioxidant effects on development in vitro of bovine and ovine embryos." Thesis, University of Aberdeen, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401191.
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This thesis provides evidence that in vitro produced ruminant embryos accumulate lipid during culture in the presence of serum; the increase due to serum in the area occupied by lipid droplets was 30 to 40% (P<0.001). Innovative HPLC techniques revealed similar increases in fatty acid content of bovine (from 57 to 74 ng embryo-1, P<0.05) and ovine (from 73 to 84 ng embryo-1, P<0.001) embryos when foetal calf serum (FCS) was used as a media supplement. For ovine embryos, their total fatty acid content reached 130 ng embryo-1 in a polyunsaturated fatty acids (PUFA)-enriched medium (P<0.001). Serum-exposed embryos benefited from supplementation of their culture medium with Vitamin E, an inhibitor of lipid peroxidation. Despite a >20% increase in the total fatty acid content of embryos cultured in serum-supplemented medium, Vitamin E significantly improved blastocyst yields and cell proliferation rates. For bovine embryos cultured with FCS-supplemented medium, yields increased from 20 to 33% (P<0.001) and, for ovine embryos in a PUFA-enriched medium, from 47 to 64% (P<0.001). It was only when PUFA-enriched serum was used in the culture of ovine embryos, that Vitamin E prevented a decline in pyruvate oxidation (P<0.05), indicating maintenance of mitochondrial activity. It also precluded increased generation of H2O2 and lipid peroxides (P<0.05). Lipid accumulation increased from 111 to 130 ng embryo-1 when ovine embryos were cultured in the presence of albumin supplemented with docosahexaenoic acid (DHA, C22:6n-3, P<0.05). DHA increased the production of peroxides (P<0.001). It also inhibited embryo development, completely, beyond the 9- to 16-cell stage (DHA concentration = 1.7 mg ml-1 medium) and partially when its concentration was lowered (0.5 mg ml-1 medium, P<0.001). Partial inhibition involved a reduction in cell proliferation (P<0.05) and pyruvate oxidation (P<0.001) and increased apoptosis (P<0.05), features avoided by inclusion of Vitamin E or Trolex, a water-soluble analogue, in the culture media.
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Orozco, Lucero Ernesto. "Role and modulation of maternal transcripts during the first cleavage divisions in bovine embryos." Doctoral thesis, Université Laval, 2016. http://hdl.handle.net/20.500.11794/27002.
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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.